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Journal of Drug Delivery and Therapeutics

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Open Access  Full Text Article                                                                                                                                                                                     Research Article 

GCMS and FTIR analysis of ethanol and methanol leave extract of Urena lobata (Caesar weed) for bioactive phytochemical constituents

Collins Obinna Keke*¹, Winifred Njideka Nsofor¹, Francis Koku Ras Kumabia², Godian Chima Iloabuchi¹, Juliet Chioma Ejiofor¹, Olachi Lilian Osuagwu¹

¹ Department of Biochemistry, School of Biological Sciences, Federal University of Technology, Owerri, Imo State, Nigeria  

² Department of Science Education, St. Francis College of Education, Hohoe, Ghana

INTRODUCTION

Plants, both in their natural state and as extracts, have provided significant benefits to human health for centuries. The pharmacological benefits of these plants come from the wide variety of secondary and primary metabolites they contain, each of which has its own complicated chemical makeup and is responsible for their pharmacological effects¹. For their low or nonexistent price tags and lack of negative side effects, natural remedies routinely top the list of those recommended over today's synthetic pharmaceuticals. The chemical make-up of these plants is useful not only in traditional therapy but also in the characterization and finding of novel sources of phyto-components for pharmacological research and development^{2, 3.} Recently, the use of Gas chromatography-mass spectrometry and FTIR spectroscopy for target identification of functional groups and determination of these different phyto components that are present in medicinal plants even in small concentrations has expanded substantially ¹. GC/MS remains superior among effective, rapid, and precise methods for identifying plant-based substances such as amino acids, alkaloids, esters, long-chain hydrocarbons, steroids, organic acids, nitro compounds and alcohols^4,3.

The sub-shrub Urena lobata is a member of the Malvaceae family. They attain heights of 0.6-3m and diameters of 7cm at the base⁵. The plant is mostly found in the tropics and sub-tropics of South and North America, Philippines, Asia, Indonesia as well as in several African nations like Nigeria, Senegal, Ghana and the Democratic Republic of the Congo⁶. In Nigeria, U. lobata is also popular and known with different local names among different ethnic groups. The Igbos of Eastern Nigeria particularly those in Imo state call it Achara /Udo⁷the Hausas call it Rama-rama, in Efik, it is known as Rideri, the Binis and some other parts of Edo state call it Oronhon while the Yorubas of Western Nigeria call the plant different names such as; Ilasa-omode, Akeri, Ilasa-agbunrin⁸. U. lobata is employed in forkloric medicine in the treatment of dysentery, hematemesis, edema, carbunicle, bleeding due to trauma, leucorrhea, gonorrhea, fever, pain, cold, numbness resulting from rheumatism and other disease conditions⁹. The inhabitants of Nigeria's Katsina State utilize the leaves and petals of this plant to make local delicacies and also offer them to lactating women to increase their milk supply¹⁰. Numerous studies have reported significant pharmacological activities of various extracts of this plant parts to include anti-inflammatory¹¹, anti-diabetic¹², anti-tumor¹³,antimicrobial¹⁴, anti-oxidant and cytotoxic¹⁵,wound healing effect¹⁶, anti-diarrheal¹¹, neuro-pharmacological activities¹⁷, anti-fertility in female rats as a result of reduced myometrial and epimetrial thickness accompanied by reduced uterine diameter¹⁸as well as reversibly inhibiting spermatogenesis and steroidogenesis serving as  male contraceptives¹⁹.

In order to better understand the therapeutic effects of Urena lobata, this study used GC/MS and FTIR to identify the many bioactive phytoconstituents and functional groups present in ethanol and methanol extracts of the plant.

MATERIALS AND METHODS

Collection and processing of plant materials

Healthy and mature leaves of the plant were freshly picked from a farm in Umuode village in Osisioma Ngwa L.G.A of Abia state, Nigeria and identified as Urena lobata.

Extraction of plant material

The picked leaves underwent a thorough cleaning with distilled water before being air dried for two weeks. Using an electric blender (ES-BL-090/350W/220-240V/50Hz/China), the dry leave materials were crushed into a fine powder. Soxhlet extraction was performed on the dried and powdered materials using ethanol and methanol as extraction solvents. A rotary evaporator(Heidolph Rotavapor, Germany) was used to filter and concentrate the extracts. When the dried extracts were ready for analysis, they were put into airtight containers, corked, and kept in a refrigerator at 4°C.

Gas Chromatography –Mass Spectrometry (GC-MS) Analysis

Using a BUCK M910 Gas chromatograph with an HP-5MS column (30 m in length, 250μmin diameter, and 0.25μm in film thickness), we were able to conduct the GC-MS analysis of the bioactive components. High-energy electrons were used in a system of electron ionization for spectroscopic detection by GC-MS (70 eV). Helium gas, purified to 99.995% purity, was employed as the carrier gas at a flow rate of 1 mL/min. We started with a temperature of 50°C, ramped it up by 3°Cevery minute, and held it there for around 10 minutes.

At last, a 10°C/min increase in temperature brought the total to 300 °C. In splitless mode, 1μm of the produced 1% extracts diluted with acetonitrile was injected. Based on the chromatographic peak area, the relative amount of bioactive chemicals in each extract was calculated and expressed as a percentage. Based on GC retention time on HP-5MS column and spectrum matching with software data of standards, bioactive chemicals were identified from various extracts (Replib and Mainlab data of GC–MS systems). The name, molecular weight and structure of the compounds of the test extracts were determined 

Fourier Transform Infrared Spectroscopic (FT-IR) Analysis

A Buck Scientific M530 USA FTIR was used for the analysis. A deuterated triglycine sulfate detector and a potassium bromide beam splitter were used in this device. The spectra were acquired and adjusted with the help of the Gram A1's software. About 1.0g of samples and 0.5ml of nujol were used; they were carefully mixed and then placed on the salt pellet. FTIR spectra were acquired during the measurement in frequency ranges of 4,000 - 600 cm-1 and co-added at 32 scans and 4 cm-1 resolution. Transmitter values were shown as FTIR spectra.²⁰.

RESULTS AND DISCUSSION

Among the 41 phytocompounds identified by GC-MS in the ethanol leave extract of U. lobata (Table 1), the following were found to be most predominant: 9-Octadecenoic acid, (E)-(16.80%), Dodecanoic acid (13.43%), n-Hexadecanoic acid (11.73%), Octadecanoic acid (9.78%), 1-Docosene (9.57%), Cyclohexadecane, 1,2-diethyl-(4.85% and  2.03%).), Oleic acid (4.74%), Phenol,3,5-bis(1,1-dimethylethyl) (2.42%), Tetradecanoic acid (2.14%). Conversely, the least abundant bioactive phytocompounds in the extract were Nonadecyltrifluoroacetate (0.12%), 6,11-Dimethyl-2, 6,10-dodecatrien-1-ol (0.16%), Decane (0.19%), Dodecyl acrylate (0.22%), 1-Eicosanol (0.22%) and Methyl stearate (0.24%). In a similar study, ⁷⁶Fadillah and his groupfrom Jawa Barat, Indonesia investigated the active compounds in both 50% and & 70% ethanol extracts of U. lobata. Their findings revealed 5 and 17 bioactive compounds in 50% and 70% ethanol extract of U. lobata leaves respectively. They went further to report high concentration of n-Hexadecanoic acid (19.97%) as well as Oleic acid (9.42%) in 50% ethanol leave extract of U. lobata. Again, the most prevalent bioactive constituent in the 70% ethanol leave extract of U. lobata was discovered to be n-Hexadecanoic acid (18.91%).These findings are comparable with ours, particularly in methanol extract which had, n-Hexadecanoic acid (26.65%) as the most abundant bioactive compound, nevertheless, their study revealed the presence of other bioactive phytocompounds which were not identified in ours and vice versa. As demonstrated in table 5, ethanol leave extract of U.lobata has been shown to contain a number of biologically active phytocompounds with diverse biological and pharmacological effects. 9-Octadecenoic acid has been reported to exhibit antimicrobial activities³¹. 

Dodecanoic acid (also kmown as lauric acid), a medium-chain as well as  saturated fatty acid found abundantly in oils of coconut and palm kernel is known to exhibit antibacterial and antioxidant activities³². Among many other bioactivities, n-hexadecanoic acid (also known as palmitic acid) has inflammatory, antioxidant, hypocholesteromic, and cancer-preventive properties38. Some common and natural sources of n-Hexadecanoic are palm oil, butter, palm kernel oil, milk, meat and cheese. 1-Docosene has antibacterial activities⁴³. Albrathy also reported the antimicrobial activities of Oleic acid, a common monounsaturated fatty acid seen naturally in numerous animal an vegetable fats and oil and in plants such as Gladiolus italicus and Prune mume⁴³. 

In addition to the above mentioned biological roles, oleic acid has been shown to have antifungal, anti-inflammatory, antioxidant, and antibacterial properties⁴¹. The phenolic compound, Phenol,3,5-bis(1,1-dimethylethyl) possesses antibacterial and antimicrobial activities^30,29.Least abundant bioactive compounds such as; 1-Eicosanol, methyl stearate, Dodecyl acrylate and 6,11-Dimethyl-2,6,10 dodecatrien-1-ol are not exempted  in exhibiting different biological activities as many researchers have also demonstrated their biological activities. For example, 1-Eicosanol, an alcohol has been reported to possess anti­malarial, antifungal, and antioxidant activities³¹while Methyl stearate has proven to be a potent anti-inflammatory,intestinal lipid metabolic regulator, GABA aminotransferase inhibitor,antinociceptive, anthelmintic, and a potent gastric inhibitor⁴². On the other hand, Dodecyl acrylate, a typical acrylate ester is known to be antioxidant and antimicrobial³⁶while 6,11-Dimethyl-2,6,10 dodecatrien-1-ol has been researched to possess antimicrobial activities⁴⁶.

The most bioactive phytocompounds (47) were found in the methanol extract of U. lobata. This was illustrated by the GC-MS chromatogram of the methanol extract, which is shown in Figure 2.The following were the prevailing bioactive phytocompounds identified; n-Hexadecanoic acid (26.65% and 9.11%), Dodecanoic acid (6.89%), 1-Docosene (6.06%), Erucic acid(4.09%), 1-Octadecene (3.54%), 9-Octadecenoic acid (3.45%), Tetradecanoicacid (3.01%) and Diisooctylphthalate (2.97%).The least abundant compounds were; 3-Eicosene (2.97%), 6-Octadecanoic acid methyl ester(0.14%), Dodecane(0.15%), 1-Docosene(0.17%), Trifluoroacetic acid pentadecyl ester (0.18%), Cyclohexane, 1,1`-(1,4-butanediyl)bis (0.22%), Tridecanoic acid,12-methyl-methyl ester ( 0.22%) and Cyclotetradecane (0.23%).These bioactive compounds and many more in methanol extract of U. lobata have been reported to possess many biological activities just like those identified in ethanol extract of the leaf . One of them is Erucic acid; an omega-9 and monosaturated fatty acid that is common among brassicaceae family of flowering plants has been reported to regulate mesenchymal stem cell differentiation into osteoblasts and adipocyte⁷². Another study reported it to have a broad-spectrum antiviral activity against Influenza A. Virus (IAV), anti-inflammatory and pro-inflammatory amplification effect as well as the ability to inhibit NF-κB and p38 MAPK⁷³. 1-Octadecene, a long –chain hydrocarbon and an alkene which has been found in appreciable quantity in Vacciniummacrocarpon, a North American variant of cranberry known to be used by women in the treatment of recurrent urinary tract infection has also shown excellent antibacterial, antioxidant and anticancer activities^39,59.60. 9-Octadecenoic acid has been reported to possess antimicrobial activities³¹.An ample amount of 9-Octadecenoic acid is found naturally in Eleutherococcus sessiliflorus and Dipteryxlacunifera, an oleaginous leguminous plant native to Piaui and Maranhao state in North East of Brazil⁷⁷. Diisooctyl phthalate has antimicrobial, antifouling⁷⁴and antibacterial activities⁷⁵. 3-Eicosene has also shown to be antimicrobial, antihyperglycemic, cytotoxic, antioxidant and insecticidal³⁵. Another fatty acid methyl ester (FAME) that has been shown to have important biological activities is 6-octadecanoic acid methyl ester. It has been researched to possess strong analgesic, anti-inflammatory and antipyretic activities⁶⁸. Pantadecanoic acid, 9-Hexadecanoic acid and Heptadecanoic acid are among the fatty acids uniquely found in methanol extract of U.lobata leaves with tremendous biological significance. Pantadecanoic acid, an essential fatty acid found in butter whose biological activities promote long-term metabolic and cardiovascular health⁷⁸has been shown to possess anticancer activity⁶¹. 9-Hexadecanoic acid from methanol extract of Tribolium castaneum also showed good antibacterial activity against Escherichia coli, a Gram-negative, enteropathogenic bacterium implicated in diarrheal disease and urinary tract infections⁶⁵. Heptadecanoic acid has antioxidant properties⁶⁶.

Tables 3 and 4 show the findings of the FTIR investigation of the ethanol and methanol leaf extracts of U. lobata.FTIR, a vibration spectroscopic technique⁷⁹is renowned for its ability to pin-point important functional groups embedded in plant extracts, biological and synthetic compounds. For identifying types of bonds (functional groups) in compounds, FT-IR remains unmatched⁸⁰. In this study, the FTIR spectra of ethanol and methanol extract shown in figure 3 and 4, unraveled twelve peaks indicative of 12 functional groups. For the ethanol extract, the peaks are in the range of 3704.812, 3498.917, 3181.879, 3013.238, 2782.866, 2665.173, 2571.721, 222.731/2018, 1622.32, 1416.411, 1235.417 and 843.5038 cm^-1 whereas the peaks of the methanol extract are in the range of; 3664.219/3415.311,  3155.608, 3056.931, 2915.574, 2814.952, 2500.399, 2117.752, 1830.701, 1614.028, 1393.314, 1295.721 and 852.1639cm^-1. The 12 functional groups revealed in ethanol extract of the leave are: alcohols/phenols, aromatic amines, primary amides, aromatic/unsaturated hydrocarbons, ether/amine, aldehydes, phosphorus oxyacids, alkynes, primary amines and trisubstituted benzene. In the methanol extract, the functional groups revealed were: alcohols/phenols, amino acids, aromatic/saturated hydrocarbons, aliphatic compounds, hydrohalides, isonitriles, β-lactones, vinyl ethers, t-butyl groups, sulfones and primary amines. The following vibrational functional groups: alcohols/phenols, aromatic/unsaturated hydrocarbons, ether/amine are all common to both extracts and produce, OH stretch, =CH-H stretch and CH stretch respectively. With its hydrogen-bonding capabilities, the OH group is likely responsible for the inhibitory effect against pathogenic microbial agents observed in both methanol and ethanol extract of the leave⁸⁰.

Figures 5 and 6 illustrate, respectively, the percentage abundance of the classes of bioactive chemicals found in the ethanol and methanol extracts. In the ethanol extract, fatty acids had a total abundance of 57.58%, alkene, 12.58%, cycloalkane, 11.62%, monounsaturated fatty acids (MUFA), 6.78% while alkane had a total abundance of 4.31%. The least abundant class of bioactive compounds was acrylate ester (0.22%) and alcohol (0.22%). For the methanol extract, as seen in ethanol extract, fatty acid has a total abundance of 56.15%, followed by alkene (20.01%), MUFA (10.02%), phthalate esters (2.97%) and delta-lactams (2.66%). The least abundant class of bioactive compounds were cycloalkane (0.8%) and fatty acid esters (0.18%).

Table 7 showed the bioactive compounds common to both ethanol and methanol extracts of U. lobata.The fatty acids were more prevalent in the methanol extract (26.65%) than in the ethanol (11.73%), including n-Hexadecanoic acid, 9-Octadecenoic acid, Dodecanoic acid, and Tetradecanoic acid. In ethanol extracts, 9-octadecenoic acid predominated (16.8%) compared to methanol extracts (3.45%).Dodecanoic acid was prevalent in ethanol extract (13.43%) compared to the methanol extract (6.89%). The varied concentrations of these bioactive compounds in the two extracts underscored the importance of choosing the right extraction solvents during research bearing in mind the bioactive compound of target.

Figure 1: GC-MS Chromatogram of ethanol extract of U. lobata leaves

Figure 2: GC-MS Chromatogram of methanol extract of U. lobata leaves

TABLE 1: BIOACTIVE COMPOUNDS PRESENT IN  U. LOBATA   ETHANOL LEAVE EXTRACT BY GC-MS

TABLE 2: BIOACTIVE COMPOUNDS PRESENT IN  U. LOBATA   METHANOL LEAVE EXTRACT BY GC-MS

Figure 3: FTIR Spectrum of ethanol extract of U. lobata leaves

Figure 4: FTIR Spectrum of methanol extract of U. lobata leaves

Table 3: FTIR peak values and functional groups of ethanol extract of U.lobata leaves

Table 4: FTIR peak values and functional groups of methanol extract of U.lobata leaves

TABLE 5: ACTIVITIES OF BIOACTIVE COMPOUNDS PRESENT  IN  U. LOBATA  ETHANOL LEAVE EXTRACT

TABLE 6: ACTIVITIES OF BIOACTIVE COMPOUNDS PRESENT  IN  U. LOBATA  METHANOL LEAVE EXTRACT 

Figure 5:  Percentage Abundance of Classes of phytocompounds in of U. lobata ethanol leave extract

Figure 6:  Percentage Abundance of Classes of phytocompounds in U. lobata methanol leave extract

Table 7: Bioactive compounds common to ethanol and methanol extracts of U.lobata leaves

Figure 7: Whole plant of U. lobata

CONCLUSION

GC-MS and FT-IR investigation of ethanol and methanol leave extracts of Urena lobata demonstrated the abundance of bioactive phytochemicals and their inherent functional groups. These bioactive phytocompounds have a wide range of biological and therapeutic actions, lending credence to the plant's ethnomedicinal use for the treatment of various disease conditions amongst ethnic groups.

CONFLICT OF INTEREST

No conflict of interest exist

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