PHYTOCHEMICAL SCREENING, ANTIMICROBIAL PROPERTIES AND ESSENTIAL OIL CONSTITUENTS OF COMBRETUM SORDIDUM EXELL

PHYTOCHEMICAL SCREENING, ANTIMICROBIAL PROPERTIES AND ESSENTIAL OIL CONSTITUENTS OF COMBRETUM SORDIDUM EXELL

O. Olaoluwa* and F. Ogunbor

Department of Chemistry, Faculty of Science, University of Ibadan, 200284, Ibadan, Oyo state, Nigeria

ABSTRACT: Plants in Combretaceae family are traditionally reputed to be anthelmintics and antimicrobial agents. Dried aerial parts of Combretum sordidum (Exell) were ground to fine powder and extracted using ethanol. Ethanol extract was partitioned to obtain hexane and ethylacetate extracts successively. These extracts were screened for secondary metabolites using standard methods and evaluated for their antimicrobial potentials against two Gram positive bacteria; Bacillus subtilis (ATCC 14579) and Bacillus cereus (ATCC 33923); two Gram negative bacteria; Proteus mirabilis (ATCC 21784), Salmonella typhi (ATCC 25179) and a fungus; Candida albicans (NCTC 227) by tube dilution method. Essential oil from its leaves was obtained by hydrodistillation method and analyzed by GC and GC-MS. Preliminary phytochemical screening revealed the presence of alkaloids, saponins, tannins, flavonoids, cardiac glycosides, steroids and anthraquinones in the ethylacetate and ethanol extracts. Both extracts displayed broad spectrum activities against the organisms used with minium inhibitory concentration (MIC) at 400µg/mL. Five compounds comprising 99.99% of the total peak area were identified from the volatile oil. The most abundant constituent was 7- octadecenoic acid methyl ester (51.26%) and others were dodecanoic acid (21.12%), hexadecanoic acid (11.58%), tetradecanoic acid (8.94%) and octadecanoic acid (7.09%) methyl esters. These secondary metabolites might be responsible for the antimicrobial activities of C. sordidum. The result obtained from this study further justifies the use of this plant as antimicrobial agent by the traditional healers.

Combretum sordidum, phytochemicals, antimicrobial, essential oil, 7- octadecenoic acid methyl ester

INTRODUCTION: Members of the Combretaceae family are widely used in African traditional medicine for the treatment of microbial infection ¹. Species from the Combretum genus and to a lesser extent Terminalia are common and distributed throughout Western and Southern African ². Records indicated that majority of  the traditional healers use these species for treating abdominal disorders, backache, conjunctivitis, venereal and heart diseases, hypertension, jaundice, pneumonia, skin and eye diseases, toothache, diabetes, tumours, tuberculosis  and yellow fever ^1-7.

Compounds such as combregenin, arjunglucoside, combreglucoside, 5, 4 – dihydroxy – 7 - methoxy flavone; 5, 4`-dihydroxy-7, 5`-dimethoxyflavone; 5,7,4`-trihydroxyflavone; punicalagin, hydrolysable tannins and imberbic acid were among the constituents isolated from combretum species with antimicrobial activities ^8-9.

The medicinal usefulness of plants has been the subject of numerous chemical and microbiological studies. Some of the reported phytoconstituents of herbs include terpenoids, alkaloids, tannins, saponins, flavonoids and phenanthrenes ^{4, 5, 8}. And the reported uses include antisplasmodic, antiasthmatic, antimicrobial and anti-inflammatory ⁹. Combretum sordidum (Exell) is a scandent shrub or a creeper. Its flowers are white and easily recognized by the small red scales on the under-surface of the leaves ¹⁰. The ethnopharmacological and medicinal uses of C. sordidum has got little or no scientific record in the literature. In view of this, we report the preliminary phytochemical screening, antimicrobial properties and essential oil constituents of C. sordidum for the first time.

MATERIALS AND METHODS:

Plant Collection and Identification: Fresh aerial parts of Combretum sordidum were collected in the year 2013, from the botanical garden of the University of Ibadan, Ibadan, Nigeria. It was identified and authenticated at the herbarium unit of the department of Botany, University of Ibadan, where a voucher specimen was deposited (UIH-22359).

Preparation, Extraction and Partitioning of Plant Material:

The aerial parts were cut into small pieces, air-dried and ground to a fine powder. Plant material (1335 g) was soaked in 10 L round bottom flask with 8 L of ethanol for 72 h. Extract was concentrated using rotary evaporator at 40 ⁰C and crude extract (160 g) was obtained. Ethanol extract (100 g) was subjected to liquid-liquid partitioning using hexane and ethylacetate to obtain the respective fractions.

Phytochemical Analysis of the Extracts:

The ethanol extract and the fractions obtained were subjected to phytochemical screening. The entire tests were carried out using standard method ¹¹. 

Alkaloids:

Each extract was acidified with 1% HCl and treated with few drops of Mayer`s, Wagner`s and Dragendorff’s reagents separately in different test tubes. A creamy white (Mayer), reddish brown (Wagner) or an orange (Dragendorff) precipitate indicates the presence of alkaloids.

Saponins:

Presence of saponins were detected by adding some amount of distilled water to the extracts. After heating the mixture, the filtrate was collected and shaken. Formation of froth indicated the presence of saponins.

Steroids:  

Each extract was dissolved in 2 mL of chloroform in a test tube. Concentrated H₂SO₄ (0.2 mL) was carefully added to form a lower layer. A red colour produced in the lower chloroform layer indicated the presence of steroids.

Flavonoids:  

Each extract was dissolved in dilute NaOH. A yellow solution that turns colourless on addition of HCl acid indicates the presence of flavonoids.

Cardiac glycosides:

Each extract was dissolved in 2 mL of glacial acetic acid containing a drop of ferric chloride solution. This underplayed with 1 mL of concentrated Sulphuric acid. A brown ring at the interface indicated the presence of cardiac glycosides.

Anthraquinones:

The extract was shaken with 4 mL of benzene. The mixture was filtered and 2 mL of 10% ammonia solution was added to the filtrate. The mixture was shaken and the presence of pink red or violet colour in ammoniacal solution (lower phase) indicated the presence of free anthraquinones.

Tannins:

Each test extract was mixed with water and filtered. A dirty green precipitate or blue-black or blue green precipitate on addition of few drops of 5% ferric chloride to the test extract was taken as an indication of the presence of tannins.

Determination of Antimicrobial Activity:

Microorganisms used were standard strains of two Gram positive bacteria; Bacillus subtilis (ATCC 14579) and Bacillus cereus (ATCC 33923); two Gram negative; Proteus mirabilis (ATCC 21784) and Salmonella typhi (ATCC 25179) and a fungus; Candida albicans (NCTC 227), these obtained from Centre for Drug Research Institute (CDRI) Lucknow, India. Standard microbial cultures were prepared by sub-culturing a loopful of each microbe into sterile nutrient broth and incubated for 24 h at 37 ⁰C for the bacteria and 48 h for the fungus. The suspensions were adjusted to a turbidity of 10⁵ colony forming unit (cfu/mL).

Minimum inhibitory concentrations (MICs) were determined using the tube dilution methods ¹². Nutrient broth (2 mL) was dispensed into each of the twelve (12) test tubes followed by an addition of the extract (1 mL) in the first tube. From the resulting mixtures, 0.5 mL of the solution was removed to make serial dilution to the nineth tube excluding the neutral, negative and positive controls. Thereafter, 0.2 mL of each organism was added into the test tubes. Gentamicin (0.3 mL) and ethylacetate: water (1:1), were used as the positive and negative controls respectively. The nutrient broth with organisms only was used as the neutral control. Lowest concentration that showed no growth was the MIC after 24 h incubation at 37 ⁰C.

Extraction of Essential Oil: Fresh leaves (250 g) were subjected to hydrodistillation in a Clevenger-type apparatus for 3 h. The essential oil was collected over hexane and stored in a sealed vial under refrigeration prior to analysis.

Analysis of the Essential Oils

Gas chromatography (GC):

The volatile oil was subjected to GC analysis on Shimadzu mol QP2010 system equipped with an AOCi-20i autosampler. The column used was DB5 (30 m×0.25 mm, 0.25 µm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL/min, linear velocity of 36 2 cm/sec and pressure 56.2 KPa. The oven temperature was set at 60 ⁰C, hold for 1 min to 180 ⁰C at 10 ⁰C/min, and hold for 3 mins, the final temperature was 280 ⁰C at 12 ⁰C, and hold for 2 mins. Both injector and detector temperatures were fixed at 250 ⁰C.

Gas chromatography- mass spectrometry (GC-MS):

The GC-MS analysis was performed on Shimadzu model QP2010 system model with split/splitless injector interfaced to a 5975 mass selective detector operated at 70 eV, ion source temperature of 200 ⁰C with a mass range of m/z 50 -700 at scan rate of 1428 amu/sec. The same temperature programme as for the GC was used. Total analysis time was 24 mins.

Identification of components:

Identification of each individual constituent of the essential oils was achieved based on their retention indices (determined with reference to a homologous series of normal alkane) and by comparison of their mass spectral fragmentation patterns (NIST data/base/chemstation data system) with data previously reported in the literature ¹³.

RESULTS AND DISCUSSION:

The ethanol crude extract gave percentage yield of 11.80 while the yield of hexane and ethylacetate fractions were 1.60% and 3.98% respectively (Table 1).

TABLE 1: PERCENTAGE YIELDS OF EXTRACT AND FRACTIONS OF C. SORDIDUM

Preliminary phytochemical screening of the extract and fractions investigated were summarized in Table 2. Saponins, flavonoids, cardiac glycosides, alkaloids, tannins and anthraquinone were present in both ethylacetate and ethanol extracts. These classes of components were reported to exhibit a variety of biological activities including antiviral, antibacterial, anti-inflammatory and analgesic, all of which were relevant to the traditional uses of some species of the genus ¹⁴. The phytochemicals also have strong antimicrobial significance against potential enteric pathogens, the presence of alkaloids in significant quantities may be used as antimalarial, analgesics and stimulants while tannins are used in treating wounds, sprains, bruises and in arresting bleeding ^15-16.

TABLE 2: PHYTOCHEMICAL ANALYSIS OF THE EXTRACT AND FRACTIONS OF C. SORDIDUM

Keys: + = Present, -   = Absent

Antimicrobial activities of the extracts of C. sordidum are presented in Table 3. The result showed that ethylacetate extract was moderately active against B. subtilis, S. typhi and C. albicans while ethanol extract was active against B. subtilis, P. mirabilis, S. typhi and C. albicans, both extracts with MIC at 400µg/mL. Ethylacetate extract exhibited strong inhibition against B.cereus with MIC at 16µg/mL.  Hexane extract showed activity against S. typhi only with MIC at 400µg/mL. This shows that the antimicrobial constituents are likely to be contained in the ethylacetate and ethanol extracts and also reaffirms the ethnopharmacological uses of this plant as an antimicrobial agent.

The essential oil yield of C. sordidum obtained from hydrodistillation of leaves was 0.24%. Five components were identified in the volatile oil accounting for 99.99% of the composition by GC-MS (Table 4). These components were fatty acid esters, the most abundant was 7- octadecenoic acid methyl ester (51.26%) and others were dodecanoic acid (21.12%), hexadecanoic acid (11.58%), tetradecanoic acid (8.94%) and Octadecanoic acid (7.09%) methyl esters  at retention time 13.0, 15.4, 20.8, 22.7 and 23.0 mins respectively.  The fatty acid esters identified in the essential oil have been reported to have antibacterial and antifungal activities ^{1, 17-19} and these constituents might have contributed to the antimicrobial properties of C. sordidum.

TABLE 3: MINIMUM INHIBITORY CONCENTRATION (MIC) OF ETHANOL EXTRACT, ETHYL ACETATE AND HEXANE FRACTIONS OF COMBRETUM SORDIDUM

Key: + = Growth; - = No growth

TABLE 4: CHEMICAL COMPOSITION OF ESSENTIAL OIL OF COMBRETUM SORDIDUM LEAVES

   ^aRetention index; ^bMolecular mass; ^cMolecular formular; ^dPercentage area

CONCLUSION: The antimicrobial activities of C. Sordidum extracts corroborate the ethnomedicinal uses of this genus. The constituents of the extracts from the phytochemical analysis confirmed the reason for the antimicrobial activities of the plant against organisms used and therefore could be harnessed as a potent antimicrobial agent. Also the results obtained could form a good basis for further investigation in the discovery of new natural bioactive compounds. This report can be considered as the first scientific information on the phytochemical, antimicrobial properties and chemical composition of the essential oil constituents of C. sordidum.

ACKNOWLEDGMENTS: The authors are grateful to Dr. I. O. Oladosu, Department of Chemistry, Faculty of Science, University of Ibadan, for his assistance during the antimicrobial analysis.

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    How to cite this article:

    Olaoluwa OO and Ogunbor F: Phytochemical Screening, Antimicrobial Properties and Essential oil Constituents of Combretum Sordidum exell. Int J Pharm Sci Res 2015; 6(3): 1176-80.doi: 10.13040/IJPSR.0975-8232.6(3).1176-80.

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