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The Long Chain Alcohols of the n-hexane Fraction of Water Hyacinth (Eichhornia crassipes): Extraction, Estimation, GC-MS Analysis and Antimicrobial Activity.

Byline: Zahid Hussain, Khalid Mohammed Khan, Shahnaz Perveen, Khair Zaman, Ghazanfar Hayat, Aneela Karim, Momin Khan and Shad Ali Khan

Summary: Oily fraction was extracted from the water hyacinth (Eichhornia crassipes) using n- hexane by both cold and hot extraction in 5.5 0.05% and 9.30 0.05%, respectively, however, no difference was found in the nature of the compounds extracted by the two processes. The analysis of this oil was carried out using GC-FID and GC-MS in separate experiments and the nature of the compounds was investigated by library matching of the spectra obtained through GC-MS. This extract was found to contain 33.267% of the nonadecan-4-ol and 20.51% of a long chain ketone in addition to various quantities of the long chain nitrogenous compounds. The antibacterial activity of the extract was investigated against eight pathogenic bacteria and fungus Candida albican using disc diffusion method. The extract was found to be highly active against all tested bacterial strains except S. typhi. For most of the strains the activity of the extract was near the reference.

Ciprofloxacin, clotrimazole and erythromycin were used as reference antibiotics.

Keywords: Water hyacinth (Eichhornia crassipes), Non polar fraction, Long chain, alcohols, Oily fraction, Antimicrobial

Introduction Most of the aquatic plants are considered as best biomass due to less quantity of lignin and their greater productivity in terms of their photosynthesis activity. Although, these plants may have invasive character but still considered as useful in many applications. They may be used for the treatment of the waste water and may also be used for the treatment of water logged area due to their potential of absorbing large quantities of water. Among these water hyacinth (Eichhornia crassipes) is a floating plant which is native to Brazil but found throughout the world [1]. It is better known as an invasive plant and in many cases it is considered as causing damage to the water bodies and aquatic life by choking the water channels [2]. It may be used as resource by its utilization as biomass. It is reportedly used for the removal of toxic metals and chemicals from water [3-5]. It was also used for the synthesis of silver nano- particles [6].

The extraction of useful compounds from this plant is another considerable option. Water hyacinth contains allelophatic compounds which are reportedly extracted from the leaves of water hyacinth [7, 8]. The extraction of phenyl phenalines, polyamines, phenols, hemicelluloses, and proteins are also reported [9-15]. The extract from the water hyacinth in various solvents may show antibacterial, antifungal and antialgal activities [5, 6, 11, 16-18]. The analysis of methanol, acetone and crude extract by various techniques indicates the presence of alkaloids, proteins, fatty acids and alcohols [6, 8, 9, 16-18]. No significant work is reported on the chemical composition and the antimicrobial activity of the n-hexane fraction of this plant [19]. The n- hexane extract of most of the plants mainly contains oil and non-polar long chain compounds. These compounds are important as nutritional supplement, medicine and fuel. In the present study the n-hexane fraction of the water hyacinth was investigated for their chemical nature and antimicrobial activity.

Experimental Material and Method Plant Material

Eichhornia crassipes was collected from a three (3) feet deep and 5 feet wide channel of water near the Mardan city of Khyber Pakhtunkhwa Pakistan in the month of March 2012, where there was plenty of water due to rains. The plant along with roots was properly cleaned and then dried it was cut into pieces of about 2 cm for acceleration of drying at the ambient temperature of the laboratory which was 30 C. The dried plant was converted into fine powder using the grinder.

Extraction Process

A crude extract was obtained by soaking 100 g of the finely powdered water hyacinth in ethanol. The same plant material was subjected to extraction for three times to ensure maximum recovery. The extract was obtained in the form of a viscous liquid and treated with n-hexane for the extraction of non-polar fraction. The fractions were concentrated using a BA1/4chi rotary evaporator. This concentrated extract was analyzed and tested against the bacteria. In another experiment the same quantity of the finely powdered water hyacinth was subjected to hot extraction process using soxhelt apparatus by using n-hexane as a solvent.

GC-MS analysis of the sample

The non-polar n-hexane soluble fraction of the water hyacinth was analyzed using a 600 H Jeol GC-MS. A fused capillary column HP.5 of 30 meter length and 0.32 mm internal diameter having a film thickness 0.25 m was used for separation of the components of the extract. The column contains polydimethylsiloxane as the stationary phase and the carrier gas was helium at a flow rate of 1.8 mL/minute. The mode of injection was split at the split rate of 35. The temperature of the injector was 250 C. The column was kept at 60 C for 2 min and then heated to 250 C at the rate of 5 C per minute.

Bioassay

The n-hexane extract of the water hyacinth was tested for its antimicrobial against eight pathogenic bacteria i.e. Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Bacillus subtilis, Bacillus atrophaeus, Agrobacterium tumifaciens, and Pseudomonas aeruginosa. The antibacterial assays were carried out using disc diffusion method [20]. The samples were dispersed in DMSO and applied on the microbes in the concentration of 1 mg/12 L of the DMSO. The samples were applied by the use of a micropipette on the dry and sterilized discs which were placed on the agar medium. Standard antibiotic discs and blank impregnated with solvent used as the positive and negative control. This study was conducted using erythromycin as reference for S. aureus, B. subtilis and B. atrophaeus, clotrimazole for A. tumifaciens and P. aeruginosa and ciprofloxacin as positive control for each of the Klebsiella pneumoniae, E. coli, P. aeruginosa and S. typhi.

The blank, control and test discs were kept at low temperature for 24 h for maximum diffusion followed by incubation period of 24 h at 37 C for the growth of the organisms. A clear inhibition zone was observed for the test material and control. The solution of extract was applied in 12 l doses. The antibacterial activity was determined by measuring the diameter of the zone of inhibition in millimeter. For checking the reproducibility of our results all these experiments were carried out in three sets.

Results and Discussions

Yield and Analysis of the Extract

It was assumed that water hyacinth may contain large quantities of the non polar fraction due to the non-wetting nature of its stem and leaves [21]. The yield and extraction efficiency was determined using 100 g of the dry powder of the leaves of water hyacinth. The extraction of the non-polar compounds was carried out both by hot and cold extraction processes. The cold extraction was initially carried out using ethanol as solvent followed by extraction of the non-polar part using n-hexane as the solvent. This cold process gives a yield of 5.5 0.05%, however, the yield increased to 9.30 0.05% when n-hexane was used as solvent and soxlet apparatus was used for extraction.

Analysis of the Extract

Despite of the difference in yield, the nature of extract obtained by hot and cold extraction processes were found to be same. The nature of the compounds of the extract was determined by GC-MS analysis. The components of the extract were identified on the basis of the fragmentation pattern and library matching. The spectra obtained for the components of the mixture were compared with mass spectral library of our institute and the library of the National Institute of Standards and Technology (NIST). The conditions for the GC-MS analysis were optimized using GC equipped with FID. The relative concentration data of the components was obtained from the GC-FID data. The results of these investigations are given in Table-1 and can be observed from that the n-hexane fraction of water hyacinth contains eighteen (18) compounds in addition to small quantity of undetectable compounds.

Most of these compounds are long chain alcohols the most abundant of which is nonadecan-4- ol in a relative concentration of 33.26%. It also contains 17-methoxydocosa-1,4,7,10-tetraene-6,9- dione in as high as 20.50% relative concentration. A compound of molecular weight 279, however have a highly complicated spectrum and found to be in 10.30% relative concentration. There were compounds like 9,16-dimethylnonadec-1-en-9-ol, tricosane-4-ol and 5-methoxyhenicosane. In addition to these compounds the extract was also found to contain nitrogenous compounds like 1-amino- octadeca-8,10,12-trien-7-ol and 6-(6-(octadeca- 1,3,7,12,14,16-hexaenyl)pyridin-2-yl)hex-5-en-1-ol in addition to various quantities of ether functional group. The extract was also found to have small quantities of unresolved spectra, the concentration of which are not included in this list.

This fraction is soluble in the non polar n-hexane may be due to the presence of long chain saturated and long chain unsaturated compounds.

Antimicrobial Activities of the Non Polar Extract of the Water Hyacinth

The n-hexane fraction of the water hyacinth was investigated for its antimicrobial activities against eight (8) bacteria and one fungal strain Candida albican. Only Candida albican was used due to the non-availability of other fungal strains in our laboratory. The extract were screened against K. pneumonia, E. coli, S. typhi, S. aureus, B. subtilis, B. Atrophaeus, A. tumifaciens, and P. aeruginosa bacterial strains and results are summarized in Table- 2. It was observed that this n-hexane fraction of water hyacinth have significant activity against all the bacterial strains except E. coli. The extract was also active against Candida albican. The antibacterial activity was almost comparable to the standards against most of the strains. It may be due to two reasons, firstly the long chain and secondly the effective functional groups. The long chain may be responsible for change in surface tension of the medium and inside the cell which make the media unfavorable for these bacteria [22, 23].

Major fraction of extract is composed of the alcohols and ketones, non-polar nature of this fraction is due to long chain nineteen carbons containing alcohol. The extract contains both monohydroxy and polyhydroxy alcohols and it had been reported that alcohols have antimicrobial properties [24-26]. Most of the long chain alcohols like 1-tridecanol and C9-C12 are reported as antimicrobial [27-29]. This activity of the alcohol was described due to the fact that alcohols and phenols alter the properties of membrane and disrupt the native membrane-associated function of the integral proteins [26, 29]. In addition to alcohols, ketones of natural and synthetic nature are reportedly having antibacterial and antifungal activities [30, 31]. It was reported that some of the ketones containing oils are antimicrobial due to the ability of these to penetrate the phospholipids bi-layer of cell membrane and interference with enzymes of the microbes [32, 33].

This extract contains 20.50% of 17-methoxydocosa-1,4,7,10-tetraene-6,9-dione. This diketone is believed to have significant contribution to the antimicrobial activity of the extract. The antimicrobial activity is due to long chain two ketonic sites and the presence of an ethereal site in addition to the unsaturated sites. It can be seen from Table-2 that the extract contains various concentrations of three long chain ethers. Most of the ethers of natural origin were found to be antimicrobial [34-36].

Table-1: Relative concentrations and list of the compounds present in the extract.

###S. No.###Name###Molecular Weight###% Concentration

###1###Hexadec-4-ene-4,6-diol###282###1.75

###2###9-Ethylheptadec-16-en-1-ol###256###1.05

###3###Nonadecan-4-ol###284###33.26

###4###Eicosan-7-ol###298###1.05

###5###11-Butoxy-1-ethoxy-3-methyldodec-1-ene###298###2.73

###6###Unknown###279###10.30

###7###16-Ethoxy-10-propylhexadeca-1,4-diene###208###2.73

###8###17-Methoxydocosa-1,4,7,10-tetraene-6,9-dione###306###20.50

###9###9,16-Dimethylnonadec-1-en-9-ol###310###8.32

###Henicosan-5-ol###312###1.63

###10###5-Methoxyhenicosane###326###4.03

###11###Tricosane-4-ol###340###4.28

###12###11-Hydroxy-18-methylhenicosan-10-one###340###1.11

###13###1-Aminooctadeca-8,10,12-trien-7-ol###279###3.87

###14###Pentacosan-7-ol###368###2.06

###15###6-Hydroxypentacosane-8-one###382###2.56

###16###19-(Heptyloxy)nonadec-7-en-6-ol###396###0.99

###17###6-(6-(Octadeca-1,3,7,12,14,16-hexaenyl)pyridin-2-yl)hex-5-en-1-ol###419###0.42

###18###4-Methyltetracosa-2,5,14,19,21-pentaen-16-yne-4,8,12-triol###386###1.11

Table-2: Summary of the antimicrobial activities.

###Zone of Inhibition (mm)

###Organisms###Sample###Ciprofloxacin 30 g###Clotrimazole 50 g###Erythromycin 15 g

###K. pneumonia###20###32.0###-###-

###E. coli###13###36.0###-###-

###S. typhi###-###38.0###-###-

###S. aureus###17###-###-###23.0

###B. subtilis###18###-###-###24.0

###B. Atrophaeus###17###15.0###-###25.0

###A. tumifaciens###13###-###24###-

###P. aeruginosa###18###-###18###-

###C. albicans###19###-###38###42

The extract contains smaller quantities of nitrogenous compounds having long chain. It can be seen from the Table-1 that 1-aminooctadeca-8,10,12- trien-7-ol have a concentration of 3.87%. It may have a significant contribution to the antibacterial and antifungal activity due to the reported use of these types compounds as antimicrobial agents The antimicrobial activity of amines is due to the ability of these compounds to penetrate the hydrophobic membrane of the microbes [37-41]. In addition to the chemical nature of the compounds of the extract, its oil like properties may also play role in the antimicrobial activity.

Conclusion

The n-hexane fraction of water hyacinth was found to contain large quantities of the long chain compounds. Most of the compounds are alcohols of saturated and unsaturated side chains. The major compound was nonadecan-4-ol which contributes 33.27% of total extract and next in concentration was a ketonic compound. The extract was also found to contain various quantities of ethers and nitrogenous compounds. This extract was found to have significant activity against all the organisms except S. typhi. The antimicrobial activities of the extract are believed to be due to synergistic effects of the components of the mixture.

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