1. INTRODUCTION:
Mosquitoes constitute a major public health problem as vectors of serious human diseases 1 . Several mosquito species belonging to genera Anopheles, Culex and Aedes are vectors for pathogens of various diseases like malaria, filariasis, Japanese encephalitis, dengue fever, dengue hemorrhagic fever and yellow fever2.
Hubalek and Halouzka2 reported that Culex pipiens is the vector of West Nile virus that causes encephalitis or meningitis, which is known to affect the brain tissue, finally resulting in permanent neurological damage. Aedes aegypti is known to transmit the viruses that cause dengue fever3. The inefficiency of the organophosphate and carbamate insecticides4, along with the need for safer methods regarding toxicity to man and the environment has stimulated the search for new means of vector control. Oil-resin or plant extracts are an alternative with potential for use. Although some diseases such as yellow fever have been reasonably brought under control by vaccination, no effective vaccine is available for malaria5. Several drugs, most of which are also used for treatment of malaria, can only be taken preventively. Modern drugs used include mefloquine (Lariam), doxycycline (available generically), and the combination of atovaquone and proguanil hydrochloride (Malarone). Doxycycline and the atovaquone and proguanil combination are the best tolerated with mefloquine associated with higher rates of neurological and psychiatric symptoms6. The choice of which drug to use depends on which drugs the parasites in the area are resistant to, as well as side-effects and other considerations. Use of prophylactic drugs is seldom practical for full-time residents of malaria-endemic areas, and their use is usually restricted to short-term visitors and travellers to malarial regions. This is due to the cost of purchasing the drugs, negative side effects from long term use, and because some effective anti-malarial drugs are difficult to obtain outside of wealthy nations. Quinine was used historically; however the development of more effective alternatives such as quinacrine, chloroquine, and primaquine in the 20th century reduced its use. Today, quinine is not generally used for prophylaxis. The use of prophylactic drugs where malaria bearing mosquitoes are present may encourage the development of partial immunity 7. One strategy of the world health organization (WHO) in combating tropical diseases is to destroy their vectors or intermediate hosts. Malaria is a parasitic disease from which more than 300 million people suffer yearly throughout the world. It is one of the main causes of infant and young child mortality 8. Consequently, control of A. gambiae s.s is of particular interest because it is the prime carrier of malaria parasite plasmodium falciparum. Hence a suitable method used to protect individuals in malaria endemic areas is to eradicate mosquitoes and thereby prevent mosquito bites. The prevention of malaria may be more cost-effective than treatment of the disease in the long run, although the capital costs required are out of reach of many of the world's poorest people. Currently, the only efficacious approaches of minimizing the incidence of this disease are to eradicate or control mosquito vectors mainly by application of insecticides to larval habitats. It has been shown that plant- derived natural products used as larvicides have the advantage of being harmless to non-target organisms and no vector resistance has been observed so far 9,10. In the recent years, the emphasis to control the mosquito populations has shifted steadily from use of conventional chemicals towards more specific and environmentally friendly materials, which are generally of botanical origin. For this purpose, many phytochemicals extracted from various plants species have been tested for their larvicidal and repellant actions against mosquitoes 11,12. As part of continued search of the biodiversity resource available in Kenya for natural products with utilizable bioactivity, L. alata was considered for this work. This plant has shown good bioactivity from ethenopharmacological point of view but no larvicidal activity of these plants has been reported. The oils and non-volatile extracts of L. alata were assayed for larvicidal activity towards A. gambiae s.s.
2. MATERIALS AND METHODS:
2.1 Collection and Identification of Plants
Laggera alata is in the well-known medicinal plant family of Asteraceae and grows wildly in the outskirts of Mau forest complex near Molo at an altitude range of 2127 -2137m in Kenya. It is from here that fresh aerial parts of the plant were collected. The average temperatures remain similar throughout between 15 - 28 °C. A taxonomist identified the plant materials and a voucher specimen was deposited at the department of biological sciences of Egerton University Njoro Campus, Nakuru, Kenya.
2.2 Extraction
2.2.1 Non – volatile compounds
The plant materials were dried under shade to constant
weight and ground to a fine powder. A powder weighing 600 g of the plant
powder was extracted sequentially with hexane (3 
1.5 L), ethyl acetate (3 1.5 L), chloroform (3
1.5 L), acetone (31.5 L) and methanol (3 
1.5 L) after soaking the sample in each solvent for
24 hours. The extracts were filtered through a Buchner funnel fitted to a
vacuum pump with a thin layer of activated charcoal, and then concentrated
using a rotary evaporator and the solvent recovered. All crude extracts were
partitioned between equal volumes (250 ml each) of distilled water and
chloroform to remove sugars. The chloroform fraction was concentrated under
reduced pressure. The dry sample was then subjected to column chromatography
using hexane (4 x 200 ml), ethyl acetate (4 200 ml), chloroform (4 200 ml), acetone (4 200 ml), and methanol (4 200 ml). The solvents were recovered using rotor
evaporator to obtain 17.10 g, 10.60 g, 14.95 g, 15.40 g and 14.20 g of dry
hexane, chloroform, ethyl acetate, acetone, and methanol soluble fractions of L.
alata respectively. The extracts were then subjected to larvicidal
assays. The flow chart in Fig. 1 (A) shows the flow diagram for the extraction
of non-volatile secondary metabolites.
2.2.2 Essential oils
A 180g of fresh whole plants of L. alata was subjected to hydro-distillation in a modified Clevenger-type apparatus for at least four hours according to the British pharmacopoeia (Fig 1 (B)). The essential oil obtained was 5g (2.78% w/w) of L. alata after drying over anhydrous sodium sulphate. The oil was stored in sealed glass vial (Bijoux bottle) at 4 °C.
Figure 1: Extraction of (A) non – volatile compounds5 and (B) essential oil and larvicidal activity test13
2.3 GC, GC-MS analysis
Samples of essential oils were diluted in
methyl-t-butylether (MTBE) (1:100) and analysed on an Agilent GC-MSD
chromatograph equipped with an Rtx-5SIL MS (‘Restek’) (30 m 0.25 mm i.d. 0.25 μm film thickness)
fused-silica capillary column. Helium (0.8 mL/min) was used as a carrier gas.
Samples were injected in the split mode at a ratio of 1:10 – 1: 100. The
injector was kept at 250 °C and the transfer line at 280 °C. The column was
maintained at 50 °C for 2 min and then programmed to rise to 260 °C at 5 °C/min
and held for 10 min at 260 °C. The MS was operated in the EI mode at 70 eV, in
m/z range 42-350. The identification of the compounds was performed by
comparing their retention indices and mass spectra with those found in
literature14
then supplemented by Wiley and QuadLib 1607 GC-MS libraries. The relative
proportions of the essential oil constituents were expressed as percentages
obtained by peak area normalization, all relative response factors being taken
as one.
2.4 Larvicidal assays
The extracts were solubilized in analytical reagent grade dimethyl-sulphoxide (DMSO) obtained from Lobarchemi and diluted to give 2 mg/ml of stock solution with DMSO kept at a concentration of 1%. The bioassay experiments were conducted mainly according to standard WHO procedure15 with slight modifications. The bioassays were conducted at the Kenya Medical Research Institute (KEMRI), Centre for Disease Control (CDC), Kisumu, Kenya, where the larvae were reared in plastic and enamel trays in spring river water. The larvae were maintained, and all experiments carried out at 26 ± 3°C and the humidity ranged between 70 to 75%. The bioassays were performed with third in-star larvae of A. gambiae s.s and carried out in triplicate using 20 larvae for each replicate assay. The larvae were placed in 50 ml disposable plastic cups containing 15 ml of test solution and fed on tetramin fish feed during all testing. Larvae were considered dead if they were unrousable within a period, even when gently prodded. The dead larvae in the three replicates were combined and expressed as the percentage mortality for each concentration. The negative control was spring river water while the positive control was the pyrethrum-based larvicide, pylarvex.
2.5 Isolation, purification and structure elucidation of larvicidal compounds
To isolate, purify and elucidate the structures of larvicidal compounds from L. alata the following analytical techniques were employed.
2.5.1 Chromatographic techniques
The hexane extracts of L. alata were found
sufficiently bioactive and therefore considered for further analysis. These
extracts were chromatographed on a silica gel column using gradient elution of
hexane - ethyl acetate solvent system to give four fractions. The crude
extracts were firstly analyzed using the TLC (Merck, 60F254) to establish
suitable solvent system (silica gel, 20 20 cm, 0.20 mm thick, cut into 5 15 cm for use). All solvents were distilled before
use. The main solvents used as the mobile phase were hexane and ethyl acetate.
The ratios of the solvent were changed while using the hexane as the main
solvent in the following percentages: 0, 10, 20 and 30% (v/v) of ethyl acetate
in hexane. The TLC analysis with the above solvent systems showed that hexane
and ethyl acetate in a ratio of 7:3 gave the most pronounced separation with
distinct spots. Column chromatography was then performed using Merck silica
gel 60 (70-230 mesh). The column used was of the dimension 50 cm height by 19
mm internal diameter. Silica gel used was about 65 g per column to give 45 cm
of gel height.
2.5.2 Preparative TLC analysis and Isolation of compound 1 and 2
The extracts that showed bioactivity were subjected to
preparative thin layer chromatographic analysis. This was done on silica gel
plates using the solvent system Hex-EtOAc, 7:3. The visualization and
identification of spots of the compounds was done using an ultra violet lamp at
a wavelength of 254 nm. The retention factor (Rf) values were then
determined. A mass of 17.10 g of hexane fraction was suspended in 250 ml of
distilled water and extracted with 250 ml chloroform using a separating
funnel. The chloroform extract was dried using anhydrous sodium sulphate. The
solvent was then recovered on vacuum rotary evaporator. The dry sample was
dissolved in hexane and re-eluted on a column packed with 65 g of silica gel.
Isolation was carried out using the solvents: hexane, ethyl acetate by
increasing polarity. A total of 25 fractionswere collected. Fractions that
showed the same Rf value and the same characteristic colour on TLC observed
using UV lamp operating at 254 nm were combined and subjected to preparative
thin layer chromatography on 20 2 0 cm plates. Fractions collected with 100% hexane
and hex-EtOAc (9:1) ratios were discarded because their TLC result did not show
spots. However fraction with 80 % hexane gave very many close spots indicating
several compounds with relatively same polarity. The 70% hexane fraction was
concentrated under reduced pressure (on a rotary evaporator ) to yield 2.86 gm
(1.67%) and was applied on a preparative thin layer chromatography plate and
developed in 7:3 ratio of hexane- ethyl acetate as the mobile phase. Two
distinct band separations were observed in this solvent system: a more mobile
yellow band and a grey band only visible under UV lamp operating at 254 nm.
The bands were carefully scraped and re-extracted using the same solvent
system. Concentrating this fraction under reduced pressure yielded 680 mg of
pure compound 1. Compound 1 was a yellow gummy substance with Rf
value of 0.50 on TLC. The second band yielded 275 mg of pure white substance,
compound 2. On TLC this compound had Rf value of 0.24. The other
combined fractions on preparative TLC showed rather very close and
superimposing bands hence no further work was done on them.
2.5.3 NMR Spectroscopic analysis of the compounds
All the spectra were measured on a Bruker Advance 400 spectrometer, which operated at 400 MHz for 1H and 100 MHz for 13C NMR analyses. 1H and 13C NMR (Appendices 1and 2) spectra were performed in deuterated solvent and chemical shifts were assigned by comparison with the residue proton and carbon resonance of the solvent and tetramethylsilane (TMS) as internal reference (δ = 0). 2D-NMR spectroscopy was used to elucidate the structures and especially establish the connectivities in the molecules. The proton-carbon connectivity (three bonds) was identified using 1H-13C COSY and HMBC (Heteronuclear Multiple Bond Correlation) spectrum (Appendices 1, 2, 3, 4, 5, 6, 7 and 8) in which there was one- dimensional 13C NMR spectrum along the left and the 1H NMR spectrum along the top. The two-dimensional array of spots forming a ‘‘square box’’ identified the proton-carbon connectivity.
2.6 Statistical analysis
The lethal concentrations were determined using SPSS package version 11.5. The bioassay data was subjected to probit regression analysis according to Finney16,17. Probit analysis of concentration-mortality data was conducted to estimate the LC50 and LC99.
3. RESULTS AND DISCUSSION:
3.1 Essential Oils of L. alata
The oils were dominated by sesquiterpenes which accounted for 50.3% of the oils (Table 1). Considering components with concentrations of about 2 % and above, the sesquiterpenes were 2,5-dimethoxy-para-Cymene (24.4%) δ-germacrene (8.4%), α-humulene (6.2%), (E)-caryophyllene (2.3%) and β-bourbonene (2.5%). Monoterpenes accounted for 31.5% of the constituents’ compounds in the oils, main components were cis-chrysanthenol (11.8%), chrysanthenone (8.7%), thymol methyl ether (4.6%), filifolone (3.5%) and sabinene (3.6%). A total of 15.4% were compounds whose identity was unkown.
Table 1: Laggera alata Oils Chemicals Composition
|
ID |
R.T. |
% of total |
RI |
ID Method |
|
Monoterpenes α-Pinene Sabinene Filifolone Chrysanthenone Chrysanthenol<cis> Thymol methyl ether |
5.6 6.6 10.2 10.8 12.1 14.0 |
1.1 3.6 3.5 8.7 11.8 4.6 |
932 971 1100 1122 1165 1229 |
MS, RI MS, RI MS MS, RI MS, RI MS, RI |
|
|
Total |
30.3% |
|
|
|
Sesquiterpenes α-Copaene β-Bourbonene β-Elemene Cymene<2,5-dimethoxy-para> Caryophyllene(E) γ-Elemene α-Humulene Germacrene-D Muurola-4(14),5-diene<trans> Bicyclogermacrene δ-Cadinene Germacrene-D-4-ol Caryophyllene oxide α-Cadinol |
18.2 18.4 18.5 19.2
19.3 19.6 20.3 21.0 21.2 21.3 21.9 23.4 23.5 25.3 |
1.2 2.5 0.8 24.4
2.3 0.6 6.2 8.4 0.8 0.8 1.9 0.5 0.5 0.6 |
1376 1384 1389 1414
1420 1430 1456 1482 1492 1496 1519 1578 1583 1657 |
MS, RI MS, RI MS, RI MS
MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI |
|
|
Total |
51.5% |
|
|
|
Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown |
10.3 10.4 12.6 24.2 24.7 25.0 25.4 26.0 27.8 28.5 28.6 31.1 31.4 |
1.8 4.1 1.5 0.9 1.1 0.8 1.0 1.2 0.5 0.6 0.3 1.1 0.5 |
1104 1107 1181 1611 1632 1644 1660 1685 1767 1796 1801 1964 |
|
|
|
|
|||
|
|
Total |
15.4% |
|
|
3.2 Larvicidal Activity of the oils
The LC50 and LC99 of L. alata was 273.38 mg/l and 507.75 mg/l respectively. A constituent α-Pinene (1.1%) though found in small amount has been reported to be the cause of the antifungal activity of oils from Pistacia lentiscus (ana-cardiaceae)18. The unknown compounds may not to have significant influence but we cannot ignore their probable synergistic larvicidal effect. The unknown compounds therefore need to be carefully isolated, structures elucidated and their larvicidal activity studied. However, a compound like β-caryophyllene (2.3%) is a common sesquiterpene widely distributed in plants, possesses anti-inflammatory and ant-carcinogenic activities19,20. Its oxygenated form caryophyllene oxide is present in a minor quantity of 0.50 %, (Table 1) it is known to possess antimicrobial properties against a wide range of bacteria and fungi21. The difference in larvicidal activity of the essential oils and the standard larvicide can be explained in terms of the fact that the active components in the oils comprise of only a fraction of the oils used. Therefore, the concentration of the active components could be much lower than the standard larvicide; pylarvex used.
3.3 Hexane fraction of L. alata
Laggera alata oils showed much better activity compared to the active hexane fraction (Table 2). The log probit analysis gave LC50 and LC99 as 273.38 mg/l and 507.75 mg/l respectively. The hexane fraction of L. alata demonstrated reasonable activity (Table 2) against A. gambiaes.s. Larvae. This fraction gave an LC50 and LC90of 1161.30 mg/l and of 2734.91 mg/l respectively. These values are significantly higher compared to the oil’s larvicidal activities these difference could be due to lower available bioactive secondary metabolites in the non-volatile fraction. A sizeable amount of the active metabolites may have been lost during drying session. These compounds also have been shown to partition exclusively in particular solvents22. The LC50 of standard pylarvex was 30 mg/l the big difference in activity of the extracts compared with the reference larvicide could be because the active compounds are only a small percentage of the extracts since; no purification was done at this stage.
Table 2: Larvicidal assay for L. alata oils and hexane extract
|
Concentration (mg/l) |
|||
|
4000.00 3500.00 3000.00 2500.00 2000.00 1500.00 1000.00 |
100 100 100 98.35 80.00 78.90 55.00 |
||
3.4 Chloroform, Acetone and Methanol fractions
Chloroform, acetone and methanol fractions did not show notable activity for L. alata as they did not give 100% mortality at very high concentration of 4000 mg/l. The medicinal properties of plant extracts normally depend upon the presence of active compounds22 possessing specific functional groups that are soluble only in solvents of particular polarity. The active compounds in these extracts of the L. alata were therefore not soluble appreciably in these solvents.
S1: 1HNMR Analysis of Compound 1
S2: H/H COSY of Compound 1
S3: 13C NMR, DEPT 135 and DEPT 90 spectra of compound 1
S4: HSQC spectra of compound 1
S5: HSQC Spectra of Compound 1 Cont’d
S7: HMBC of Compound 1 Cont’d
S8: 1H NMR spectra of compound 2
S9: H-H COSY of compound 2
S10: 13C NMR and DEPT 90 Spectra of compound 2
Cq = 9, CH = 4, CH2 = 3, CH3 = 8
S11: 13C NMR spectra of compound on 600MHZ
S12: DEPT 135 spectra of compound 2
Appendix13: HSQC spectra of compound 2
S14: HMBC spectra of compound 2
4.0 Eudesmane sesquiterpenoids 1 and 2
Bioassay-guided fractionation of active hexane fraction of L. alata led to isolation of 30 mg of compounds 1 and 28 mg of compound 2. Elucidation of the structures of the pure compounds was determined using spectroscopic 1D and 2D NMR methods. The NMR data is shown in the Tables 3 and 4. High-resolution mass spectrometry established the molecular formulas of compounds 1 as C20H30O4 and molecular as 357.2034090 the calculated mass was 357.2041791, with double bond equivalence of six. Similarly, the molecular formula of compound 2 was established as C24H36O9 with corresponding mass of 491.2261970. However, the calculated mass was 491.2257024 with double bond equivalence of seven.
4.1 Larvicidal activity of L. alata compounds 1and 2
The eudesmane sesquiterpenoids 1 and 2 were isolated from very active hexane fraction of L. alata but themselves did not show appreciable activity. The hexane fraction gave LC50 and LC99 as 1161.30 mg/l and 2734.91mg/l at 95% confidence interval respectively.
4.2 Structure Elucidation of Eudesmane Sesquiterpenoid 1
Both one- (ID) and two-dimensional (2D) NMR were used to determine the structure of the pure compound 1. 13C-NMR and DEPT (Supplementary material S1 and 6), 1H-1H COSY (S7, Table 3, 1H-13C NMR (HMBC) (S6 and 7, Table 3), HSQC (S3 and 5) techniques achieved the structural elucidation and complete proton and carbon assignments. The comparisons of DEPT spectrum with a broadband decoupled carbon spectrum, made the carbon peaks to be firstly classified into methyl, methylene, methine and quaternary carbon atoms (Table 3). The proton decoupled 13C NMR spectrum (S1, Table 3) of 1 showed well resolved resonance of the 20 carbon atoms. The multiplicity of each carbon atom was determined using DEPT-135 and DEPT 90 experiment, which revealed the presence of six methyl groups, four methylene groups, three methine groups and seven quaternary carbon (two carbonyl carbon atoms, three vinylic carbon atoms and two saturated carbon atoms, indicating 30 hydrogen atoms attached to carbon atoms. Based on 1H NMR (S1 and 2) and proton decoupled 13C NMR spectrum (S1, Table 3) data of 1 the proposed structure of the compound is shown below and the corresponding fragmentation. The H-H correlation is also shown below.
The above prediction was supported by using its 2D NMR spectral data as follows. 1H-1H correlation spectroscopy (COSY) (Appendix 8, Table 9) showed strong correlation between H-3 (δ 4.8) and H-2 (δ 1.7) indicating methylene protons at C-2 are in the same environment as hydrogen atom on the oxygen containing carbon which is chiral. There are also coupling between H-3’ (δ 6.1) and H-4’ (δ 1.9) Heteronuclear Single Quantum Correlation (HSQC) experiment correlates the chemical shift of proton with the chemical shift of directly bonded carbon atom. In the HSQC spectral data (appendices 9 and 10), showed three protons at δ 1.0 (s) connected with C-14 (δ19.31). Three protons at δ 1.3 (s) connected with C-15, (δ 17.85) three protons at δ 1.8 (s) connected with C-13, (δ 23.13). Three protons at δ 2.0 (s) connected with C-5’, (δ 16.27), six protons at 1.98-2.00 connected with C-4’, (δ 21.02) and C-12, (δ 23.82). Two protons at δ 1.5 (m) connected with C- 1 (δ 38.60), two protons at δ 2.2 (m) connected with C-2 (δ 25.85). One proton at δ 4.8 (dd) correlates to C-3, (δ 81.69) and C-5, (δ 51.44) correlates with a single proton at δ 1.9 respectively. Two protons at δ.2.2 (d) connected with C-9, (δ 60.21). One proton at δ 4.8 (dd) attached to C-3, (δ 81.69), one proton at δ 1.9 (dd) connected with C-5, (δ 51.44) and one proton at δ 6.1 (q) connected with C-3’, (δ 139.08). Heteronuclear multiple bond correlation (HMBC) experiment gave information about coupling of hydrogen atoms and carbon atoms that are two or three bonds away. In the HMBC (appendix 10 and 11), the methyl protons at δ 1.9 (s) (H-5’) correlated with δ 139.08 (C-3’); δ 128.21 (C-2’) methylene proton at δ 1.56 (H-1) correlated with δ 81.69 (C-3); δ 60.21(C-9) and δ 19.31 (C-14). The methylene protons at δ 2.2 (m) (H-2) correlated with δ 74.76 (C-4). Methine proton at δ 4.8 (dd H-3) correlated with the δ 38.60 (C-1); δ 74.76 (C-4) and δ168.73 (C-1’). Another methane proton at δ 1.9 (dd H-5) correlated with δ 81.69 (C-3); δ 74.76 (C-4); δ 25.85 (C-6); δ 60.21 (C-9); δ 36.76 (C-10); δ 19.31 (C-14); δ 17.85 (C-15). The methylene proton at δ 2.68 (dd H-6) correlated with δ 51.44 (C-5); δ 130.64 (C-7); δ 202.64 (C-8); δ 36.76 (C-10); δ 145.29 (C-11). The methylene protons at δ 2.2 (d) H-9 correlated with δ 51.44 (C-5); δ130.64 (C-7); δ 202.64 (C-8); δ 36.76 (C-10); δ19.31 (C-14). The methyl protons at δ 2.0 (s) (H-12) correlated with δ 23.13 (C-13) and methyl proton at δ 1.8 (s) (H-13) correlated with δ 23.82 (C-12). The methyl protons at δ 1.0 (s) (H-14) correlated with C-1 δ 38.60; (C-5) δ 51.44; (C-9) δ 60.21 and C-10 δ 36.76. The methyl protons at δ 1.3 (s) (H-15) correlated with C-3 δ 81.69; C-4 δ 74.76 and C-5 δ 51.44. Methine proton at δ 6.1 (q) (H-3’) correlated with δ 16.27 C-5’ and δ 166.99 C-1’. The vinylic proton at δ 6.1 (q) (H-3’) correlated with δ 16.27 C-5’; δ 168.73 C-1’ and δ 21.02 C-4’. The configuration of 1 was proposed to be β-substituted at C-3 and C-4. The coupling constant of H-5 and H-6 confirm the configuration.