Larvicidal activity of extracts from three Plumbago spp against Anopheles gambiae

ARTICLES

Larvicidal activity of extracts from three Plumbago spp against Anopheles gambiae

Barasa M Maniafu^I; Lwande Wilber^II; Isaiah O Ndiege^III; Cornelius C Wanjala^IV; Teresa Ayuko AkengaIV, ⁺

^IChemistry Department, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

^IIBehavioural and Chemical Ecology Department, International Centre of Insect Physiology and Ecology, Nairobi, Kenya

^IIIChemistry Department, Kenyatta University, Nairobi, Kenya

^IVDepartment of Applied Chemistry, Kigali Institute of Science and Technology,BP 3900, Kigali, Rwanda

ABSTRACT

Three Plumbago spp have been tested for mosquito larvicidal activity. The crude extracts exhibiting the highest larvicidal activity against Anopheles gambiae were hexane (LC₅₀ = 6.4 μg/mL) and chloroform (LC₅₀ = 6.7 μg/mL) extracts from Plumbago zeylanica Linn, chloroform (LC₅₀ = 6.7 ug/mL) extract from Plumbago stenophylla Bull and ethyl acetate (LC₅₀ = 4.1 μg/mL) extract from Plumbago dawei Rolfe. These LC₅₀ values were within 95% confidence limits. 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin) 1 (LC₅₀ = 1.9 μg/mL) and β-sitosterol 2 were characterised from ethyl acetate extract of root bark of P. dawei, a native medicinal plant growing in Kenya, based on spectral analysis and comparisons with data in literature.

Key words:Plumbago zeylanica - Plumbago stenophylla - Plumbago dawei - larvicidal activity - 5-hydroxy-2-methyl-1,4-naphthoquinone - Anopheles gambiae

The repeated use of synthetic insecticides for mosquito control has disrupted natural biological control systems and led to resurgences in mosquito populations. It has also resulted in the development of resistance (Brown 1986), undesirable effects on non-target organisms and fostered environmental and human health concern (Cheng et al. 2003). These side effects have initiated a search for alternative control measures. Natural products of plant origin with insecticidal properties have been assayed in the recent past for the control of a variety of insect pests and vectors. These include essential oils (Harve & Kamath 2004) and azadiractins (Obomanu et al. 2006). Phytochemicals derived from plant sources can act as larvicides, insect growth regulators, repellents and ovipositor attractants, as observed by many researchers (Venketachalam & Jebasan 2001, Thomas et al. 2004).

Plumbaginaceae is a plant family consisting mostly of shrubs that is found in scarcely distributed grassland steppes of arid and coastal regions. They are slow growing, highly branched evergreen shrubs (Kokwaro 1993). Extracts of several Plumbaginacea family members have been investigated for anti-malarial, anti-bacterial and anti-protozoal properties (Saraswathy et al. 2003, Parekh & Chanda 2006, Dorni et al. 2007). Here we report the larvicidal activity of organic extracts of three Plumbago spp commonly found in the Coastal and Eastern Regions of Kenya. The species investigated were Plumbago zeylanica Linn, Plumbago dawei Rolfe and Plumbago stenophylla Bull. P. zeylanica is traditionally used topically for skin disorders and orally for hookworms. Additionally, in some communities, this species is used as a traditional medicine against malaria. The decoction of the leaf of P. dawei is consumed as a purgative and P. stenophylla root extracts are used for the treatment of wounds (Kokwaro 1993). In addition to the characterisation of larvicidal activity, phytochemical investigation of the most active extracts was carried out.

MATERIALS AND METHODS

Plant material - The roots of P. zeylanica, P. dawei and P. stenophylla were collected from Kibwezi, Mwatate and Arabuko, respectively, in June 2005, and identified by SG Mathenge of the Nairobi University Botany Department. Voucher specimen 2005/10, 2005/11 and 2005/12, respectively, were deposited at the University Herbarium, Botany Department, University of Nairobi.

Extraction - The air dried root bark of the plant (1 kg) was crushed and extracted sequentially with hexane, chloroform, ethyl acetate and methanol at RT for three days. The extract was filtered and the solvent removed at low temperature (35-40⁰C) and reduced pressure on a rotary evaporator.

Fractionation and purification- All solvents used were of analytical grade (E. Merck, D-6100 Darmstadt, F. R. Germany). Melting point was determined on micro-capillary melting point apparatus, Yanaco model MP 500D and is uncorrected. Analytical thin layer chromatography (TLC) was performed using pre-coated silica gel 60 F₂₅₄ plates (5 x 10 cm x 0.20 mm) with fluorescence indicator. For preparative TLC (PTLC), 20 x 20 cm x 0.25 mm plates were used. The spots were first visualised using a multiband UV-254/366 nm lamp (UV GL-58). The TLC plates were then sprayed with 2% concentrated H₂SO₄ in methanol and kept in the oven at 105⁰C for 10 min. ¹H NMR was recorded at 200 MHz and ¹³C NMR (DEPT) recorded at 50 MHz on Varian Gemini 200 MHz instrument at RT; chemical shifts are reported in ppm upfield of tetramethylsilane and multiplicities are reported as singlet (s), doublet (d), triplet (t) and quartet (q). MS analyses were carried out in Finigan Mat SSQ700 single quadrupole instrument and Autospec time of flight spectrometer EI-MS and ES-MS.

The root bark of P. dawei (410 g) was extracted (RT) with ethyl acetate (1.5 L) for three days. The extract (3 g) was fractionated by column chromatography on silica gel 230-400 mesh eluting with C₆H₁₄: EtOAc (9:1, 8:2, 7:3 and 100% EtOAc) to afford 56 fractions (Fr_1-56). The first solvent system yielded fractions Fr_1-11(76 mg, 0,0253%). The second system gave fractions Fr_12-34 (401 mg, 13,37%) while the third gave fractions Fr _35-56 (106 mg, 0,0353%). Fraction Fr_12-34 gave compound 1, which was obtained as bright yellow needle-like crystals with melting point 77.8-77.4ºC. PTLC purification (using 8: 2 C₆H₁₄: EtOAc) of fraction Fr_35-56 gave 2 as a colourless crystalline solid with melting point 130-132ºC.

Larvicidal bioassays - Larvicidal tests were carried out on late 3rd and newly emerged 4th instar Anopheles gambiae larvae. The larvae were reared under standard conditions in the mosquito insectary of the International Centre of Insect Physiology and Entomology, Nairobi, Kenya. A. gambiae larvae were reared on fish food and maintained at 27 ± 1ºC, > 70% relative humidity, with a photoperiod of 16 h. Twenty-five larvae were used (5 insects per 30 mL vial) for each experiment in five replicates. The larvae were considered dead if they were immobile, unable to reach the water surface and lacked head to tail flexion in response to tapping the beaker with the end of a pencil. The larvae were scored for percent mortality after 24, 48 and 72 h. Deltamethrin was used as a positive control.

Larvicidal activity of extracts/compounds was determined on the basis of the percentage mortality of the larvae introduced into the test vials. Mortality was monitored after 24, 48 and 72 h until emergence of adults in the test at different concentration ranges. Percentage mortality (PM) was determined using the equation:

% PM = [mean death count/initial larvae population] 100

Relative potency values LC₅₀ were determined by probit analysis.

RESULTS AND DISCUSSION

The yields of the crude extracts from the three species are presented in Table I. The MeOH extracts gave the highest yields from each species.

Larvicidal bioassays on crude extracts and isolated compounds - Larvicidal activities of crude organic extracts from Plumbago spp are summarised in Tables II-IV. They are presented as percentage mortalities (% mortality).

From the results, all crude extracts (except that of methanol) (Table II) achieved maximum mortality of 100% at 100 μg/mL; 84-98% at 10 μg/mL; 26-98% at 5 μg/mL and 6-88% at 1 μg/mL (24-72 h). Mortality increased when the larvae were exposed for a longer period. P. dawei extracts had the highest activity, followed by P. zeylanica (Table IV) and finally P. stenophylla (Table III). The results reveal that the extracts of different plants varied significantly in their larvicidal potential.

The larvicidal activity of the organic extracts was found to be species-dependent. For P. dawei, EtOAc extract showed the highest larvicidal activity (Table V). For P. zeylanica, the extract of C₆H₁₄ was the most active, while for P. stenophylla the CHCl₃ extract was the most active.

From the data in Table V, the ethyl acetate extract from the root bark of P. dawei displayed LC₉₀ and LC₅₀ values of 10.6 and 4.10 μg/mL, respectively, making it the most active of all extracts tested. This extract was selected for further analysis and was subjected to chromatographic fractionation. The fractions obtained were then tested for their larvicidal activity to establish the chemicals responsible in these species.

Three major fractions were obtained when the EtOAc extract of P. dawei was subjected to column chromatography. Table VI shows the lethal concentration values of these fractions. Fr_1-11 (LC₅₀ = 110.0 μg/mL) and F_12-34 (LC₅₀ = 1.9 μg/mL) exhibited the strongest larvicidal activities, while Fr_35-56 with (LC₅₀ = 16.1 μg/mL) was the least active.

The larvicidal activities obtained in this study are in agreement with reported data. For example, Sosan et al. (2001) reported the larvicidal activities of essential oils of Ocimum gratissium, Cymbopogon citrus and Ageratum conyzoides against Aedes aegypti, achieving 100% mortality at concentrations of 120, 200 and 300 ppm, respectively. Das et al. (2007) reported that the LC₉₀ values of methanol and ethanol extracts from Aristolochia saccata roots, Annona squamosa leaves and Gymnopetelu cochinchinensis fruits/pericarp against Aedes albopictus and Culex quinquefasciatus larvae ranged from 31.80-155 ppm. These results show that the larvicidal activities of plant extracts vary according to the plant species, part of the plant used, application method and geographical location where the plants were grown. However, ours is the first report of the larvicidal activities of these Plumbago spp.

The naphthoquinone, plumbagin, 1 was obtained from fractions F_12-34, while β-sitosterol 2 was obtained from Fr_35-56. These two compounds were subjected to larvicidal assays. The concentration of 1 used ranged from 1-5 μg/mL over a period of 24 h. The results confirm that the larvicidal activity of P. dawei is due to 1. Plumbagin has been isolated in a range of Plumbago species (Croft et al. 1989, Panichayupakaranant & Tewtrakul 2002, Ribeiro de Paiva et al. 2003) and also in the leaves, roots and stem bark of different plant species (Borges-Argáez et al. 2007, Dzoyem et al. 2007, Ganapaty et al. 2006). This compound has been reported to exhibit anti-protozoal, anti-fungal, cytotoxic and anti-bacterial effects (Cai et al. 2000, Wang et al. 2001, Inbaraj & Chignell 2003). In contrast, α-sistosterol, 2 did not show any larval mortality at the tested concentration ranges.

In conclusion, it is evident that all three Plumbago species tested and the isolated naphthoquinone 1 possess remarkable larvicidal activity. Larvicides of great value could be developed from these plants as an alternative means to control mosquitoes and thus help combat malaria.

Identification of isolated and purified compounds

5-Hydroxy-2-methyl-1,4-naphthoquinone (Plumbagin) - This was obtained from fraction Fr_12-34 and identified from 1D and 2D NMR data, mass spectral data and by comparing these spectroscopic data with those reported in literature. Plumbagin (Figure) was obtained as bright yellow needles (401 mg), mp 77.8-77.4°C [literature value 76.8-77.4°C (Dinda et al. 1998, Hazra et al. 2002)]; ¹H-NMR (CDCl₃, 200 MHz,), δ 2.20 (3H, s, CH₃), 6.81 (1H, s, H-3), 7.24 (1H, d, J = 7.8 Hz, H-8), 7.61 (2H, t, J = 7.8, 2.4 Hz, H-6 and H-7), 11.98 (1H, s, OH). ¹³C-NMR (CDCl_3, 50 Hz), δ: 16.8 (C-9, q), 115.3 (C-4a, s), 119.6 (C-8, d), 124.4 (C-6, d), 132.3 (C-8a, s), 135.7 (C-7, d), 136.4 (C-3, d), 149.9 (C-2, s), 161.4 (C-5, s), 185.1 (C-1, s), 190.5 (C-4, s). EIMS: m/z 188 (M^+, 100%, C₁₁H₈O₃), 173 (M-CH_3, 2.9), 160 (3.5), 131 (71.4), 120 (4.7), 92 (72.9), 77 (3.4) and 63 (85).

17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol (β-Sitosterol) - This compound (Figure) was identified by comparing the spectral data obtained with those reported in the literature (Dey & Harbone 1991). The stereochemistry at the C-24 asymmetric centre was investigated by a careful comparison with the ¹H NMR spectra of both epimers at C-24 in similar steroids (Gamze et al. 2002). The steroid was obtained as colourless crystals (13 mg), mp 130-132ºC; ¹H-NMR (CDCl₃, 200 MHz) δ 0.67 (3H, s, H-18), 1.0 (3H, s, H-19), 0.81 (3H, dd, H-27), 0.84 (3H, t, H-29), 0.82 (3H, d, H-26), 0.93 (3H, d, H-21), 3.50 (1H, m, H-3), 5.34 (1H, d, 6-H); the rest of the protons were found in a complex multiplet signal between α 1.13-2.34. ¹³C NMR (CDCl₃, 50 Hz) δ 37.2 (C-1), 31.6 (C-2), 71.8 (C-3), 42.3 (C-4), 140.7 (C-5), 121.7 (C-6), 31.9 (C-7), 31.6 (C-8), 50.1 (C-9), 36.5 (C-10), 23.0 (C-11), 39.7 (C-12), 42.3, (C-13), 56.6 (C-14), 24.3 (C-15), 28.2 (C-16), 56.0 (C-17), 11.8 (C-18), 19.4 (C-19), 36.1 (C-20), 19.0 (C-21), 33.9 (C-22), 26.0 (C-23), 45.7 (C-24), 29.7 (C-25), 18.8 (C-26), 19.8 (C-27), 21.1 (C-28), 12.0 (C-29); EIMS: m/z, 414 ([M]⁺, 4.2, C₂₉H₅₀O), 396 ([M-H₂O]⁺, 1.8), 381 ([M-H₂O-CH₃]⁺, 1.2), 303 ([M-C₇H₁₁O]⁺, 1.6), 301 ([M-C₈H₁₇]⁺, 1.0), 273 ([M-C₁₀H₂₀]⁺, 1.4), 231 ([M-C₁₃H₂₅]⁺, 1.0), 213 ([231-H₂O]⁺, 1.7), 255 (M-H₂O-C₁₀H₂₁]⁺, 14), 43 (C₃H₇]⁺ , 100).

Though compounds 1 and 2 have been reported elsewhere in literature (Dey & Harbone 1991, Hazra et al. 2002), this is the first time they are being reported from the Plumbago species growing in Kenya.

ACKNOWLEDGEMENTS

To Kenya Wildlife Services, for permission to collect plants in the protected areas.

REFERENCES

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Received 5 December 2008

Accepted 30 June 2009

Financial support: WHO/TDR/MIM (99056)

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Publication Dates

• Publication in this collection
  22 Oct 2009
• Date of issue
  Sept 2009

History

• Received
  05 Dec 2008
• Accepted
  30 June 2009