Toxicity of Individual and Blends of Pure Phytoecdysteroids Isolated from Vitex Schiliebenii and Vitex Payos against Anopheles Gambiae S.S. Larvae

Mokua Gladys Nyamoita

Department of Pharmacy and Complementary/Alternative Medicine, Kenyatta University, Nairobi, Kenya

Abstract: Four phytoecdysteroids: (20-hydroxyecdysone-20, 22-monacetonide (1), 20-hydroxyecdysone (2), stigmasterol (3), gamma-sitosterol (4), were investigated for toxic effect against 3rd /4th instar larvae of Anopheles gambiae under laboratory conditions as individual compounds and in blends. The test larvae were treated with solutions containing the phytoecdysteroids of concentrations 1, 5 and 10ppm. The blends were prepared in the ratio of 1:1. Compounds 1 and 2, isolated from acetone extracts of Vitex payos caused 100% mortality at 10ppm. Compound 3 isolated from acetone leaves of V. schiliebenii and compound 4 isolated from acetone stem bark of V. schilebenii also showed potent activity against the larvae at 10ppm. At the lower concentrations, abnormal mobility and impaired development was observed. Phytoecdysteroids (20-hydroxyecdysone-20, 22-monacetonide (1) and 20-hydroxyecdysone (2) are larvicidal against An. gambiae. stigmasterol (3), gamma-sitosterol (4) also show potent IGR activities against An. gambiae. Also addition of compounds 1 and 2 to stigmasterol (3) and gamma-sitosterol (4) seperately improved the activity of the two compounds.

Keywords: anopheles gambiae, phytoecdysteroids vitex schiliebenii, V. payos

Received December 31, 2012; Revised February 23, 2013; Accepted March 12, 2013

Citation: Mokua Gladys Nyamoita, Toxicity of Individual and Blends of Pure Phytoecdysteroids Isolated from Vitex Schiliebenii and Vitex Payos against Anopheles Gambiae S.S. Larvae. World Journal of Organic Chemistry, 1 (1). 1-5. January 2013. doi:10.12691/wjoc-1-1-1

1. Introduction

Phytoecdysteroids are plant analogues of insect growth hormones whose role in insect molting is understood, but their function in plants is uncertain [1]. However, it has been suggested that these chemicals are synthesized by the plants for their defense against phytophagous insects [1]. It is therefore expected that their production will be enhanced by insect attack. In the search for bioactive secondary metabolites with potential in the control of disease vectors and/or the diseases they transmit, detrimental effects including reduced weight, molting disruption and/or mortality have been reported in insects which ingest phytoecdysteroids [2]. These structurally diverse compounds have also been reported to affect protozoa and mollusks by impairing the digestion of protein and uptake of vitamins and minerals in their gut [2,3].

One of the main benefits of phytoecdysteroids is their therapeutic effects in mammals including humans. Their claimed medical properties include adaptogenic [4], anabolic [3], hypoglycemic [5], hepatoprotective [3], immunoprotective, wound-healing and anti-tumor. Other pharmacological properties associated with phytoecdysteroids include anti-proliferative, anti-microbial and anti-oxidant activities [6]. Ecdysteroids are also considered as nutraceutical additives to food products [7].

Phytoecdysteroids are found in a wide range of species in the genus Vitex and may be used as taxonomic markers. An extensive literature search on the genus Vitex revealed the presence of ecdysteroids in a number of Vitex species [8] found in tropical and subtropical regions [9]. They affect a wide range of insects at low concentrations and are not harmful to human and animal cells [10]. Literature reports indicate that ingested phytoecdysteroids affect several insect species for example Spodoptera frugiperda [11], Bombyx mori [12,13], Lobesia botrana [14], Inachis io, Alglass urticae [15] and Bradysia impatiens [16], resulting in significant growth and developmental disruption. However, some insect species such as Heliothis virescens [17], H. armigera [18], S. litturalis [19], Locusta oleracea, Ostrinia nubilalis [20], and Lacanobia oleracea [21] were reported not to be affected after phytoecdysteroid ingestion by developing a detoxifying mechanism [22].

In this study, the author reports the effects of four phytoecdysteroids namely (20-hydroxyecdysone-20,22-monacetonide (1), 20-hydroxyecdysone (2), Stigmasterol (3), Gamma-sitosterol (4) on 3rd and 4th instar larvae of An. gambiae under laboratory conditions. The compounds were isolated from two Vitex species namely, V. schiliebenii and V. payos collected from the Kenyan coastal region.

2. Materials and Methods

2.1. Plant Materials

Leaves and stem bark of V. schiliebenii and root bark of V. payos used in this study were collected from the Kenyan coastal region in 2009. The plants were authenticated at the field by a botanist from the National Museum of Kenya (NMK), who preserved a voucher specimen (GMN/22) at the museum. The materials were air-dried at room temperature under the shade for three weeks and ground into powder in an electric miller.

2.2. Extraction of Plant Materials

Each powdered material was extracted three times using acetone (5-fold volume) for 24h with occasional stirring at room temperature. The extracts were filtered and concentrated to dryness using a rotary evaporator at 40°C. The extraction process was repeated three times and the combined extracts were stored at 4°C. This procedure was repeated with methanol in the same proportions and for the same periods of time.

2.3. Isolation of Phytoecdysteroids

2.3.1. From Acetone Extract of V. payos Root Bark

Powdered root bark (30g) of V. payos was extracted using acetone and the extract (8.0g) was subjected to column chromatography on silica gel (105g) eluting with 100% dichloromethane and gradually increasing acetone to 100% then methanol to 30%. The 60% acetone eluent gave a mixture of two compounds, which were separated by repeated preparative Thin Layer Chromatography (PTLC) on silica gel (eluting with dichloromethane-acetone, 1:1) to give 20-hydroxyecdysone-20, 22-monacetonide (1) (16mg of a white amorphous powder which was soluble in methanol, with melting point 257-259oC; lit. 256°C, [23] ) and impure 20-hydroxyecdysone (2). This was further subjected to repeated PTLC (eluting with dichloromethane-acetone, 4:1) followed by sephadex LH₂₀ (DCM: MeOH 1:1). Ten milligrams of need-like crystals of melting point 234-236°C (lit. 230-233°C, [24] were obtained which were soluble in methanol.

2.3.2. Acetone Extract of V. schiliebenii Leaves

The acetone extract (10g) was subjected to column chromatography on silica gel (eluting with 100% dichloromethane and gradually increasing acetone to 100%). The 40% acetone eluent gave a mixture of two compounds, which were subjected to repeated column chromatography using DCM: MeOH to yield gamma sitosterol (4) (6mg with a melting point of 142-144°C) which was then re-crystallized using the same solvent into a white star shaped crystal soluble in DCM. The other compound was too little to be analyzed.

2.3.3. Acetone Extract of V. schiliebenii Stem Bark

Sixteen grams (16g) of the extract was subjected to column chromatography on silica gel with Dichloromethane: Acetone gradient (100:0 - 0:100). The 50% acetone eluent gave a mixture of two compounds (47mg), which were separated by repeated column chromatography followed by PTLC. The fraction yielded two compounds 20-hydroxyecdysone (2) (16mg) isolated as a crystalline solid and stigmasterol (3) (25mg of white crystals with a melting point of 165-7°C) lit. 163-6°C, [25].

2.4. Liebermann-Burchard Test for Steroids

The isolated compounds were examined for the presence of steroids using Liebermann-Burchard test. Acetic anhydride (2ml) was added to the compounds (2mg) and the mixture was thoroughly heated and stirred for 2 minutes on a water bath and allowed to stand at room temperature. Sulphuric acid (2ml) was gently added to 1ml acetic acid layer. The blue to green color of the upper layer suggested the presence of phytosterols.

2.5. Mosquito

Larvae of An. gambiae Giles s.s. used in bioassays were obtained from a colony maintained at the International Centre of Insect Physiology and Ecology (ICIPE) Insect Mass Rearing Unit. This strain of mosquitoes originated from ICIPE’s Thomas Odhiambo Campus (Mbita Point) near in 2003. Larvae were allowed to emerge from eggs in plastic containers filled with distilled water and were transferred to larger pans (37 × 31 × 6) at densities of 200-300 at 2nd instar stage. They were fed on Tetramin® fish food (Terta GmbH, Germany) and the water temperature was maintained at 28±2°C throughout larval development.

2.6. Larvicidal Assays

Larvicidal and insect growth regulatory (IGR) activities of the individual and blends of the four phytoecdysteroids (20-hydroxyecdysone-20, 22-monacetonide (1), 20-hydroxyecdysone (2), stigmasterol (3) and gamma-sitosterol (4) were conducted in accordance to the World Health Organization method [26]. Batches of twenty freshly moulted late 3rd and early 4th instar larvae of An. gambiae s.s. were transferred by means of dropper to glass beakers containing 100ml of tap water. Appropriate volume of stock solution where the pure compounds or blends were dissolved in 5% dimethylsuphoxide (DMSO) was added to 100ml water in the glass beakers to obtain 1, 5, 10ppm dose levels. The blends were mixed in the ratio of 1:1. Three replicates were set up for each concentration and two negative controls (treated with DMSO-distilled water) were set up simultaneously. Larval mortality, abnormal behavior and/or morphological deformations were recorded at 24h intervals until the death of the last larva or emergence to adult. The bioassay room was kept at a temperature of 30°C, an average humidity of 78 % and a photo period of 12 hours of light and 12 hours of darkness. The larvae were fed on Tetramin® fish food (Terta GmbH, Germany) at about 1mg per beaker every 24h.

2.7. Calculation of Larval Mortality

The average number of larvae or pupae collected for each replicate of each treatment and the control were recorded after 24h. Percentage larval mortality was estimated for each treatment according to the formula:

Where: Z=Initial number of larvae introduced into each test beaker and Y=Mean death defined by the difference between the mean test deaths and the mean control deaths.

3. Results

Chromatographic separation of the acetone extracts of V. payos root bark, V. schiliebenii leaves and V. schiliebenii stem bark yielded four known phytoecdysteroids [(20-hydroxyecdysone-20, 22-monacetonide (1), 20-hydroxyecdysone (2), stigmasterol (3), gamma-sitosterol and (4)]. The identification was done by physical, spectroscopic and chemical analysis as well as literature data comparisons [8,27,28] (Table 1). These compounds were tested for larvicidal and/or IGR activities and the results revealed that they were toxic to An. gambiae larvae. Compounds 20-hydroxyecdysone-20, 22-monacetonide (1) and 20-hydroxyecdysone (2) caused 100% mortality at 10ppm (Table 2). Similarly, high mortality was obtained at 1 and 5 ppm (≥ 80%) with about 15-18 % showing impaired development. Stigmasterol (3) and gamma-sitosterol (4) caused 55±2.5 and 65±2.4 % mortality respectively at 10ppm (Table 2). A blend of 20-hydroxyecdysone-20, 22- monacetonide (1) and 20-hydroxyecdysone (2) was moderately active (65±2.1%) at 1ppm but the activity was high at 5 and 10 ppm (85±2.9 and 90±2.5) respectively. These results therefore indicated a slight drop in the activity of the resulting blend. On the other hand, a combination of stigmasterol (3) and gamma-sitosterol (4) which were less active improved the activity of the individual compounds. Addition of 20-hydroxyecdysone-20, 22-monacetonide (1) to stigmasterol (3) and gamma-sitosterol (4) improved the activity of the two compounds to 90 and 100% at 1 and 10 ppm while individually each caused 25±2.4, 55±2.5 and 35±2.5 65±2.4% mortality, respectively. It was also interesting to note that addition of 20-hydroxyecdysone (2) to stigmasterol (3) and gamma-sitosterol (4) also improved their activity as shown in (Table 2).

Table 1. ¹³CNMR spectral data for compounds 1, 2, 3 and 4

Table 2. Percentage mortality of An. gambiae larvae exposed to pure compounds isolated from V. payos and V. schiliebenii individually and in blends at 1, 5, and 10ppm

4. Discussion

In the present study, 20-hydroxyecdsone (2) exhibited larvicidal activity against An. gambiae larvae. This observation compares with previous studies where 20-hydroxyecdysteroid (2) was used as the sole or major component in bioassays. Results indicated a range of detrimental effects on development and survival of several insect species including Bombyx mori [11], Pectinophora gossypiella [11,29], Spodoptera frugiperda [30], Acrolepiopsis assectella [29,30] and Agrius convolvulus [31]. The blend effect observed in the current study indicated that the activity of stigmasterol (3) and gamma-sitosterol (4) can be improved by preparing a formulation of the two compounds containing either 20-hydroxyecdysone-20, 22-monacetonide (1), or 20-hydroxyecdysone (2). In addition, the synergism observed in the blend of compounds 3 and 4 also indicated that a better formulation can be prepared by using the two compounds as a blend instead of using them as seperate compounds. Reported experiments [32,33,34,35,36] working on some plant extracts having potential larvicides also note that botanical blends provide better effect in reducing vector population. This is attributed to the adaptive value of phytochemical diversity in ecological interactions among plants and their associated herbivores and pathogens.

The purpose of this study was to elucidate the role and relative importance of steroids in Vitex species in controlling An. gambiae larvae and to use this information in guiding effective development of formulations to be used in integrated pest management programmes. Three variants of blend effects were noted from these results. First, production of a less active blend from active constituents; secondly, enhancement of the activity of a moderately active compound by an active constituent and; thirdly, synergism between moderately active compounds to give a blend that was more active than the individual constituents. The first variant was illustrated by the high lethal activity of compounds 1 and 2 while the second was illustrated by the enhancement of the activity of compounds 3 and 4 in blends with 1 and 2. The third variant was illustrated by the combination of compounds 3 and 4.

5. Conclusion

It can be concluded that phytoecdysteroids (20-hydroxyecdysone-20, 22-monacetonide (1) and 20-hydroxyecdysone (2) possess high larvicidal activity against An. gambiae. Stigmasterol (3) and gamma-sitosterol (4) also show potent insect growth regulatory (IGR) activities against An. gambiae. Consequently, V. payos and V. schiliebeinii have important practical implications in the search for and use of plants and their phytochemicals for mosquito larvae control.

Acknowledgements

The author is grateful to the International Centre of Insect Physiology and Ecology (ICIPE-Kenya) for providing the necessary bioassay requirements. Muhimbili University of Health and Allied Sciences is also acknowledged for providing the necessary chromatographic equipment for fractionation and purification of the crude plant materials. The author also acknowledges DAAD-NAPRECA for funding the work reported in this paper.

References

[1]   | Klein, R, “Guild, Fall/Water,” J. Am. Herb. 2004.
[2]   | Dinan, L., Savchenko, T. and Whiting, P, “On the distribution of phytoecdysteroids in plants,” Cellular and Molecular Life Sci. 58 (8). 1121-1132. 2001.
[3]   | Syrov, V.N. and Khushbaktova, Z.A, “Experimental study of pharmacotherapeutic effect of phytoecdysteroids and nerobol in toxic liver damage”. [Russian]. Eksp Klin Farmakol., 64. 56-58. 2001.
[4]   | Kholodova, Y.D, “Phytoecdysteroids: biological effects, application in agriculture and complementary medicine,” Ukr Biokhim Zh, 73. 21-29. 2001.
[5]   | Kutepova, T.A., Syrov, V.N., Khushbaktova, Z.A. and Saatov, Z, “Hypoglycemic activity of the total ecdysteroid extract from Ajuga turkestanica” [Russian]. Pharm. Chem. J. (Khim Farm Zhur), 35. 608-609. 2001.
[6]   | Nilufar ,Z., Mahmoud, Z.E., Elisa, O. Mohamed, L.A., Razan, H., Shamansur ,S.S. Sakhnoza, S.A., Antonio, T. and Michael, W, “Antiproliferative, antimicrobial and antioxidant activites of the chemical constituents of Ajuga turkestanica,” Phytopharmacology, 4 (1). 1-18. 2012.
[7]   | Lafont, R. and Dinan, L, “Practical uses for ecdysteroids in mammals including humans”: an update. J. Insect. Sci., 3 (7). 2003.
[8]   | Sena Filho,J.G., Duringer, J., Maia, G.L., Tavares, J.F., Xavier, H.S., da Silva, M.S., da-Cunha, E.V. and Barbosa-Sena Filho, J.M, “Ecdysteroids from Vitex species: distribution and compilation of their 13C-NMR spectral data,” Chem. Biodiverse, 5 (5). 707-713. 2008.
[9]   | Correa, M.P, “Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas” Imprensa Nacional, Rio de Janeiro. 1926, Vol. IV, p 208.
[10]   | Bathori, M, “Phytoecdysteroids effects on mammalians, isolation and analysis”. Mini Reviews in Medicinal Chemistry, 2: 285-293. 2002.
[11]   | Kubo, I., Klocke, J.A. and Asano, S. “Effects of ingested phytoecdysteroids on the growth and development of two lepidopterous larvae,” J. Insect Physiol., 29. 307-316. 1983.
[12]   | Tanaka, Y. and Takeda, S, “Ecdysone and 20-hydroxyecdysone supplements to the diet affect larval development in the silkworm, Bombyx mori, differently”. J Insect Physiol., 20. 805-809. 1993.
[13]   | Tanaka, Y., Asaoka, K. and Takeda, S, “Different feeding and gustatory responses to ecdysone and 20-hydroxyecdysone by larvae of the silkworm, Bombyx mor,I” J. Chem. Ecol., 20. 125-133. 1994.
[14]   | Mondy, N., Ca¨ıssa, C., Pitoizet, N., Delbecque, J.P. and Corio-Costet, M.F, “Effects of the ingestion of Serratula tinctoria extracts, a plant containing phytoecdysteroids, on the development of the vineyard pest Lobesia botrana (Lepidoptera: Tortricidae),” Arch. Insect Biochem., 35. 227-235. 1997.
[15]   | Blackford, M. and Dinan, L, “The effects of ingested 20-hydroxyecdysone on the larvae of Aglais urticae, Inachis io, Cynthia cardui (Lepidoptera: Nymphalidae) and Tyria jacobaeae (Lepidoptera: Arctiidae),” J. Insect Physiol. 43. 315-327. 1997.
[16]   | Schmelz, E., Grebenok, R., Ohnmeiss, T. and Bowers, W. “Interactions between Spinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids,” Arch. Insect Biochem Physiol., 51. 204-221. 2002.
[17]   | Kubo, I., Komatsu, S., Asaka, Y. and De Boer, G, “Isolation and identification of a polar metabolites of ingested 20-hydroxyecdysone in frass of Heliothis virescens larvae,” J.Chem. Ecol., 13.785-794. 1987.
[18]   | Robinson, P.D., Morgan, E.D., Wilson, I.D. and Lafont, R. “The metabolism of ingested and injected [3H]ecdysone by final instar larvae of Heliothi armigera,” Physiol. Entomol., 12. 321-330 1987.
[19]   | Blackford, M., Clarke, B., Dinan, L, “Tolerance of the Egyptian cotton leafworm Spodoptera littoralis Lepidoptera: Noctuidae) to ingested phytoecdysteroids,” J. Insect Physiol. 42. 931-936. 1996.
[20]   | Rharrabe, K., Alla, S., Maria, A., Sayah, F. and Lafont, R, “Diversity of detoxification pathways of ingested ecdysteroids among phytophagous insects,” Arch. Insect Biochem. Physiol., 65. 65-73. 2007.
[21]   | Kubo, I., Klocke, J.A. and Asano, S, “Insect ecdysis inhibitors from the East African medicinal Plant Ajuga remota (Labiatae),” Agric. Biol. Chem. 45. 1925-1927. 1981.
[22]   | Dinan, L. and Hormann R.E. Ecdysteroid agonists and antagonists. In: Comprehensive Molecular Insect Science (Eds. Gilbert, L.I., Iatrou, K. and Gill, S), Elsevier, 3, 197-242. 2005.
[23]   | Jiaju, Z., Guirong X. and Xiajin Y. Encyclopadia of traditional Chinese Medicines. Molecular Structures, Pharmacological Activities, Natural Sources and Applications. SpringerVerlag, Berlin Heideberg, 2011.
[24]   | Kavel, V.M., Budesnsky, J.H and Jitka, K. Ecdysteroid constituents of the mushroom Tapinella panuoides. Phytochemistry 49. 2109-2114. 1998.
[25]   | Greca, M.D., Monaco, P. and Previta L. Stigmasterol from Typha latifolia. J. Nat. Prod. 53. 1430-3. 1990.
[26]   | WHO Report of the WHO informal Consultation on the Evaluation and Testing of Insecticides. WHO 1996; Geneva. pp 37.
[27]   | Maria do, C.E., Mara, A.D.F., Adebal, F.M., va G.M. and Ana, L.T.G. Steroids and triterpenes from Eleocharis acutangula and E. sellowiana (Cyperaceae). Pyhtochemistry Anal. 15. 125-129. 2004.
[28]   | McCarthy, F.O., Chopra, J., Ford, A., Hogan, S.A., Kerry, J.P., O’Brien, N.M., Ryan, E. and Maguire, A.R. Synthesis, isolation and characterization of β-sitosterol and β-sitosterol oxide derivatives. J.Org.Biomol. Chem. 3, 3059-65. 2005.
[29]   | Arnault, C. and Sláma K.knowle, “Dietary effects of phytoecdysone in the leek-moth, Acrolepiopsisassectella Zell. (Lepidoptera: Acrolepiidae),” J. Chem. Ecol. 12. 1979-1986. 1986.
[30]   | Harmatha, J. “Chemo-ecological role of spirostanol saponins in the interaction between plants and insects”. In: Saponins in Food, Feedstoffs and Medicinal Plants (Eds. Oleszek W, Marston A), Kluwer, Dordrecht, Proceedings of the Phytochemistry Society of Europe, 45, 129-141. 2000.
[31]   | Tanaka, Y. and Naya, S. “Dietary effect of ecdysone and 20-hydroxyecdysone on larval Development of two lepidopteran species,” Appl. Entomol. Zool,. 30. 285-294. 1995.
[32]   | Soriano, I. R., Riley, I. T., Potter, M. J. and Bowers, W.S. “Phytoecdysteroids: a novel defense against plant-parasitic nematodes,”. J. Chem. Ecol., 30 (10). 1885-1899. 2004.
[33]   | Dinan, L, “Phytoecdysteroids: biological aspects,” Phytochemistry, 57. 325-339. 2001.
[34]   | Berenbaum, M.R. and Zangerl, A.R, Phytochemical diversity: adaptation or random variation? In: Romeo, J.T., Saunders J.A. and Barbosa P. (Eds.). Phytochemical Diversity and Reductancy in Ecological Interactions. Plenum Press, New York, 1-24, 1996.
[35]   | Cates, R.G, The role of mixtures and variation in the products of terpenoids in conifer-insect-pathogen interactions. In: Romeo J.T., Saunders J.A. and Barbosa P. (Eds.), Phytochemical Diversity and Reductancy in Ecological Interactions. Plenum Press, New York, 179-216, 1996.
[36]   | Anupam, G., Nandita, C. and Goutam C. Plant extracts as potential mosquito larvicides. Indian J Med Res. 135. 581-598. 2012.