Utilization of electrocoagulation for the isolation of alkaloids from the aerial parts of Stemona aphylla and their mosquitocidal activities against Aedes aegypti
Graphical abstract
Introduction
The roots of plants from the Stemona-genus are widely known in Thailand and Southeast Asian countries for their insecticidal and antitussive properties. The roots and their extracts have been traditionally used as an insecticide, larvicide and scabicide and to treat headlice and respiratory diseases (Mungkornasawakul et al., 2003, Mungkornasawakul et al., 2004; Greger, 2006; Kongkiatpaiboon and Gritsanapan, 2012). A recent taxonomic revision indicates that Stemona in Thailand comprises 11 species (Inthachub et al., 2010). Stemona aphylla is an important species widely found in the North and Northeast of Thailand. Its roots contain a specific group of Stemona alkaloids, including (2′S)-hydroxystemofoline, stemofoline, (2′S)-hydroxy-(11S,12R)-dihydrostemofoline, (11Z)-1′,2′-didehydrostemofoline, stemaphylline and stemaphylline-N-oxide (Mungkornasawakul et al., 2009; Sastraruji et al., 2011). However, the chemical constituents of the aerial parts of this plant have not been investigated. In addition, synthetic insecticides such as the organochlorine, organophosphate, carbamate, and pyrethroid are a current major public health problem because of their toxicity and persistence in the environment. The use of botanical insecticides is an alternative way for solving this problem. The advantage of the botanical insecticides is a wide range of chemical defenses types with different mechanisms of action against a variety of insects, including phenols and polyphenols, terpenoids and alkaloids. Moreover, these bioactive compounds are often less toxicity and have shorter half-lifes compared to the synthetic compounds, resulting in decreased amounts of the harmful residues in the environment and the life cycles of human and animals. However, because of the development of resistance to insecticides by agricultural pests and diseases carrying insects, research and development of new active compounds with high effectiveness and environmental safety to control agricultural pests and vector-borne diseases is necessary (World Health Organization (WHO), 2012; Pavena, 2016; Muangmoon et al., 2018). Previous research has indicated that the root extracts of Stemona species have been widely used as insecticides on agricultural pests, and insects acting as disease vectors. In 1978, Sakata et al. have reported that the Stemona alkaloids stemonine, stemospironine and stemofoline have insecticidal activity against the fourth instar Bombyx mori (silkworm larvae). Additionally, other Stemona alkaloids such as neostemonine and isoproto-stemonine, were also reported to have antifeedant activities against last-instar larvae of Spodoptera litura. In 2001, Jiwajinda et al. found that the stemofoline alkaloids, 16-17-didehydro-16(E)-stemofoline and its isomer at C-4, 16-17-didehydro-4(E)-16(E) stemofoline displayed insecticidal and antifeedant activities against the diamondback moth larvae. Greger (2006) found that the roots of Stemona species containing certain protostemonine derivatives, especially didehydro-stemofoline exhibited, very high toxicity against larvae of Spodoptera littoralis. Additionally, 1′-2′-didehydrostemofoline, which was the major compound of the root of S. collinsae, and stemofoline displayed contact toxicity and antifeedant activity. In 2016 Sakulpanich et al., reported that didehydrostemofoline was highly effective against third-instar larvae of Chrysomya megacephala with a LD50 value of 1.59 mg/larva using the direct contact toxicity method. While, the ethanolic extract of S. collinsae roots showed a more moderate effect with a LD50 of 101.82 mg/larva. Mungkornasawakul et al. (2004) reported that the pure alkaloids, oxyprotostemonine, stemocurtisine and stemocurtisinol and the crude ethanolic extract of S. curtisii, showed strong larvicidal activities on mosquito larvae (Anopheles minimus HO).
Generally, Stemona alkaloids have been isolated using organic solvent or acid-base extraction technique (conventional technique) including, chromatographic separation techniques. Large amounts of highly toxic or carcinogenic organic solvents have often been used in these process. These organic solvents, finally, end up as foul volatile chemical wastes. Long term exposure to these wastes can lead to deleterious effects on respiratory, hematological and thyroid functioning. Furthermore, the spills or leaks of these organic solvents that reach to the underlying soil are the cause of soil contamination and deleteriously affect aquifers (Jumpatong et al., 2007, Rama koteswararao et al., 2014). In order to decrease the use of harmful solvents in this process, the electrocoagulation (EC) technique is an alternative method. EC is capable of fractionating, in a rather efficient manner, a number of both organic and inorganic substances by selective electrochemically coagulating some of them, while leaving the other components free in solution. The application of the EC method to isolate seven known alkaloids from Camellia sinensis, Nicotina tabacum, Piper nigrum, Areca catechu and Capsicum frutescens have been reported. The results indicated that these alkaloids could be extracted successfully with yields comparable to that obtained by conventional extraction methods but with a decrease in the amounts of organic solvents consumed (Phutthawong et al., 2007).
One objectives of this current study was to examine the alkaloid components of the aerial parts of S. aphylla and compare these with those isolated earlier from the roots (Mungkornasawakul et al., 2009; Sastraruji et al., 2011). A comparison of the amount of the alkaloid components isolated using the EC and the conventional solvent extraction method was made by quantitative HPLC analysis of the crude plant extracts and from isolation of the individual alkaloids by chromatographic techniques. A comparison of the amounts of organic solvents used in these two isolation processes was also be examined. Moreover, the isolated alkaloids were used to study their ovicidal, larvicidal and oviposition-deterrent activities on Ae. aegypti, a vector for dengue fever. The morphology of the alkaloid treated larvae were also studied.
Section snippets
Plant material
S. aphylla was collected from Lampang province, Thailand. A voucher specimen was deposited at the Herbarium (number 09–111) of the Department of Biology, Chiang Mai University. Plant material was identified by Mr. James F. Maxwell from the Department of Biology, Chiang Mai University.
Extraction and isolation using the solvent extraction method
The dry, aerial parts of S. aphylla (1.0 kg) were extracted with 95% EtOH (4 × 2000 ml) over 4 days at room temperature. The ethanolic solution was evaporated to give a dark residue (43.1 g). A portion of this
Extraction and isolation by the conventional method versus electrocoagulation method
Stemona alkaloids were isolated from aerial parts of S. aphylla by the conventional solvent extraction and the EC method. (2′S)-Hydroxystemofoline (1) was isolated after column chromatography as a colorless, amorphous solid. The structure of (2′S)-hydroxystemofoline was established from a comparison of its 1H and 13C NMR spectroscopic data with those of (2′S)-hydroxystemofoline reported (Fig. 1) (Brem et al., 2002). Stemofoline (2) was isolated as a yellow, amorphous solid. The structure of
Conclusions
In conclusion, (2′S)-hydroxystemofoline and stemofoline were isolated from aerial part of S. aphylla by the conventional solvent extraction method and an EC method. It has been demonstrated that EC can be successfully applied to the isolation of Stemona alkaloids, with an important advantage over conventional isolation methods. This is the reduction in both the number and amount of toxic organic solvents involved in the isolation process. This method is also friendly to the environment and
Acknowledgements
We are grateful thank to the Science Achievement Scholarship of Thailand (SAST) for the grant of a Ph.D. student. A CMU Short-Term Research Fellowships in Overseas, Chiang Mai University, Thailand; the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation, Thailand and the School of Chemistry and Molecular Biosciences, University of Wollongong, Australia are deeply thanked for their financial support. We are also acknowledge
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