Research Article: Bioactivity-guided isolation of rosmarinic acid as the principle bioactive compound from the butanol extract of Isodon rugosus against the pea aphid, Acyrthosiphon pisum

Date Published: June 24, 2019

Publisher: Public Library of Science

Author(s): Saira Khan, Clauvis Nji Tizi Taning, Elias Bonneure, Sven Mangelinckx, Guy Smagghe, Raza Ahmad, Nighat Fatima, Muhammad Asif, Mohammad Maroof Shah, Ahmed Ibrahim Hasaballah.


Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control practices involving the excessive use of synthetic insecticides over many years have resulted in aphid resistance to a number of pesticides. In nature, plants produce secondary metabolites during their interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon pisum. To further explore this finding, the current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid, as the bioactive compound in I. rugosus. Insecticidal potential of rosmarinic acid against A. pisum was evaluated using standard protocols and the data obtained was analyzed using qualitative and quantitative statistical approaches. Considering that a very low concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and development of formulations for effective field application.

Partial Text

Aphids are among the most important agricultural pest insects of many crops worldwide. They feed exclusively on plant phloem sap by inserting their needle-shaped mouthparts into sieve elements, usually resulting to plant discoloration, stunting and deformation. Honey dew produced by aphids promotes the growth of sooty molds which further reduces the economic value of the crop [1, 2]. Moreover, aphids are also vectors of many important plant viruses [3–5]. The pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae), adversely affects economically important legume crops worldwide. It is oligophagous, comprising of a number of biotypes or races living on a number of legume hosts (red clover, pea, broad bean and alfalfa races) [6–9]. Current aphid control strategies predominantly rely on the use of insecticides such as carbamates, organophosphates, pyrethroids, neonicotinoids and pymetrozine [10]. However, the repeated use of these insecticides for many years has resulted in aphid resistance to most insecticides, making it very difficult to control aphids [11].

Screening candidate plants, purifying active ingredients, isolating and identifying the active plant constituents is required to discover new bioactive natural products [33]. We applied this methodology to identify rosmarinic acid as an active principle from the plant I. rugosus. Based on our previous study on the insecticidal activity of botanical extracts from various plant species, we found that the extract from I. rugosus was the most toxic to A. pisum [27] Further fractionation showed that the butanol fraction most likely contained the active principle. In this study we used the bioactivity-guided strategy to isolate and identify the active compound as rosmarinic acid. This strategy is interesting and has been used in previous studies to identify bioactive compounds. For example, the butanol fraction from Citrullus colocynthis was reported to be active against the black legume aphid, Aphis craccivora, and through the bioactivity-guided isolation strategy, the active principle, 2-O-ß-D-glucopyranosylcucurbitacin E, was successfully isolated [34]. Similarly, in another study involving bioactivity-guided isolation, the active principle, ailanthone, was isolated from the aqueous fraction of Ailanthus altissima against A. pisum [35].

In this study, I. rugosus was identified as an interesting source for a botanical insecticide against A. pisum. Following bioactivity-guided selection, rosmarinic acid was isolated and identified through spectroscopic analysis as the bioactive compound in the I. rugosus extract for the first time. Based on the bioassay results, either the extracts from I. rugosus or the isolated insecticidal compound, rosmarinic acid could be used to develop effective aphicides, because of the high mortality of aphids caused at very low rosmarinic acid concentrations in 24 h. This potential botanical insecticide may fit well in integrated pest management programs intended to control aphids. Considering that I. rugosus is known to be used for medicinal purposes, it is expected to be safer as compared to the current conventional pesticides used for controlling aphids. Also, rosmarinic acid is known to reduce genotoxic effects induced by chemicals, which is contrary to some currently used toxic synthetic pesticides that could induce genotoxic effects in consumers. An interesting avenue to follow for future studies will be the analyses of the underlying molecular mechanisms responsible for the cause of mortality in rosmarinic acid-treated aphids. While this study highlights the potential of I. rugosus as a possible biopesticide source against a notorious insect pest such as A. pisum, it also provides the basis for further exploration and development of a formulation for effective field application. However, more experiments under field conditions are required to further verify the applicability of rosmarinic acid for the insect’s control and in future contact application tests can be performed to broaden its effects on crops under field conditions. From an implementation point of view, genes involved in the biosynthesis of rosmarinic acid could be transformed under the control of a phloem-specific promoter to produce resistant crops towards aphids.