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

Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control involving the excessive use of synthetic insecticides over decades has led to multiple forms of aphid resistance to most classes of insecticides. 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. It was however not known as which compound was responsible for such activity. To further explore this finding, 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 activity of rosmarinic acid was carried out using standard protocols on A. pisum. The data 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.


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Aphids are among the most important agricultural pest insects of many crops worldwide. They feed exclusively on 39 plant phloem sap by inserting their needle-shaped mouthparts into sieve elements, usually resulting to the stunting, 40 discoloration and deformation of plants, while the growth of sooty molds on honeydew produced by these insects 41 reduces the economic value of crops [1,2]. Moreover, aphids are also vectors of many important plant viruses [3][4][5].
most classes of insecticides, making it very difficult to control this insect pest [11].

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The use of botanical pesticides could present a safe alternative compared to the use of broad spectrum 49 chemical insecticides in crop protection [12,13]. In nature, plants produce secondary metabolites during their 50 interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and 51 deterrents towards the insects [14][15][16]. Because of such wide insecticidal properties, the study of secondary 52 metabolites and the development of new potent formulations based on them has become increasingly important.

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Screening of plant extracts followed by bioactivity-guided fractionation, isolation and identification of active 54 principles is considered to be one of the most successful strategies for the discovery of bioactive natural products     [28]. All the bioassays were performed under these conditions. Newly born nymphs (˂ 24 h old) of A. pisum were 76 used for all the bioassays. By gentle probing of the aphids with a brush and also by observing post-mortem color 77 change of the body, mortality was assessed after 24 h of treatment.

Plant collection and extraction 79
The aerial parts of I. rugosus were collected from lower Northern areas of Pakistan in the month of October, 2012.

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The plant material was shade-dried for up to 3 months and ground to powder using an electric grinder. 1 kg of the 81 dried powder was soaked in a glass jar containing 3 L of methanol at room temperature. After two days, the solvent 82 layer was filtered with Whatman filter paper No. 1 and this process was repeated three times. The resulting filtrate 83 was concentrated by using a rotary evaporator at 35 °C and the obtained crude methanolic extract was stored at 4 °C 84 [29,27]. For fractionation, 90 g dried crude methanolic extract was mixed with five parts of water and then 85 extracted successively by n-hexane (4 × 150 mL), dichloromethane (4 × 150 mL), ethyl acetate (4 ×150 mL) and n-86 butanol (4 × 150 mL) as described by Khan et al. [27]. All the fractions were concentrated using a rotary evaporator 87 under reduced pressure at 40 °C. The resulting extracts were stored in a refrigerator at 4 °C until further use.

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Isolation of the bioactive principle 89 Based on bioassays conducted by Khan et al,[27] the butanol extract presented the best biological activity against A.

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pisum and was hence selected in this study for further bioactivity-guided fractionation and identification of the 91 active principle. The butanol extract (500 mg) was eluted with a Reveleris automated flash chromatography 92 instrument on a 12 g C18 pre-packed column (GRACE, Columbia, MD, US) starting with 100% water. The gradient 93 was ramped to 100% methanol over 60 column volumes (CV) and after collection of 95 fractions, the solid phase 94 was flushed with 5 CV acetonitrile. The flow rate was set to 30 mL/min (Table 1). Based on the UV spectral data,

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Additionally, the growth of the surviving aphids exposed to 0.4 ppm of the active compound for 24 h was 152 followed for 9 days (on the same treated diet) in comparison to the untreated aphids.

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pisum. This fraction 3A-3 was analyzed through 1 H NMR which confirmed that the bioactive fraction 3A-3 201 contained rosmarinic acid. Different gradients were used to purify the compound but during different Prep-LC runs, 202 the chromatographic behavior, that is, peak shape and position, of this fraction was inconsistent. Therefore, the  (Table 7).

Identification of the most bioactive compound 234
Out of the two phases of acidic extraction, the ethyl acetate phase fraction was the most active. After removing ethyl 235 acetate azeotropically, this fraction was analyzed and the active compound was identified as rosmarinic acid through 236 HPLC-MS, optical rotation measurement and 1 H and 13 C NMR spectroscopy.

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Both isolated and commercial rosmarinic acid (Sigma Aldrich) had the same peak appearance in the HPLC-MS 239 chromatograms with the same solvent gradient. Both had a pseudo-molecular ion with an m/z value of 359 with 240 negative mode electrospray ionization which confirmed that it was rosmarinic acid (Fig 1).   (Table 9). Table 9. Toxicity of isolated rosmarinic acid (RA) and commercial rosmarinic acid (RA) against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs 259 following 24 h exposure to artificial diet containing different concentrations of isolated rosmarinic acid and commercial rosmarinic acid 260 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 261 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Comparison of the growth of surviving aphids exposed to 263 rosmarinic acid-treated and untreated diet after 24 h of bioassay 264

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After incorporating the rosmarinic acid in aphid's diet at 0.4 ppm, its effect on A. pisum that survived after 24 h 265 treatment, was analyzed every day for up to 9 days (on same treated diet). It was confirmed that rosmarinic acid had 266 a drastic effect on their growth. Firstly, most aphids exposed to treated diet were dead while the survivors did not 267 grow further to become adults and were thus not able to reproduce further.    for future studies will be the analyses of the underlying molecular mechanisms responsible for the cause of mortality 313 in rosmarinic acid-treated aphids.

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Additionally, a comparison between the growth of surviving aphids exposed to rosmarinic acid-treated and 315 untreated diet after 24 h of bioassay was analyzed. It was clearly observed that the growth of surviving A. pisum 22 316 nymphs stopped after 48 h of exposure to rosmarinic acid-treated diet, resulting in a size reduction and ultimately 317 death as compared to aphids exposed to an untreated diet. A similar observation was made by Sadeghi et al, [30] 318 who observed that the aphid size was reduced after 48 h of exposure to novel biorational insecticides, flonicamid 319 and pymetrozine, and mortality was observed after 72 h.

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In this study, I. rugosus was identified as an interesting source for a botanical insecticide against A. pisum.

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Following bioactivity-guided selection, rosmarinic acid was isolated and identified through spectroscopic analysis as