Compositional Analysis and Potent Insecticidal Activity of Supercritical CO2 Fluid Extracts of Alcea nudiflora L. Leaves.

To mitigate potentially severe food shortages due to the exponential growth of the global population, it is of paramount importance to improve the yield and quality of globally harvested food crops. As pest control contributes to both these aspects, the development of safe and effective pesticides is one of the main strategies pursued in this direction in the context of agricultural chemistry. During our investigation of natural pesticides, a supercritical CO2 fluid extract of Alcea nudiflora L. was found to exert extremely potent insecticidal activity against aphids (Macrosiphum euphorbiae) and cowpea seed beetles (Callosobruchus maculatus) with LC50 values of 0.03 mg/mL (24 h exposure, contact method). The facts that their insecticidal activity is in the most potent class among the essential oils known to date, and that the extract did not show any toxicity toward beneficial insects such as ladybugs (Coccinella magnifica) and European honeybees (Apis mellifera Linnaeus), indicate that this extract could be a good, natural, and safe new pesticide candidate. A compositional analysis of this extract was carried out using GC/MS.

Introduction

Due to the improved quality of life, and especially medical care, the global population has reached 7.8 billion and continues to grow.[1] One study estimates that it will grow to 10 and 11 billion in 2050 and 2100, respectively.[2] To manage the associated potentially severe shortages of food, improving crop yield and quality are of paramount global importance. There are many strategies to increase the yield and quality of crops, among which pest control to decrease crop loss is one of the most essential. Thus, to date, hundreds of artificial pesticides have been developed and made commercially available.[3] Chemical pesticides are powerful, scalable, and inexpensive, but they have historically caused health problems in humans, although recently developed ones are often much less harmful.[4]

On the other hand, various plant-derived materials, such as essential oils, plant extracts, and fermentation products (e.g., vinegars) have also been used as natural pesticides[5] and medicines;[6] these are believed to be superior to their chemical counterparts on account of their greater safety for both humans and the environment. Among these, supercritical CO2 extracts (SCE) of plants have attracted great attention, as CO2 can achieve the extraction of unique substances that conventional solvents (e.g., organic solvents and water) cannot afford.[7] Furthermore, unlike organic solvents, CO2 is easily removed by evaporation, and even if it remains in the products until their distribution, it is completely harmless.

During our investigations to identify useful plant extracts, we found that a frequently observed hollyhock, Alcea nudiflora, contains a variety of natural substances such as astragalin and polyprenols, which can be extracted using organic solvents.[8] Although there is no evidence that plants belonging to the Alcea genus have historically been used to control pests, we have previously detected some insecticidal compounds such as β-amyrin, β-sitosterol, phytol, and lupeol[9] in the EtOH infusion extract (EIE) and EtOH sonication extract (ESE) of A. nudiflora leaves, which prompted us to further investigate the extracts of this plant. In particular, the SCE, if sufficiently insecticidal, might represent a good candidate for new, safe, and inexpensive natural pesticides, since A. nudiflora is commonly encountered in Central Asia, Western China, and Western Siberia. Herein, we report the insecticidal activity of SCE of A. nudiflora L. against pests such as aphids (Macrosiphum euphorbiae) and cowpea weevils (Callosobruchus maculatus). Furthermore, we examined its insecticidal activity against beneficial insects such as ladybugs (Coccinella magnifica) and European honeybees (Apis mellifera Linnaeus). Identification of molecular components in the SCE was accomplished using gas chromatography/mass spectrometry (GC/MS) and comparison of the MS characteristics and retention indices (RIs) with data in databases.[10] As far as we know, this work is the first to demonstrate the insecticidal activity of extracts of A. nudiflora.

Materials and Methods

Plant Materials

Leaves of A. nudiflora L. {identified by an experienced botanist, Dr. Alimjan M. Nigmatullaev, and kept at the herbarium of the S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan (administrative number: 1338)} were collected in the vicinity of Chortak village, Namangan region, Uzbekistan (August 2015), dried in the shade, and crushed (4–6 mm) before the extraction.

Extraction

The crushed dry leaves of A. nudiflora L. (1 kg) were extracted with fluidic supercritical CO2 at 40–50 °C under 28–30 MPa for 100 min to give 3.00 g of SCE. Additional samples of dry leaves (10.0 g each) were extracted three times with EtOH (96%, 80 mL, 60 mL, and 60 mL, respectively) under sonication (90 min) or by infusion (24 h) at room temperature. Evaporation at 40 °C afforded an ESE (1.73 g, dark green oily mass) and an EIE (1.44 g, dark green oily mass), respectively.

Further Separation of the CO2 Extract

The SCE (2.00 g) was further separated into three fractions using column chromatography on silica gel. Fraction (Fr) 1 (450 mg, yellow oil after the removal of solvents), Fr 2 (275 mg, colorless amorphous mass), and Fr 3 (720 mg, colorless oil) were eluted using hexane–CHCl3 = 1:1, 1:5, and 1:10, respectively.

Insects

Dozens of C. maculatus were collected from the institutional storage house and kept separately in a cage with tap-water-washed and dried mung beans (Vigna radiata) to prevent infestation. Adult C. maculatus were reared at 25 ± 1 °C and 55 ± 5% relative humidity with a photoperiod of 12 h. Three-day-old C. maculatus adults were selected and used in assays. The laboratory population of M. euphorbiae was kept on seedlings of the potato variety Sante at 22 ± 2 °C with a photoperiod of 16 h. Adult C. magnifica and A. mellifera Linnaeus were collected at a farm in Tashkent, Uzbekistan and kept in separate cylindrical glass bottles closed with a mosquito net.

Insecticidal Assays

Each extract (1.00 g) and a drop of Tween 80 or OP-10 were diluted with distilled H2O to give 100 mL of stock solution (10 mg/mL). These stock solutions were further diluted with H2O to prepare samples with lower concentrations. The various sample solutions (1.00 mL) were added to Petri dishes (diameter: 9 cm) in which a filter paper had been placed. After 10 min, insects (M. euphorbiae and C. maculatus, 10 adults each), together with their feed plants [potatoes (Solanum tuberosum L.) and mung beans (Vigna radiate), respectively] were placed on each dish, and the insect mortality was determined 5, 10, 30, and 60 min and 24 h after exposure. After obtaining three independent replicates at each concentration, the insecticidal activity of each extract was determined as percentage mortality of the insects.

Identification of the Components in the CO2 Extract

A solution of the SCE (1.0 mg) in hexane (1 mL) was injected (1.0 μL) into a GC/MS (5975C inert MSD/7890A GC system, Agilent Technologies, USA, 2009), equipped with a quartz capillary column: HP-INNOWax, 30 m (length) × 0.25 mm (diameter) × 0.25 μm (thickness), Agilent Technologies. The separation and analysis were carried out under the following conditions: injector temperature: 220 °C; temperature mode: 50 °C (1 min) → 200 °C (heating rate: 4 °C/min followed by a constant temperature of 200 °C for 6 min) → 250 °C (heating rate: 15 °C/min followed a constant temperature of 250 °C for 25 min); recorded EI-MS m/z range: 10–550; carrier gas: helium; flow rate: 1.1 mL/min. The components were identified by comparison of the observed MS characteristics with MS libraries, that is, the Wiley Registry of Mass Spectral Data, 9th Ed., and the National Institute of Standards and Technology (USA) Mass Spectral Library (2011). The Kratz RIs of compounds, which were determined relative to a mixture of n-alkanes (C9–C20), were also compared with a previous literature report.[10]

Results and Discussion

Insecticidal Activity of the EtOH and CO2 Extracts

We first examined the insecticidal activity of the EtOH and CO2 extracts (10 mg/mL each) against aphids, M. euphorbiae (Figure ), with negative and positive control experiments using H2O and the known synthetic pyrethroid cypermethrin (0.10 mg/mL).

Figure 1

Insecticidal activity of the ESE, EIE, and SCE of A. nudiflora against M. euphorbiae (5 min exposure).

After 5 min of exposure, only SCE exhibited potent insecticidal activity, whereas both EtOH extracts, that is, ESE and EIE, were inactive. These results were surprising given that we have detected some insecticidal compounds (e.g., β-amyrin, β-sitosterol, phytol, and lupeol) in our preliminary study of ESE and EIE. We concluded that neither ESE nor EIE were active due to the (very) low concentration of insecticidal compounds. On the other hand, these results were also very interesting as they indicate that supercritical CO2 is able to extract more/other insecticidal compounds than those mentioned above and/or different unique insecticidal plant substances that EtOH cannot.

Insecticidal Activity of the CO2 Extract against M. euphorbiae and C. maculatus

As preliminary insecticidal activity was only detected for SCE, we further investigated the dose responses of M. euphorbiae and C. maculatus to SCE. Treatment of M. euphorbiae with systematically diluted SCE solutions (0.050–5 mg/mL) revealed that the SCE of A. nudiflora L. exhibits potent pesticidal activity (Figure ). At the highest concentration of 5.0 mg/mL, 87% of the aphids were dead within 10 min (blue bar), with the death rate reaching 100% after 30 min of exposure (red bar). Even at concentrations of 0.10–1.0 mg/mL, the potent insecticidal activity was maintained especially after 60 min (green bar) and 24 h (purple bar) of exposure, with mortality rates of >80%. At 0.050 mg/mL, the activity dropped to <50%. The lethal concentration (LC50) value after 24 h was calculated to be 0.03 mg/mL.

Figure 2

Insecticidal activity of SCE of A. nudiflora against M. euphorbiae.

The SCE of A. nudiflora also exhibited potent insecticidal activity against the larger pest C. maculatus (Figure ). The activity was almost identical to that against M. euphorbiae, with its LC50 value for C. maculatus after 24 h also being found to be 0.03 mg/mL. Ikbal and Pavela have reported in their excellent review article that potent insecticidal essential oils show LC50 values on the order of 101 to 102 μL/mL (≈mg/mL for solids) against aphids in contact applications.[11] Only 9% (7 examples) of the 77 examples in the literature showed an LC50 of <0.1 μL/mL, and only 4% (3 examples) were more potent than the SCE of A. nudiflora. This comparison clearly shows that among materials of plant origin, the SCE of A. nudiflora exhibits excellent potency against aphids. Moreover, it is noteworthy that the SCE of A. nudiflora is completely harmless to the beneficial insects C. magnifica and A. mellifera L. as well as to human skin, even at a concentration of 10 mg/mL (data not shown). These results indicate that the SCE of A. nudiflora is a good candidate as a novel natural insecticide, although the scalability of the extraction should further be addressed.

Figure 3

Insecticidal activity of SCE of A. nudiflora against C. maculatus.

Insecticidal Activity of Further-Fractionated SCE

To investigate the active species, we further separated the SCE using column chromatography on silica gel to give three fractions. The obtained Frs. 1 (eluted with hexane/CHCl3 = 1/1), 2 (hexane/CHCl3 = 1/5), and 3 (hexane/CHCl3 = 1/10) were subsequently subjected to insecticidal assays and the mortality (%) was calculated for insects exposed to the fractions for 24 h. Frs. 1 and 2 exhibited potent insecticidal activity against both M. euphorbiae (Figure ) and C. maculatus (Figure ), while Fr. 3 did not. The LC50 values of Fr. 1 against M. euphorbiae and C. maculatus were 0.09 and 0.08 mg/mL, while those of Fr. 2 were 0.1 and 0.08 mg/mL, respectively. Since these LC50 values are almost identical to those of the parent SCE mixture, these results indicate that less-polar organic compounds extracted by supercritical CO2 fluid are responsible for the insecticidal activity.

Figure 4

Insecticidal activity of fractionated SCE against M. euphorbiae.

Figure 5

Insecticidal activity of fractionated SCE against C. maculatus.

Components in the SCE Detected by GC/MS and a Comparison of Their Insecticidal Activity with Literature Precedents

Finally, we analyzed the chemical components of the SCE of A. nudiflora using GC/MS. Each component was identified via comparison of its Kratz RI with literature values, as well as its EI-MS fragmentation pattern. As a result, 32 compounds were identified (Table ). Among these, thymol is known to be pesticidal against many pests, such as tobacco cutworms (Spodoptera litura Fab),[12] bed bugs (Cimex lectularius L),[13] houseflies (Musca domestica),[14] red spider mites (Tetranychus urticae),[14] Western corn rootworms (Diabrotica virgifera), and click beetles (Agriotes obscurus L).[15] Pulegone is known to be toxic against S. litura Fab.,[12] maize weevils (Sitophilus zeamais),[16]M. domestica,[14]D. virgifera[14] and vine mealybugs (Planococcus ficus).[17] Menthone acts pesticidally against C. lectularius L.,[13]C. lectularius L.,[13]S. zeamais,[16]M. domestica,[14] and D. virgifera,[14] while citral is pesticidal against M. domestica,[14] and D. virgifera.[14] Therefore, the main active insecticidal compounds in the SCE of A. nudiflora are most likely menthone, thymol, pulegone, and citral which differ from those in the EIE and ESE (β-amyrin, β-sitosterol, phytol, and lupeol), albeit that the presence of novel, structurally unknown natural products and/or synergistic effects with some minor components cannot be ruled out at this point. The good insecticidal selectivity of the SCE of A. nudiflora against beneficial/pest insects might be derived from the presence of thymol and pulegone, given that they are safe for European honeybees, which has recently been reported.[18]

Table 1

Composition of SCE of A. nudiflora Analyzed by GC/MS

Conclusions

In summary, A. nudiflora L. leaves have been extracted with supercritical CO2 under fluidic conditions and been subjected to insecticidal assays for the first time. The supercritical CO2 extract (SCE) of A. nudiflora exhibited potent insecticidal activity against aphids (M. euphorbiae) and cowpea seed beetles (C. maculatus) with LC50 values of 0.03 mg/mL (24 h exposure, contact method). It is worth noting that the EtOH extract of A. nudiflora is not pesticidal and that the SCE is harmless to beneficial insects such as ladybugs (C. magnifica) and European honeybees (A. mellifera Linnaeus) at the tested concentrations (<10 mg/mL). These findings indicate that the SCE of A. nudiflora exhibits promising potential as an excellent lead for a safe pesticide of natural origin. Further investigations into the development of practical methods to enable the large-scale production of A. nudiflora SCE and the application of supercritical CO2 extraction to various other plants native to Uzbekistan are currently in progress in our laboratory.