Extraction, Purification, Quantification, and Stability of Bioactive Spilanthol from Acmella oleracea

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• General experimental procedures
• Plant material
• Isolation and characterization of spilanthol (1)
• Procedure of NMR quantitation of spilanthol in extracts
• Procedure of HPLC quantitation of spilanthol in extracts
• Extraction of spilanthol from Acmella oleracea
• Purification of the crude ethanolic extract
• Contributorsʼ Statement
• References

Abstract

Acmella oleracea is an ethnobotanically significant plant with a relatiwely high content of spilanthol. Due to its broad spectrum of activity, including anti-inflammatory, antioxidant, analgesic, antifungal, and bacteriostatic properties, it is considered a valuable bioactive natural product. In addition, spilanthol as its main bioactive component inhibits facial muscle contractions, making it an attractive ingredient in anti-wrinkle and anti-aging cosmetics. Due to its muscle paralyzing effects, it is called herbal botox. The commercial interest in spilanthol encourages the development of effective methods of isolating it from plant material. The methodology used in this paper allows for the obtaining of extracts from Acmella oleracea with a relatively high content of spilanthol. An effective method of spilanthol extraction from all aerial parts of Acmella oleracea as well as methods of enriching spilanthol concentration in extracts achieved by removing polar and acidic substances from crude extracts was developed. To quantify the concentration of spilanthol, a simple, fast and economically feasible quantification protocol that uses nuclear magnetic resonance (HNMR) was developed. In addition, it has been proven, that oxidation of spilanthol by air gives (2E,7Z)-6,9-endoperoxy-N-(2-methylpropyl)-2,7-decadienamide. The studies on spilanthol solutions stability were carried out and the conditions for the long-time storage of spilanthol solutions have also been developed. Additionally, for confirmation of obtained results a sensitive (LOQ=1 ng/mL), precise (RSD lower than 7%) and accurate (RE lower than 7.5%), new HPLC-MS/MS method was applied.

Introduction

The constant interest in natural cosmetics and pharmaceuticals has motivated bioprestive discovery in the plant kingdom. Acmella oleracea (L.) R. K. Jansen (syn. Spilanthes acmella (L.) L., Spilanthes oleracea L.) is a culturally significant plant that belongs to the family Compositae. It is known by a variety of vernacular names including toothache plant, jambu, and paracress. This species has a long history of traditional use in cuisines and medicines across several different civilizations [1]. In medicine it was used for toothache, rheumatism, and maladies of throat and gums [2], [3], [4].

The multidirectional biological activity of Acmella oleracea, confirmed by extensive scientific research, is due to the presence of a variety of bioactive substances. The compound with the widest spectrum of biological effects is (2E,6Z,8E)-N-(2-methylpropyl)-2,6,8-decatrienamide (1, C₁₄H₂₃NO, 221.339 g/mol), is commonly known as spilanthol (syn. affinin), and is classed as a N-alkylamide (NAA, [Fig. 1]) [3], [5], [6], [7].

Other bioactive substances such as 3-acetylaleuritolic acid (8), trans-ferulic acid (9), stigmasterol (10), vanillic acid (11), sitosterone (12), and trans-isoferulic acid (13) are also present in extracts from A. oleraceaʼs ([Fig. 2]) [6].

Spilanthol displays a broad spectrum of biological activity, mainly including anti-inflammatory [2], [6], [8], [9], analgesic [2], [6], [8], [9], [10], [11], antioxidant [6], [7], [8], [9], antifungal [2], [6], [8], [12], bacteriostatic [6], [7], [8], [12], anti-larvicidal, and insecticidal effects [6], [8], [10], [13], [14] and therefore has been the subject of great interest. Importantly, spilanthol displays muscle relaxing and skin smoothing effect. Its cosmetic and dermatological properties have been documented by several authorities. In addition, in vitro and in vivo studies show that spilanthol has excellent migration properties across all layers of the skin, giving it the ability to reach the muscles transdermally [15]. Spilanthol inhibits contractions in subcutaneous muscles, notably face muscles, meaning that it can be used as an ingredient in cosmetic products. It exhibits activity similar to that of botulinum toxin, and the difference lies only in the length and strength of muscle deactivation, making it popular as a natural herbal botox [2], [3], [16], [17], [18], [19], [20], [21], [22]. Due to its analgesic and bacteriostatic properties, extracts containing spilanthol have been used to treat toothaches, stomatitis, and skin diseases. Spilanthol, or extracts of plants that contain it, may be added to toothpaste and used as an oral analgesic in gels (e.g., Buccaldol and Indolphar) [2], [16], [17], [23], [24], [25].

In recent years, numerous analytical approaches have been proposed for the determination of spilanthol in plants. An overview of these analytical methods can be found in a comprehensive review by Barbosa et al. [2]. For quantifiction of spilanthol in plant samples high performance liquid chromatography (HPLC) with UV detection [3], [22], [25], [26] and MS or MS/MS have been reported [16], [27]. Separation was performed in all cases using reversed phase HPLC (C18 column). Some HPLC method used an isocratic mobile phase and some gradient mobile phase. One chromatographic method is based on gas chromatography coupled with FID detection [9]. The mentioned publications describe the analytical methods for quantification of spilanthol in Spilanthes acmella [9], [16], [22], [25], [26], [27] and Acmella oleracea flower [3], [9], [28].

Sample extraction and clean-up are normally required prior to chromatographic analysis due to the matrix effect. For some extraction methods, including maceration [22], [25], [26], solid-liquid extraction (SLE) [3], [16], [28] and Soxhlet extraction [28] were applied. Reports have shown that ethanol [3], [16], [22], [29], methanol [25], [26] and mixtures of methanol, ethanol, acetonitrile, water [28] are crucial solvents for the extraction of spilanthol from plants. Supercritical CO₂ with added ethanol and water was also used to try to extract spilanthol from S. acmella flowers, leaves, and stems [9].

Acmella oleracea is a plant with a relatively high content of spilanthol. Due to its broad spectrum of bioactivity, it is considered a valuable natural product. In addition, the commercial interest in spilanthol by the cosmetics industry means that methods for its isolation from plant material are still being sought. In this paper, we present research aimed at developing a simple and effective method for obtaining spilanthol-rich extracts. Our goal was to create a scalable protocol for extraction with satisfactory yield and to develop an analytical method that enables the determination of the spilanthol content at every stage of the procedure.

Results and Discussion

Determination of spilanthol content in plant extracts is a difficult analytical issue due to the complex chemical composition of the studied samples. According to literatures in addition to spilanthol, there are at least 15 substances (e.g. NAAs) with a very similar structure and molecular weight in the Acmella oleracea extract ([Fig. 1], [2]) [3], [6], [17]. Mostly, spilanthol have been investigated in Acmella oleracea extracts using high performance liquid chromatography coupled with mass spectrometry [16], [22], [23] and UV detection [3]. However, accurate quantification is a labour-consuming process that requires construction of a calibration curve using high purity spilanthol standard samples that are difficult to acquire. A particularly difficult problem is the determination of spilanthol content in mixtures whose main components are non-volatile substances (e.g., capron-caprylic triglyceride used in cosmetic preparations).

To the best of our knowledge, spilanthol is not commercially available as an analytical standard. To obtain spectral data (NMR, IR), spilanthol was isolated and purified by column chromatography (Fig. 1S–3S, Supporting Information). A pre-purified ethanolic extract containing approximately 10% spilanthol was used. The spilanthol was obtained with a purity of 97% with 78% recovery from the crude extract. A method for the determination of spilanthol content in multi-component mixtures was then developed as an alternative to chromatographic methods. The method involves the use of proton magnetic resonance spectroscopy (¹H NMR). Calculation of spilanthol content in the sample is based on the integration values of characteristic proton signals corresponding to spilanthol compared to the integration values of proton signals corresponding to the internal standard (e.g., dimethyldiphenylsilane), as shown in [Fig. 3].

Non-crowded proton signals (H₂, H₃, H₇ and H₈) with the characteristic coupling pattern of a doublet (d), doublet of doublet (dd) or doublet of triplet (dt) were selected as analytical signals. Identification of spilanthol with this method is beyond doubt, as it is made on the basis of four characteristic multiplets, each of which has specific chemical shifts, integrals, multiplicities and characteristic coupling constants as shown in [Fig. 4]. This makes it possible to confirm the presence of spilanthol in compound mixtures, and in the presence of substances of unknown structure following by quantification relative to an internal standard.

HPLC-ESI-MS/MS was then applied to confirm the results obtained by following the HNMR method. Positive ESI was employed as the ionization mode in this study and the MS analysis showed the presence of the protonated molecular ion [M + H]⁺. Continuous mass spectra were obtained by scanning from m/z 50 to 250 (Fig. 4S, Supporting Information). In the MS/MS spectrum a [M + H]⁺ molecular ion peak at m/z 222.04, corresponding to a molecular formula of [C₁₄H₂₃NO + H]⁺, and two main fragment ions at m/z 81.04 and 141.01 respectively, were observed, corresponding to the fragments generated from cleavage at the amidic bond, which is normally observed in compounds with an amide moiety (Fig. 4S, Supporting Information). After optimization of the MS/MS conditions, multiple reaction monitoring (MRM) mode was used to quantify spilanthol in plant extracts. Two MRM transitions were monitored for spilanthol; the more sensitive ion was used for quantitation and the second for confirming the presence of spilanthol. The use of this methodological approach significantly increased the sensitivity and selectivity of the analysis (Fig. 5S, Supporting Information). In this way interference from coeluting components in the mixture was prevented.

To obtain the best chromatographic conditions, the mobile phase composition, flow rate, type of column, and column temperature were judiciously selected. The Kinetex C18 (75 mm × 2.1 mm, 2.6 µm) column was found to be optimal compared with the tested columns, including including Zorbax Rapid Resolution High Definition (RRHD) SB-C18 column, Hypersil GOLD column and Chromolith Fast Gradient C18e column, due to the unique, superficially porous particles and 2.6 µm particle size providing high and symmetry peak of analyte. Different mobile phases (methanol–water and acetonitrile–water with different additives, such as formic acid, acetic acid, ammonium acetate, and ammonium formate) with isocratic and gradient elution modes were explored. It was determined that the optimal mobile phase consisted of acetonitrile and 0.1% formic acid in water in a isocratic elution and with flow rate 0.5 mL/min. Acetonitrile, rather than methanol, was chosen as the organic modifier due to its better peak shape and lower pressure. The effect of column oven temperature on the analysis of spilanthol was evaluated in the range of 25 – 40 °C, and the best results in terms of retention factor were observed at 25 °C. Under these conditions, retention times of the spilanthol was constant (t_r = 1.00 min), with a relative standard deviation lower than 0.3%. Hence, the chromatogram of the plant extract displayed only the peak from spilanthol (t_r = 1.00 min), and not the other components in the mixture.

The developed HPLC-MS/MS procedure was validated as following:

Linearity and sensitivity – The linear range of the method was obtained in the range of 1.0 – 500 ng/mL with a correlation coefficient of 0.9994. The LOD and LOQ were 0.27 ng/mL and 0.8 ng/mL, respectively.

Precision and accuracy – the RSD of QC samples was in the range of 1.1 – 4.0% (intra-day) and 1.7 – 6.9% (inter-day). RE was between − 3.8% and 4.9% (intra-day) and between − 5.5% and − 7.4% (inter-day).

Dilution integrity – the mean precision and accuracy after 10-fold dilution of a spilanthol solution were less than 5.2% and within ± 4.6%, respectively.

Carry-over effect – The carry-over is negligible since the peak areas of the methanol samples re-analyzed immediately after the calibration standard at the ULOQ were always lower than 3.9% of the areas of the ULOQ for spilanthol.

The validated HPLC-MS/MS procedure was used to quantify spilanthol in fresh extracts and to monitor its stability in plant extracts. The results obtained for extracts of A. oleracea extracts using HPLC-MS/MS were consistent with those obtained using the new ¹H NMR procedure. This confirms the possibility of the application of ¹H NMR for spilanthol quantification. The application of a complementary technique is especially important in laboratories that do not have a selective mass spectral detector. Furthermore, the low selectivity of a UV detector can often overestimate the quantity of spilanthol, due to interference from other components in the mixture. Therefore, this new, fast, and economically viable ¹H NMR procedure is an excellent tool for confirming quantifying spilanthol content independently of HPLC-UV or HPLC-MS/MS. This may be especially important for laboratories that do not have easy access to the tandem mass spectrometry.

The standard method of obtaining spilanthol from raw material consists of its extraction with organic solvents, e.g., ethanol, methanol, ethyl acetate, acetonitrile, dichloromethane, or hexane, and mixtures thereof. Most of these procedures require a long extraction time or the use of specialized equipment. Moreover, in most cases, authors focus on studying the biological activity of A. oleracea extracts, without assessing the main active ingredient (spilanthol) content in the crude or final extract, meaning optimized extraction protocols have not been made available [3], [10], [27], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39].

Like other N-alkylamides, the long fatty acid chain of spilanthol makes it a low to moderately amphiphilic compound, brought about by the relatively polar amide group and the less polar fatty chain. Due to the amphiphilic nature of spilanthol, ethanol was chosen as the extraction medium, which is relatively safe, and it presents low toxicity and cost and also is widely used in the extraction of natural products. The following extraction procedures were then tested: Soxhlet extraction (Procedure A), extraction at elevated temperature with stirring (Procedure B), extraction at room temperature with stirring (Procedure C) and extraction at room temperature without stirring (Procedure D). Each of the tested procedures has been repeated 3 times, and the results presented in [Table 1] are averaged results.

Thus, in the case of extraction carried out using the Soxhlet apparatus, procedure A was maintained for 15 h. Using a ratio of dried aerial parts of A. oleracea to ethanol (m_AO/V_EtOH: 1/30, g/mL), a concentration of spilanthol in a crude ethanolic extract (EE) of 194 mg spilanthol/1 L was obtained. Following procedure A, the highest amount of spilanthol was obtained with plant material (4.9 mg SPL/1 g AO, [Table 1]). However, the drawback of this method is the danger of thermal degradation of spilanthol and the high costs associated with the necessity of multiple evaporation of large amounts of ethanol.

As an alternative procedure B, dried aerial parts of the plant were placed directly in the flask and heated at boiling point ethanol (with reflux), stirring constantly, at the ratio of m_AO/V_EtOH: 1/40, g/mL. Due to the difficulty of mixing, a slightly larger amount of ethanol was used. After only 4 h, a crude EE containing 110 mg spilanthol/1 L was obtained. In procedure C, the extraction methodology was simplified by maintain the extraction at room temperature. Using the ratio of plant material to ethanol (m_AO/V_EtOH: 1/30, g/mL) and extending the extraction time to 72 h, a spilanthol concentration of 142 mg spilanthol/1 L was obtained.

Procedures B and C, despite using different extraction conditions, allow for obtaining comparable amounts of spilanthol from 1 g of plant material, respectively: procedure B – 4.4 mg SPL/1 g AO, procedure C – 4.3 mg SPL/1 g AO ([Table 1]). It was shown that the use of a lower temperature of the process (25 °C instead of 78 – 80 °C) can be compensated by extending the extraction time to 72 h. In addition, it was eliminated is the danger of thermal degradation of spilanthol.

Obtaining a relatively high concentration of spilanthol in the crude EE has a key impact on the yield of the whole process. Therefore, an attempt was made to apply a minimum amount of solvent, assuming that the reduced rate of extraction, resulting from the lower diffusion gradient, will be compensated for by extending the extraction time. Thus, after 6 days of extraction at room temperature, using the ratio of dried aerial parts of Acmella oleracea to ethanol (m_AO/V_EtOH: 1/2.8, g/mL), a crude EE containing 1478 mg spilanthol/1 L and 4.1 mg SPL/1 g AO was obtained. This concentration is approximately ten-fold higher than that obtained in procedures A – C.

According to the literature, in addition to spilanthol the crude EE of Acmella oleracea contains at least 15 other substances of different polarity and chemical nature ([Fig. 1] and [Fig. 2]). Thus, in optimization of the extraction protocol, attempts were made to enrich the spilanthol content by removing polar substances, as well as substances of an acidic nature. In these studies, dry residues (DEE) obtained after evaporation of solvent from crude EE produced according to procedure D were used. Re-extraction of the condensed extract with dichloromethane at room temperature excludes substances with higher polarity than spilanthol. The spilanthol content in the dry secondary extract (DE2) increased to 9.2, 10.0 or 14.7%, depending on the spilanthol concentration in the dry primary extracts (DEE). Moreover, it was shown that there is no substantial loss of spilanthol in this process, and the extraction recovery efficiency is 95 – 98% ([Table 2]).

To further enrich the extract, a 5% aqueous NaOH solution was used to remove of the acidic substances from the secondary extracts. Most of the secondary condensed extract remained undissolved in the aqueous solution, and the spilanthol content increased by almost two-fold ([Table 2]). Unfortunately, a loss of spilanthol was observed during alkaline extraction (yield 48 or 53%), probably due to its instability under the alkaline conditions. Alternatively, chromatography performed with the use of an alkaline ion exchanger (Amberlite IRA 67) turned out to be a more efficient method of removing acid substances from the secondary condensed extracts from A. oleracea (DE2), without substantial loss of spilanthol. This method produced a condensed residue containing 15.9 – 18.2% of spilanthol with a recovery efficiency of spilanthol over 96% ([Table 2]).

In the course of the current study it was observed that leaving spilanthol solutions in DCM or CDCl₃ in contact with the air and light leads to the gradual disappearance of spilanthol, with the simultaneous formation of a new substance. Our study proved that compound (16) is probably the oxidation product of spilanthol identified as (2E,7Z)-6,9-endoperoxy-N-(2-methylpropyl)-2,7-decadienamide (16, [Fig. 5]).

We recorded the ¹H NMR spectrum of pure spilanthol as shown [Fig. 6]: 6.83 (dt, 1H, J₁ 15.0 Hz, J₂ 6.6 Hz, H-3), 6.32 – 6.26 (m, 1H, H-8), 5.97 (dd~t, 1H, J₁ = J₂  = 10.8 Hz, H-7), 5.80 (dt, 1H, J₁ 15.6 Hz, J₂ 1.8 Hz, H-2), 5.70 (dq, 1H, J₁ 12.0, J₂ 6.0, H-9), 5.52 (br, s, 1H, NH), 5.26 (dt, 1H, J₁ 10.8 Hz, J₂ 7.2 Hz, H-6). After 60 days the signals of spilanthol disappeared, while new signals ([Fig. 6]: 5.80 (dd, 1H, J₁ 1.5, J₂ 1.5, H-2), 5.82 (dd, 1H, J₁ 1.5, J₂ 1.5, H-7), 5.86 (ddd, 1H, J₁ 7.5, J₂ 1.8, J₃ 1.5, H-8), 6.81 (ddd, 1H, J₁ 15.0, J₂ 7.2, J₃ 6.6, H-3) characteristic of the product of its oxidation 16, we observed. An identical compound was isolated by Biavatti et al. in 2016 from an extract of Acmella ciliata (Kunth) Cass. In tests against multiresistant P. falciparum K1 peroxide (16) was found to be more active then spilanthol (IC₅₀: 2.1 µM) [40].

Our study provedthat compound (16) is the oxidation product of spilanthol. Oxidation of the conjugated C=C bond system usually requires the presence of a photosensitizing substance, which, when excited by absorption of a light quantum, in turn excites an oxygen molecule from the unreactive triplet state (³O₂) to a more reactive singlet state (¹O₂). In this state, the oxygen molecule is able to attach to the coupled system of bonds to form a cyclic six-membered system of 4,5-dehydro-1,2-dioxane [41]. It seems that analogous processes may be responsible for the slow disappearance of spilanthol in contact with air. Chlorophyll present in plant extraxts, can act as a photosensitizing substance [42], [43]. A fragment of the spilanthol molecule sensitive to oxidation is a system of two conjugated C = C double bonds at positions C6 – C9 cisoid conformation ([Fig. 5]).

Our studies on the stability of spilanthol have shown that when the crude EE was stored at room temperature, in an air-filled bottle, it loses about 30% of the original mass of spilanthol after 30 days. On the other hand, a dry EE dissolved in chloroform or dichloromethane, stored in the presence of air at room temperature after 60 days, contained mainly the oxidation product and contains only traces of spilanthol as shown in [Fig. 6]. An attempt was made to solve the problem of the stability of spilanthol by removing most of the chlorophyll from the extract with the use of activated carbon. It has been shown that storing the decolorized condensed extract under a nitrogen atmosphere and reduced temperature (5 – 10 °C) significantly slows down the oxidation process of spilanthol, ensuring its stability for up to 24 months. Probably, the period of stability of spilanthol with proper storage is longer, because 24 months is the time that has elapsed since the preparation of our first archival sample.

Material and Methods

General experimental procedures

NMR spectra were recorded on Varian spectrometer at operating frequencies of 600 and 150 MHz, respectively using TMS as the resonance shift standard and NMR solvent (CDCl₃-d₁) which was purchased from ACROS Organics (Geel, Belgium). Infrared spectra were measured on a Nicolet 6700 FT-IR spectrophotometer (Thermo Scientific, USA). Column chromatography fractions were monitored by TLC on precoated plates of silica gel 60 F254 (Merck Millipore). TLC plates were inspected under UV light (λ = 254 nm). All used chemicals were purchased from Fluka and ACROS Organics and were used without purification. The formic acid, water, and acetonitrile for LC-MS were obtained from Merck (Darmstadt, Germany).

Plant material

The plant of Acmella oleracea was collected in June 2019, in the Grodzisk in the Podlaskie Voivodship (administrative unit) in Poland. The plant was authenticated by Angielczyk (number 01 092 019) and was deposited in the Biotechnology Center of Silesian University of Technology (Gliwice, Poland). The dried aerial parts were cut into small pieces and were air dried.

Isolation and characterization of spilanthol (1)

A dry secondary extract from AO (DE2, 4.45 g), containing 10% of SPL, was purified by column chromatography under a gradient elution Hx/AcOEt (5 : 1 to 2 : 1) using silica gel (70 – 230 mesh, Fluka). Spilanthol (0.35 g) with a purity of 97% was isolated with a recovery efficiency of 78% from the crude extract.

¹H NMR (CDCl₃, 600 MHz) δ_H: 6.83 (dt, 1H, J₁ 15.0 Hz, J₂ 6.6 Hz, H-3), 6.32 – 6.26 (m, 1H, H-8), 5.97 (dd~t, 1H, J₁ = J₂  = 10.8 Hz, H-7), 5.80 (dt, 1H, J₁ 15.6 Hz, J₂ 1.8 Hz, H-2), 5.70 (dq, 1H, J₁ 12.0, J₂ 6.0, H-9), 5.52 (br, s, 1H, NH), 5.26 (dt, 1H, J₁ 10.8 Hz, J₂ 7.2 Hz, H-6), 3.15 (dd~t, 2H, J₁ = J₂ 6.3 Hz, H-1′), 2.36 – 2.22 (m, 4H, H-4 and H-5), 1.82 – 1.77 (m, 2H, H-2′ and H-10), 0.92 (d, 1H, J 6.0 Hz, H-3′and H-4′) ppm; ¹³CNMR (CDCl₃, 150 MHz) δ_C: 165.9 (C=O), 143.4 (C-3), 129.9 (C-9), 129.4 (C-7), 127.8 (C-6), 126.6 (C-8), 124.1 (C-2), 46.8 (C-1′), 32.1 (C-4), 28.6 (C-2′), 26.4 (C-5), 20.1 (C-3′ and C-4′), 18.3 (C-10) ppm; IR (ATR) ν: 3276, 2958, 2928, 2871, 1669, 1629, 1552, 1247, 1159, 980, 947, 820 cm⁻¹.

Procedure of NMR quantitation of spilanthol in extracts

Crude ethanolic extract, secondary, or final extract (2 mL) was placed in a round bottom flask and the sample weight was determined (m_P ). An internal standard solution in EtOH (1 mL, 30.0 mg Ph₂ Me₂Si/100 mL EtOH) was then added and weighed again to calculate the weight of the added standard (m_IS). The solvent was then evaporated under reduced pressure at 40 – 50 °C using a rotatory evaporator and the residue was dried under reduced pressure at 50 mmHg until a constant weight was reached. The dry residue was extracted with CDCl₃ (0.75 mL) and then the nuclear magnetic resonance spectrum (¹H NMR) was recorded. The spilanthol content in the extract was calculated from [Fig. 3].

Procedure of HPLC quantitation of spilanthol in extracts

HPLC-MS/MS analyses were performed using the Dionex HPLC system (Dionex Corporation, Sunnyvale, CA, USA) connected with an API 4000 QTRAP mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA, USA). HPLC system was equipped with an UltiMate 3000 RS (Rapid Separation) pump, an UltiMate 3000 autosampler, an UltiMate 3000 column compartment with a thermostable column area, and an UltiMate 3000 variable wavelength detector. The chromatographic analysis of plant extracts were performed on Kinetex C18 column (75 mm × 2.1 mm, 2.6 µm) (Phenomenex, Germany) at 25 °C with a mobile phase consisting of 0.1% formic acid in water: acetonitrile (45 : 55; v/v) at a flow rate 0.5 mL/min. The injection volume was 2 µL. The mass spectrometer was equipped with an electrospray ionization (ESI) source operated in positive mode. MS/MS analysis was performed in multiple reaction monitoring (MRM) using mass transition m/z 222.04 → 81.183 (MRM1; quantitative) and m/z 222.04 → 126.178 (MRM2; qualitative) for spilanthol. Compound dependent parameters set for spilanthol were as following: declustering potential DP=86 eV; entrance potential EP=7 eV; collision energy CE=21 (MRM1)/29 eV (MRM2); and collision cell exit potential CXP=6 (MRM1)/10 eV (MRM2). The source temperature (TEM) was maintained at 500 °C, and the ionization voltage (IS) was set at 4000 V. Q1 and Q3 were maintained at unit resolution and the dwell time was kept at 250 ms. The nebulizer gas (GS1), heater gas (GS2), curtain gas (CUR), and collision activated dissociation gas (CAD) were set at 60, 50, 20, and 6 psi, respectively. Analyst 1.5.1 software (Applied Biosystems, Foster City, CA, USA) was applied for instrumental control, data acquisition and quantitative analysis.

The established HPLC-MS/MS method was evaluated for calibration curve linearity, sensitivity (limit of detection; LOD and limit of quantification; LOQ), intra- and inter-day precision and accuracy, dilution integrity, and carry-over.

The linearity of the calibration curve was evaluated by analyzing spilanthol solutions at eight concentrations ranging from 1.0 to 500 ng/mL. Each concentration level was prepared with three replicates. The calibration curve was prepared using a regression weighted by a factor of 1/x². The LOQ was defined as the lowest amount of analyte which could be quantified reliably, while complying with the criteria for accuracy and precision (lower than 20%).

Precision and accuracy were determined for the three QC levels (LQC 5 ng/mL, MQC 50 ng/mL, and HQC 400 ng/mL). Six replicates of each QC level were processed the same day for the intra-day assay, whereas each QC level was processed six times on three different days over a period of one week for the inter-day assay. Precision was evaluated as the relative standard deviation (%RSD) and accuracy was expressed as the relative error (%RE).

In order to analyze the samples at the concentration above the upper limit of quantification (ULOQ), methanol samples spiked with spilanthol at 500 ng/mL were prepared in 10-fold dilution with MeOH and then analyzed. Precision and accuracy from the nominal concentrations for 6 replicates after dilution should be ≤ 15%.

Methanol, which did not contain analyte, was injected after the ULOQ samples to investigate the carry-over of this method in each validation batch. A value less than 20% of the limit of quantification was considered to confirm the absence of any significant carry-over.

Extraction of spilanthol from Acmella oleracea

Procedure A. EtOH at 96.5% purity (750 mL) was added to a round bottom flask, which was attached to a Soxhlet extractor and a condenser for reflux. The dried and crushed aerial parts (leaves, stem, and flowers) of AO (25.0 g) were loaded into the thimble, which was placed inside the Soxhlet extractor. It was heated to reflux for 15 h, then the crude EE obtained as a green liquid, was separated (V_EE = 675 mL). The SPL content was determined at 194 mg SPL/1 L EE.

Procedure B. Dried aerial parts of AO (50.0 g) and 95.6% EtOH (2.0 L) heated under reflux for 4 h, with stirring. The crude EE obtained as a green liquid, was then separated from the post extract leaves by centrifugation (10 000 rpm for 6 min), yielding 1.7 L of supernatant. The SPL content was determined to be 110 mg SPL/1 L EE.

Procedure C. Dried plant material (50.0 g) and 95.6% EtOH (1.75 L) was stirred at room temperature for 72 h. The crude EE obtained as a green liquid, was then separated from the plant material by gravity filtration. SPL content in the crude EE (V_EE = 1.5 L) was determined at 142 mg SPL/1 L.

Procedure D. Aerial parts of AO (500.0 g) were crushed and placed into an Erlenmeyer flask with 95.6% EtOH (1.4 L). This was left for 144 h at room temperature and protected from light, then the crude EE was separated by filtration. SPL content was determined at 1478 mg SPL/1 L. All crude extracts were stored at low temperature (5 – 10 °C) and under an atmosphere of inert gas until used for further isolation of spilanthol. SPL content was determined following the procedure described in the section Procedure Quantitation of spilanthol in extracts ([Table 1]).

Purification of the crude ethanolic extract

DCM Extraction. The crude EE was concentrated under reduced pressure at 50 °C to give a resinous residue (20.0 g). Dichloromethane (350 mL) was added and stirred vigorously under nitrogen for 0.5 h, then the upper layer was decanted. The procedure was repeated twice. The spilanthol content in the secondary extract (E2, dissolved in dichloromethane) was determined by ¹H NMR ([Table 2]).

5% NaOH Extraction. The DE2 (100 mL) was extracted three times with 5% NaOH (150 mL) and water (150 mL) at room temperature. The spilanthol content in the remaining resin was determined by ¹H NMR ([Table 2]).

Ion Exchange Chromatography. An Amberlite IRA67 (4.0 g) resin and system 96% EtOH/H₂O (8/2, v/v) were left at room temperature for 24 h. Then, the resin was used to produce an ion exchange filler by placing it into a glass column. The condensed dichloromethane extract (DE2, 2.1 g) was dissolved into a small volume of the eluent and loaded to the ion exchange filler. Solvent was passed through the column and fractions were collected. The progress of the ion exchange chromatography was monitored by TLC (Hx : AcOEt, 2 : 1). The spilanthol content of combined fractions was determined by ¹H NMR ([Table 2]).

Contributorsʼ Statement

Conceptualization and methodology: M. Grymel, R. Mazurkiewicz; supervision: M. Grymel; extraction and quantification: M. Grymel, S. Bajkacz, S. Kowalczyk; analysis and interpretation of the data: M. Grymel, R. Mazurkiewicz, J. Bilik; writing – original draft preparation: M. Grymel, R. Mazurkiewicz, S. Bajkacz; writing – review and editing: M. Grymel and S. Bajkacz. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported in part by Silesian University of Technology (Poland) Grant BK No. 04/050/BK_22/0139.

• References

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• 15 De Spiegeleer B, Boonen J, Malysheva SV, Mavungu JDD, De Saeger S, Roche N, Blondeel P, Taevernier L, Veryser L. Skin penetration enhancing properties of the plant N-alkylamide spilanthol. J Ethnopharmacol 2013; 148: 117-125
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  CrossrefPubMedSearch in Google Scholar
• 18 Veryser L, Wynendaele E, Taevernier L, Verbeke F, Joshi T, Tatke P, De Spiegeleer B. N-Alkylamides: From plant to brain. Funct Foods Health Dis 2014; 4: 264-275
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  PubMedSearch in Google Scholar
• 20 Belfer WA. Cosmetic compositions comprising peptides and Acmella oleracea extract to accelerate repair of functional wrinkles. US Patent 2007048245, 2007
  PubMedSearch in Google Scholar
• 21 Demarne F, Passar G. Use of an Acmella oleracea extract for its botox-like effect in an antiwrinkle cosmetic composition. FR Patent 286513, 2005
  PubMedSearch in Google Scholar
• 22 Bae SS, Ehrmann BM, Ettefagh KA, Cech NB. A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem Anal 2010; 21: 438-443
  CrossrefPubMedSearch in Google Scholar
• 23 Pandey HK, Rawut PS, Kumar N, Verma GS. A herbal formulation for toothache and related disorders and a process for preparation thereof. IN Patent 2004DE00260, 2007
  PubMedSearch in Google Scholar
• 24 Adler RJ. Compositions for the acute and/or long term treatment of periodontal diseases using herb extracts. WO Patent 2006059196, 2006
  PubMedSearch in Google Scholar
• 25 Singh M, Chaturvedi R. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr. Ind Crops Prod 2012; 36: 321-328
  CrossrefPubMedSearch in Google Scholar
• 26 Singh M, Chaturvedi R. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr. Bioprocess Biosyst Eng 2012; 35: 943-951
  CrossrefPubMedSearch in Google Scholar
• 27 Sharma V, Boonen J, Chauhan NS, Thakur M, De Spiegeleer B, Dixi VK. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine 2011; 18: 1161-1169
  CrossrefPubMedSearch in Google Scholar
• 28 Baliero OC, Pinheiro MSS, Silva SYS, Oliveira MN, Silva SC, Gomes AA, Pinto L. Analytical and preparative chromatographic approaches for extraction of spilanthol from Acmella oleracea flowers. Microchem J 2020; 157: 105035
  CrossrefPubMedSearch in Google Scholar
• 29 Shimada T, Gomi T. Spilanthol-rich essential oils for manufacturing toothpastes or other oral compositions. JP Patent 07090294, 1995
  PubMedSearch in Google Scholar
• 30 Santana de Freitas-Blanco V, Franz-Montan M, Groppo FC, de Carvalho JE, Figueira GM, Serpe L, Oliveira Sousa IM, Guilherme Damasio VA, Yamane LT, de Paula E, Ferreira Rodrigues RA. Development and evaluation of a novel mucoadhesive film containing Acmella oleracea extract for oral mucosa topical anesthesia. PLoS One 2016; 11: e0162850
  CrossrefPubMedSearch in Google Scholar
• 31 Rao BG, Rao YV, Rao TM. Hepatoprotective activity of Spilanthes acmella extracts against CCl₄-induced liver toxicity in rats. Asian Pac J Trop Dis 2012; 2: S208
  CrossrefPubMedSearch in Google Scholar
• 32 Abeysiri GRPI, Dharmadasa RM, Abeysinghe DC, Samarasinghe K. Screening of phytochemical, physico-chemical and bioactivity of different parts of Acmella oleraceae Murr. (Asteraceae), a natural remedy for toothache. Ind Crops Prod 2013; 50: 852-856
  CrossrefPubMedSearch in Google Scholar
• 33 Cruz PB, Barbosa AF, Zeringóta V, Melo D, Novato T, Fidelis QC, Fabri RL, de Carvalho MG, Oliveira Sabaa-Srur AU, Daemon E, Monteiro CMO. Acaricidal activity of methanol extract of Acmella oleracea L. (Asteraceae) and spilanthol on Rhipicephalus microplus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae). Vet Parasitol 2016; 228: 137-143
  CrossrefPubMedSearch in Google Scholar
• 34 Wongsawatkul O, Prachayasittikul S, Isarankura-Na-Ayudhya C, Satayavivad J, Ruchirawat S, Prachayasittikul V. Vasorelaxant and antioxidant activities of Spilanthes acmella Murr. Int J Mol Sci 2008; 9: 2724-2744
  CrossrefPubMedSearch in Google Scholar
• 35 Castro KNC, Lima DF, Vasconcelos LC, Leite JRSA, Santos RC, Paz Neto AA, Costa-Júnior LM. Acaricide activity in vitro of Acmella oleracea against Rhipicephalus microplus . Parasitol Res 2014; 113: 3697-3701
  CrossrefPubMedSearch in Google Scholar
• 36 Mbeunkui F, Grace MH, Lategan C, Smith PJ, Raskin I, Lila MA. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879: 1886-1892
  CrossrefPubMedSearch in Google Scholar
• 37 Pandey V, Chopra M, Agrawal V. In Vitro isolation and characterization of biolarvicidal compounds from micropropagated plants of Spilanthes acmella . Parasitol Res 2011; 108: 297-304
  CrossrefPubMedSearch in Google Scholar
• 38 Ahmed S, Rahman A, Muslim T, Sohrab MH, Akbor MA, Siraj S, Sultana N, Mansur MA. Antimicrobial cytotoxicity and phytochemical activities of Spilanthes acmella . Bangladesh J Sci Ind Res 2012; 47: 437-440
  CrossrefPubMedSearch in Google Scholar
• 39 Mayara TP, Deisiane Del Castilo B, Lobato AB, da Silva R, da Silva SSM. Antioxidant and cytotoxic potential of aqueous crude extract of Acmella oleracea (L.) R. K. Jansen. J Chem Pharm Res 2015; 7: 562-569
  PubMedSearch in Google Scholar
• 40 Silveira N, Saar J, Santos ADC, Barison A, Sandjo LP, Kaiser M, Schmidt TJ, Biavatti MW. A new alkamide with an endoperoxide structure from Acmella ciliata (Asteraceae) and its in vitro antiplasmodial activity. Molecules 2016; 21: 765-774
  CrossrefPubMedSearch in Google Scholar
• 41 Arbuzov YA. The Diels-Alder reaction with molecular oxygen as dienophile. Russ Chem Rev 1965; 34: 558-572
  CrossrefPubMedSearch in Google Scholar
• 42 Krieger-Liszkay A. Singlet oxygen production in photosynthesis. J Exp Bot 2005; 56: 337-346
  CrossrefPubMedSearch in Google Scholar
• 43 Krieger-Liszkay A, Fufezan C, Trebst A. Singlet oxygen production in photosystem II and related protection mechanism. Photosynth Res 2008; 98: 551-564
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 16 January 2022

Accepted after revision: 27 June 2022

Article published online:
31 August 2022

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Spelman K, Depoix D, McCray M, Mouray E, Grellier P. The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and Undeca-2E-ene-8, 10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity. Phytother Res 2011; 25: 1098-1101
  CrossrefPubMedSearch in Google Scholar
• 2 Barbosa AF, de Carvalho MG, Smith RE, Sabaa-Srur AUO. Spilanthol: Occurrence, extraction, chemistry and biological activities. Rev Bras Farmacogn 2016; 26: 128-133
  CrossrefPubMedSearch in Google Scholar
• 3 Cheng YB, Liu R, Ho MC, Wu TY, Chen CY, Lo IW, Hou MF, Yuan SS, Wu YC, Chang FR. Alkylamides of Acmella oleracea . Molecules 2015; 20: 6970-6977
  CrossrefPubMedSearch in Google Scholar
• 4 Prachayasittikul V, Prachayasittikul S, Ruchirawat SPV. High therapeutic potential of Spilanthes acmella Murr.: A review. EXCLI J 2013; 12: 219-312
  PubMedSearch in Google Scholar
• 5 Tiwari K. An updated review on medicinal herb genus Spilanthes . J Chin Integr Med 2011; 9: 1170-1178
  CrossrefPubMedSearch in Google Scholar
• 6 Dubey S, Maity S, Singh M, Saraf SA, Saha S. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: A review. Adv Pharmacol Sci 2013; 2013: 1-9
  PubMedSearch in Google Scholar
• 7 Prachayasittikul S, Suphapong S, Worachartcheewan A, Lawung R, Ruchirawat S, Prachayasittikul V. Bioactive metabolites from Spilanthes acmella Murr. Molecules 2009; 14: 850-867
  CrossrefPubMedSearch in Google Scholar
• 8 Sharma R, Arumugam N. N-alkylamides of Spilanthes (syn: Acmella): Structure, purification, characterization, biological activities and applications – a review. Future Foods 2021; 3: 100022
  CrossrefPubMedSearch in Google Scholar
• 9 Dias AMA, Santos P, Seabra IJ, Júnior RNC, Braga MEM, de Sousa HC. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J Supercrit Fluids 2012; 61: 62-70
  CrossrefPubMedSearch in Google Scholar
• 10 Sharma A, Kumar V, Rattan RS, Kumar N, Singh B. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. against Plutella xylostella L. Am J Plant Sci 2012; 03: 1568-1572
  CrossrefPubMedSearch in Google Scholar
• 11 Déciga-Campos M, Rios MY, Aguilar-Guadarrama AB. Antinociceptive effect of Heliopsis longipes extract and affinin in mice. Planta Med 2010; 76: 665-670
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Molina-Torres J, Salazar-Cabrera CJ, Armenta-Salinas C, Ramírez-Chávez E. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: Affinin and reduced amides. J Agric Food Chem 2004; 52: 4700-4704
  CrossrefPubMedSearch in Google Scholar
• 13 Ramsewak RS, Erickson AJ, Nair MG. Bioactive N-isobutylamides from the flower buds of Spilanthes acmella . Phytochemistry 1999; 51: 729-732
  CrossrefPubMedSearch in Google Scholar
• 14 Moreno SC, Carvalho GA, Picanço MC, Morais EGF, Pereira RM. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag Sci 2012; 68: 386-393
  CrossrefPubMedSearch in Google Scholar
• 15 De Spiegeleer B, Boonen J, Malysheva SV, Mavungu JDD, De Saeger S, Roche N, Blondeel P, Taevernier L, Veryser L. Skin penetration enhancing properties of the plant N-alkylamide spilanthol. J Ethnopharmacol 2013; 148: 117-125
  CrossrefPubMedSearch in Google Scholar
• 16 Boonen J, Baert B, Burvenich C, Blondeel P, De Saeger S, De Spiegeleer B. LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behaviour of its main bio-active spilanthol. J Pharm Biomed Anal 2010; 53: 243-249
  CrossrefPubMedSearch in Google Scholar
• 17 Boonen J, Baert B, Roche N, Burvenich C, De Spiegeleer B. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts. J Ethnopharmacol 2010; 127: 77-84
  CrossrefPubMedSearch in Google Scholar
• 18 Veryser L, Wynendaele E, Taevernier L, Verbeke F, Joshi T, Tatke P, De Spiegeleer B. N-Alkylamides: From plant to brain. Funct Foods Health Dis 2014; 4: 264-275
  CrossrefPubMedSearch in Google Scholar
• 19 Demarne F, Passaro G. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent 7531193, 2009
  PubMedSearch in Google Scholar
• 20 Belfer WA. Cosmetic compositions comprising peptides and Acmella oleracea extract to accelerate repair of functional wrinkles. US Patent 2007048245, 2007
  PubMedSearch in Google Scholar
• 21 Demarne F, Passar G. Use of an Acmella oleracea extract for its botox-like effect in an antiwrinkle cosmetic composition. FR Patent 286513, 2005
  PubMedSearch in Google Scholar
• 22 Bae SS, Ehrmann BM, Ettefagh KA, Cech NB. A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem Anal 2010; 21: 438-443
  CrossrefPubMedSearch in Google Scholar
• 23 Pandey HK, Rawut PS, Kumar N, Verma GS. A herbal formulation for toothache and related disorders and a process for preparation thereof. IN Patent 2004DE00260, 2007
  PubMedSearch in Google Scholar
• 24 Adler RJ. Compositions for the acute and/or long term treatment of periodontal diseases using herb extracts. WO Patent 2006059196, 2006
  PubMedSearch in Google Scholar
• 25 Singh M, Chaturvedi R. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr. Ind Crops Prod 2012; 36: 321-328
  CrossrefPubMedSearch in Google Scholar
• 26 Singh M, Chaturvedi R. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr. Bioprocess Biosyst Eng 2012; 35: 943-951
  CrossrefPubMedSearch in Google Scholar
• 27 Sharma V, Boonen J, Chauhan NS, Thakur M, De Spiegeleer B, Dixi VK. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine 2011; 18: 1161-1169
  CrossrefPubMedSearch in Google Scholar
• 28 Baliero OC, Pinheiro MSS, Silva SYS, Oliveira MN, Silva SC, Gomes AA, Pinto L. Analytical and preparative chromatographic approaches for extraction of spilanthol from Acmella oleracea flowers. Microchem J 2020; 157: 105035
  CrossrefPubMedSearch in Google Scholar
• 29 Shimada T, Gomi T. Spilanthol-rich essential oils for manufacturing toothpastes or other oral compositions. JP Patent 07090294, 1995
  PubMedSearch in Google Scholar
• 30 Santana de Freitas-Blanco V, Franz-Montan M, Groppo FC, de Carvalho JE, Figueira GM, Serpe L, Oliveira Sousa IM, Guilherme Damasio VA, Yamane LT, de Paula E, Ferreira Rodrigues RA. Development and evaluation of a novel mucoadhesive film containing Acmella oleracea extract for oral mucosa topical anesthesia. PLoS One 2016; 11: e0162850
  CrossrefPubMedSearch in Google Scholar
• 31 Rao BG, Rao YV, Rao TM. Hepatoprotective activity of Spilanthes acmella extracts against CCl₄-induced liver toxicity in rats. Asian Pac J Trop Dis 2012; 2: S208
  CrossrefPubMedSearch in Google Scholar
• 32 Abeysiri GRPI, Dharmadasa RM, Abeysinghe DC, Samarasinghe K. Screening of phytochemical, physico-chemical and bioactivity of different parts of Acmella oleraceae Murr. (Asteraceae), a natural remedy for toothache. Ind Crops Prod 2013; 50: 852-856
  CrossrefPubMedSearch in Google Scholar
• 33 Cruz PB, Barbosa AF, Zeringóta V, Melo D, Novato T, Fidelis QC, Fabri RL, de Carvalho MG, Oliveira Sabaa-Srur AU, Daemon E, Monteiro CMO. Acaricidal activity of methanol extract of Acmella oleracea L. (Asteraceae) and spilanthol on Rhipicephalus microplus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae). Vet Parasitol 2016; 228: 137-143
  CrossrefPubMedSearch in Google Scholar
• 34 Wongsawatkul O, Prachayasittikul S, Isarankura-Na-Ayudhya C, Satayavivad J, Ruchirawat S, Prachayasittikul V. Vasorelaxant and antioxidant activities of Spilanthes acmella Murr. Int J Mol Sci 2008; 9: 2724-2744
  CrossrefPubMedSearch in Google Scholar
• 35 Castro KNC, Lima DF, Vasconcelos LC, Leite JRSA, Santos RC, Paz Neto AA, Costa-Júnior LM. Acaricide activity in vitro of Acmella oleracea against Rhipicephalus microplus . Parasitol Res 2014; 113: 3697-3701
  CrossrefPubMedSearch in Google Scholar
• 36 Mbeunkui F, Grace MH, Lategan C, Smith PJ, Raskin I, Lila MA. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879: 1886-1892
  CrossrefPubMedSearch in Google Scholar
• 37 Pandey V, Chopra M, Agrawal V. In Vitro isolation and characterization of biolarvicidal compounds from micropropagated plants of Spilanthes acmella . Parasitol Res 2011; 108: 297-304
  CrossrefPubMedSearch in Google Scholar
• 38 Ahmed S, Rahman A, Muslim T, Sohrab MH, Akbor MA, Siraj S, Sultana N, Mansur MA. Antimicrobial cytotoxicity and phytochemical activities of Spilanthes acmella . Bangladesh J Sci Ind Res 2012; 47: 437-440
  CrossrefPubMedSearch in Google Scholar
• 39 Mayara TP, Deisiane Del Castilo B, Lobato AB, da Silva R, da Silva SSM. Antioxidant and cytotoxic potential of aqueous crude extract of Acmella oleracea (L.) R. K. Jansen. J Chem Pharm Res 2015; 7: 562-569
  PubMedSearch in Google Scholar
• 40 Silveira N, Saar J, Santos ADC, Barison A, Sandjo LP, Kaiser M, Schmidt TJ, Biavatti MW. A new alkamide with an endoperoxide structure from Acmella ciliata (Asteraceae) and its in vitro antiplasmodial activity. Molecules 2016; 21: 765-774
  CrossrefPubMedSearch in Google Scholar
• 41 Arbuzov YA. The Diels-Alder reaction with molecular oxygen as dienophile. Russ Chem Rev 1965; 34: 558-572
  CrossrefPubMedSearch in Google Scholar
• 42 Krieger-Liszkay A. Singlet oxygen production in photosynthesis. J Exp Bot 2005; 56: 337-346
  CrossrefPubMedSearch in Google Scholar
• 43 Krieger-Liszkay A, Fufezan C, Trebst A. Singlet oxygen production in photosystem II and related protection mechanism. Photosynth Res 2008; 98: 551-564
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material