Enhanced production of ‘spilanthol’ through elicitation of cell suspension cultures in Acmella ciliata (Kunth) Cass. and spilanthol characterization by HPLC-HRMS analysis

doi:10.21203/rs.3.rs-4297961/v1

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Enhanced production of ‘spilanthol’ through elicitation of cell suspension cultures in Acmella ciliata (Kunth) Cass. and spilanthol characterization by HPLC-HRMS analysis

https://doi.org/10.21203/rs.3.rs-4297961/v1

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The study presented here has established a cell suspension culture system for the in vitro production of the high value bioactive alkamide ‘spilanthol’ in Acmella ciliata. White, purple, friable callus procured from leaf explants in MS medium containing 0.5 mgl^-1 BA and 1.0 mgl^-1 NAA have succeeded in fourfold increase in cell mass after 25 days of culture upon 120 rpm agitation in a gyratory shaker and the presence of ‘spilanthol’ in the harvested cells was detected using HPLC coupled with HRMS. Here, high spilanthol content (239.512 µgg^-1) was noticed in embryogenic callus cultures established in NAA-BA combination followed by in vitro plant (193.935 µgg^-1) as well as cells harvested from suspension culture (173.702 µgg^-1). In A. ciliata flower heads 102.730 µgg^-1 spilanthol content was recorded, while the leaves and stem contained 96.69 and 61.146 µgg^-1 spilanthol respectively. The least quantity of spilanthol was noticed in the in vivo plant (92.198 µgg^-1) that was the absolute control. Thus quantification of spilanthol in the in vitro system revealed more amount of the same in it especially in embryogenic callus than in vivo plant (control). Elicitation was attempted using both biotic and abiotic elicitors to scale up the production of spilanthol in cell suspension culture system. Elicitation using biotic elicitors showed maximum 34.61-fold increase with Yeast extract (YE) in 24 hrs exposure; while the abiotic elicitor MeJA (Methyl jasmonate) treatment evoked the production of spilanthol to 41.02 fold in 72 hrs treatment. Comparatively more time of exposure was required in abiotic elicitation than with biotic elicitors. By considering the merit of perceiving maximum yield in short duration, treatment with YE for 24 hrs period can be suggested as the choice of elicitation for improved production of the alkamides particularly spilanthol in cell suspension cultures of A. ciliata. The established system can be extended for the bioproduction of the bioactive alkamide ‘spilanthol’ using bioreactor technology with suitable refinement thus benefiting the phyto-pharma needs.

Acmella ciliata

cell suspension culture

spilanthol

elicitation

yeast extract

methyl jasmonate

HPLC-HRMS

This is the first report on enhanced production of ‘spilanthol’ in Acmella ciliata using cell suspension culture followed by the characterisation of spilanthol by HPLC-HRMS analysis and the system established can be extended for the bioproduction of this high value bioactive alkamide using appropriate bioreactor technology thus benefiting the phyto-pharmacological applications of this taxa.

Medicinal plants are the key sources of pharmaceutically as well as industrially important bioactive secondary compounds. However, population growth, habitat destruction, urbanization, climate change and the unrestricted collection and over-exploitation of these genetic wealth from the natural habitat has resulted in the threatening of their natural resources. Hence it is recommended by the conservation authorities to formulate methods for the appropriate management of these important plant resources and it has become a matter of urgency. The conservation and multiplication of several medicinal species using biotechnological techniques viz., plant tissue, organ and cell suspension cultures have been investigated as a promising tool for bioproduction of desired natural products (Hasnain et al. 2022). A constantly increasing demand of economically important plant-based products warrants their mass propagation through plant tissue culture strategy, an essential component of plant biotechnology which also offers novel approaches to micropropagation, conservation and secondary metabolite extraction. Since most of the plant cells are totipotent, it should be possible to make a culture of cells from a plant that naturally produces a certain biochemical and cause the culture to produce that specific chemical substance under in vitro conditions. A surprising number of cell cultures have been established that produce specialized biochemicals, including alkaloids, their precursors and derivatives, cardiac glycosides, steroids, benzoquinones, latex, phenolics, anthocyanins, organic acids, antitumor, antibacterial, flavoring and odouring agents (Ozyigit et al. 2023). The commercial improvement of the technology will benefit the production of pharmaceuticals and high valued biochemicals (Chandran et al., 2020). Many studies have been undertaken like manipulation of nutrient media, optimization of culture conditions, identification of the most effective elicitors, the use of hairy root culture and cell suspension culture techniques with the objective of improving the in vitro production of plant secondary compounds.

The targeted species for the present study Acmella ciliata (Kunth) Cass. Commonly known as ‘fringed pod tooth ache plant’ is a medicinally important plant belonging to Asteraceae which is over-exploited for its pharmacological properties. The plant possesses diverse bioactive properties and immense utilization in medicine, health care, cosmetics as well as health supplements. It is used as an active ingredient in several anti-ageing herbal tonics and in mouth washes (Sharma et al. 2022). The species is of African origin and brought to Brazil during the colonial period (Risso et al. 2010) and is considered as sacred in some African-Brazilian religions. The leaf extract is used in Brazilian folk medicine due to its analgesic and anti-inflammatory properties (Perez-Amador et al. 2008) and is used in the treatment of heartburn, gastro-intestinal disorders, gastritis, headache, diarrhoea, hypertension (Alves and Snow 2003; Nergard et al. 2004; Perez-Amador et al. 2008). Besides, it is used in folk medicine to treat liver ailments (Binu 1999). The different species of Acmella are well known in folklore remedy as a potential medicinal plant used for the culinary purposes, by the tribes in various parts of the world. Apart from its traditional usage, it is commercially cultivated and marketed in different parts of the world that indicates the increasing economic potential of this herbaceous medicinal species. Spilanthes is mentioned as Acmella in some of the literature. However, monographs have been written about both genera Acmella and Spilanthes (Jansen 1981) and the “toothache plant” was placed in the genus Acmella (Jansen 1981). N-alkylamides or alkamides are the most abundant phytochemicals present in the genus Spilanthes/ Acmella which account for its biological activity. Spilanthol (N-Isobutyl-2(E),6(Z),8(E)-decatrienamide) is a high value bioactive alkamide in this taxa along with other bioactive metabolites viz. phenolic, flavanoid, coumarin and triterpenoid compounds and at present there are about twenty-three pharmaceutical companies manufacturing spilanthol containing products. Spilanthol, the key component of the plant is responsible for most of its pharmaceutical applicability especially its antimalarial property (Silveira et al. 2016). Additionally, it has an efficient analgesic, anti-inflammatory and antimicrobial properties (Rios et al. 2007). As the awareness towards safe, natural compounds are increasing, the demand of the natural source is also increasing continuously to the extent that the current supply of spilanthol from Spilanthes/ Acmella plant is insufficient to meet the same and this scenario necessitates the formulation of appropriate in vitro production methods of this high demanding compound. The perusal of literature reveals that there are reports on in vitro propagation, direct and indirect organogenesis, in vitro flowering as well as secondary metabolite production particularly in Spilanthes acmella, and very scarce in Acmella cilita. In vitro culture establishment and alkamide profiling was reported in another species Acmella radicans var. radicans (Rios-Chavez et al. 2003). In a recent study we have demonstrated the anti-proliferative efficacy of the cell biomass of Acmella ciliata against SK Mel 28 cell lines (Neethu Mohan et al. 2022) and concluded that the demonstrated potentiality is attributed to the presence of the bioactive alkamide spilanthol in it. Also, we have reported the in vitro flowering phenomenon in this taxa (Neethu Mohan et al. 2022). The development of an efficient in vitro system for the synthesis of ‘spilanthol’ in A. ciliata is the very recent report solely by us regarding spilanthol production through somatic embryogenesis (Mohan et al. 2023). In this backdrop, we here explain the setting of a model in vitro system for the scale up production of ‘spilanthol’ from cell suspension cultures of A. ciliata using elicitation and this optimized protocol can be extended further for benefiting the phyto-pharma industry.

Plant material

Acmella ciliata (Kunth) Cass. plants were collected from the natural population in Aruvikkara, Thiruvananthapuram District of Kerala State, India and maintained in the green house of Department of Botany, University College, Thiruvananthapuram, Kerala, India. The specimens were identified by taxonomy experts and the herbarium (Voucher numbers TBGT 32710–32711) was authenticated from Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, Kerala, India.

Establishment of shoot culture and shoot biomass

Shoot cuttings bearing shoot tip and first nodal segments from A. ciliata plants growing in the greenhouse were collected, the leaves were removed and the explants were cut into appropriate sizes, slightly larger than the size to be inoculated. They were washed in running tap water for 30 minutes to remove all the dust particles followed by washing with 0.5% (v/v) Labolene for 20 minutes and washed again in running tap water to remove the detergent remaining. Then the explants were rinsed 5–7 times with distilled water and subsequent surface sterilization procedures were done inside the laminar airflow chamber, where the explants were sterilized in 0.1% (w/v) HgCl₂ for 5–10 minutes that depend upon the hardness of the tissues. Subsequently the explants were rinsed 3–4 times with sterile distilled water and transferred to pre-sterilized petridishes. The surface sterilant exposed ends were dissected apart and the shoot tips (0.5–0.7 cm) and nodal segments (0.8-1.0 cm) were then inoculated vertically into MS (Murashige and Skoog) medium (Murashige and Skoog 1962) supplemented with 0.5 mgl^− 1 BA in culture tubes. All the cultures were incubated at 25 ± 2 ºC under 12 hour photoperiod at a photon flux intensity of 50–60 µEm^− 2s^− 1 provided by cool, white, fluorescent tubes (Philips, India) under 50–60% RH. In vitro shoot biomass was raised through the nodal culture and multiplied through subsequent subculturing which were harvested after the fourth subculture period.

Establishment of embryogenic callus culture

For callus induction, in vitro leaves dissected out from in vitro-raised shoot cultures were cut in to appropriate sizes (1–2 cm²) and inoculated on MS medium fortified with 0.5, 1.0, 2.0 mgl^-1 NAA/ IAA/ IBA/ 2,4-D either individually or in combination with 0.5 mgl^-1 BA. MS basal solid medium served as the control. MS medium supplemented with 1.0 mgl^-1 NAA along with 0.5 mgl^-1 BA induced white, slightly purplish, friable callus that upon subculturing to fresh medium of the same combination turned embryogenic, finally resulting in the formation of somatic embryos.

Establishment of cell suspension culture system

Callus cultures established in MS medium containing 0.5 mgl^-1 BA in combination with 1.0 mgl^-1 NAA was used for establishing cell suspension culture. For the same, approximately 500 mg callus was transferred to 100 ml liquid MS medium with the same concentration of BA-NAA. The cultures were incubated at 25 ± 2 ºC with a photoperiod of 8/16 hours light/dark period on a gyratory shaker (Orbitek, Scigenics) at varying rotatory speeds (60, 90, 120 and 150 rpm) in order to standardize the suitable rotation speed for the growth of cells. At regular time interval (4, 7, 10, 13, 16, 19, 22, 25, 28 and 31 days), the cells were collected and the fresh weight and dry weight were recorded for analyzing the growth kinetics. Finally, growth curve was drawn using the parameters viz. culture period (days) in X-axis and fresh weight of the cells (g) in Y-axis. During the establishment phase of cell suspension culture, the pH of the medium was routinely observed to check whether some exudations are coming in to the medium. The cells collected during the course of time were analysed under a microscope (Labomed) to study the viability and cell division capacity during these periods. After 25 days of inoculation, the cells were harvested by vacuum filtration for further HPLC analysis. Also, subculturing was made by taking 1000 µl of inoculum to fresh medium containing the same concentration and combinations of phytohormones for the maintenance of cultures.

HPLC analysis for the quantification of spilanthol

The in vitro shoot biomass, embryogenic callus, in vivo plant and cells harvested from suspension cultures were utilised for the extraction and analysis of bioactive alkylamide spilanthol keeping the in vivo mother plant as the control. The isolation and characterisation were performed by HPLC coupled with High Resonance Mass spectrometry (HRMS) and the amount of spilanthol present in the samples were determined. In addition, spilanthol content in various plant parts viz. stem, leaves, flower heads and in roots were also quantified (Neethu 2022).

Quantification of spilanthol was carried out on Varian Prostar HPLC system (Varian, USA) consisting of Ultraviolet (UV) detector, a prostar binary pump and a 20-lL injection loop. Hypersil BDS RP-18 column (Thermo, USA) of dimensions 4.6×250 mm was used with acetonitrile:water (93:7) as mobile phase at a flow rate of 0.5 ml min^− 1. The eluted samples were detected at 237 nm. Spilanthol peaks obtained in HPLC was identified by comparing with published data and was tentatively quantified on the basis of the reference compound, dodeca-2(E),4(E)-dienoic acid isobutylamide (Chromadex, USA). Stock solutions (1000 µgml^− 1) of dodeca-2(E), 4(E)-dienoic acid isobutylamide, was prepared by dissolving 5 mg of the compound in 5 ml of HPLC-grade methanol. The solution was then stored at -20°C. Quantification was carried out using five levels of external standards obtained by serial dilutions of stock solutions at a concentration range of 250 − 15 µgml^− 1. Each concentration of standard was filtered through a 0.22 µm nylon membrane filter (Millipore, USA) before HPLC analysis. Method linearity was demonstrated by determining a calibration curve, injecting the standard at different concentrations and calculating the regression coefficient (r²). Slope equation obtained was used to calculate the amount of the spilanthol in unknown samples and was reported as µgg^− 1 DW of sample.

Characterization of spilanthol by High Resonance Mass Spectrometry

For spilanthol characterisation, the peaks eluted from HPLC were collected and analysed by Mass spectrometry (THERMOSCIENTIFIC EXACTIVE). LC-HRMS was carried out on Hypersil C18 Column with methanol: water (0.1% formic acid) (97:3) as mobile phase for 5 minutes with a flow rate of 150 µl per minute. 2 µl of sample was injected in to the loop and finally, the source was operated in both positive and negative mode at an ion spray voltage of 3 KV and oven temperature was set to 30°C. Spilanthol was identified by its fragmentation profile and confirmed with literature data.

Elicitation in cell suspension cultures

For enhancing the production of spilanthol, elicitation experiments were conducted in the established cell suspension culture system. Cells in suspension cultures were treated with two types of biotic and abiotic elicitors viz. yeast extract (0.5, 1.0, 2 and 4%), chitosan (25, 50, 100 and 200 mgl^-1) as biotic elicitors, while salicylic acid (10, 50, 100 and 200 µl) and methyl jasmonate (10, 50, 100 and 200 ppm) constituted the abiotic elicitors in the initial day of log phase i.e., 21st day of inoculation. The elicitor treatments were carried out for 24, 48 and 72 hours exposure periods. Finally, the cells were collected after these time intervals, both the fresh weight and dry weight of the cells were recorded and further analysed for the alkamide content in them.

Establishment of shoot culture and shoot biomass

Shoot cultures were initiated from the surface sterilized explants of A. ciliata. Single shoots isolated from the initiated shoot cultures were transferred to the respective medium for further subculture passages and as the subculture passages went on, two fold increase in the frequency of multiplication was noticed. MS medium fortified with 1.0 mgl^-1 BA in subsequent subculture passages produced a mean number of 7.02 ± 0.66, 11.24 ± 0.23, 20.71 ± 0.52, 37.42 ± 0.23 shoots per explant, during first, second, third and fourth subculture passages respectively (data not shown). Thus, starting from a single node, it is possible to produce approximately 37.42 shoots within 20 weeks.

Establishment of embryogenic callus culture

In the present study, callus formation was observed in leaf segments of A. ciliata after 14–20 days of inoculation in MS medium supplemented with different combinations of 2,4-D, NAA, IAA, IBA and BA (Table 1) while MS basal medium (control) failed to evoke callogenesis. While auxins like NAA, IAA and IBA (0.5, 1.0, 2.0 mgl^-1) were supplemented individually, after 14th day of inoculation, the leaf segments showed callusing and on the 20th day, rhizogenesis occurred from the callus tissue (Figs. 1a&b). In MS medium fortified with 0.5, 1.0 and 2.0 mgl^-1 2,4-D, friable cream callus was formed after 14 days which turned to black after 24 days of culture. Callus was not proliferating when 0.5 and 1.0 mgl^-1 2,4-D was aided in combination with 0.5 mgl^-1 BA, and white friable callus was produced in this combination which suddenly changed to black (Fig. 1c&d). When the concentration of 2,4-D was increased to 2.0 mgl^-1 in this combination, the explants eventually died and no response was noticed (Table 1). Better response of explants and comparatively more amount of callus formation was observed in MS medium supplemented with 1.0 mgl^-1 NAA along with 0.5 mgl^-1 BA, wherein white, slightly purplish, friable callus was produced (Fig. 1f). When NAA was substituted with IAA in NAA-BA combination, white purplish callus was produced in which shoot organogenesis was noticed after 26 days (Fig. 1e). The white, purplish, friable callus, induced in in MS medium supplemented with 1.0 mgl^-1 NAA along with 0.5 mgl^-1 BA subcultured to fresh medium of the same combination turned embryogenic, and ultimately resulted in the formation of somatic embryos.

Table 1

Effect of plant growth regulators on callus induction in *A. ciliata*
Plant growth regulators (mgl^-1)					% of callus induction	Morphology of callus	Colour	Amount of callus
BA	2,4-D	NAA	IAA	IBA	% of callus induction	Morphology of callus	Colour	Amount of callus
-	0.5	-	-	-	65.24 ± 0.12^h	Friable	Black	++
-	1.0	-	-	-	72.36 ± 0.58^g	Friable	Black	++
-	2.0	-	-	-	25.41 ± 0.40^k	Friable	Black	+
0.5	0.5	-	-	-	63.28 ± 0.57ⁱ	Friable	Black	++
0.5	1.0	-	-	-	58.43 ± 0.64^j	Friable	Black	++
0.5	2.0	-	-	-	---	---	---	---
-	-	0.5	-	-	91.54 ± 0.45^c	Compact, rhizogenic	White	++
-	-	1.0	-	-	95.24 ± 0.84^b	Compact, rhizogenic	White	++
-	-	2.0	-	-	92.48 ± 0.36^c	Compact, rhizogenic	White	+
0.5	-	0.5	-	-	75.63 ± 0.84^f	Compact	Off white	++
0.5	-	1.0	-	-	97.28 ± 0.53^a	Friable	White purplish	+++
0.5	-	2.0	-	-	95.36 ± 0.64^b	Friable	White purplish	+++
-	-	-	0.5	-	89.57 ± 0.72^d	Compact more rhizogenic	White	++
-	-	-	1.0	-	92.42 ± 0.84^c	Compact more rhizogenic	White	++
-	-	-	2.0	-	88.63 ± 0.45^d	Compact more rhizogenic	White	++
0.5	-	-	0.5	-	72.27 ± 0.94^g	Compact	White	++
0.5	-	-	1.0	-	80.18 ± 0.49^e	Compact caulogenic	White, purplish	+++
0.5	-	-	2.0	-	82.43 ± 0.63^e	Compact	White, purplish	+++
-	-	-	-	0.5	91.68 ± 0.62^c	Compact rhizogenic	Cream	++
-	-	-	-	1.0	94.47 ± 0.35^b	Compact rhizogenic	Cream	++
-	-	-	-	2.0	90.18 ± 0.17^c	Compact rhizogenic	Cream	++
0.5	-	-	-	0.5	81.34 ± 0.24^e	Compact	Cream	++
0.5	-	-	-	1.0	76.49 ± 0.15^f	Compact	Cream	+++
0.5	-	-	-	2.0	79.25 ± 0.48^e	Compact	Cream	+++
+ ~ 0.25 mg callus; ++ ~ 0.5 mg callus; +++ ~ 1.0 mg callus; --- indicates no response
^{Data represents mean values ± SE of 10 replicates repeated thrice, recorded after 4 weeks of culture. The mean values followed by the same letter in the superscript in a column do not differ significantly based on ANOVA & t-test at p≤0.05}.

Establishment of cell suspension culture system

Here, in this study, embryogenic callus induced in 0.5 mgl^-1 BA and 1.0 mgl^-1 NAA (Fig. 2a) served as the stock for the suspension inoculum. Approximately 500 mg callus when transferred to fresh liquid medium (100 ml) of the same composition in 250 ml Erlenmeyer flasks and kept in a gyratory shaker (Scigenics) at 120 rpm started establishing fine cell suspension after 5 days (Fig. 2b&c).

Growth kinetics of cells during cell suspension culture

Growth analysis was carried out periodically by taking an inoculum of cells after every 3 days interval from 4th day onwards. The fresh weight and dry weight of the cells harvested during different time periods are given in Table 2. Maximum cell division and biomass production was noticed after 25th days of inoculation and after that stationary phase occurred. The cells grown in liquid medium exhibited better multiplication with 4 fold increase in the cell biomass after 28 days of culture (Table 2). The growth of cells exhibited a characteristic S- shaped curve (Fig. 3).

Table 2

Cell growth during cell suspension culture of *A. ciliata*
Days of culture	Cell growth during cell suspension culture
Days of culture	Fresh weight cells (g)	Dry weight of cells (g)
4	0.0345 ± 0.12ⁱ	0.0024 ± 0.34^j
7	0.0716 ± 0.31^h	0.0034 ± 0.54ⁱ
10	0.1843 ± 0.42^g	0.0076 ± 0.24^h
13	0.3891 ± 0.28^f	0.0134 ± 0.38^g
16	0.9342 ± 0.36^e	0.0549 ± 0.47^f
19	1.7659 ± 0.57^d	0.1437 ± 0.29^e
22	2.9452 ± 0.62^bc	0.2891 ± 0.75^d
25	3.7542 ± 0.32^ab	0.3641 ± 0.46^c
28	3.9857 ± 0.68^ab	0.3972 ± 0.40^b
31	4.0076 ± 0.21^a	0.4007 ± 0.26^a
^{Data represents mean values ± SE of 10 replicates repeated thrice, recorded after 4 weeks of culture. The mean values followed by the same letter in the superscript in a column do not differ significantly based on ANOVA and t-test at p ≤ 0.05}.

Optimization of rotation speed in cell suspension culture

It was observed that from the 5th day itself, the cell division started (Fig. 4a). Various rotation speeds (60, 90, 120 and 150 rpm) were tried in order to optimize the agitation speed during suspension culture and the cells grown at 60 rpm and 90 rpm showed clumping of cells characterized by delayed cell division (Fig. 4b); while those grown at 150 rpm exhibited rupturing of cell wall (Fig. 4c) because of the high agitation speed. The cells grown at 120 rpm was suitable for the fine establishment of cell suspension to grow and in this, single cells were abundant which exhibited normal cell division features (Fig. 4d).

pH analysis of medium during cell suspension culture

The routine analysis of the pH of the medium for the specified time periods showed a decrease in values during the first 9 days, then a slight increase for up to 25 days after which, the medium pH again decreased (Fig. 5). Interestingly, HPLC analysis of the medium left after harvesting of cells after 25 days revealed the presence of spilanthol in it.

HPLC analysis for the quantification of spilanthol

Spilanthol was quantified using HPLC analysis in the selected samples viz. in vivo entire plant (control), plant parts, in vitro shoots, callus and cells harvested from suspension culture. A characteristic chromatogram of spilanthol was obtained at retention time 1.05 ± 0.25 (Fig. 6). The amount of spilanthol was estimated by calibration curve generated from standard (Dodeca-2(E),4(E)-Dienoic acid isobutylamide). The linear curve obtained was Y = 0.85x + 2.82 with regression coefficient (r²) (0.99); (x is the concentration of standard and y is the total peak area). Among the different plant parts of A. ciliata, 102.730 µgg^− 1 spilanthol was noticed in the flower heads, while in the stem and leaves it was 61.146 µgg^− 1 and 96.697 µgg^− 1 respectively, and least content was observed in the roots. i.e., 30.655 µgg^− 1 (Fig. 7). However, high content of spilanthol was noticed in callus cultures established in NAA-BA medium combination (239.512 µgg^− 1) followed by in vitro plant (193.935 µgg^− 1); whereas, the control exhibited 92.19 µgg^− 1 spilanthol in it. Cells harvested from cell suspension culture recorded 173.702 µgg^− 1 spilanthol content (Fig. 7).

Characterization of spilanthol by High Resonance Mass spectrometry

The eluted peaks in HPLC were collected and the presence of spilanthol was confirmed based on collision induced dissociation fragmentation pattern by Mass spectrometry. The peak eluted at 1.05 ± 0.25 minute in HPLC showed characteristic fragmentation pattern of spilanthol (Fig. 8). In the mass spectrum, a peak at m/z 222.02003 (in positive mode) indicated the presence of spilanthol in the sample analysed here in accordance with the previous findings.

Elicitation in cell suspension cultures

Elicitation experiments conducted in cell suspension culture system of A. ciliata scaled up the production of the bioactive alkamide and the amount of ‘spilanthol’ varied in different elicitors for different exposure time. The findings are detailed below.

Effect of biotic elicitors on spilanthol production

When the cells of A. ciliata were treated with biotic elicitors viz. chitosan and yeast extract (YE) at different concentrations, the amount of spilanthol in the cultures went on increased (Fig. 9). Regarding the elicitation with chitosan, maximum amount of spilanthol was noticed at 25 mgl^− 1 chitosan after 72 hours of exposure (4696.337µgg^− 1) that exhibited 27.04 fold increase in the production of this alkamide (Table 3). However, after 24 hours of chitosan treatment, the amount of spilanthol was comparatively lower than that in other exposure periods. In contrast, when YE was used for elicitation response, the spilanthol content was found to be higher in 24 hours of elicitation, after that there was a drop in the quantity in most of the YE concentrations tested. Maximum 6011.431 µgg^− 1 spilanthol was observed in 24 hours of elicitation in 0.5% YE augmented medium, after which it dropped to 4345.010 µgg^− 1 and then to 3483.945µgg^− 1 at 48 and 72 hours of exposures respectively (Table 3). The results denotes that among the two biotic elicitors, YE was comparatively best to evoke the elicitation response in enhancing the production of spilanthol to approximately 34.61 fold upon 24 hrs exposure period (Fig. 9).

Table 3

Effect of biotic elicitors on spilanthol production in *A. ciliata* cell suspension culture
Treatments		Amount of spilanthol (µgg^− 1) [control − 173.702 µgg^− 1]
Treatments		24 Hours	Fold increase	48 Hours	Fold increase	72 Hours	Fold increase
Chitosan (mgl^− 1)	10	216.425^c	1.25	289.823^b	1.67	3375.245^a	19.43
	25	203.709^c	1.17	367.837^b	2.12	4696.337^a	27.04
	50	129.605^c	-	509.217^a	2.93	216.683^b	1.25
	100	129.060^b	-	395.740^a	2.28	62.340^c	-
	200	125.609^b	-	819.035^a	4.71	33.280^c	-
Yeast extract (%)	0.2	5975.875^a	34.41	3595.040^b	20.69	3085.235^c	17.76
	0.5	6011.431^a	34.61	4345.010^b	25.02	3483.945^c	20.06
	1.0	2670.067^a	15.37	2509.226^b	14.45	187.820^c	1.08
	2.0	953.766^b	5.49	3288.096^a	18.93	774.937^c	4.46
	4.0	205.767^c	1.18	2172.853^a	12.51	708.939^b	4.08
^{Data represents mean values of triplicate measurements. The mean values followed by the same letter in the superscript in a row do not differ significantly based on ANOVA and t-test at p ≤ 0.05}.

Effect of abiotic elicitors on spilanthol production

When abiotic elicitors like methyl jasmonate (MeJA) and salicylic acid (SA) were aided to the liquid medium after 21st day of culture, it also succeeded in the enhanced production of spilanthol after 24, 48 and 72 hours of elicitation (Figs. 10). The addition of MeJA at 200 ppm facilitated the enhanced production of spilanthol in the cell culture system which after 72 hours of elicitation yielded maximum 7125.668 µgg^− 1 spilanthol (Table 4). However, in SA amending, maximum spilanthol content (5356.826 µgg^− 1) was noticed at 50 µl concentration after 48 hours treatment. With the various concentrations of SA tested for elicitation, 10 and 50 µl was found to be appropriate in scaling up the production of spilanthol at 24 and 48 hours of exposure period. The findings reveals that among the two abiotic elicitors, MeJA was comparatively effective to evoke the elicited response in enhancing the production of spilanthol to approximately 33.13 to 41.02 fold after 72 hrs exposure period (Table 4) (Fig. 10). Comparatively more time of exposure was needed with regard to abiotic elicitation than biotic elicitors.

Table 4

Effect of abiotic elicitors on spilanthol production in *A. ciliata* cell suspension culture
Treatments		Amount of spilanthol (µgg^− 1) [control 173.702 µgg^− 1]
Treatments		24 Hours	Fold increase	48 Hours	Fold increase	72 Hours	Fold increase
MeJA (ppm)	10	289.432^b	1.67	2650.058^a	15.26	71.562^c	-
	50	249.352^c	1.43	3432.221^a	19.76	515.840^b	2.97
	100	326.510^c	1.88	1778.001^b	10.24	5754.545^a	33.13
	200	270.011^c	1.55	5893.462^b	33.93	7125.668^a	41.02
SA (µl)	10	1959.676^b	11.8	2685.118^a	15.46	1582.771^c	9.11
	50	3922.336^b	22.58	5356.826^a	30.84	73.531^c	-
	100	2313.302^a	13.32	1912.948^b	11.02	740.637^c	4.26
	200	1278.859^b	7.36	1989.997^a	11.45	1041.307^c	5.99
^{Data represents mean values of triplicate measurements. The mean values followed by the same letter in the superscript in a column do not differ significantly based on ANOVA and t-test at p ≤ 0.05}.

Plant cell technology has been explored for the production of natural or recombinant compounds of commercial interest and has received increasing consideration currently as well as in the past decades. It is a potential tool for triggering the plant metabolism for the production of valuable bioactive compounds (Selwal et al., 2023). Cell suspension culture is more preferable than any other techniques for the large scale production of secondary metabolites naturally due to its rapid growth cycles. This system is widely used for generating large amount of cells for improving the production of novel chemicals from the cell cultures within a limited time and space and is a promising approach compared to conventional methods (Bapat et al. 2023). Besides, cell suspension based bioreactor system tremendously facilitated the production of secondary metabolites via elicitation or by adding precursors or additives. Till to date, several cell culture techniques have been developed for the production of valuable secondary metabolites with high medicinal values. Enhanced production level is crucial for the utilization of cell suspension cultures via some culture parameters like elicitation, medium alteration and precursor feeding (Khan et al. 2018). In this study, cell suspension culture system was established in A. ciliata for the in vitro bioproduction of the important N-alkylamide ‘spilanthol’ by means of elicitation.

Estimation of spilanthol in shoot biomass and/or embryogenic callus culture

In this study, high spilanthol content was noticed in callus cultures established in NAA-BA medium combination (239.512 µgg^− 1) followed by in vitro plant (193.935 µgg^− 1), cells harvested from suspension culture (173.702 µgg^− 1) and the least quantity was found in control, i.e., in vivo plants (92.198.µgg^− 1) of A. ciliata. Regarding plant parts, in the flower heads, 102.730 µgg^− 1 spilanthol was noticed, and the quantity in leaves and stem were 96.69 µgg^− 1 and 61.146 µgg^− 1 respectively. Earlier Singh and Chaturvedi (2012) conducted study in S. acmella and reported that 2703 µgg^− 1 spilanthol content was noticed in in vivo leaves, 3294.36 µgg^− 1 in in vitro leaves, 998.03 µgg^− 1 in callus and 91.4 µgg^− 1 in cell suspension cultures upon HPLC analysis. Variation in the values noticed in the study presented here may be attributed to species difference as well as extraction procedures. Substantiating this, Dias et al. (2012) reported high temperature and low pressure are essential for obtaining high spilanthol content in the extracts, whereas Barbosa et al. (2016) stated that extract taken by supercritical CO₂ of Acmella oleracea which are lyophilized showed maximum spilanthol content (1.07%) than the dried samples. However, the present study reported a more effective bioproduction system for the biosynthesis of spilanthol in A. ciliata via callus culture and cell suspension culture for the first time.

Estimation of spilanthol in cell suspension culture

A cell suspension culture system was established in A. ciliata, wherein, the friable callus procured from MS medium containing 0.5 mgl^− 1 BA and 1.0 mgl^− 1 NAA have succeeded in producing four-fold increase in cell mass after 25 days of culture as in Scrophularia striata (Ardestani and Sharifi 2015) at 120 rpm agitation in a gyratory shaker. Agreeing with this, maximum viable cultures in Spilanthes acmella were obtained at a rotation speed of 120 rpm (Singh and Chaturvedi 2012). Besides, according to Mishra et al. (2022) 120 rpm was most appropriate speed of agitation in suspension cultures for the production of 2-hydroxy-4-Methoxybenzaldehyde from the germinated root of Decalepis hamiltonii.

pH analysis of medium during cell suspension culture

In the case of S. acmella, during the establishment of cell suspension cultures, the pH of culture medium increased from 6th day to 21st day of inoculation after which it remains the same till 24th day (Singh and Chaturvedi, 2012). However, here in A. ciliata there was a decrease in pH till 9th day after which it got increased which again decreased as the stationary phase attained and it may be due to the elucidation of some components including spilanthol in to the medium as the culture has attained its maturity period. The detection of spilanthol in the used up medium confirmed by HPLC analysis explains the changes in pH values in different culture periods in this study. Thus the system also offers a two-in-one alternative for the isolation of spilanthol even from the spent medium after harvesting the cells for the extraction of secondary metabolites.

Analysis of spilanthol by High Resonance Mass spectrometry

While the peaks eluted in HPLC were collected and the presence of spilanthol was checked based on collision induced dissociation fragmentation pattern by HRMS in positive mode, the peak eluted at 1.05 ± 0.25 minutes in HPLC showed characteristic fragmentation pattern of spilanthol and this was used for spilanthol estimation in further experiments. The mass spectrum peak at m/z 222.02003 (in positive mode) indicated the presence of spilanthol in the samples analysed here in accordance with the previous findings (Boonen et al. 2010; Singh and Chaturvedi 2012) and this further confirms that spilanthol contain isobutylamide group.

Elicitation of cell suspension cultures

Plant cell culture is a promising alternative approach for the efficient production of valuable secondary metabolites and enhanced production by means of elicitation, Change in phytohormones, culture medium and precursor feeding is vital for the commercial utilization of the cell suspension cultures. Among these, the elicitors are stress inducers that stimulate secondary pathways resulting in the synthesis of bioactive compounds. They have the capacity to activate the defense mechanisms and also to enhance the production of secondary metabolites by creating stress. Both abiotic and biotic elicitors were employed in vitro to induce large quantity of different bioactive secondary metabolites such as alkaloids, alkamides, glucosinolates, terpenes, saponins, flavonoids, steroids, phenolics and coumarins within a short period of time (Al-Khayri and Naik 2020). Elicitors of fungal, bacterial and yeast extract, heavy metals and hormonal origin were studied for various bioactive secondary metabolite synthesis in vitro (Naik and Al-Khayri 2016; Song et al. 2017; Shakya et al. 2019; Acikgoz 2020; Jalota et al. 2024). In A. ciliata, elicitation experiments by means of biotic and abiotic elicitors were conducted in cell suspension culture system for enhancing the production of the bioactive N-alkamide spilanthol.

Effect of biotic elicitors on spilanthol production

In this study, when the cells of A. ciliata were treated with the biotic elicitors viz. chitosan and YE at different concentrations, the amount of spilanthol in the cultures went on increased. Ahmad and Baig (2014) reported that fungal elicitation which includes yeast and chitosan, acts as a prominent elicitor for the production of psoralen in Psoralia corylifolia. In A. ciliata, upon chitosan treatment, maximum amount of spilanthol (4696.337 µgg^− 1) was noticed at 25 mgl^− 1 chitosan for 72 hours exposure time. In previous studies chitosan elicitation enhanced the production of alkamides in Echinacea purpurea hairy root cultures and cell suspension cultures (Romeiro et al. 2009; Acikgoz et al. 2018). However, with YE, maximum 6011.431 µgg^− 1 spilanthol was observed in 24 hours exposure in 0.5% YE augmented medium, after which it dropped to 4345.010 µgg^− 1 and then to 3483.945µgg^− 1 after 72 hours exposure. There are several studies indicating the promotive effect of yeast elicitors on the accumulation of phytochemicals wherein the concentration varies according to the plant species. For instance, Bandhakavi and Kamarapu (2016) reported that when 2% (v/v) YE was treated with cell cultures, it produced 19 mgl^− 1 oleanolic acid. Also, in Echinacea purpurea, the alkamide content raised from 24.2 µgg^− 1 to 57.7 µgg^− 1 (1.33 fold increase) with the application of 50 mgl^− 1 YE (Acikgoz et al. 2018) thus substantiating the elevated synthesis of alkamides especially spilanthol in A. ciliata as observed here.

Effect of abiotic elicitors on spilanthol production

When abiotic elicitors like MeJA and SA were aided to the liquid medium, the scaled up production of spilanthol was noticed after 24, 48 and 72 hours of elicitation. The addition of MeJA at 200 ppm was optimum for the enhanced production of spilanthol in this cell culture system in A. ciliata. MeJA at 200 ppm after 72 hours produced 7125.668 µgg^− 1 spilanthol, while in SA amending, maximum spilanthol (5356.826 µgg^− 1) was noticed at 50 µl concentration after 48 hours period. MeJA elicitation has improved the synthesis of alkamides in E. purpurea also, wherein increased accumulation of alkamides was observed in transformed hairy roots in response to jasmonic acid treatments (Romero et al. 2009).

Thus the present study has analyzed the effect of biotic and abiotic elicitors in cell suspension culture system of A. ciliata for enhancing the production of the bioactive N-alkylamide ‘spilanthol’. Maximum 34.61 fold increase was achieved with biotic elicitor YE in 24 hrs exposure; while the abiotic elicitor MeJA treatment evoked the production to 41.02 fold in 72 hrs treatment. Also, extended time of exposure was needed with regard to abiotic elicitation than that of biotic elicitors. So, considering the merit of perceiving maximum yield in short duration, treatment with YE for 24 hrs period can be suggested as the choice of elicitation treatment for the enhanced production of spilanthol in cell suspension cultures of A. ciliata.

Due to the diverse pharmacological properties of N-alkamides especially ‘spilanthol’, plants synthesizing them are exploited at industrial scale in various phyto-remedial applications. As the taxa targeted in the present investigation Acmella ciliata is one of the most potent source of this high demanding alkamide, it needs to be explored to fulfil the emerging market demand. A novel system for the in vitro production of the high value pharmaceutical compound ‘spilanthol’ from A. ciliata accomplished in this study can be extended for the bioproduction of this bioactive alkamide using bioreactor technology with proper refinement thus benefiting the pharmaceutical industry for phyto-pharma applications. The findings established and the bioproduction system demonstrated here would probably serve as a model system for the bioreactor mediated industrial scale production of spilanthol, as an ingredient in multi-dimensional products especially functional foods and therapeutics. Besides, the bioproduction system developed constitutes a better approach for the conservation and sustainable utilization of this medicinal herb.

BA 6-benzyl adenine

HPLC High Performance Liquid Chromatography

HRMS High Resonance Mass Spectrometry

MeJA Methyl jasmonate

MS Murashige and Skoog

NAA α-naphthaleneacetic acid

PGR Plant growth regulator

SA Salicylic acid

YE Yeast extract

Author contribution statement

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dr TS Preetha, Dr S NeethuMohan and Dr AS Hemanthakumar. The first draft of the manuscript was written by Dr S NeethuMohan and all authors made necessary corrections on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are thankful to the Director, CSIR-NIIST, Pappanamcode, Thiruvananthapuram, Kerala, India for providing the facility for HPLC-HRMS analysis of the samples.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Acikgoz MA (2020) Establishment of cell suspension cultures of Ocimum basilicum L. and enhanced production of pharmaceutical active ingredients. Industrial Crops and Products 148:112278. https://doi.org/10.1016/j.indcrop.2020.112278.
Acikgoz MA, Kara SM, Ay EB, Odabas S (2018) Effect of light on biosynthesis of alkamide, caffeic acid derivatives and echinacoside in Echinacea purpurea L. callus cultures. Akademik Ziraat Dergisi 7(2):179-184. https://doi.org/10.29278/azd.476349.
Ahmed SA, Baig MMV (2014) Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L. Saudi Journal of Biological Sciences 21(5):499-504. https://doi.org/10.1016/j.sjbs.2013.12.008.
Al-Khayri JM, Naik PM (2020) Elicitor-induced production of biomass and pharmaceutical phenolic compounds in cell suspension culture of Date Palm (Phoenix dactylifera L.). Molecules 25:4669. https://doi.org/10.3390/molecules25204669.
Alves VFG, Neves LJ (2003) Anatomia foliar de Vernonia polyanthes Less (Asteraceae). Rev. Univ. Rural Ser. Cien. da Vida 22:1–8.
Ardestani NK, Sharifi M (2015) Establishment of callus and cell suspension culture Scrophularia striata Bioss: an in vitro approach for acetoside production. Cytotechnology 67:475-485. https://doi.org/10.1007/s10616-014-9705-4.
Bandhakavi S, Kamarapu P (2016) Production of Oleanolic acid by plant tissue culture - A review. Journal of Pharmacognosy and Phytochemistry 4(3):1-4.
Barbosa AF, Carcalho de MG, Smith RE, Sabaa-srur AU (2016) Spilanthol: Occurrence, extraction, chemistry and Biological activities. Revista Brasileira de Farmacognosia 26:128-133. https://doi.org/10.1016/j.bjp.2015.07.024.
Binu S (1999) Ethnobotany of Pathanamthitta District, Kerala, India. PhD Thesis. University of Kerala, Trivandrum, India.
Boonen J, Baert B, Roche N, Burvenich C, De spiegeleer B (2010) Transdermal behavior of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts. Journal of Ethnopharmacology 127:77-84. https://doi.org/10.1016/j.jep.2009.09.046.
Chandran H, Meena M, Barupal T, Sharma K (2020) Plant tissue culture as a perpetual source for production of industrially important bioactive compounds Biotechnology Reports 2020, e00450.
https://doi.org/10.1016/j.btre.2020.e00450.
Dias AMA, Santos P, Seabra IJ, Junior RNC, Braga MEM, De Sousa HC (2012) Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. Journal of Supercritical Fluids 61:62-70. https://doi.org/10.1016/j.supflu.2011.09.020.
Hasnain A, Naqvi SAH, Ayesha SI, Khalid F, Ellahi M, Iqbal S, Hassan MZ, Abbas A, Adamski R, Markowska D, Baazeem A, Mustafa G, Moustafa M, Hasan ME and Abdelhamid MMA (2022) Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches. Front. Plant Sci. 13:1009395. https://doi.org/10.3389/fpls.2022.1009395.
Jalota K, Sharma V, Agarwal C and Jindal S (2024). Eco-friendly approaches to phytochemical production: elicitation and beyond. Natural Products and Bioprospecting 14:5 https://doi.org/10.1007/s13659-023-00419-7.
Jansen RK (1981) Systematics of Spilanthes (Compositae: Heliantheae). Systematic Botany 6(3):231-257. https://doi.org/10.2307/2418284.
Khan SA, Siddhiqui MH, Osama K (2018) Bioreactors for hairy roots culture: A Review. Current Biotechnology 7:417-427. http://dx.doi.org/10.2174/2211550108666190114143824.
Mishra VK, Goswami R and Naidu RT (2022) Establishment of in vitro cell suspension culture, kinetics of cell growth, pH, nutrient uptake and production of 2-hydroxy-4- Methoxybenzaldehyde from the germinated root of Decalepis hamiltonii Wight & Arn. -An endangered plant. Current Applied Science and Technology 22(1): 1-13. https://doi.org/10.55003/cast.2022.01.22.013.
Mohan SN, HemanthakumarAS, Preetha TS (2023) A novel system for the production of the bioactive N-alkylamide ‘Spilanthol’ through somatic embryogenesis in Acmella ciliata Kunth (Cass.). Journal of Tropical Life Science 13(3): 461–472. http://doi.10.11594/jtls.13.03.05.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473-493. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.
Naik PM, Al-Khayri JM (2016) Abiotic and biotic elicitors-role in secondary metabolites production through in vitro culture of medicinal plants. In: Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives, Shanker AK, Shankar C (Eds.), InTech: Rijeka, Croatia, pp. 247–277.
Neethu Mohan S, Hemanthakumar AS, Preetha TS (2021) In vitro flowering for the production of bioactive alkamides in Acmella ciliata (Kunth) Cass.: A promising herb in pharamceuticals. Journal of Advanced Scientific Research 12(1):151-165.
Neethu Mohan S (2022) Standardization of in vitro protocol for enhanced production of ‘spilanthol’ from Acmella ciliata (Kunth) Cass. PhD Thesis, University of Kerala.
Neethu Mohan S, Hemanthakumar AS, Preetha TS (2022) Evaluation of inhibition of proliferation of SK-Mel-28 cell lines by Acmella ciliata (Kunth) Cass. cell biomass. International Journal of Pharmaceutical Sciences and Drug Research 14(2):181-188. https://doi.org/10.25004/IJPSDR.2022.140204.
Nergard CS, Diallo D, Michaelsen TE, Malterud KE, Kiyohara H, Matsumoto T, Yamada H, Paulsen BS (2004) Isolation, partial characterisation and immunomodulating activities of polysaccharides from Vernonia kotschyana Sch. Bip. ex Walp. Journal of Ethnopharmacology 91:141-152. https://doi.org/10.1016/j.jep.2003.12.007.
Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, Yalcin B, Erdogan A, Yalcin IE, Cabi E and Kaya Y (2023) Production of secondary metabolites using tissue culture-based biotechnological applications. Front. Plant Sci. 14:1132555. https://doi.org/10.3389/fpls.2023.1132555.
Perez-Amador MC, Ocotero VM, Benitez SP, Jimenez FG (2008) Vernonia patens kunth, an Asteraceae species with phototoxic and pharmacological activity. Pyton 77:275-282.
Rios-Chavez P, Ramirez- Chavez E, Armenta-Salinas C, Molina-Torres J (2003) Acmella radicans var. radicans: In vitro culture establishment and alkamide content. In Vitro Cell Dev Biol- Plant 39:37-41. DOI: https://doi.org/10.1079/IVP2002354.
Rios MY, Aguilar-Guadarrama AB, Gutierrez MD (2007) Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). Journal of Ethnopharmacology 110:364-367. https://doi.org/10.1016/j.jep.2006.09.041.
Romero FR, Delate K, Kraus GA, Solco AK, Murphy PA, Hannapel DJ (2009) Alkamide production from hairy root cultures of Echinacea. In Vitro Cell Dev Biol- Plant 45(5):599-604. https://doi.org/10.1007/s/11627-008-9187-1.
Enhancing secondary metabolite production in plants: Exploring traditional and modern strategies Selwal N, Rahayu F, Herwati A, Latifah E, Supriyono, Cece Suhara, Suastika IBK, Mahayu WM, Wani AK (2023) Journal of Agriculture and Food Research 14 (2023) 100702. https://doi.org/10.1016/j.jafr.2023.100702.
Shakya P, Marslin G, Siram K, Beerhues L, Franklin G (2019) Elicitation as a tool to improve the profiles of high-value secondary metabolites and pharmacological properties of Hypericum perforatum. Journal of Pharmacy and Pharmacology 71:70–82. https://doi.org/10.1111%2Fjphp.12743.
Sharma, R., Karunambigai, A., Gupta, S. et al. (2022) Evaluation of biologically active secondary metabolites isolated from the toothache plant Acmella ciliata (Asteraceae). Advances in Traditional Medicine 22: 713–722. https://doi.org/10.1007/s13596-021-00584-5.
Silveira N, Saar J, Santos A, Barison A, Sandjo L, Kaiser M (2016) A new alkamide with an endoperoxide structure from Acmella ciliata (Asteraceae) and its in vitro antiplasmodial activity. Molecules 21:765. https://doi.org/10.3390%2Fmolecules21060765.
Singh M, Chaturvedi R (2012a) Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr. Bioproces Biosyst Eng 35:943-951. https://doi.org/10.1007/s00449-012-0679-3.
Singh M, Chaturvedi R (2012b) Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr. Industrial Crops and Products 36:321-328. https://doi.org/10.1016/j.indcrop.2011.10.029.
Song X, Wu H, Yin Z, Lian M, Yin C (2017). Endophytic bacteria isolated from Panax ginseng improves ginsenoside accumulation in adventitious ginseng root culture. Molecules 22:837. https://doi.org/10.3390%2Fmolecules22060837.
Bapat VA, Kavi Kishor PB, Jalaja N, Jain SM, Penna S (2023). Plant Cell Cultures: Biofactories for the Production of Bioactive Compounds. Agronomy13(3):858. https://doi.org/10.3390/agronomy13030858

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Enhanced production of ‘spilanthol’ through elicitation of cell suspension cultures in Acmella ciliata (Kunth) Cass. and spilanthol characterization by HPLC-HRMS analysis

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Abstract

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Key Message

INTRODUCTION

MATERIALS AND METHODS

Plant material

Establishment of shoot culture and shoot biomass

Establishment of embryogenic callus culture

Establishment of cell suspension culture system

HPLC analysis for the quantification of spilanthol

Characterization of spilanthol by High Resonance Mass Spectrometry

Elicitation in cell suspension cultures

RESULTS

Establishment of shoot culture and shoot biomass

Establishment of embryogenic callus culture

Establishment of cell suspension culture system

Growth kinetics of cells during cell suspension culture

Optimization of rotation speed in cell suspension culture

pH analysis of medium during cell suspension culture

HPLC analysis for the quantification of spilanthol

Characterization of spilanthol by High Resonance Mass spectrometry

Elicitation in cell suspension cultures

Effect of biotic elicitors on spilanthol production

Effect of abiotic elicitors on spilanthol production

DISCUSSION

Estimation of spilanthol in shoot biomass and/or embryogenic callus culture

Estimation of spilanthol in cell suspension culture

pH analysis of medium during cell suspension culture

Analysis of spilanthol by High Resonance Mass spectrometry

Elicitation of cell suspension cultures

Effect of biotic elicitors on spilanthol production

Effect of abiotic elicitors on spilanthol production

Abbreviations

Declarations

References

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