Comparative analysis of bioactive N-alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS)

Accepted Manuscript Title: Comparative analysis of bioactive N-Alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS) Author: Zubair Shanib Bhat Neeharika Jaladi Ravi Kant Khajuria Zeeshan Hamid Shah Neelakantan Arumugam PII: DOI: Reference:

S1570-0232(16)30102-7 http://dx.doi.org/doi:10.1016/j.jchromb.2016.02.023 CHROMB 19896

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

26-6-2015 10-2-2016 14-2-2016

Please cite this article as: Zubair Shanib Bhat, Neeharika Jaladi, Ravi Kant Khajuria, Zeeshan Hamid Shah, Neelakantan Arumugam, Comparative analysis of bioactive N-Alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS), Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.02.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Comparative analysis of bioactive N-Alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS) Zubair Shanib Bhat1,2, Neeharika Jaladi1, Ravi Kant Khajuria2, Zeeshan Hamid Shah2, Neelakantan Arumugam1* 1

Pondicherry University, Department of Biotechnology, Kalapet, Puducherry, Tamil Nadu-605014,

India 2

Indian Institute of Integrative Medicine (IIIM), Sanatnagar, Srinagar-190005, J & K, India

*

Corresponding author: [email protected], +91- 9944338491

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HIGHLIGHTS  High shoot regeneration (81%) for Spilanthes Ciliata in 6 weeks of culture period was achieved using leaf discs as explants.  Maximum number of compounds eluted out in 1:2 (Plant :Solvent) ratio of extraction with Ethanol.  Comparative analysis of 14 distinct Alkylamides in tissue culture verses field grown plants, tentatively identified by LC-HRMS.

Abstract Spilanthes ciliata (S.ciliata) is a perennial herb of global importance owing to its luscious source of bioactive fatty acid derived amides known as N-alkylamides .It finds application in skin creams, mouth gels and toothpastes. Despite multifaceted applications, a major limitation associated for its commercial application is the scarcity of contamination free plant source, fluctuations in active metabolites due to variation in extraction procedures, and lack of rapid qualitative method for alkylamide profiling. In the current work, attempts were made to 1) optimize conditions for mass propagation of contamination free plants of S.ciliata by tissue culture using leaf discs as explants, 2) establish an optimum extraction ratio of plant/solvent (w/v) for maximum elution of alkylamides and 3) develop a rapid method for qualitative estimation of alkylamide from in vitro raised plants in comparison with that of the field grown counterpart by using LC-Q-TOF (HRMS). To the best of our knowledge this is the first qualitative report on alkylamide profile of micropropagated whole plant of Spilanthes. The correlation pattern reported in this study may form the basis for using tissue culture raised plantlets of S.ciliata as potential source of bioactive alkylamides on industrial scale.

Abbreviations: LC-ESI-QTOF- (HRMS), liquid chromatography quadrapole time of flight– high resolution mass spectrometry; MW, molecular weight; BAP, 6-Benzylaminopurine; NAA αNaphthaleneacetic; IBA, Isobutylamide; MBA,2-Methylbuylamide; PEA,2 Phenylethylamide

Keywords: Tissue culture; Spilanthes ciliata; Alkylamides; Ethanol extract; LC-MS

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1.

Introduction

Spilanthes ciliata H.B.K (syn: Acmella ciliata; Asteraceae), is a perennial herb of global precedence owing to its multiple medicinal uses. Ethno-pharmacological claims associated with it include its use as sialogogue and in the treatment of toothache, gum infection, tongue paralysis and sore throat. Spilanthes attained pharmacological importance owing to its ability to display a unique property that elicits a distinct tingling sensation when applied to mucosal surfaces [1]. This property is bestowed to the plant by the amide based predominant secondary metabolites called N-alkylamides. They are endogenous group of molecules with promising bioactivity reflected in their broad structural diversity [2,3]. Their basic structures are closely similar to the amides found naturally in Echinacea and Xanthoxylum species. Current surge of interest in Spilanthes is triggered by pre-clinical tests associated with the most prominent N-alkylamide, (2E, 6Z, 8E) –deca-2, 6, 8-trienoic acid N-isobutyl amide, commonly known as Spilanthol [4] . Spilanthol has the ability to permeate the skin and buccal mucosa thereby finding application in anti-wrinkle skin creams, mouth gels and toothpastes [5,6]. Spilanthes is distributed in tropical and sub-tropical regions of the world but has recently been declared as threatened in Red data book because of its over exploitation from the wild [3]. In India, it is now confined to Chhattisgarh, Jharkhand, Western Ghats, and few pockets of south India. The plants preference to grow in damp pastures, rocks near the river, or at roadside places with little or no exposure to direct sunlight renders it susceptible to contamination and other infections [7]. Dependence on season and slow germination rate are some of the other limiting factors common in conventional propagation of Spilanthes [8]. This disrupts the balance between its production and demand in the pharmaceutical sector. In vitro production of secondary metabolites of pharmacological importance has given a major impetus in the research and development of plant tissue culture [9]. To reduce the pressure on natural populations and to mass produce elite plants as potential source of secondary metabolites, the in vitro method would be a reliable alternative. This would not only allow its conservation and nullify the effect of seasonal variation but also facilitate a constant supply of uninfected material for production of bioactive compounds irrespective of season and region [10,11]. The use of leaf explants to raise in vitro cultures is convenient for enhancing cultivation and metabolite production as the foliar explants are easy to obtain and do not require the sacrifice of mother plant. The Combitorial use of cytokinin and auxin (Table 1) for inducing shoot-bud differentiation is well established in plants such as Aegle marmelos[12], Pistacia vera[13],

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Emblica officinalis[14] and Arnebia hispidissima [15]. The promotive role of BA on shoot proliferation has been previously documented in Centaurea ultreiae[16] Schinopsis balansae[17], Eclipta alba[18] ,Holarrhena antidysenterica[19] .In this context, during past few years, some efforts were made for in vitro propagation of this important plant through organogenesis using 6-Benzylaminopurine (BAP) and α-Naphthaleneacetic (NAA) as growth regulators[20–23]. However, till date no attempts were made towards qualitative analysis of in vitro raised plantlets of Spilanthes. Furthermore, although many studies have focussed on LCMS profiling of alkylamides from wild growing plants[5,24,25], there are no alkylamide qualitative analysis reports for comparison with field growing counterparts available in the literature. Alkylamides have relatively simple molecular architecture, but they display a broad structural variability as the plants containing them act as a source of generating combinatorial libraries of compounds. The minor differences in structure makes characterization of complex mixtures of these compounds challenging[2]. Previous studies have documented profound effect of the type of solvent and plant: solvent ratio(w/v) used for the extraction of the active constituents[10] .But there are few investigations comparing the efficiency of extracting active constituents under different extracting conditions. Analysis of ethanolic extracts under different plant: solvent ratio (w/v) by a highly sensitive LC-MS system could provide insight into the similarities and differences in the overall pattern of alkylamides present in invitro raised plantlets of Spilanthes. If validated they can serve as alternative source of raw material for extraction of potential bioactive alkylamides for commercial exploitation. Therefore, in the current research work, our first attempt was to analyze the effect of different combinations of Benzyl amino purine (BAP) and Naphthalene acetic acid (NAA) to establish the optimised conditions for the maximum denovo regeneration of normal in vitro raised plantlets of S.ciliata using leaf discs as explants. Next, we directed our attention to select the optimum extraction ratio of plant: solvent (w/v) for maximum elution of alkylamides. For this purpose, the fresh plant stocks comprising of all vegetative parts including leaves, stem, buds and roots of S.ciliata were subjected to extraction with ethanol. Finally, alkylamide profiling of an ethanolic extract of S. ciliata whole plant was initiated using a gradient reversed phase LC-ESI-Q-TOF (HRMS) due to its high resolution and the ability to analyze by mass alkylamide ions of interest[5,24].

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Our results reveal the presence of nearly14 distinct N-alkylamides from in vitro raised plantlets including the exclusively detected compound 1 and 5 in comparison to field growing counterparts (Table 2). This finding provides a new insight in the potential of exploiting tissue culture derived amides to meet the ever increasing demand of pharmaceutical sector. However, further quantification studies are needed in order to validate the use of in vitro derived alkylamides on commercial scale.

2.

Material and Methods

2.1

Chemicals and Reagents.

The chemicals used were of analytical grade obtained from Himedia (Mumbai-India). Sterilants used were Savlon Solution, 70% Ethanol and 0.05% Mercuric Chloride. The LC-MS grade solvents, Acetonitrile and Water were purchased from Merck India Ltd.

2.2

Plant Growth Regulators.

The plant growth hormones, 6-Benzyl-aminopurine (BAP) and α-Naphthalene acetic acid (NAA) were procured from Sigma Aldrich, USA. Hormone stocks (1mg/ml) were prepared by dissolving 50mg of BAP or NAA separately in few drops of (1N) NaOH and the final volume made to 50ml each with milli-Q distilled water.

2.3

Establishment of Aseptic Plant Material.

S. ciliata was maintained in Departmental nursery of Pondicherry University (India). The identity of the plant was confirmed by Dr. S. N Sharma, taxonomist, at Indian Institute of Integrative Medicine, Jammu, CSIR (India) and a voucher specimen of the same was deposited (with Accession number 22164) in the herbarium of the Institute. Nodal segments, with opposite auxiliary buds of the plant were used as explants for generating stock of in vitro plantlets. After removing the leaves, (1.5 cm) long nodal segments were cut, washed and immersed for 20 min in (1% v/v) aqueous solution of Savlon (Johnson-Johnson, India). The detergent solution was decanted and the traces of detergent were removed from the explants by a thorough rinse in sterile distilled water. All subsequent operations were carried out inside a laminar air-flow cabinet. The nodal segments were then given a quick rinse (30s) in 90% ethanol, washed in distilled water. and then surface sterilized by dipping the nodal segments in 50 ml of mercuric

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chloride (0.01% w/v) solution for 6 min . After the treatment, the nodal segments were given three washes by agitation with sterile distilled water to remove the traces of mercuric chloride [7]. The nodal explants were then dried by blotting on sterile filter paper, trimmed at the cut ends to remove the dead tissues and inoculated onto MS basal medium following Murashige and Skoog protocol, 1962. Mercuric chloride is a good surface sterilizer after sodium hypochlorite owing to its broad spectrum antibacterial and anti-fungal potential. Although use of mercuric chloride is a last choice owing to its toxicity, its preferential use in our study for surface sterilization of Spilanthes explants is due to the plants preference to grow in damp habitats contaminated with soil-borne and epiphytic fungi. However, extra care is needed in handling or dealing with mercuric chloride as it is also slightly volatile at room temperature and can cause mercury poisioning.

2.3.1 Preparation of MS medium: The Murashige and Skoog media was prepared with pH adjusted to 5.8 using (0.1 N) NaOH or (0.1 N) HCL. This was followed by addition of (8 g/l) plant culture tested agar (Hi Media Laboratories, India) before autoclaving at 121⁰C for 15 min. The cultures were maintained at 25 ± 2⁰C temperature with 50–60% relative humidity and 16h photoperiod and 1,600 Lux provided with 40W cool white tube light (Philips, India). Observations were recorded at weekly intervals.

2.4

Preparation of leaf explants: The healthy sterile plantlets were selected as a source of

leaf explants for in vitro testing. In order to identify the suitable maturity stage for callus initiation, leaves were harvested at different maturity stages i.e first, second and third fully opened leaf. Leaf segments measuring 5 mm2 were cut from the aseptic in vitro grown healthy plant and inoculated on MS medium supplemented with different combinations of Benzylaminopurine (BAP) at different concentrations (4.40µM, 13.20µM, 22.0µM, 26.4µM) and α-Naphthalene acetic acid (NAA) at (5.40 µM, 10.8 µM, and 16.2 µM). The cultures were maintained as that of the aseptic shoot cultures described before. The cultures were sub-cultured after four weeks on the media of same composition.

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2.5

Preparation and storage of extracts.

Healthy plants comprising of leaves, auxiliary buds, stem and root collected from the nursery of Biotechnology Department and in vitro raised plantlets from tissue cultures were washed thoroughly, blotted dry, weighed to the nearest gram and blended with 95% ethanol (as a solvent). Different variants of extractions were tested ranging from 1:1 to 1:15 of plant: solvent ratios. In the first variant 30g of the fresh field plant material was blended in 30ml of ethanol. The extract obtained following this procedure was labelled as “FL 1:1” (w/v). The same procedure was followed for tissue culture raised plants and labelled as “TC 1:1”. All other variants of extraction were labelled accordingly. The plant material: solvent mixture was allowed to macerate in screw capped Schott bottles for 7 days on a shaker. The liquid contents were filtered and stored at 4oC for future use.

2.6

LC-Q-TOF (HRMS) analytical N-alkylamide profiling

2.6.1. Sample Preparation. Samples were prepared by dissolving the crude extracts in HPLC grade ethanol, followed by filtration through a 0.2 µm nylon filter (Whatman, Dassel, Germany). Prior to analysis, 500µL aliquots of the sample extract were centrifuged at 14,000 rpm for 5 min. Supernatant was then diluted in the same solvent used for extraction (95% ethanol) and 300µl aliquot of the extract was transferred into auto sampler vials of (Agilent Technologies, Santa Clara, CA) for LCMS analysis.

2.6.2. Instrumentation. Agilent 1260 affinity HPLC system consisted of a quaternary pumps, auto-sampler, thermocouple column compartment and diode array detector. The HPLC was coupled to 6540 Ultra High Definition (UHD) electron spray ionisation mass spectrometer (Agilent Technologies Germany). Agilent Mass Hunter Qualitative analysis software (Version B.04.00) was used for sample analysis and Agilent Mass Hunter Workstation was used for data acquisition.

2.6.3. HPLC Conditions. Reverse phase HPLC was carried out using SB-C18 Agilent column (4.6×150mm) with 2.7 µm particle size at 30°C. An isocratic separation was conducted using mobile phase consisting of 10% of water (A) and 90% of acetonitrile (B), run in a gradient at a flow rate of 0.4ml/min for the first 10 mins. For the next 5 mins the linear gradient optimised

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was as follows:, A:B (20:80,v/v), t=10-15 mins. The gradient was reversed for the next 3 mins to: A:B (90:10 v/v) ,t=15-18 mins and finally readjusted back to: A:B (10:90,v/v), in the next 2 mins t=18-20 mins. For the last 5 mins, system was re-equilibrated to initial condition. Total run time for each sample was 25 mins.

2.6.4. Mass spectrometry conditions. The ionisation was achieved under positive ion mode of electrospray ionisation (ESI).The nebulizer temperature was optimised to 350 ⁰C and nitrogen gas flow rate was 8 litre/ minute. The values of capillary and fragmentation voltages were 3500V and 140V respectively.

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Statistical analysis. The statistical analysis of the morphological results was determined

for each set of experiments on tissue culture propagation of shoots. The measured values are expressed as the mean ± standard deviation (SD) from three independent experiments (n = 3) and data was recorded after three weeks of culture.

3. Results 3.1. Effect of BAP and NAA on the in vitro cultured leaf explants BAP and NAA are plant growth regulators that are established for induction of shoots and roots respectively in tissue culture procedures. The leaf explants of S. ciliata were induced for shoots through a callus phase by culturing the leaf discs on media containing both BAP and NAA. The effect of BAP and NAA alone or in combination on shoot regeneration from leaf explants is presented in Table1. In the presence of NAA alone, the explants showed formation of roots without an intervening callus (Table 1). On MS basal medium supplemented with BAP alone, the leaf explants expanded upto twice their size in a week time but turned brown in the next 7 days. In the presence of both BAP and NAA in culture medium, the explants at the cut ends grew into a large dark green callus that covered the entire explants within next 21 days (Fig. 1). A piece of callus from a four-week-old culture, on subculture to the media of same composition from which it was derived, differentiated pinkish shoot buds within a week. By 7 days, these buds grew into multiple shoots with dark green leaves (Fig.1). At low concentration of NAA: BAP combinations, the callus induced formation of shoots without rooting while as on higher concentrations, the callus differentiated into well developed shoots and roots (Table 1).Though

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the shoot regeneration was observed in most of the combinations, maximum response ( 81%) with a mean number of 3.4±0.62 shoots per explant in 6 weeks culture period was observed on MS medium supplemented with 4.40 µM BAP + 5.40 µM NAA (Fig 2). Also, 75% shoot regeneration with mean of 2.2±0.58 shoots/explant on medium supplemented with 13.2 µM BAP + 5.40 µM NAA was obtained (Fig.2). It was observed that although leaf disc inoculated on MS + BAP (22.0µM) + NAA (10.8µM) produced only about 37.2% of shoot regeneration; it produced highest number of shoots per explants (4.8± 0.37) with extensive rooting (Fig.3). The shoots when transferred to MS medium without any hormones produced roots within a week at 100% frequency. Though overall regeneration pattern confirm the earlier reports , the frequency of shoot regeneration observed in the present study involving S.ciliata was observed to be on lower side as compared to the shoot regeneration in the related Spilanthes acmella reported earlier[20,26]. This difference could be attributed to the variation in genetic makeup of the plant playing a role in the process of shoot regeneration.

3.2. Acclimatization of tissue culture raised S. ciliata plantlet The in vitro raised S. ciliata plantlets were washed with water to remove the adhering agar and transferred to the soil. The plants were acclimatized by covering the plants with polyethylene bags to maintain high humidity for 6–7 days and regularly irrigated. After 7 days, the polyethylene bags were removed and plants allowed to grow under natural conditions (Fig.1A.c). Gradual hardening after transfer of plantlets in the field ensured survival up to 98% under natural conditions.

3.3. LC-MS Alkylamide profile and Effect of extraction conditions LC-MS analysis of ethanolic extracts provides an insight into the similarities and differences in alkylamide composition resulting from the variations in extraction methods. As alkylamides accumulate in different organs of the plant, the use of fresh plant comprising of all vegetative parts including leaves, stem, buds and roots of S.ciliata were used for analysing the overall pattern of alkylamides present in the said species. A distinct difference was observed in the number and types of alkylamides, eluting out with different extraction ratio tested. The majority of the compounds eluted out in 1:2 ratio (w/v) of extraction with ethanol that decreased upto 1:10 ratio (w/v) after which no compounds were detected .The TIC-MS chromatograms of two end

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points of ethanolic extracts of S. ciliata are presented in Fig.2 (a-d). The peaks in Fig.2 were assigned numbers on the basis of characteristic m/z values for each N-alkylamide in the increasing order of their retention time [5,27].Extraction of plantlets with ethanol in a ratio of 1:2 w/v (plant: solvent) resulted in the elution of maximum number of compounds. On the basis of their characteristic m/z values, a total of 14 distinct alkylamides were tentatively identified (Fig. 2, & Table 2). The compounds numbered 2, 4, 6, 8, 9, 10, 11 and 14 were identified as isobutylamides (IBA), compounds 3, 5 and 13 to be methyl-butylamides (MBA) and compound 7 as phen-ethylnonenedyine-amide (PEA) corresponding to twelve N-alkylamides that have previously been reported in the genus Spilanthes [28,29].Two unidentified compounds were also detected (Compound 12 with m/z of 174.1258 detected in field plants and Compound 1 with m/z of 205.9596 detected exclusively in tissue culture plantlets .(Fig. 2A, B; Table2). Presence of Spilanthol (2E, 6Z, 8E)-N-isobutyl-2, 6, 8-decatrienamide) with an m/z value of 222 was observed in all the four samples investigated in our study. In addition to these, the elution of various isomeric analogs, that displayed the same m/z values but were detected at different retention time highlighted the complex structural diversity of N-alkylamides. In tissue culture raised plantlets (TC;1:2) ,one isomeric form (8a) for compound 8 (2E,4Z-N-isobutly-2,4undecadiene-8,10-diynamide) eluted earlier at Rt of 4.58 mins and two isomers ( 11a and 11f) eluted at Rt of 4.8 and 12.31 mins respectively. In field grown plants (FL;1:2), 11c-11e eluted at Rt of 7.0, 7.7 and 8.7 respectively for compound 11 (2E,4E,8Z,10E-N-isobutyl-dodeca 2,4,8,10tetraenamide) .Two additional isomers viz., 14c-14d of compound 14 ((2E)-N-isobutyl2- undecene-8,10-diynamide) eluted at Rt of 10.3 mins and 13.4 min respectively (Table.2).

4.

Discussion

4.1. Micropropagated plantlets as potential source of Alkylamides Accumulation of secondary metabolites in plant cell culture depends on the composition of the culture medium, the kind and concentration of plant growth regulators, mineral salts and carbon sources. Further, it has been frequently observed that specialized plant organs show different expression pattern of secondary metabolites when inducted into the in vitro systems [30,31]. In1957, Skoog and Miller demonstrated that, type and concentration of auxin and cytokinin used and the possible interaction between exogenous and endogenous concentrations of plant growth regulators have marked effect on the in vitro culture responses .Many attempts on in vitro

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regeneration of Spilanthes using plant growth regulators have been carried out through direct or indirect organogenesis in Spilanthes acmella [7,20–22,32] and Spilanthes mauritiana [33]. Furthermore, there are many reports on leaf-disc culture of Spilanthes[20,26] and a number of methods have demonstrated qualitative analysis of N-alkylamides from naturally growing plants of Spilanthes[28,29]. But till date no attempts were made to profile alkylamides in in vitro raised plantlets. We subjected ethanolic extracts of these plantlets for qualitative estimation of alkylamides. The alkylamides of pharmacological relevance were detected at different retention time points. This opens up the opportunity to study the effect of different plant growth regulators on the quantification of alkylamides in tissue culture raised Spilanthes plantlets for its full commercial exploitation in future.

4.2. Effect of extraction solvent system on Alkylamide profile. The difference in plant preparations possibly implies alterations in biological effects, toxicity and interaction characteristics that may arise due to varying plant: solvent ratios (w/v) used in extraction process. Therefore analytical profiling of bio-actives in plant preparations is prerequisite in functional cosmetics, drug pharmacology as well as in toxicology. Ethanolic extracts of genus Spilanthes are rich source of bioactive fatty acid amides. Rapid analysis to identify the presence of these amides in a variety of extracts is imperative for assuring safety and efficacy of its herbal remedies as their use is set to continue impulsively well into the estimable future. Previous studies documented minor differences in the alkylamide profiles of Echinacea purpurea due to difference in the solvent system used but major changes were observed in the quantity when the dry and fresh materials were compared[27,34]. However, profound effect of the type of solvent on extraction of the active constituents and the resulting alterations in biological effects and toxicity has been reported in Trifolium alexandrinum[10]. In our study, the solvent system used was ethanol and water and a prominent decline in the elution of alkylamides was observed when the plant: solvent ration was changed gradually from 1:1 (w/v) upto 1:15(w/v) with majority of compounds detected in 1:2 ratio and lest in 1:10 after which no compound were detected by mass analyzer. This difference may be attributed to the difference in the microenvironment that prevailed in the isolation mixture of the solvent system[27]. Furthermore it is obvious from the results that elution of maximum types of alkylamides was possible with ethanol when the plant: solvent (w/v) ratio was maintained at 1:2 rather than at 1:1

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where peaks failed to resolve due to the minute structural differences in alkylamides. The decrease in the number of alkylamides with increasing ration may be attributed to dilution factor in extraction procedure.

4.3. LC-Q-TOF (HRMS) analytical N-alkylamide profiling. 4.3.1. The technique and identification of alkylamides: Analyzing the secondary metabolites in plants is a challenging task because of their chemical diversity, usually low abundance and variability even within the same species. Considering the fact that 100,000–200,000 metabolites are estimated to occur in the plant kingdom[35–37], only sensitive and highly selective methods would be suitable for analysis of their composition and quality. Qualitative analysis of the raw material is an important aspect of drug development. A number of reports on alkylamide profile using TLC and liquid chromatography with LC-UV detection are available [38–40]. Many times there is co-elution of structurally similar molecules and UV detectors fail to detect minor alkylamides making such methods unreliable for precise qualitative analysis[41]. Few methods for analysis of alkylamide profile of Echinacea spp. [27,42,43] and Spilanthes acmella

using LCMS have been reported

recently[5,24,25]. This technique is considered to be highly powerful for tentative identification of secondary metabolites in plant extracts[41,44,45].Over the past decade, a number of biologically active compounds have been reported in Spilanthes ssp. [2,46]. Q-TOF (HRMS) is a powerful tool to detect and annotate secondary metabolites present even in crude aqueous-alcohol plant extracts. In our study, alkylamide profiling of an ethanolic extract of S. ciliata whole plant was initiated using a gradient reversed phase LC-ESI-Q-TOF (HRMS). The samples of the two main extracts (ie. 1:2 and 1:10) were run in triplicates on same day with the base line blank run between each sample run. The same procedure was repeated for three consecutive days. Analysis showed that the number of compounds identified were highly consistent across the triplicates with all peaks appearing across the samples at almost same retention time thereby displaying reproducible qualitative analysis report. The run time for analysis of a given sample was in the range of only 15 to 20 minutes and therefore could be considered as a method of choice for analysis of complex ethanol extracts.

5. Conclusion In the current work, attempts were made to standardise the use of tissue culture raised plantlets of S.ciliata for their possible commercial application by validating three basic criteria pretaining

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to:1) Optimise conditions for mass propagation of contamination free plants of S.ciliata by in vitro culture: Up to 81% of shoot regeneration (Fig. 4).with a mean of 3.4± 0.62 shoots per explants (Fig. 5) in 6 weeks of culture period was achieved on MS+BAP (4.4µM) + NAA (5.4µM) using leaf discs as explants (Table.1). 2) Establish an optimum extraction ratio of plant/solvent for maximum elution of alkylamides: The type of alkylamides detected differed when the plant to solvent ratio was changed in the extraction procedure with most of the compounds eluting out in 1:2 ratio of extraction with ethanol. 3) Develop a rapid method for qualitative estimation of alkylamide in ethanolic extracts of in vitro raised plantlets in comparison with that of the field grown counterparts. The crude ethanol extracts on analysis by SB-C18 Agilent column (2.7um, 4.6mm × 150mm), revealed the presence of nearly14 distinct compounds on the basis of their respective m/z values (Eight isobutylamides (IBA), three methylbutylamides (MBA) ,one phenethylnonene dyineamide (PEA). These findings provide a new insight in the potential of exploiting tissue culture derived amides to meet the ever increasing demand of pharmaceutical sector. However, further quantification studies are needed in order to validate the use of in vitro derived N-alkylamides on commercial scale.

Acknowledgements ZSB and NA acknowledge University Grants Commission (UGC), New Delhi for providing financial support for this research.

References 1]

B.P. Bryant, I. Mezine, Brain Res. 842 (1999) 452.

[2]

R.S. Ramsewak, A.J. Erickson, M.G. Nair, Phytochemistry 51 (1999) 729.

[3]

K.L. Tiwari, S.K. Jadhav, V. Joshi, J. Chinese Integr. Med. 9 (2011) 1170.

[4]

I. YASUDA, K. TAKEYA, H. ITOKAWA, Chem. Pharm. Bull. (Tokyo). 28 (1980) 2251.

[5]

J. Boonen, B. Baert, C. Burvenich, P. Blondeel, S. De Saeger, B. De Spiegeleer, J. Pharm. Biomed. Anal. 53 (2010) 243.

[6]

J. Boonen, B. Baert, N. Roche, C. Burvenich, B. De Spiegeleer, J. Ethnopharmacol. 127 (2010) 77.

14

[7]

M. Singh, R. Chaturvedi, Plant Cell. Tissue Organ Cult. 103 (2010) 243.

[8]

J. Dobránszki, J.A. Teixeira da Silva, Biotechnol. Adv. 28 (2010) 462.

[9]

F. Bourgaud, A. Gravot, S. Milesi, E. Gontier, Plant Sci. 161 (2001) 839.

[10] A.M. Stafford, Curr. Opin. Drug Discov. Devel. 5 (2002) 296. [11] M. Singh, R. Chaturvedi, Ind. Crops Prod. 36 (2012) 321. [12] P. Nayak, P.R. Behera, T. Manikkannan, Vitr. Cell. Dev. Biol. - Plant 43 (2007) 231. [13] E. Tilkat, A. Onay, H. Yildirim, E. Ayaz, Sci. Hortic. (Amsterdam). 121 (2009) 361. [14] P. Nayak, P.R. Behera, M. Thirunavoukkarasu, P.K. Chand, Sci. Hortic. (Amsterdam). 123 (2010) 473. [15] M.S. Shekhawat, N.S. Shekhawat, Acta Physiol. Plant. 33 (2011) 1445. [16] R. Mallón, J. Rodríguez-Oubiña, M. González, Plant Cell. Tissue Organ Cult. (2010). [17] P. Sansberro, H. Rey, L. Mroginski, C. Luna, Trees - Struct. Funct. 17 (2003) 542. [18] A. Ray, S. Bhattacharya, Plant Cell. Tissue Organ Cult. 92 (2008) 315. [19] R. Kumar, K. Sharma, V. Agrawal, Vitr. Cell. Dev. Biol. - Plant 41 (2005) 137. [20] K. V Saritha, C. V Naidu, Biol. Plant. 52 (2008) 334. [21] S.K. Singh, M.K. Rai, P. Asthana, L. Sahoo, Acta Physiol. Plant. 31 (2009) 693. [22] V. Pandey, V. Agrawal, Vitr. Cell. Dev. Biol. - Plant 45 (2009) 491. [23] M. Singh, R. Chaturvedi, Plant Cell, Tissue Organ Cult. 103 (2010) 243. [24] S.S. Bae, B.M. Ehrmann, K.A. Ettefagh, N.B. Cech, Phytochem. Anal. 21 (2010) 438. [25] V. Sharma, J. Boonen, N.S. Chauhan, M. Thakur, B. De Spiegeleer, V.K. Dixit, Phytomedicine 18 (2011) 1161. [26] V. Pandey, M. Chopra, V. Agrawal, Parasitol. Res. 108 (2011) 297. [27] K. Spelman, M.H. Wetschler, N.B. Cech, J. Pharm. Biomed. Anal. 49 (2009) 1141. [28] M. Nagashima, N. Nakatani, Lebensm.-Wiss.u.-Technol. 25 (1992) 417.

15

[29] N. Nakatani, M. Nagashima, Biosci. Biotechnol. Biochem. 56 (1992) 759. [30] M. Lucchesini, A. Bertoli, A. Mensuali-Sodi, L. Pistelli, Sci. Hortic. (Amsterdam). 122 (2009) 484. [31] T.K. McGhie, S. Hudault, R.C.M. Lunken, J.T. Christeller, J. Agric. Food Chem. 60 (2012) 482. [32] K. V Saritha, E. Prakash, N. Ramamurthy, C. V Naidu, Biol. Plant. 45 (2002) 581. [33] H.P.A.L. BAIS, J.B. GREEN, T.S. WALKER, P.O. OKEMO, J.M. VIVANCO, Vitr. Cell. Dev. Biol. Plant 38 (2002) 598. [34] K. Spelman, D. Depoix, M. McCray, E. Mouray, P. Grellier, Phyther. Res. 25 (2011) 1098. [35] K.M. Oksman-Caldentey, D. Inzé, Trends Plant Sci. 9 (2004) 433. [36] S. Ramachandra Rao, G.A. Ravishankar, Biotechnol. Adv. 20 (2002) 101. [37] S. Karuppusamy, J. Med. Plants Res. 3 (2009) 1222. [38] R. Bauer, I.A. Khan, H. Wagner, Planta Med. 54 (1988) 426. [39] R. Bauer, P. Remiger, Planta Med. 55 (1989) 367. [40] R. Bauer, S. Foster, Planta Med. 57 (1991) 447. [41] N.B. Cech, C.G. Enke, Mass Spectrom. Rev. 20 (2002) 362. [42] N.B. Cech, M.S. Eleazer, L.T. Shoffner, M.R. Crosswhite, A.C. Davis, A.M. Mortenson, J. Chromatogr. A 1103 (2006) 219. [43] A.K.L. Goey, R.W. Sparidans, I. Meijerman, H. Rosing, J.H.M. Schellens, J.H. Beijnen, J. Chromatogr. B 879 (2011) 41. [44] N.B. Cech, J.R. Krone, C.G. Enke, Anal. Chem. 73 (2001) 208. [45] N.B. Cech, C.G. Enke, Anal. Chem. 73 (2001) 4632. [46] M. Singh, R. Chaturvedi, Bioprocess Biosyst. Eng. 35 (2012) 943.

16

LEGENDS TO THE FIGURES Figure 1. Four weeks old leaf explant cultures on MS basal medium supplemented with appropriate growth regulators in different concentrations showing denovo shoot regeneration

a)

22.0 µM BAP; 10.8 µM NAA

b)

13.2 µM BAP; 5.4 µM NAA

c)

4.4 µM BAP; 5.4 µM NAA

Figure 2. Effect of different concentration ratios of NAA and BAP as supplements in basal MS media on the % of callus showing shoots regeneration after in vitro culture. A piece of callus from a four-week-old culture, on subculture to the media of same composition from which it was derived, differentiated pinkish shoot buds within a week.

Figure 3. Effect of different concentration ratio of NAA and BAP as supplements in basal MS media on the mean number of shoots per explants ± SD after in vitro culture for 6 weeks. By last week, the differentiated pinkish shoot buds grew into multiple shoots with dark green leaves

Figure 4 (a-d). Chromatogram (TIC) obtained by LC-Q-TOF (HRMS) positive ion mode analysis of Spilanthes ciliata H.B.K crude ethanol extract, extracted in plant to solvent ratio of 1:2 (a, b) and 1:10 (c, d) respectively for the field grown normal plants (a & c) and the in vitro raised plants (b & d). The peak labels correspond to the identity of compounds depicted in Table 2. Analytical conditions are described in the text

17

Fig. 1

18

Fig. 2

19

Fig. 3

20

Fig. 4a

21

Fig. 4b

22

Fig. 4c

23

Fig. 4d

24 Table 1. Effects of BAP and NAA on leaf disc of Spilanthes ciliata cultured in vitro. Data was recorded after six weeks of culture (Four weeks of explants culture followed by two weeks of subculture) S. No.

BAP (µM)

NAA (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 10 20

0.00 4.40 13.2 22.0 26.4 0.00 4.40 13.2 22.0 26.4 0.00 4.40 13.2 22.0 26.4 0.00 4.40 13.2 22.0 26.4

0.00 0.00 0.00 0.00 0.00 5.40 5.40 5.40 5.40 5.40 10.8 10.8 10.8 10.8 10.8 16.2 16.2 16.2 16.2 16.2

% of Explants with Callus 48 100 100 95 100 19 93 96 85 100 54 100 100 100 100

% of explants showing Shoot regeneration 0.000 81.00 75.00 23.68 16.60 0.000 0.000 0.000 37.29 29.00 0.000 0.000 21.40 15.70 22.70

Mean No. of Shoots / Explant ± S.E 0.0±0.00 3.4±0.62 2.2 ±0.58 2.0 ±0.63 1.6±0.24 0.0±0.00 0.0±0.00 0.0±0.0 4.8±0.37 0.6±0.40 0.0±0.00 0.0±0.00 2.2±0.37 1.8±0.48 2.8±0.37

% of explants showing roots 0.00 0.00 0.00 0.00 0.00 100 0.00 0.00 0.00 0.00 100 0.00 10.0 2.00 0.00 83.0 35.0 0.00 0.00 8.00

25

Table 2. Note: FL 1:2-Field growing plantlets extracted with ethanol in the ration of one part of plant material with two parts of ethanol, FL 1:10- Field growing plantlets extracted with ethanol in the ration of one part of plant material with ten parts of ethanol, TC 1:2- Tissue culture raised plantlets extracted with ethanol in the ration of one part of plant material with two parts of ethanol, TC 1:10- Tissue culture raised plantlets extracted with ethanol in the ration of one part of plant material with ten parts of ethanol. Nd: Not detected; Na: Not available; IBA: Isobutylamide; MBA-2-Methylbutylamide; PEAPhenylethylamide; X1 Not reported earlier; X2 Structure is a tetraene, a diene-monoyne or a diyne; a - reference [13], b - reference [21], c - reference [8] Table 2A. N-alkylamides present in the ethanol extract of field grown and in vitro raised plantlets of Spilanthes ciliata as detected by LC-Q-TOF (HRMS) (HRMS) - Precursor ions m/z MH+ (m/z ) Peak No

Identify

IUPAC Name

Reference (MW)

Rt (mins)

FL 1:2

FL 1:10

TC 1:2

TC 1:10

1

Na

Na

X1

3.30

Nd

Nd

205.9596

-

2

IBA

Undeca,2E,4Z-diene-8,10 diynoic acid isobutylamides

229.32 b

3.42

229.0720

-

Nd

Nd

MBA

Undeca,2E,4Z-diene-8,10- diynoic acid 2-methylbutylamide

243.35 b

3.95

243.1447

Nd

Nd

IBA

Dodeca-2E,4E, 8Z,10E tetraenoic Acid isobutylamide

247.38b

3.50

-

247.1741

247.1742

a

4.40

Nd

Nd

246.1864

-

a

4.29 4.43

220.1716 Nd

Nd

Nd 220.1644

Nd -

c

5.01

252.1354

252.1354

-

252.1367

4.58

Nd

Nd

230.1170

-

5.03

230.1516

230.1516

230.1520

230.1510

5.53

258.2897

258.1834

258.1799

258.1839

3

-

4

-

5 MBA

(2E)-N-(2-methyl butyl)-2-undecene8,10-diynamide

6a 6b

IBA

X2

7

PEA

(2Z)-N-phenethy l-2-nonene- 6,8-diynamide

8a 8b

IBA

246 220

252

230

a

(2,4-Undecadiene-8,10-diynamide, N(2 methylbutyl)(2E,4Z)

9 IBA

(2E,7Z)-N-isobuty l-2,7- tridecadiene10,12-diynamide

258

222

a

a

-

26 10a 10 b

IBA

(2E,6Z,8E)-N-Isobutyl-2,6,8-decatrienamide (Spilanthol)

11a 11b 11c 11d 11e 11f

IBA

(2E,4E,8Z,10Z)- N-isobutyl-dodeca2,4,8,10-tetraenamide

12

Na

Na

13 a 13b 13c

MBA

(2E,6Z,8E)-N-(2-Methylbutyl)-2,6,8 decatrienamide (Homospilanthol)

IBA

(2E)-N-Isobutyl-2-undecene8,10-diynamide

248

4.28 5.72

222.1834

222.1817 -

Nd 222.1832

Nd 222.1667

4.80 6.16 7.04 7.79 8.70 12.31

Nd 248.1985 248.1293 248.1249 248.1253 Nd

Nd 248.1986 Nd

248.1967 248.1988 Nd Nd Nd 248.2015

248.1985 Nd Nd Nd -

6.30

174.1258

Nd

Nd

a

4.01 6.03 7.03

Nd Nd 236.1652

Nd Nd -

236.1979 Nd

236.1435 Nd

a

5.93 8.92 10.36 13.40

Nd 232.1669 232.1700 232.1672

Nd -

a

-

14 a 14 b 14 c 14 d

X1 236

232

232.1312 Nd Nd Nd

Nd Nd Nd

27 Table 2B. Structures of N-alkylamides as detected by LC-Q-TOF (HRMS)

Peak No

Structure

Peak No

5

9

6

10

7

11

8

13

14

Structure