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Effect of agitation speed on growth and viability of cells. Three-week-old cells stained with 1% fluorescein diacetate solution. a Cellular clump at 60 rpm, showing aggregate of compact cells (bar 2.5 mm). b Cellular clump at 90 rpm, showing aggregate with loosely attached cells (bar 2.5 mm). c The cultures maintained at 120 rpm in the cell suspension, showing individual, live and healthy, fluorescent greenstained cells with intact wall (bar 9.3 mm). d Same at 180 rpm, showing dead (dark bodies) and sheared cells (bar 9.3 mm)
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Spilanthes acmella Murr. has a plethora of highly valuable biologically active compounds and has been listed as one of the important medicinal plants of the world. However, no perceptible biotechnological advances have been made for this genus to exploit or enhance its utility. To nullify the effect of seasonal variations, the present report is the...
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Context 1
... of agitation directly affects the growth and viability of cells in culture due to aeration and agitation. The via- bility profile of Spilanthes cells at different agitation speeds is shown in Fig. 6. Similar to many other plant species, Spilanthes cells were also found to be highly sensitive to increase in agitation speed. The maximum fresh weight (163.63 g L -1 ) (Fig. 7) and maximum viability (Fig. 6) were observed at 120 rpm. At higher rpm , the biomass and viability profile was highly unsatisfactory. At lower rpm (60-90), the ...
Context 2
... of cells in culture due to aeration and agitation. The via- bility profile of Spilanthes cells at different agitation speeds is shown in Fig. 6. Similar to many other plant species, Spilanthes cells were also found to be highly sensitive to increase in agitation speed. The maximum fresh weight (163.63 g L -1 ) (Fig. 7) and maximum viability (Fig. 6) were observed at 120 rpm. At higher rpm , the biomass and viability profile was highly unsatisfactory. At lower rpm (60-90), the cells died due to aggregation and clumping. At 60 rpm, cells were aggregated into hard clump while at 90 rpm cells were loosely attached in the clump. Only the cells at the outermost layer of the aggre- gate ...
Citations
... Thus, we found that higher carbon source levels (6%) stimulated not only dry biomass yield but also arbutin release into the culture medium, indifferent to hydroquinone concentration. Sucrose was reported to act as an external energy source that stimulates the absorption of mineral nutrients present in the culture medium, stimulating the production of the energy required for metabolite synthesis [53][54][55]. Moreover, many studies have pointed out that yields of in vitro cell cultures for secondary metabolite biosynthesis were highly dependent on the type and concentration of carbohydrates used in the medium [56,57]. ...
This study aimed to investigate the biotransformation capabilities of a hydroquinone-tolerant Digitalis purpurea cell line (DpHQ) for bioconverting hydroquinone (HQ) into arbutin, a compound with significant therapeutic and cosmetic applications. The research evaluated the influence of various HQ concentrations, feeding protocols, and carbon sources on arbutin bioconversion yield. By using HPLC-MS for the quantification of arbutin in biomass and medium, the study revealed that higher precursor (HQ) concentration led to a more pronounced growth inhibition under single dosing than sequential dosing. At lower sugar (3%) and precursor (4 mM HQ) levels, arbutin predominantly remained within the cells, whereas higher sugar (6%) and HQ (5–6 mM) levels promoted its release into the medium. Arbutin production ranged from 591 mg/L under single dosing to 3049 mg/L with sequential dosing, with the highest yield being achieved with 5 mM HQ in divided doses and 6% glucose. This study holds novelty for being the first to demonstrate the DpHQ’s tolerance to high concentrations of HQ and its efficient capabilities to bioconvert HQ to arbutin, indicating that D. purpurea is equipped with the enzymes required for this process. These aspects highlight its potential as a biotechnological source for arbutin synthesis.
... µg.g -1 ) ( Figure 5a) compared to 92.19 µg.g -1 in in vivo plant (control) (Figure 5b). The spilanthol content was 2703 µgg -1 in in vivo leaves, 3294.36 µg.g -1 in in vitro leaves, 998.03 µg.g -1 in callus and 91.4 µg.g -1 in cell suspension cultures of S. acmella [26]. Variation in the values noticed in the study presented here may be attributed to the difference in species as well as extraction procedures. ...
Acmella ciliata Kunth (Cass.), a medicinally important plant in the family Asteraceae, has high commercial value because of its traditional phytomedicinal uses. The plant contains many phytochemicals like alkyl amides, alkaloids, tannins, saponins and flavonoids accountable for most of its pharmacological applications. The study presented here reports the callus culture and somatic embryogenesis of this plant thereby raising a novel system for the subsequent production of the N-alkyl amide ‘spilanthol’, the valuable secondary metabolite presents in it. Murashige and Skoog (MS) medium supplemented with auxins either alone or in combination with cytokinins were used for the induction and maturation of somatic embryos. MS medium supplemented with 2,4-D (0.5, 1.0 and 2.0 mg.L-1) produced black friable callus whereas, 1.0 mg.L-1 NAA in combination with 0.5 mg.L-1 BA induced white, slightly purple coloured friable callus which on further subculture to fresh medium induced somatic embryos that germinated into plantlets upon transfer to MS basal medium. The mode of regeneration via somatic embryogenesis was confirmed by histological analysis through free-hand sectioning and stereomicroscopic observation. The plantlets raised through somatic embryogenesis after a short hardening period, were found to acclimatise in the field at 83.33% efficiency and exhibited genetic uniformity with 96.6% similarity in the ISSR analysis. HPLC analysis of in vitro raised embryogenic callus showed 239.512 µg.g-1 spilanthol content which was comparatively higher than the mother plants (92.19 µg.g-1). The bioproduction of the N-alkylamide ‘spilanthol’ through embryogenic callus can be extended for the scale-up production of this bioactive compound using bioreactor technology for the formulation of phytodrugs.
... Sucrose not only acts as an external energy source but also contributes to the osmotic potential of the medium. allowing the absorption of mineral nutrients present in the medium, stimulating mitochondrial activity, and, hence, production of the energy required for metabolite synthesis [19,20]. Moreover, many researchers have pointed out the effects of sucrose concentration on secondary metabolite biosynthesis, during which development, chemical profile, and yields of in vitro cell cultures were highly dependent on the type and concentration of carbohydrates used in the medium [21][22][23]. ...
Pentacyclic triterpenes, including lupeol, α- amyrin, and β-amyrin, present a large range of biological activities including anti-inflammatory, anti-cancer, and gastroprotective properties. The phytochemistry of dandelion (Taraxacum officinale) tissues has been widely described. Plant biotechnology offers an alternative for secondary metabolite production and several active plant ingredients are already synthesized through in vitro cultures. This study aimed to establish a suitable protocol for cell growth and to determine the accumulation of α-amyrin and lupeol in cell suspension cultures of T. officinale under different culture conditions. To this end, inoculum density (0.2% to 8% (w/v)), inoculum age (2- to 10-week-old), and carbon source concentration (1%, 2.3%, 3.2%, and 5.5% (w/v)) were investigated. Hypocotyl explants of T. officinale were used for callus induction. Age, size, and sucrose concentrations were statistically significant in cell growth (fresh and dry weight), cell quality (aggregation, differentiation, viability), and triterpenes yield. The best conditions for establishing a suspension culture were achieved by using a 6-week-old callus at 4% (w/v) and 1% (w/v) of sucrose concentration. Results indicate that 0.04 (±0.02) α-amyrin and 0.03 (±0.01) mg/g lupeol can be obtained in suspension culture under these starting conditions at the 8th week of culture. The results of the present study provide a backdrop for future studies in which an elicitor could be incorporated to increase the large-scale production of α-amyrin and lupeol from T. officinale.
... An overview of these analytical methods can be found in a comprehensive review by Barbosa et al. [2]. For quantifiction of spilanthol in plant samples high performance liquid chromatography (HPLC) with UV detection [3,22,25,26] and MS or MS/MS have been reported [16,27]. Separation was performed in all cases using reversed phase HPLC (C18 column). ...
... One chromatographic method is based on gas chromatography coupled with FID detection [9]. The mentioned publications describe the analytical methods for quantification of spilanthol in Spilanthes acmella [9,16,22,[25][26][27] and Acmella oleracea flower [3,9,28]. ...
... Sample extraction and clean-up are normally required prior to chromatographic analysis due to the matrix effect. For some extraction methods, including maceration [22,25,26], solid-liquid extraction (SLE) [3,16,28] and Soxhlet extraction [28] were applied. Reports have shown that ethanol [3,16,22,29], methanol [25,26] and mixtures of methanol, ethanol, acetonitrile, water [28] are crucial solvents for the extraction of spilanthol from plants. ...
Acmella oleracea is an ethnobotanically significant plant with a relatiwely high content of spilanthol. Due to its broad spectrum of activity, including anti-inflammatory, antioxidant, analgesic, antifungal, and bacteriostatic properties, it is considered a valuable bioactive natural product. In addition, spilanthol as its main bioactive component inhibits facial muscle contractions, making it an attractive ingredient in anti-wrinkle and anti-aging cosmetics. Due to its muscle paralyzing effects, it is called herbal botox. The commercial interest in spilanthol encourages the development of effective methods of isolating it from plant material. The methodology used in this paper allows for the obtaining of extracts from Acmella oleracea with a relatively high content of spilanthol. An effective method of spilanthol extraction from all aerial parts of Acmella oleracea as well as methods of enriching spilanthol concentration in extracts achieved by removing polar and acidic substances from crude extracts was developed. To quantify the concentration of spilanthol, a simple, fast and economically feasible quantification protocol that uses nuclear magnetic resonance (HNMR) was developed. In addition, it has been proven, that oxidation of spilanthol by air gives (2E,7Z)-6,9-endoperoxy-N-(2-methylpropyl)-2,7-decadienamide. The studies on spilanthol solutions stability were carried out and the conditions for the long-time storage of spilanthol solutions have also been developed. Additionally, for confirmation of obtained results a sensitive (LOQ=1 ng/mL), precise (RSD lower than 7%) and accurate (RE lower than 7.5%), new HPLC-MS/MS method was applied.
... However, rotation and aeration can exert hydrodynamic stress on cells due to the physical characteristics of suspended cells. Shear pressures cause many changes in aggregate size, shape, cell integrity, viability and ultimately, biomass accumulation and secondary metabolism in cells (19). ...
... At higher speeds (150 and 180), several cells died due to hydraulic stress. At 60 and 90 rpm, the cells also decreased viability due to clustering (19). ...
Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.
... This mixture was incubated for 5 min in a boiling water bath, and then absorbance at 629 nm was recorded. For making standard curve, glucose was utilized [8]. Phosphate uptake was calculated using a standard calibration curve made from NaH 2 PO 4 [11,12]. ...
... So, the establishment of cell suspension culture were done in liquid (without agar) MS (3% sucrose) medium + 2,4-D (1 µM) + NAA (1 µM) + BAP (10 µM) (Figure 1c) and cells were healthy and loosely attached (Figure 1d). Carbohydrates as carbon sources are the major constituents of plant cell culture [8,11]. In the present study, sucrose was used as a carbon source. ...
... 3%) sucrose gave the highest biomass yield (105.6±4.5 g/l) followed by 40 g/l (Figure 2). Similarly, in other species of cell suspension culture, sucrose was shown to significantly increase biomass [8,11]. Variation of the initial pH of the medium was also optimised, and pH 5.8 (Figure 3) was observed to be suitable as it yielded the highest biomass (105.6±4.5 g/l). ...
Decalepis hamiltonii Wight & Arn. belongs to Asclepiadaceae family which is an ethno-pharmaceutically important monogenic plant species that is native to the Deccan peninsula forest areas of India. It is endangered due to habitat loss and over exploitation for volatile 2-hydroxy-4-methoxybenzaldehyde (HMB), which is an aromatic bioactive secondary metabolite. HMB is of great biological significance and is present in the plant’s tuberous roots. Plant cell culture is a viable alternative method for in vivo plant cultivation for secondary metabolites production. Callus induction with high biomass from the germinated root as explants for HMB production was optimised on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1 μM α- naphthaleneacetic acid (NAA) and 10 μM 6-benzylaminopurine (BAP) in dark incubation. Cell suspension cultures were established in 250 ml shake flasks, and each flask contained 50 ml of the same induction medium. Moreover, the pH 5.8, 3% sugar concentration and 120 rpm agitation speed in dark condition were suitable for high biomass production and the specific growth rate (µ) was 0.086 /day. Extraction of HMB was done by steam condensate methods from the biomass of cell suspension culture. Qualitative and quantification analyses of HMB were performed with gas chromatography (GC) and observed that 0.92 ± 0.02 mg/ml (0.092%) HMB were synthesized in the cell suspension culture.
... Acmella oleracea (L.) R.K. Jansen [1], also known as Spilanthes acmella Murr. [2][3][4] or Spilanthes oleracea L. [5], is part of the Asteraceae family. It was first discovered in Peru and is now commonly found in tropical and subtropical regions across the world, especially in the north of Brazil, where it is known as jambu [6][7][8][9]. ...
... Among the alkylamides, spilanthol ((E, E, Z)-2,6,8-decatrienoic acid N-isobutylamide) is considered to be the most potent bioactive compound found in A. oleracea. First identified by Gerber in 1903 as identical to affinin 1, spilanthol has been mostly found in Acmella flowers, leaves, and stems [4,7,8,[12][13][14][15][16], but also in roots [17,18], and its accumulation in in vitro cell cultures has been documented [2,3]. Due to its pharmacological importance, based on a series of effects typical of alkylamides, such as analgesic, neuroprotective, antioxidant, antimutagenic, anti-cancer, anti-inflammatory, antimicrobial, anti-larvicidal, and insecticidal activities [19], many protocols for spilanthol production and extraction have been developed (see [19] for a review; [20]). ...
... Due to its pharmacological importance, based on a series of effects typical of alkylamides, such as analgesic, neuroprotective, antioxidant, antimutagenic, anti-cancer, anti-inflammatory, antimicrobial, anti-larvicidal, and insecticidal activities [19], many protocols for spilanthol production and extraction have been developed (see [19] for a review; [20]). Besides recent in vivo treatments with biostimulants [21], a good approach to obtain a great number of plants, in a short period of time under selected standardized conditions of cultivation, is the use of in vitro plant cultures [2,3,8,[22][23][24][25][26][27]. These strategies have allowed rapid improvement of raw material and metabolite production. ...
Acmella oleracea L. is an important medicinal plant, commonly known as the toothache plant. It is a rich source of secondary metabolites used for the treatment of different human disorders. The demand for Acmella oleracea L. has increased due to its putative health benefits (in terms of both biomass quantity and bioactive compound purification). In vitro plant cultures have allowed the rapid increase of raw material availability through the use of suitable regeneration and multiplication systems. On the other hand, there is a general lack of methods for Acmella genetic transformation as a promising new technological approach for the improvement of secondary metabolites. In this work, an efficient transformation protocol has been established using the Agrobacterium tumefaciens LBA4404 strain bearing the binary vector pBI121 containing the NPTII gene for the resistance to kanamycin. Plant genetic transformation has been verified by direct polymerase chain reaction and GUS assay on regenerants. Transformation efficiency has been affected by the high level of the selection agent kanamycin. To our knowledge, this is the first report on the genetic transformation of A. oleracea, paving the way to further studies to improve in vitro plant growth and secondary metabolite production.
... Estimating Spilanthol in the callus: It was estimated with the High-Performance Liquid Chromatography (HPLC) according to Chaturvedi and Singh (2012) with following steps: Spilanthol extraction and separation: Spilanthol compounds were extracted and separated as the same as the method formerly mentioned, nevertheless, the device conditions differed accordingly except for the constant retention time. Separation column type C-Fast liquid chromatography column (FLC) 18, DB (deactivated base) (25cm×4.6mm ...
The present study was conducted to investigate the effect of adding methyl jasmonite at 0.0, 25, 50, and 75 µm on growth and multiplication of callus induced from murri. L plant and on the secondary metabolism compounds produced from it. The Spilanthes acmella secondary metabolism compounds of samples were estimated with High-Performance Liquid Chromatography (HPLC) from the shoot tips of paracress seedlings and extracted with methanol. The callus was induced from culturing the shoot tips in Murashige and Skoog (media supplied with 2.0 mg l of 2, 4-D and 0.5 mg l of benzyl adenine). Adding methyl Jasmonit led to a significant difference between the added-1-1 levels. The highest fresh and dry weights of callus were 11.414 and 1.419 g respectively from the concentration 50 µm while the 75 µm gave the lowest fresh and dry weights of 6.495 and 1.011 g respectively for increasing the secondary metabolism compounds, chemical initiators were utilized. The significant differences between the secondary values resulted from adding the chemical initiators at 25 µm of methyl tasmonit as effective concentration providing highest amount of scopoletin and β-sitosterol (178.653 and 108.790 µg g dry weight respectively). The-1 concentration of 50 µm gave the maximum spilanthol and β-amyrins (94.903 and 220.813 µg g dry weight respectively) while the control-1 treatment gave the maximum stigma sterol and-amyrins (117.50 and 4.467 µg g dry weight respectively). α-1
... The growth kinetics is similar to that for Spilanthes acmella, in which the cell suspension culture reached a specific growth rate of 0.28 days −1 ; during the exponential phase, the doubling time was 2.50 days −1 , and the maximum biomass was 8.5-g L −1 at day 15 [32]. On the other hand, Satureja khuzistanica cell suspension cultures reached a maximum dry biomass of 19.7-g L −1 at 21 days, with a specific growth rate of 1.5 days −1 and a doubling time of 7.6 days −1 [33]. ...
... Likewise, the production of the fatty acid amide spilanthol by cell suspension cultures of Spilanthes acmella Murr. presented a trend associated with its growth, reaching a maximum yield during the exponential phase and, subsequently, decreasing rapidly due to the lack of nutrients and consequent cellular death [32]. In another species, Eurycoma longifolia, it was reported that cell suspension cultures produce the quassinoid eurycomanone; this also occurs from the beginning of the growth kinetics, reaching a maximum amount of 1.7-mg g −1 DW [44]. ...
Ageratina pichinchensis (Kunth) is a plant used in traditional Mexican medicine to treat multiple ailments. However, there have not been biotechnological studies on producing compounds in in vitro cultures. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran (2) and 3-epilupeol (3), evaluate the anti-inflammatory potential of its extracts, and perform a phytochemical analysis. Cell suspension cultures were established in a MS culture medium of 30-g L −1 sucrose, 1.0-mg L −1 α-naphthaleneacetic acid, and 0.1-mg L −1 6-furfurylaminopurine. The ethyl acetate extract of the cell culture analyzed by gas chromatography (GC) revealed that the maximum production of anti-inflammatory compounds 2 and 3 occurs on days eight and 16, respectively, improving the time and previously reported yields in callus cultures. The anti-inflammatory activity of these extracts exhibited a significant inhibition of nitric oxide (NO) production. Furthermore, a phytochemical study of the ethyl acetate (EtOAc) and methanol (MeOH) extracts from day 20 led to the identification of 17 known compounds. The structures of the compounds were assigned by an analysis of 1D and 2D NMR data and the remainder by GC-MS. This is the first report of the production of (-)-Artemesinol, (-)-Artemesinol glucoside, encecalin, and 3,5-diprenyl-acetophenone by a cell suspension culture of A. pichinchensis.
... As spilanthol is an amphiphilic compound, 47 it can be extracted from plants using solvents that vary in polarity such as Hex 18 and MeOH:H 2 O (4:1, v/v). 48 Studies report that HPLC quantification with UV detection allows the identification of the content of spilanthol in different extracts, 24,49,50,51 as it was observed in this study. Spilanthol showed solubility in chloroform and it had its structure determined by NMR, according to the data compared in the literature. ...
Hydroethanolic preparations of Acmella oleracea is used in the north of Brazil as a female aphrodisiac. Thus, the objective of this study was to evaluate the action of the hydroethanolic extract of Acmella oleracea (EHFAo) flowers (21.873 and 44.457 mg/kg) and spilanthol (3 mg/kg) administered orally on reproductive performance and effects on the embryonic development of zebrafish F1 generation. It was observed that in the groups in which males and females received EHFAo and spilanthol, the spawning was interrupted, whereas in the groups in which only the females were treated, spawning occurred during the 21 days. Thus, in the histopathological evaluation of the gonads, it was possible to observe that the percentage of mature cells in the spermatozoa and females was significantly reduced. Only the embryo groups in which parental generation was treated with EHFAo showed lethal and teratogenic effects. On the other hand, the parental groups treated with the spilanthol presented only the lethality. Spilanthol and some metabolites showed good oral availability and important toxicological properties. Thus, it is suggested that the treatment of parental generation of zebrafish with EHFAo and spilanthol caused severe changes in the gonads and on fertility. However, on the embryo, the most striking effects in the development were recorded in the groups in which the parental generation was treated with the EHFAo, while the spilanthol influenced the lethality of the embryos.
