HPLC Analysis of Alizarin and Purpurin Produced by Rubia tinctorum L. Hairy Root Cultures

We have determined the quantities of the anthraquinones alizarin and purpurin, with especial regard to their effective antigenotoxic activity, in genetically transformed hairy root cultures of Rubia tinctorum L. Hairy roots were cultured on solid and in liquid Gamborg B5 and 1/2 NMS media in a shaking cabinet and in a bioreactor. Methanolic extracts of lyophilized hairy roots were hydrolysed, and then purified by solid phase extraction with good recovery. A new HPLC method was developed for the determination of alizarin and purpurin. The analysis was performed on a Luna C8 RP column using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate-formic acid buffer (pH 3.00) as eluent. Peaks were identified by addition of standards and by diode-array detection. External standardization allowed the determination of alizarin and purpurin with good sensitivity and reliability. The maximum purpurin content was observed in cultures cultivated on solid B5 medium (5.94 mg g⁻¹). The highest alizarin content was measured in cultures cultivated on solid 1/2 NMS medium (2.14 mg g⁻¹).

     

The Antioxidant,Cytotoxic and Antimicrobial Potential of Phenolic Acids-Enriched Extract of Elicited Hairy Roots of Salvia bulleyana

Hairy root cultures are valuable sources of a range of phytochemicals. Among them, Salvia bulleyana root culture is a promising source of polyphenols, especially rosmarinic acid (RA), a phenolic acid depside with pleiotropic activity and a wide application in medicine and cosmetology. The aim of the study was to enhance the culture productivity by finding suitable elicitation protocol and to determine its biological potential in terms of antioxidant, anticancer and antimicrobial properties. The total content of phenols and the levels of particular constituents in root extracts were analyzed using HPLC-PDA. Among four elicitors tested (yeast extract; methyl jasmonate, MJA; trans-anethol; and cadmium chloride), MJA was found to be the most effective. The greatest boost in phenolic production (up to 124.4 mg/g dry weight) was observed after three-day treatment with MJA at 100 µM, with an almost 100% improvement compared to the controls (non-treated root culture). The hydromethanolic extract from the elicited culture exhibited strong antioxidant activity with IC₅₀ values of 11.1 µg/mL, 6.5 µg/mL and 69.5 µg/mL for DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)) and superoxide anion radical, respectively. Moreover, in concentrations of 0.5–5 mg/mL the extract inhibited the growth of LoVo, AGS and HeLa cell lines, but was safe for the L929 cells up to the concentration of 5 mg/mL. The extract also exhibited moderate antimicrobial activity. Thus, the results confirmed that elicitation can be a beneficial strategy for increase the phenolic acid biosynthesis in hairy roots of S. bulleyana, and that such a highly productive culture can show significant biological potential.     

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.     

Antioxidant Assessment of Prenylated Stilbenoid-Rich Extracts from Elicited Hairy Root Cultures of Three Cultivars of Peanut (Arachis hypogaea)

Peanut produces prenylated stilbenoids upon biotic stress. However, the role of these compounds against oxidative stress have not been thoroughly elucidated. To this end, the antioxidant capacity of extracts enriched in prenylated stilbenoids and derivatives was studied. To produce these extracts, hairy root cultures of peanut cultivars Hull, Tifrunner, and Georgia Green were co-treated with methyl jasmonate, cyclodextrin, hydrogen peroxide, and magnesium chloride and then the stilbenoids were extracted from the culture medium. Among the three cultivars, higher levels of the stilbenoid derivatives arachidin-1 and arachidin-6 were detected in cultivar Tifrunner. Upon reaction with 2,2-diphenyl-1picrylhydrazyl, extracts from cultivar Tifrunner showed the highest antioxidant capacity with an IC₅₀ of 6.004 µg/mL. Furthermore, these extracts had significantly higher antioxidant capacity at 6.25 µg/mL and 3.125 µg/mL when compared to extracts from cultivars Hull and Georgia Green. The stilbenoid-rich extracts from peanut hairy roots show high antioxidant capacity and merit further study as potential nutraceuticals to promote human health.     

Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

Stimulation of Lignan Production in Schisandra rubriflora In Vitro Cultures by Elicitation

The study investigated the effect of elicitation with: chitosan (CH) (200 mg/L), yeast extract (YeE) (3000 mg/L), ethephon (ETH) (25 µM/L), and methyl jasmonate (MeJA) (50 µM/L), on lignan accumulation in agitated and bioreactor (Plantform temporary immersion systems) microshoot cultures of female (F) and male (M) Schisandra rubriflora Rehd. et Wils. (Schisandraceae) lines. The elicitors were supplemented on the 10th day of culture. Biomasses were collected at 24 h and 48 h, and 4, 6, and 8 days after the addition of each elicitor. The 24 compounds from the dibenzocyclooctadiene, aryltetralin, dibenzylbutane, and tetrahydrofuran lignans and neolignans were determined qualitatively and quantitatively in biomass extracts using the UHPLC–MS/MS method. The highest total contents [mg/100 g DW] of lignans were: for CH-95.00 (F, day 6) and 323.30 (M, 48 h); for YeE 104.30 (F, day 8) and 353.17 (M, day 4); for ETH 124.50 (F, 48 h) and 334.90 (M, day 4); and for MeJA 89.70 (F, 48 h) and 368.50 (M, 24 h). In the biomass extracts of M cultures grown in bioreactors, the highest total lignan content was obtained after MeJA elicitation (153.20 mg/100 g DW). The maximum total lignan contents in the biomass extracts from agitated and bioreactor cultures were 3.29 and 1.13 times higher, respectively, than in the extracts from the non-elicited cultures. The poor understanding of the chemical composition and the lack of studies in the field of plant biotechnology of S. rubriflora emphasize the innovativeness of the research.     

Effects of abiotic inducers on sesquiterpene synthesis in hairy root and cell-suspension cultures ofhyoscyomus muticus

The effects of hydrogen peroxide (H₂O₂), salicylic acid (SA), ethanol (EtOH), and methyl jasmonate (MJ) on the lubimin and solavetivone production in hairy root and cell suspension cultures ofHyoscyamus muticus were investigated. H₂O₂ (0.194–1.94) mM did not show any positive effect of the lubimin and solavetivone production. Lubimin production of cellsuspension cultures increased 50% in the presence of 40 ΜM SA. For hairy root cultures, lubimin and solavetivone production increased 5 and 48%, respectively, in the presence of 4 ΜM SA ?EtOH (20 mM) increased the lubimin and solavetivone concentrations 13% for hairy roots suggesting a potential rule for wounding or membrane disruption. MJ (4 ΜM) and 20 mM EtOH increased the lubimin and solavetivone concentrations 25 and 85%, respectively for hairy roots ofH. muticus.     

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg?L^?1) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg?L^?1) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.     

The Evaluation of Phenolic Acids and Flavonoids Content and Antiprotozoal Activity of Eryngium Species Biomass Produced by Biotechnological Methods

Three species from the Eryngium L. genus—E. campestre, E. maritimum, and E. planum, plants with a rich chemical composition, were selected for phytochemical and biological studies. The applied biotechnological methods allowed to obtain the biomass of these rare or protected species in the form of multiplied shoots (stationary system) and roots cultured in a liquid medium (agitated system). In the extracts from the raw material obtained under in vitro conditions, the content of selected phenolic acids and flavonoids (HPLC-DAD method) as well as the total of polyphenols (Folin–Ciocalteu assay) were quantified. The highest amount of all phenolic compounds was found in extracts from E. planum roots (950.90 ± 33.52 mg/100 g d.w.), and the lowest from E. campestre roots (285.00 ± 10.07 mg/100 g d.w.). The quantitatively dominant compound proved to be rosmarinic acid. The highest amounts were confirmed for E. planum root extract (694.58 mg/100 g d.w.), followed by E. planum (388.95 mg/100 g d.w.) and E. campestre (325.85 mg/100 g d.w.) shoot extracts. The total content of polyphenols was always increased in the biomass from in vitro cultures in comparison to the analogous organs of intact plants of each species. The obtained extracts were assessed for antiprotozoal activity against Acanthamoeba sp. The strength of biological activity of the extracts correlated with the content of phenolic compounds. To our knowledge, this is the first report on the amoebicidal activity of E. campestre, E. maritimum, and E. planum extracts from biomass produced by biotechnological methods.     

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA^®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.     

Gibberellic Acid Increases Secondary Metabolite Production in Echinacea purpurea Hairy Roots

Gibberellic acid (GA₃) is reported to have diverse effects on hairy root cultures of many plant species; therefore, the effects of GA₃ on the growth, secondary metabolite production (caffeic acid derivatives and lignin), phenylalanine ammonia lyase (PAL) activity, and free radical scavenging activity of light-grown Echinacea purpurea L. hairy roots were investigated. Eight concentrations of GA₃, ranging from 0.005 to 1.0 μM, were added to shake flask cultures. The moderate GA₃ concentration, 0.025 μM, resulted in the highest concentrations of cichoric acid, caftaric acid, and chlorogenic acid, as well as increased PAL activity, cell viability, and free radical scavenging activity, while higher and lower GA₃ concentrations resulted in reduced levels compared to the control (lacking GA₃). The moderate GA₃ concentration also affected root morphogenesis; supplementation with 0.025 μM GA₃ resulted in the development of thick, dense, purple-colored roots, while roots exposed to the higher and lower concentrations of GA₃ were thin and off-white. This study demonstrates that supplementation with GA₃ may be an excellent strategy to optimize the production of secondary metabolites from E. purpurea hairy root cultures; however, the GA₃ concentration is a critical factor.     

Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae)

Daidzin (7-O-glucoside of daidzein) has several pharmacological benefits in herbal remedy, as antioxidant and shown antidipsotropic activity. Hairy root culture of Psoralea corylifolia L. was developed for biomass and enhanced daidzin production using signalling compounds such as jasmonic acid (JA) and acetyl salicylic acid (ASA). Best response of 2.8-fold daidzin (5.09% DW) with 1 μM JA treatment after second week and 7.3-fold (3.43% DW) with 10 μM JA elicitation after 10th week was obtained from hairy roots compared to untreated control. ASA at 10 μM promoted 1.7-fold increase in daidzin (1.49% DW) content after seventh week compared to control (0.83% DW). Addition of 25 μM ASA resulted in 1.44% DW daidzin (1.5-fold increase) with 0.91% DW in control after fifth week and 1.44% DW daidzin (2.3-fold increase) after eighth week when compared to untreated control (0.62% DW). Reduced biomass with increased daidzin content was facilitated by elicited hairy root cultures.     

HPLC Determination of Flavonoids in Hairy-Root Cultures of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

A simple and sensitive liquid chromatographic method has been established for determination of the main flavonoid components of Scutellaria baicalensis Georgi (Lamiaceae) roots and hairy-root cultures. Wogon, the dried root of the plant, is used in traditional Chinese medicine for treatment of bronchitis, hepatitis, tumors, and inflammatory diseases. Lyophilized hairy roots were extracted with methanol. The crude extracts were purified by SPE on Supelco LC-8 cartridges. HPLC separations were performed on a Eurospher 100-C₈ reversed-phase column. The mobile phase was a gradient prepared from mixtures of acetonitrile and 0.1% trifluoroacetic acid. Peaks were identified by addition of standards and/or by diode-array detection. Baicalein 7-O-glucuronide (baicalin), wogonin 7-O-glucuronide (wogonoside), baicalein, wogonin, and acteoside were determined by the external standard method at 280 nm. We found that the aglycon (baicalein and wogonin) content of the transformed roots was consistently higher than that of the intact root from Siberia.     

Effects of inoculum conditions on growth of hairy roots of Panax ginseng C. A. Meyer

Plants have a potential to produce a large number of important metabolites such as pharmaceuticals, food additives, pigments, flavors, fragrances, and fine chemicals. Large-scale plant cell and tissue cultures for producing useful products has been considered an attractive alternative to whole plant extraction for obtaining valuable chemicals. In plant cell and tissue cultures, cell growth and metabolite production are influenced by nutritional and environmental conditions as well as physical properties of the culture system. To obtain a high growth rate of plant cell and tissue cultures, the culture tem. To obtain a high growth rate of plant cell and tissue cultures, the culture conditions should be maintained at an optimum level. We studied the relationship between inoculum conditions and the growth of Panax ginseng hairy root culture, and found that the growth rate varied with the inoculum conditions such as the number of root tips, the length of root tips, the part of root tips, and the inoculum size and age of hairy roots.     

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l^?1dry weight and azadirachtin yield of 3.2 mg g^?1 leading to a volumetric productivity of azadirachtin as 1.14 mg l^?1 day^?1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).     

Methyl jasmonate influence on silymarin production and plant stress responses in Silybum marianum hairy root cultures in a bioreactor

In this article our aim was to evaluate mass cultivation of S. marianum hairy roots in a bioreactor to produce silymarin. The effects of methyl jasmonate (MJ) elicitation on the accumulation of silymarin and the extent of the MJ-induced oxidative damage were investigated in bioreactor hairy root cultures of S. marianum. The growth rate of the bioreactor hairy root cultures was higher than that of those in a shake flask after 3 weeks. Silymarin accumulation was increased from 0.13?mg?g?1 DW in non-treated hairy roots to 0.22?mg?g?1 DW in hairy roots 72?h after 100?μM?MJ treatment. Guaiacol peroxidase and ascorbate peroxidase were activated by MJ?72?h after treatment, being 3.2- and 1.3-fold higher, respectively, than that of the control. An increase in enzymatic activity suggests increased scavenging of reactive oxygen species, indicating the tolerance to MJ stress. These results suggest that MJ elicitation is beneficial for silymarin production using bioreactor hairy root cultures.     

Volatile oils from the plant and hairy root cultures of Ageratum conyzoides L

Two lines of hairy root culture of Ageratum conyzoides L. induced by Agrobacterium rhizogenes ATCC 15834 were established under either complete darkness or 16 h light/8 h dark photoperiod conditions. The volatile oil yields from aerial parts and roots of the parent plant, the hairy root culture photoperiod line and the hairy root culture dark line were 0.2%, 0.08%, 0.03% and 0.02%, (w/w), respectively. The compositions of the volatiles from the hairy roots, plant roots and aerial parts were analysed by GC and GC-MS. The main components of the volatiles from the hairy root cultures were β-farnesene, precocene I and β-caryophyllene, in different amounts, depending on light conditions and also on the age of cultures. Precocene I, β-farnesene, precocene II and β-caryophyllene were the main constituents of the volatile oils from the parent plant roots, whereas precocene I, germacrene D, β-caryophyllene and precocene II were the main constituents of the aerial parts of the parent plant. Growth and time-course studies of volatile constituents of the two hairy root lines were compared. Qualitative and quantitative differences were found between the volatile oils from the roots of the parent plant and those from the hairy roots.     

Cerium-Promoted Ginsenosides Accumulation by Regulating Endogenous Methyl Jasmonate Biosynthesis in Hairy Roots of Panax ginseng

Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce³⁺-mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce³⁺ improved the elongation and biomass of hairy roots. The Ce³⁺-induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes (PgSS, PgSE and PgDDS). At a Ce³⁺ concentration of 20 mg L⁻¹, the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce³⁺ concentration range of 10 to 30 mg L⁻¹. The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce³⁺ in the range of 20–40 mg L⁻¹. The Ce³⁺ exposure induced transient production of superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX, PgAOS and PgJMT. Our results also revealed that Ce³⁺ did not directly influence PgSS, PgSE and PgDDS activity. We speculated that Ce³⁺-induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce³⁺-mediated signal transduction.     

HPLC-DAD Based Polyphenolic Profiling and Evaluation of Pharmacological Attributes of Putranjiva roxburghii Wall.

The current study was intended to explore the phytochemical profiling and therapeutic activities of Putranjiva roxburghii Wall. Crude extracts of different plant parts were subjected to the determination of antioxidant, antimicrobial, antidiabetic, cytotoxic, and protein kinase inhibitory potential by using solvents of varying polarity ranges. Maximum phenolic content was notified in distilled water extracts of the stem (DW-S) and leaf (DW-L) while the highest flavonoid content was obtained in ethyl acetate leaf (EA-L) extract. HPLC-DAD analysis confirmed the presence of various polyphenols, quantified in the range of 0.02 ± 0.36 to 2.05 ± 0.18 μg/mg extract. Maximum DPPH scavenging activity was expressed by methanolic extract of the stem (MeOH-S). The highest antioxidant capacity and reducing power was shown by MeOH-S and leaf methanolic extract (MeOH-L), respectively. Proficient antibacterial activity was shown by EA-L extract against Bacillus subtilis and Escherichia coli. Remarkable α-amylase and α-glucosidase inhibition potential was expressed by ethyl acetate fruit (EA-F) and n-Hexane leaf (nH-L) extracts, respectively. In case of brine shrimp lethality assay, 41.67% of the extracts (LC₅₀ < 50 µg/mL) were considered as extremely cytotoxic. The test extracts also showed mild antifungal and protein kinase inhibition activities. The present study explores the therapeutic potential of P. roxburghii and calls for subsequent studies to isolate new bioactive leads through bioactivity-guided isolation.