Abscisic Acid Regulates the 3-Hydroxy-3-methylglutaryl CoA Reductase Gene Promoter and Ginsenoside Production in Panax quinquefolium Hairy Root Cultures

Panax quinquefolium hairy root cultures synthesize triterpenoid saponins named ginsenosides, that have multidirectional pharmacological activity. The first rate-limiting enzyme in the process of their biosynthesis is 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In this study, a 741 bp fragment of the P. quinquefolium HMGR gene (PqHMGR), consisting of a proximal promoter, 5′UTR (5′ untranslated region) and 5′CDS (coding DNA sequence) was isolated. In silico analysis of an isolated fragment indicated a lack of tandem repeats, miRNA binding sites, and CpG/CpNpG elements. However, the proximal promoter contained potential cis-elements involved in the response to light, salicylic, and abscisic acid (ABA) that was represented by the motif ABRE (TACGTG). The functional significance of ABA on P. quinquefolium HMGR gene expression was evaluated, carrying out quantitative RT-PCR experiments at different ABA concentrations (0.1, 0.25, 0.5, and 1 mg·L−1). Additionally, the effect of abscisic acid and its time exposure on biomass and ginsenoside level in Panax quinquefolium hairy root was examined. The saponin content was determined using HPLC. The 28 day elicitation period with 1 mg·L−1 ABA was the most efficient for Rg2 and Re (17.38 and 1.83 times increase, respectively) accumulation; however, the protopanaxadiol derivative content decreased in these conditions.

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The Increase of Triterpene Saponin Production Induced by Trans-Anethole in Hairy Root Cultures of Panax quinquefolium

Molecules ◽

10.3390/molecules23102674 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2674 ◽

Cited By ~ 10

Author(s):

Ewa Kochan ◽

Piotr Szymczyk ◽

Łukasz Kuźma ◽

Grażyna Szymańska ◽

Anna Wajs-Bonikowska ◽

...

Keyword(s):

Hairy Root ◽

Fold Increase ◽

In Vitro Cultivation ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium ◽

Saponin Content ◽

Ginsenoside Rc ◽

Pharmaceutical Production

In vitro cultivation is an effective way to increase pharmaceutical production. To increase ginsenoside production in hairy root cultures of American ginseng, the present study uses trans-anethole as an elicitor. The content of nine triterpene saponins was determined: Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Re and Rf. Trans-anethole was found to stimulate saponin synthesis regardless of exposure time (24 and 72 h). Twenty-four hour exposure to 1 μmol trans-anethole in the culture medium resulted in the highest increase of total saponin content (twice that of untreated roots), and optimum accumulation of Rb-group saponins, with ginsenoside Rc dominating (8.45 mg g−1 d.w.). In contrast, the highest mean content of protopanaxatriol derivatives was obtained for 10 μmol trans-anethole. The Re metabolite predominated, reaching a concentration of 5.72 mg g−1 d.w.: a 3.9-fold increase over untreated roots. Elicitation with use of trans-anethole can therefore be an effective method of increasing ginsenoside production in shake flasks.

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The activity of different extracts from Panax quinquefolium L. cultures against pathogenic Staphylococcus aureus with respect to ginsenoside content

Archives of Biological Sciences ◽

10.2298/abs150330104s ◽

2015 ◽

Vol 67 (4) ◽

pp. 1277-1284 ◽

Cited By ~ 2

Author(s):

Monika Sienkiewicz ◽

Anna Głowacka ◽

Edward Kowalczyk ◽

Ewa Kochan

Keyword(s):

Staphylococcus Aureus ◽

Hairy Root ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Diffusion Method ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium ◽

Disk Diffusion Method ◽

Ginsenoside Content

Ginsenosides can be isolated from various cultures of Panax quinquefolium L., American ginseng. The aim of the study was to determine the antibacterial activity of extracts from leaves, stalks, hairy root cultures and field roots of P. quinquefolium L. containing ginsenosides against Staphylococcus aureus isolates obtained from various clinical materials. The agar well diffusion assay was used to evaluate microbial growth inhibition at various concentrations of extracts. The susceptibility of the clinical isolates to recommended antibiotics was determined with the disk-diffusion method. The results showed that the tested extracts inhibited the growth of all S. aureus clinical isolates, including MRSA (methicillin-resistant S. aureus) with MIC values ranging from 0.5 mg/mL to 1.7 mg/mL. The level of antimicrobial activity of extracts depends on the ginsenoside content. Both field roots and hairy root cultures represent excellent sources of these metabolites. Extracts with ginsenosides were found to inhibit multidrug-resistant staphylococci and can be a valuable complement to antistaphylococcal therapy.

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High Level Expression of Chorismate Pyruvate-Lyase (UbiC) and HMG-CoA Reductase in Hairy Root Cultures of Lithospermum erythrorhizon

Plant and Cell Physiology ◽

10.1093/pcp/pcf106 ◽

2002 ◽

Vol 43 (8) ◽

pp. 894-902 ◽

Cited By ~ 20

Author(s):

Annegret Köhle ◽

Susanne Sommer ◽

Kazufumi Yazaki ◽

Albert Ferrer ◽

Albert Boronat ◽

...

Keyword(s):

Hairy Root ◽

Hairy Root Cultures ◽

Root Cultures ◽

Lithospermum Erythrorhizon ◽

High Level Expression ◽

Hmg Coa Reductase ◽

High Level ◽

Coa Reductase

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Nitrogen and phosphorus as the factors affecting ginsenoside production in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

Acta Physiologiae Plantarum ◽

10.1007/s11738-016-2168-9 ◽

2016 ◽

Vol 38 (6) ◽

Cited By ~ 10

Author(s):

E. Kochan ◽

P. Szymczyk ◽

Ł. Kuźma ◽

G. Szymańska

Keyword(s):

Hairy Root ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium ◽

Nitrogen And Phosphorus ◽

Factors Affecting ◽

Shake Flasks ◽

Nutrient Sprinkle Bioreactor

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Panax quinquefolium L. Ginsenosides from Hairy Root Cultures and Their Clones Exert Cytotoxic, Genotoxic and Pro-Apoptotic Activity towards Human Colon Adenocarcinoma Cell Line Caco-2

Molecules ◽

10.3390/molecules25092262 ◽

2020 ◽

Vol 25 (9) ◽

pp. 2262 ◽

Cited By ~ 1

Author(s):

Ewa Kochan ◽

Adriana Nowak ◽

Małgorzata Zakłos-Szyda ◽

Daria Szczuka ◽

Grażyna Szymańska ◽

...

Keyword(s):

Cell Line ◽

Hairy Root ◽

Biologically Active ◽

Hairy Root Cultures ◽

Dependent Manner ◽

Root Cultures ◽

Panax Quinquefolium ◽

Adenocarcinoma Cell Line ◽

Concentration Dependent Manner ◽

Adenocarcinoma Cell

American ginseng, Panax quinquefolium (L.), is traditionally used in folk medicine. It exhibits a range of anti-inflammatory, hepatoprotective, anti-diabetic, anti-obesity, anti-hyperlipidemic and anti-carcinogenic effects. Its main components are ginsenosides, also known as panaxosides or triterpene saponins. In order to obtain high yields of ginsenosides, different methods of controlled production are involved, i.e., with hairy root cultures. However, they are still employed under in vitro conditions. Our studies revealed that hairy root cultures subjected to an elicitation process can be considered as a potent source of ginsenosides. The present study examines the biological activity of ginseng hairy root cultures against the Caco-2 human adenocarcinoma cell line. Among our six different clones of P. quinquefolium hairy roots, extracts B and Be (treated with elicitor) were the strongest inhibitors of the cellular metabolic activity. While all extracts induced DNA damage, B and Be also generated reactive oxygen species (ROS) in a concentration-dependent manner, which was correlated with the depletion of the mitochondrial membrane potential and induction of apoptosis. These findings indicate that further research concerning P. quinquefolium hairy root cultures should focus on the activity of rare ginsenosides and other biologically active compound profiles (i.e., phenolic compounds).

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The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity

In Vitro Cellular & Developmental Biology - Plant ◽

10.1007/s11627-012-9469-5 ◽

2012 ◽

Vol 49 (1) ◽

pp. 24-29 ◽

Cited By ~ 13

Author(s):

Ewa Kochan ◽

Małgorzata Wasiela ◽

Monika Sienkiewicz

Keyword(s):

Antimicrobial Activity ◽

Hairy Root ◽

American Ginseng ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium

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Alterations in oleanolic acid and sterol content in marigold (Calendula officinalis) hairy root cultures in response to stimulation by selected phytohormones

Acta Physiologiae Plantarum ◽

10.1007/s11738-021-03212-6 ◽

2021 ◽

Vol 43 (3) ◽

Author(s):

Abdulwadood S. M. Alsoufi ◽

Klaudia Staśkiewicz ◽

Michał Markowski

Keyword(s):

Oleanolic Acid ◽

Hairy Root ◽

Hairy Roots ◽

Naphthaleneacetic Acid ◽

Hairy Root Cultures ◽

Root Cultures ◽

Calendula Officinalis ◽

Plant Metabolites ◽

Triterpenoid Saponins

AbstractHairy root cultures are an efficient tool for the biotechnological production of plant metabolites and a convenient experimental model for analyzing the effect of various compounds on plant metabolism. In contrast to many other types of in vitro plant cultures, hairy roots do not require an external supply of phytohormones to the medium. Consequently, plant growth regulators such as auxins and cytokinins are rarely used as elicitors in hairy root in vitro cultures; however, they can strongly influence plant defense responses. The aim of this study was to investigate the influence of two auxins: natural indole-3-acetic acid (IAA) and synthetic 1-naphthaleneacetic acid (NAA), as well as two cytokinins: natural kinetin and synthetic 6-benzylaminopurine (BAP) at a concentration of 0.75 mg/L on the metabolism of sterols and triterpenoids in Calendula officinalis hairy roots. Auxins prevented the accumulation of triterpenoid saponins (oleanolic acid glycosides), while cytokinin BAP increased their accumulation by 17% and their release into the culture medium by a factor of 10. Other cytokinins and kinetins increased the sterol levels by 17%, the level of stigmasterol by 15%, and the level of isofucosterol by 7 times.

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Panax quinquefolium hairy root extracts and their effect in connections with antibiotics against pathogenic bacteria – preliminary study

Annales Universitatis Mariae Curie-Sklodowska sectio C – Biologia ◽

10.17951/c.2018.73.1.7-17 ◽

2019 ◽

Vol 73 (1) ◽

pp. 7

Author(s):

Ewa Kochan

Keyword(s):

Hairy Root ◽

Pathogenic Bacteria ◽

Root Culture ◽

Biologically Active ◽

Root Cultures ◽

Panax Quinquefolium ◽

Bacterial Isolates ◽

Root Extracts ◽

Saponin Content ◽

Preliminary Study

The aim of the present study was to determine the level of ginsenosides in extracts from hairy root A, B, G clones of Panax quinquefolium and their action with antibiotics against clinical bacterial isolates. The content of ginsenosides (the key biologically active compounds) were determined in tested extracts using HPLC. The activity of extracts with antibiotics was established by micro-dilution broth method. Total triterpene saponin content was 14.68, 14.32 and 10.07 mgg-1 d.w. for root culture clones B, A and G, respectively. Our research indicates that the addition of extracts mainly from B and G clone hairy root cultures to antibiotics allow to reduce the ampicillin and tetracycline effective concentration respectively against Enterococcus faecalis and both Escherichia coli and Acintobacter baumannii.

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Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

Acta Physiologiae Plantarum ◽

10.1007/s11738-013-1439-y ◽

2013 ◽

Vol 36 (3) ◽

pp. 613-619 ◽

Cited By ~ 12

Author(s):

E. Kochan ◽

G. Szymańska ◽

P. Szymczyk

Keyword(s):

Hairy Root ◽

Sugar Concentration ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium ◽

Ginsenoside Biosynthesis ◽

Shake Flasks ◽

Nutrient Sprinkle Bioreactor

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Production of Triterpenoid Sapogenins in Hairy Root Cultures of Silene vulgaris

Natural Product Communications ◽

10.1177/1934578x1501001129 ◽

2015 ◽

Vol 10 (11) ◽

pp. 1934578X1501001

Author(s):

Yeon Bok Kim ◽

Darwin W. Reed ◽

Patrick S. Covello

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Leaf Explants ◽

Hairy Root Cultures ◽

Root Cultures ◽

Silene Vulgaris ◽

In Vitro Production ◽

Triterpenoid Saponins ◽

Meja Treatment

Silene vulgaris (Moench) Garcke (Caryophyllaceae) is widely distributed in North America and contains bioactive oleanane-type saponins. In order to investigate in vitro production of triterpenoid saponins, hairy root cultures of S. vulgaris were established by infecting leaf explants with five strains of Agrobacterium rhizogenes (LBA9402, R1000, A4, 13333, and 15834). The A. rhizogenes strain LBA9402 had an infection of 100% frequency and induced the most hairy roots per plant. Methyl jasmonate (MeJA)-induced changes in triterpenoid saponins in S. vulgaris hairy roots were analyzed. Accumulation of segetalic acid and gypsogenic acid after MeJA treatment was 5-and 2-fold higher, respectively, than that of control root. We suggest that hairy root cultures of S. vulgaris could be an important alternative approach to the production of saponins.

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