Panax quinquefolium hairy root extracts and their effect in connections with antibiotics against pathogenic bacteria – 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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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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Abscisic Acid Regulates the 3-Hydroxy-3-methylglutaryl CoA Reductase Gene Promoter and Ginsenoside Production in Panax quinquefolium Hairy Root Cultures

International Journal of Molecular Sciences ◽

10.3390/ijms20061310 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1310 ◽

Cited By ~ 5

Author(s):

Ewa Kochan ◽

Ewa Balcerczak ◽

Piotr Szymczyk ◽

Monika Sienkiewicz ◽

Hanna Zielińska-Bliźniewska ◽

...

Keyword(s):

Abscisic Acid ◽

Hairy Root ◽

Tandem Repeats ◽

Proximal Promoter ◽

Hairy Root Cultures ◽

Root Cultures ◽

Panax Quinquefolium ◽

Triterpenoid Saponins ◽

Saponin Content ◽

Coa Reductase

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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Comparative investigation of sesquiterpene components of essential oils originating from intact plants and hairy root chamomile cultures

GSC Advanced Research and Reviews ◽

10.30574/gscarr.2021.6.2.0016 ◽

2021 ◽

Vol 6 (2) ◽

pp. 028-049

Author(s):

Éva Szőke ◽

Éva Lemberkovics

Keyword(s):

Essential Oil ◽

Essential Oils ◽

Hairy Root ◽

Folk Medicine ◽

Root Culture ◽

Hairy Root Culture ◽

Comparative Investigation ◽

Biologically Active

The importance of chamomile (Chamomilla recutita) inflorescence is widely known in classical and folk medicine, with the largest group of its effective constituents forming the essential oil (chamazulene, a-bisabolol, α-farnesene, trans-β-farnesene, spathulenol, cis/trans-en-in-dicycloethers). Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its a-bisabolol. Both components have important antiinflammatory activities. Wild populations can be easily distinguished from cultivated ones by their high amount of bisaboloides, particularly the flower of Hungarian Szabadkígyós wild type, which contained on average 48 % of the biologically active (-)-a-bisabolol. The population of Szabadkígyós has good salt tolerance which is important owing to global warming, because the proportion of saline areas is increasing worldwide. To keep the genome of Szabadkígyós having high (-)-a-bisabolol content, Szőke and research team used biotechnological methods. Sterile plantlets, were infected by Agrobacterium rhizogenes strains #A-4, #15834, #R-1601. The hairy root clones possessing the best growing and biosynthetical potential were multiplied for phytochemical investigations. Pharmacologically important compounds of their essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. GC-MS studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the mother plant. Berkheyaradulene, geranyl-isovalerat and cedrol as new components were identified in these sterile cultures. The main component of hairy root cultures (D/400, D/1, D/100 and Sz/400) was tr-b-farnesene and in addition one new compound: a-selinene was identified. Hairy root culture originated from chamomile collected in Szabadkígyós was intensive increased the essential oil content and pharmacological active compounds: (-) -α-bisabolol and β-eudesmol was also synthetized in large quantity. Furthermore, in vitro organized cultures were made from this population to obtain propagation material containing numerous active substances.

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Hairy Roots

Advances in Environmental Engineering and Green Technologies - Molecular Plant Breeding and Genome Editing Tools for Crop Improvement ◽

10.4018/978-1-7998-4312-2.ch008 ◽

2021 ◽

pp. 219-252

Keyword(s):

Secondary Metabolites ◽

Hairy Root ◽

Hairy Roots ◽

High Growth ◽

Root Culture ◽

Hairy Root Culture ◽

High Growth Rate ◽

Hairy Root Cultures ◽

Root Cultures ◽

Transformed Root Cultures

Agrobacterium rhizogenes induces hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection, when cultured in hormone free medium, show high growth rate and genetic stability. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Several elicitation methods can be used to further enhance the production and accumulation of secondary metabolites. Thus, hairy root culture offer promise for high production and productivity of valuable secondary metabolites in many plants. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for pharmaceutical industry. Hairy root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites, and for phytoremediation due to their abundant neoplastic root proliferation property. Various applications of hairy root cultures and potential problems associated with them are discussed in this chapter.

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Hairy Root Cultures of Eurycoma longifolia and Production of Anti-inflammatory 9-Methoxycanthin-6-one

Natural Product Communications ◽

10.1177/1934578x1801300507 ◽

2018 ◽

Vol 13 (5) ◽

pp. 1934578X1801300

Author(s):

Trang Thu Tran ◽

Nam Trung Nguyen ◽

Ngoc Bich Pham ◽

Huy Nhat Chu ◽

Trong Dinh Nguyen ◽

...

Keyword(s):

Hairy Root ◽

Root Culture ◽

Dry Weight ◽

Hairy Root Culture ◽

Peritoneal Macrophages ◽

High Yield ◽

Hairy Root Cultures ◽

Root Cultures ◽

Eurycoma Longifolia ◽

Anti Inflammatory

The content of bioactive alkaloid 9-methoxycanthin-6-one in the roots of Eurycoma longifolia is rather small. In the current study, a high yield of 9-methoxycanthin-6-one was obtained from transgenic hairy roots (1.139 ± 0.20% dry weight, DW) compared to wild roots (0.164 ± 0.25% DW) of E. longifolia. Three liquid basal media supplemented with 3% sucrose were tested for the growth and accumulation of 9-methoxycanthin-6-one. The fastest growth was obtained in the Shenck and Hildebrandt (SH) medium while the highest 9-methoxycanthin-6-one content was observed in McCown's woody plant (WP) medium. In this study, compared to controls, the production of 9-methoxycanthin-6-one was increased by the maximum of 2.6 and 4.0 times after the addition of jasmonic acid and yeast extract, respectively, into hairy root cultures. In addition, pro-inflammatory cytokines IL-6 and TNF-α were significantly inhibited by 9-methoxycanthin-6-one in lipopolysaccharide-stimulated murine RAW264.7 cells, peritoneal macrophages and human THP-1 macrophages These results suggest that the elicited hairy root culture of E. longifolia is an alternative system for the production of an abundant source of anti-inflammatory 9-methoxycanthin-6-one.

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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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Optimization of conditions for the induction of Tagetes patula L. hairy roots

Ukrainian Journal of Ecology ◽

10.15421/2019_119 ◽

2019 ◽

Vol 9 (3) ◽

pp. 415-420

Author(s):

L. P. Khlebova ◽

E. S. Brovko ◽

O. V. Bychkova ◽

N. V. Pavlova

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Wild Strain ◽

Leaf Explants ◽

Biologically Active ◽

Infected Leaf ◽

Hairy Root Cultures ◽

Root Cultures ◽

Tagetes Patula ◽

Time Of Infection

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.

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Effects of methyl jasmonate on the growth and triterpenoid production of diploid and tetraploid Centella asiatica (L.) Urb. hairy root cultures

Scientific Reports ◽

10.1038/s41598-019-54460-z ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Khoa Van Nguyen ◽

Benyakan Pongkitwitoon ◽

Thanika Pathomwichaiwat ◽

Unchera Viboonjun ◽

Sompop Prathanturarug

Keyword(s):

Methyl Jasmonate ◽

Hairy Root ◽

Hairy Roots ◽

Centella Asiatica ◽

Root Culture ◽

Hairy Root Culture ◽

Asiatic Acid ◽

Hairy Root Cultures ◽

Root Cultures ◽

Meja Treatment

AbstractIn this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

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