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

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).

Download Full-text

Synthesis, Characterization and Biological Evaluation of Novel Triazolyl - Acridine Derivatives as Cytotoxic Agents

Research Journal of Pharmacy and Technology ◽

10.52711/0974-360x.2021.00889 ◽

2021 ◽

pp. 5101-5107

Author(s):

Mandeep Singh ◽

D. N Prasad ◽

Supriya Agnihotri

Keyword(s):

Cell Line ◽

Biological Evaluation ◽

Biologically Active ◽

Cytotoxic Agents ◽

Breast Adenocarcinoma ◽

Dependent Manner ◽

Adenocarcinoma Cell Line ◽

Adenocarcinoma Cell ◽

Ht 29 ◽

Mcf 7

Acridine and Triazols both are biologically active heterocyclic rings with cytotoxic potential. Triazolyl- acridine adduct attract the attention in the field of medicinal chemistry. Here we synthesized a series of triazolyl- acridine compounds by the appropriate procedures. Structure of all synthesized compounds was confirmed by the various spectroscopic methods e.g. FT-IR, proton NMR and Mass spectrograms. All synthesized triazolyl-acirdines compounds were assayed in-vitro for cytotoxic activity against MCF-7 (human breast adenocarcinoma cell line) and HT-29 (human colon adenocarcinoma cell line) cells by MTT- assay. All target compounds shows increasing activity in dose dependent manner. However MPSP-9 was sensitive against MCF-7 but compound MPSP-1 was most sensitive with minimum inhibitory concentration (IC50 value) against MCF-7 and HT-29 both cell lines.

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Download Full-text

SYNTHESIS OF BIOLOGICALLY ACTIVE COMPOUNDS IN HAIRY ROOTS OF ASTRAGALUS PENDULIFLORUS LAM.

chemistry of plant raw material ◽

10.14258/jcprm.2020026284 ◽

2020 ◽

pp. 209-221

Author(s):

Elena Valerievna Ambros ◽

Ol'ga Viktorovna Kotsupiy ◽

Tat'yana Abdulkhailovna Kukushkina ◽

Tat'yana Vital'yevna Zheleznichenko ◽

Tat'yana Ivanovna Novikova

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Virulent Strain ◽

High Growth ◽

Growth Index ◽

Biologically Active ◽

Pcr Analysis ◽

Hairy Root Cultures ◽

Root Cultures ◽

Active Metabolites

Agrobacterium rhizogenes – mediated genetic transformation of medicinal plant A. penduliflorus Lam. using A4-RT, R-1601, 15834 SWISS strains was performed. The competences for transformation of three types of explants: hypocotyls, cotyledons, and primary shoots were tested. The virulent strain (15834 SWISS) and types of explants for transformation (primary shoots and cotyledons) with high growth index (I) were determined. The frequency of transformation of cotyledons by strain 15834 SWISS after 4 weeks of cultivation was 15.4% (I = 59.6), hypocotyls – 9.1% (I = 7.3) and primary shoots – 37.5% (I = 21.0). After 8 weeks of cultivation I increased 4.5 times for primary shoots (I = 94.5 ± 0.20) and cotyledons (I = 265.8 ± 0.35), for cultures from hypocotyls – 5.97 times (I = 43.6 ± 0.30). The roots’ transgenic status and the absence of agrobacterium contamination were confirmed by PCR analysis using rolB-, virC-specific primers. The lines of hairy roots characterized by active increases of biomass with high content of biologically active metabolites were selected, moreover, the content of metabolites in hairy root cultures exceeded their content in the roots of introduced plants. The maximum accumulation of compounds was found in hairy roots obtained from primary shoots (pectins – up 7.8%, protopectins – up 15.3%) and cotyledons (tannins – up 16.1%, triterpenic saponins – up 30.5%) after 8 weeks of cultivation. High perfor-mance liquid chromatography (HPLC) analysis demonstrated that hydrolysates of extracts of hairy roots from primary shoots contained 2 flavonol aglycones – quercetin and isorhamnetin whereas ethanol extracts were characterized by presence of quercetin and 4 flavonoid components. In hairy roots from cotyledons the maximum of phenolic compounds (PСs) content did not differ significantly at 8 and 12 weeks of cultivation (1.38 ± 0.01 and 1.49 ± 0.06% of dry weight, respectively). The content of PCs in hairy roots from primary shoots increased two-fold from 4 to 12 weeks of cultivation (up 1.24 ± 0.18%). To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in A. penduliflorus using A. rhizogenes.

Download Full-text

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.

Download Full-text

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.

Download Full-text