Polyurethane Foam Rafts Supported In Vitro Cultures of Rindera graeca Roots for Enhanced Production of Rinderol, Potent Proapoptotic Naphthoquinone Compound

Abstract

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaphthoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 ± 10.47 µg/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity.

HPLC-PDA-ESI-HRMS-Based Profiling of Secondary Metabolites of Rindera graeca Anatomical and Hairy Roots Treated with Drought and Cold Stress

“…Authors suggest that in specific culture conditions, in LBA and shikonin biosynthetic pathway, in which they share its early steps, the phenylpropanoid unit is further favorably used for LBA synthesis. Nevertheless under conditions of this study in two samples of 28-day old roots: RgAR and hairy of RgTR17 line was detected rinderol, a furano-naphthoquinone compound demonstrating antiapoptotic potential [13]. Previously rinderol was determined both in RgTR7 and RgTR17 28-old-day root lines cultivated in vitro [11], but in further subcultures its biosynthesis was induced only in cultures carried out on polyurethane rafts [11].…”

“…These studies revealed the presence of phenolic compounds, pyrrolizidine alkaloids, naphthoquinone shikonin-type compounds and among them rinderol, a potent cytotoxic agent [7,11,12]. Rinderol production was optimized in root cultures of R. graeca and its proapoptotic activity was demonstrated [13]. Other various biological activities were also reported for plants of Rindera genus, including antiinflammatory [14], anti-viral [15], and antimicrobial attributed to the presence of essential oils distilled from aerial part [16], in addition the latter were also demonstrated for methanolic and hexane extracts of shoots and hairy roots of R. graeca cultivated in vitro as well as rinderol [11].…”

Selective Impact of MTMS-Based Xerogel Morphology on Boosted Proliferation and Enhanced Naphthoquinone Production in Cultures of Rindera graeca Transgenic Roots

“…In this study, Rindera graeca hairy roots were originally introduced to the in vitro conditions and were established as a stable culture by Katarzyna Sykłowska-Baranek et al [ 43 ]. Before the developing culture systems supported with samples of xerogels, the inoculum of R. graeca hairy roots was maintained in 250 mL Erlenmeyer flasks filled with 50 mL of hormone-free DCR medium (PhytoTech Labs, Inc., Lenexa, KS, USA) [ 14 ]. The flasks were agitated on an oscillatory shaker ISS-7100 (Lab Companion, Billerica, MA, USA) at 105 rpm and 24 °C in darkness for 28 days.…”

“…Moreover, the unique but end-user-defined parameters of a strictly controlling bioprocess environment allow for various culture system modifications. Among reported techniques, much attention is dedicated to in situ separation of bioproducts, defined as a process of directed metabolite accumulation within an additional phase (i.e., liquid extractant or solid-state adsorbent) added into the culture medium [ 13 , 14 , 15 ]. The desired selectivity of in situ applied extractant/adsorbent toward a specific metabolite is favorable for production efficiency because it enables enhanced secretion of bioproducts through its selective separation from the culture medium, resulting in the cancellation of separated metabolite biosynthesis inhibition, which finally resulted in increased yield of its production [ 16 ].…”

Biotechnology and In Vitro Culture as an Alternative System for Secondary Metabolite Production

“…New extraction methods, including ultrasonic-assisted extraction [28][29][30], supercritical fluid extraction [29][30][31], and accelerated solvent extraction [32], are fast and efficient for extracting chemicals from plant matrices. In addition, in situ extraction is considered an efficient method to recover secondary metabolites; moreover, it allows both to improve the yield of the product and to orient the secondary metabolite pathway's in vitro culture system [33][34][35]. As the results revealed, the use of perfluorodecalin in the in situ extraction system improved the performance of the cells' culture as well as increased the production of targeted molecules [36,37].…”