Glycyrrhizin, also known as glycyrrhizinic or glycyrrhizic acid, accumulates in the roots and rhizomes of Glycyrrhiza glabra, the classic liquorice plant, as well as related species such as G. uralensis and G. inflata. On hydrolysis it yields 18β-glycyrrhetinic acid, also called glycyrrhetic acid or enoxolone, which is itself biologically active. The molecule’s commercial and clinical portfolio is remarkably broad. Its monoammonium salts serve as high-intensity sweeteners, and it is the chemical precursor for carbenoxolone, a hemisuccinyl ester derivative with mineralocorticoid activity that is marketed as an antiulcer drug. The review emphasizes that pharmacological studies have documented antiviral activity against hepatitis B and influenza A/H1N1 viruses, anti-inflammatory effects demonstrated through albumin denaturation assays and in ulcerative colitis, anticancer activity in colorectal cancer cell lines SW620 and HT29, and growth suppression of A549 lung adenocarcinoma cells through inhibition of thromboxane synthase.

Clinical evidence further strengthens the case for scaling up production. Glycyrrhizin has been tested alone and in combination therapies for chronic hepatitis C, including in interferon-resistant patients, where injection therapy has been associated with reduced hepatocellular carcinogenesis. A randomized placebo-controlled trial explored glycyrrhizic acid as an adjunctive treatment for depression through anti-inflammatory mechanisms. Against this backdrop of expanding medical relevance, the natural content of glycyrrhizin in even the richest licorice roots remains very low, and wild plants typically need three to four years of growth before harvest, creating a pressing supply problem that the review’s authors set out to address systematically.

Understanding the biosynthetic pathway is the foundation of every enhancement strategy. Glycyrrhetinic acid is an oleanane-type triterpenoid built through the mevalonate pathway from repeated units of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The pivotal first committed step is the cyclization of 2,3-oxidosqualene into β-amyrin, catalyzed by β-amyrin synthase. From there, sequential oxidation reactions driven mainly by the cytochrome P450 enzymes CYP88D6 and CYP72A154 generate a series of intermediates that culminate in glycyrrhetinic acid. The glycoside is then assembled by glycosyltransferases: UDP-glucose dehydrogenase converts UDP-glucose to UDP-glucuronic acid, and the enzyme UGT73P12 transfers a glucuronic acid moiety to the third carbon of glycyrrhetinic acid, forming glycyrrhetic acid 3-O-mono-β-D-glucuronide, or GAMG. A second glucuronosylation event on GAMG completes glycyrrhizin, with UDP-glucose pyrophosphorylase, known as UGP1, regenerating the UDP-glucose donor.

The most extensively explored strategy for boosting glycyrrhizin is elicitation, in which cultured plant tissues are deliberately stressed with biotic or abiotic agents to switch on secondary metabolism. The earliest in vitro attempt, by Shabani and colleagues in 2009, treated Glycyrrhiza glabra cultures with methyl jasmonate and salicylic acid at concentrations from 0.01 to 2.0 millimolar. Glycyrrhizin peaked at 0.1 millimolar methyl jasmonate after eight hours, while salicylic acid raised content up to 1 millimolar but depressed it at 2 millimolar, an early demonstration that elicitor dose and timing follow narrow windows of benefit. In 2010, Karwasara and colleagues extended the approach to cell cultures of Abrus precatorius, a related legume, finding that culture filtrate of Aspergillus niger at 7.5 percent and dried cell powder of Rhizopus stolonifer at 0.5 percent, together with yeast extract and 50 micromolar ascorbic acid, maximized both biomass and glycyrrhizin accumulation.

Subsequent work has diversified the elicitor toolkit considerably. Srivastava and colleagues subjected Agrobacterium rhizogenes-induced hairy roots of G. glabra to drought-mimicking PEG 6000, the heavy metal cadmium chloride, and the biotic elicitors cellulase and mannan. Polyethylene glycol at 1 percent produced the highest glycyrrhizin after 24 hours, cellulase at 200 micrograms per milliliter progressively raised content over seven days, and remarkably the lowest mannan dose of 10 milligrams per liter yielded the peak concentration of 3.3089 micrograms per milligram after ten days. In Taverniera cuneifolia root cultures, Awad and colleagues screened six fungal and five bacterial elicitors and found that Rhizobium leguminosarum drove glycyrrhizic acid to 6 milligrams per gram, while methyl jasmonate at 100 micromolar delivered a 2.5-fold increase. Jaiswal and colleagues, meanwhile, showed that adenine sulphate outperformed biotin, salicylic acid and polyamines in G. glabra callus, reaching 35.44 micrograms per gram, likely because sulphate assimilation supports amino acid and metabolite synthesis.

Microbial partners and physical stimuli have added further dimensions. Li and colleagues raised glycyrrhizin in adventitious roots of G. uralensis with low-dose salicylic acid, optimal sucrose at 4 percent and one-strength MS salts, and later showed that Aspergillus niger treatments in 5-liter balloon-type bubble bioreactors increased glycyrrhizin up to 200 milligrams per liter of elicitor, though higher doses reversed the effect. Xie and colleagues reported that the plant growth-promoting bacterium Bacillus pumilus, inoculated into drought-stressed G. uralensis plants, reduced antinutritional factors, improved protein digestibility and boosted antioxidants, cumulatively enhancing glycyrrhizin biosynthesis. In G. inflata hairy roots, methyl jasmonate at 100 micromolar produced 5.7 times more glycyrrhizin than controls after five days, whereas chitosan proved ineffective. Allahdou and colleagues found cellulase from Aspergillus niger at 200 micrograms per milliliter optimal for both glycyrrhizin and glycyrrhetinic acid in G. glabra hairy roots, and Afsharzadeh and colleagues combined hairy root transformation with red and blue LED light, recording antioxidant capacity gains over 55 days of exposure. Most strikingly, Yamamoto and colleagues documented overwhelming glycyrrhizin induction through symbiosis with the nitrogen-fixing rhizobium Mesorhizobium sp. J8, which elevated chlorophyll, nitrogenase activity and the expression of genes for glycyrrhizin and jasmonic acid synthesis.

Beyond elicitation, the review highlights genome editing as a fundamentally different lever. Chiyo and colleagues in 2023 used CRISPR/Cas9 in G. uralensis hairy roots to knock out the genes CYP93E3 and CYP72A566, which divert flux toward soyasaponins, along with CYP716A179 for oleanolic acid and LUS1 for betulinic acid. By eliminating these competing branches of β-amyrin metabolism, more precursor was channeled toward glycyrrhetinic acid and ultimately glycyrrhizin. When pathway blocking was paired with overexpression of CYP88D6, the oxidation step toward glycyrrhetinic acid, glycyrrhizin production rose again. Although the absolute gains were modest, the authors note that the results were achieved within a single month of culture, a dramatic contrast to the three to four years required by wild licorice, illustrating how pathway rationalization could compress production timelines.

The third pillar is heterologous biosynthesis in microbes, which began when Zhu and colleagues engineered 11-oxo-β-amyrin and glycyrrhetinic acid synthesis into Saccharomyces cerevisiae in 2017. In 2019, Wang and colleagues integrated codon-optimized CYP88D6 and CYP72A154, together with β-amyrin synthase and an Arabidopsis thaliana NADPH-cytochrome P450 reductase, into the yeast chromosome, producing a strain that yielded 2.5 milligrams per liter of β-amyrin and 14 micrograms per liter of glycyrrhetinic acid. Introducing a cytochrome b5 from G. uralensis multiplied glycyrrhetinic acid output eightfold, and combining both strategies achieved a 40-fold improvement to 0.5 milligrams per liter in batch fermentation, extended to a 630-fold improvement reaching 8.78 milligrams per liter in fed-batch mode. The review cautions that plant P450 enzymes often show reduced catalytic activity in yeast and can interact poorly with reductases, generating reactive oxygen species that impair cell growth and product yield.

Taken together, the review paints a picture of a field converging on integrated solutions. Every elicitation regime, whether chemical, microbial or physical, works by provoking the plant’s in vitro defense machinery, and every approach shows a concentration ceiling beyond which toxicity erases the gains. The glycyrrhizin pathway is multifaceted and compartmentalized, limiting metabolite flux, and many of its genes and regulators remain unknown, while gene editing can sometimes merely redirect accumulation into other unintended metabolites. Even so, the authors conclude that the enhancements already demonstrated carry genuine commercial value, both for glycyrrhizin and its carbenoxolone derivative, and that the same elicitation, editing and engineering playbook is readily transferable to other plant secondary metabolites, positioning licorice biotechnology as a template for the sustainable production of high-value natural products worldwide.

Subject of Research: Biotechnological strategies for enhancing glycyrrhizin biosynthesis in licorice and engineered microbes

Article Title: An overview of strategies used for increasing Glycyrrhizin biosynthesis

Article References: Patil, S. P., Patil, R. R., Kore, S. D., & Raut, M. K. (2026). An overview of strategies used for increasing Glycyrrhizin biosynthesis. Discover Industrial Chemistry and Materials, 1(1), Article 7. https://doi.org/10.1007/s44508-026-00008-9

Image Credits: AI Generated

DOI: 10.1007/s44508-026-00008-9

Keywords: glycyrrhizin, licorice, Glycyrrhiza glabra, elicitation, CRISPR, Saccharomyces cerevisiae, secondary metabolites, metabolic engineering, hairy root cultures, triterpenoid saponin, glycyrrhetinic acid, plant tissue culture

The problem begins with how C. asiatica is currently sourced. Wild harvesting remains the dominant supply model, and the review documents staggering variability: centelloside content in wild populations fluctuates up to five- to ten-fold depending on geography, environment, and harvest timing. Because the plant naturally favors swamp and marsh ecosystems, wild-collected material faces elevated risks of heavy metal contamination, pathogen exposure, and adulteration. Field surveys in Peninsular Malaysia found significant accumulation of cadmium, copper, nickel, lead, and zinc in wild-harvested gotu kola, with estimated daily intakes suggesting potential lead toxicity risk from plants gathered at polluted sites. Standards bodies such as the World Health Organization expect high-quality herb to contain at least two percent triterpene saponins, while the European Scientific Cooperative on Phytotherapy reports saponin and sapogenin content ranging from one to eight percent—a spread that makes quality control a persistent headache for phytopharmaceutical manufacturers.

Cultivation has not solved the problem either. The review highlights a paradox familiar to anyone working with medicinal plants: more biomass does not mean more medicine. Unlike conventional crops where yield predicts output, C. asiatica can produce lush growth while delivering disappointingly dilute metabolite profiles. Multiple factors shape both growth and centelloside accumulation, including cultivation system, propagation method, light regime, genotype, soil type, farming practice, and even the ploidy status of planting material. Researchers exploring aquaponics, co-cultivation with the root endophyte Piriformospora indica, and polyhouse cultivation of rooted cuttings have reported cultivar-specific differences and dynamic metabolite trends, reinforcing the need for extensive genotypic screening before any field program can deliver consistent quality.

This is where plant tissue culture enters the picture. Callus cultures initiated from leaves, petioles, and nodal segments using auxins such as 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid, often paired with cytokinins like benzylaminopurine, have reliably produced triterpenoid- and flavonoid-rich biomass. Cell suspension cultures and hairy root cultures—induced through transformation with Agrobacterium rhizogenes—have emerged as the most scalable platforms because they grow rapidly in liquid media, remain genetically stable, and do not require exogenous hormones. Notably, Baek and colleagues demonstrated that petiole-derived hairy roots produced 1.4 times more triterpenoids than leaf-derived lines, a reminder that even the choice of starting explant can decisively shape biosynthetic output. In suspension cultures, asiaticoside accumulation peaked at 1.7-fold above baseline between 21 and 25 days of culture.

The heart of the review is a systematic comparison of elicitation strategies, and the numbers are striking. Methyl jasmonate, the field’s workhorse elicitor, increased asiaticoside by 494 percent in cell suspensions, by 5.6- to 71-fold in hairy roots, and by 69-fold in callus, depending on genotype and treatment conditions. Coronatine delivered 116 milligrams per gram dry weight of madecassoside in elicited hairy roots at day 14 post-elicitation—one of the highest absolute yields ever reported. But the authors issue a crucial warning about the so-called fold-increase paradox: a two-fold rise from a 20 milligram per gram baseline yields 40 milligrams per gram, which is pharmaceutically far more relevant than a 50-fold rise from 0.1 milligrams per gram yielding just 5. Tetraploid hairy roots responded more dramatically to methyl jasmonate than diploid lines, likely because their untreated controls were extremely low to begin with. Heavy metal elicitors such as cadmium and lead could drive 24- and 49-fold increases in asiaticoside and madecassoside respectively, but the authors dismiss these as unusable for pharmaceutical production because of phytotoxicity and contamination risk.

Among biotic elicitors, the toolkit is expanding rapidly. Yeast extract delivered a 3.5-fold boost in asiaticoside, chito-oligosaccharide achieved a five-fold increase in hairy roots at 30 parts per million, and pectin raised asiaticoside content by 31 percent in callus cultures. Endophytic symbionts add another layer of sophistication: Piriformospora indica colonization triggered a 2.5-fold increase in asiaticoside through activation of root-associated stress responses, while rhizobacteria such as Azospirillum and Pseudomonas promote triterpenoid biosynthesis by stimulating jasmonic acid and ethylene signaling pathways. Combined elicitor treatments—methyl jasmonate plus salicylic acid, or coronatine plus methyl jasmonate—consistently outperform single agents, suggesting synergistic activation of jasmonate- and salicylate-dependent signaling, though optimal ratios and staged application sequences remain largely untested.

Beyond elicitation, the review maps several emerging enhancement strategies. Precursor feeding with squalene at 2.5 micromolar boosted total triterpenoids 3.1-fold to 57.53 milligrams per gram dry weight, while higher concentrations triggered feedback inhibition—a classic concentration-dependent regulatory signature. Pyruvic acid supplementation increased triterpenoids 1.9-fold with preferential enhancement of madecassoside. Cell permeability enhancement through ultrasound-assisted extraction has proven remarkably effective: optimized conditions yielded 83.14 milligrams per gram of asiatic acid and 19.71 milligrams per gram of asiaticoside, and combining ultrasound with natural deep eutectic solvents pushed asiaticoside recovery to 229.92 milligrams per gram. Reversible electroporation could theoretically enable repeated, non-destructive metabolite harvesting from viable cultures, transforming batch processes into semi-continuous bioreactor-compatible systems—though this remains untested in C. asiatica.

The genomic era is now catching up with the chemistry. A haplotype-resolved genome assembly published in The Crop Journal confirmed that CaCYP716C11 catalyzes the conversion of 23-hydroxyursolic acid to asiatic acid, and identified CaUGT73CL69 as a glucosyltransferase that converts asiatic acid and madecassic acid to their respective monoglucosides. Tandem duplicate clusters of CaUGT73 genes on chromosome 8 reveal that gene duplication and neofunctionalization have shaped the plant’s glycosylation capacity. Earlier transcriptomic work flagged CaHDR1, CaIDI2, and CaβAS1 as key regulators, while UGT73AH1 and the glycosyltransferases CaUGT73C7 and CaUGT73C8 appear to catalyze the rate-limiting steps that assemble the characteristic sugar chains of asiaticoside and madecassoside. Yet the review’s authors caution that transcript abundance establishes correlation rather than causation, and no peer-reviewed study has yet reported CRISPR/Cas9-mediated editing of centelloside biosynthetic genes in this species.

Scale-up remains the field’s stubborn bottleneck. A 5-liter stirred bioreactor achieved 60.08 milligrams per gram dry weight of asiaticoside with optimized agitation and aeration, while a Plantform temporary immersion system combined with methyl jasmonate elicitation delivered centelloside levels 2.8-fold higher than elicited shake flasks—and 12.2-fold higher than untreated controls. A twin-bottle temporary immersion system more than tripled biomass compared to conventional semi-solid culture, though it did not quantify centellosides. Hairy root cultures, despite their biosynthetic promise, resist scale-up because their dense branching architecture and sensitivity to mechanical stress limit mass transfer. The review identifies mist reactors, wave-mixed bioreactors, and low-shear stirred tanks as untested but promising alternatives. On the translational front, preliminary estimates suggest bioreactor production only becomes economically competitive when centelloside yields exceed 5 percent dry weight and process volumes surpass 500 liters—thresholds that current elicited cultures approach but rarely achieve.

Perhaps the most forward-looking suggestion concerns extracellular vesicles. Membrane-bound nanoparticles secreted by C. asiatica cell cultures have recently been characterized and shown to carry high levels of polyphenols, reduce intracellular reactive oxygen species, suppress pro-inflammatory genes such as COX2, and promote skin repair by inhibiting tyrosinase activity and upregulating barrier-related genes including filaggrin and aquaporin-3. These vesicles outperformed conventional cell culture extracts in stability, cellular uptake, and precision. Because centellosides are packaged during vesicle biogenesis from the endomembrane system, elicited suspension cultures may serve as a platform for generating centelloside-enriched vesicles directly—skipping the extraction step entirely. Combined with the review’s proposed research framework, which prioritizes complete pathway elucidation, enzyme characterization, and systems-level regulatory mapping before engineering intervention, the picture that emerges is one of a field standing at an inflection point: the biological machinery is increasingly understood, the culture platforms are proven, and the remaining gaps—functional validation of candidate genes, standardized elicitor dosing, and validated industrial-scale bioprocesses—are now clearly defined targets rather than open questions.

Subject of Research: Centelloside biosynthesis enhancement in tissue cultures of Centella asiatica

Article Title: Advancements on centelloside biosynthesis in tissue cultures of Centella asiatica (L.) Urban

Article References: Hundare, A., & Joshi, N. (2026). Advancements on centelloside biosynthesis in tissue cultures of Centella asiatica (L.) Urban. Discover Plants, 3(1), Article 396. https://doi.org/10.1007/s44372-026-00867-8

Image Credits: AI Generated

DOI: 10.1007/s44372-026-00867-8

Keywords: Centella asiatica, centellosides, asiaticoside, madecassoside, plant tissue culture, hairy root cultures, elicitation, methyl jasmonate, bioreactor, metabolic engineering, CRISPR, extracellular vesicles

With the world facing mounting challenges in sustainable agriculture and natural resource management, the need for effective biotechnological solutions has never been more crucial. Hairy roots, known for their vigorous growth and high capacity for secondary metabolite production, offer a versatile platform for studying plant physiology, biochemistry, and genetics. The authors emphasize that harnessing the potential of hairy roots could pave the way for significant breakthroughs in crop improvement, plant-based pharmaceuticals, and even bioremediation strategies.

Historically, the exploration of hairy roots began in the mid-1980s when researchers discovered that certain strains of Agrobacterium could induce these peculiar structures in a wide range of plant species. This discovery marked a paradigm shift, transitioning from traditional propagation methods to innovative techniques that facilitate genetic manipulation and compound production. The methodology proposed by Stepanova and colleagues advances this legacy by providing a systematic approach to selecting and cultivating hairy root lines with distinct biological functions.

Central to the proposed methodology is the criterion for selecting the appropriate donor plant species. The researchers detail their process of evaluating various taxa, considering factors such as growth rates, metabolite production, and overall adaptability to sterile culture conditions. This thorough selection process is critical, as the characteristics of the donor plants directly influence the viability and productivity of the hairy root cultures.

Once the donor plants are selected, the researchers move into the transformation phase, where Agrobacterium is utilized to introduce genetic material into the plant tissue. This integration of foreign genes can enhance specific traits in the hairy roots, such as improved resistance to diseases or increased levels of desired phytochemicals. The effective transformation technique not only increases the efficiency of the process but also ensures higher yields of secondary metabolites, which are of immense value in industries ranging from cosmetics to pharmaceuticals.

Post-transformation, the initiation of hairy root cultures requires careful optimization of growth conditions. The authors lay out parameters such as the composition of the growth media, light exposure, and temperature, highlighting that maintaining these conditions is essential for the successful proliferation of hairy roots. Understanding these environmental factors allows researchers to maximize the biomass yield while also prioritizing the production of bioactive compounds.

A significant aspect of this methodology is the concept of screening different hairy root lines for functional diversity. By assessing various lines, researchers can identify those with unique biosynthetic capabilities or enhanced growth characteristics. This not only aids in understanding the genetic and biochemical pathways operative within the hairy roots but also enables initiatives aimed at plant breeding and metabolite extraction.

Furthermore, the research underscores the importance of characterizing the biochemical profiles of the resulting hairy root cultures. Advanced analytical techniques, such as spectrometry and chromatography, are employed to ascertain the levels of secondary metabolites produced. This data is invaluable, providing insights into potential applications in drug development, wherein specific compounds can be isolated and tested for therapeutic efficacy.

The innovative approach presented by Stepanova and colleagues holds considerable promise for the field of synthetic biology. Given the rise of bioengineering in producing rare and valuable compounds, the ability to cultivate specific hairy root lines tailored for unique production goals could revolutionize supply chains in pharmaceuticals. Not only does this methodology foster the creation of a diverse repository of hairy root cultures, but it also aligns with the principles of sustainable development by reducing reliance on wild-harvested plant materials.

In addition to the pharmaceutical potential, the methodology allows for extensive applications in agricultural biotechnology. By creating hairy root cultures with enhanced traits, researchers can develop crops that exhibit improved stress tolerance or higher nutritional content. The adaptability of these engineered roots could lead to innovations in food security, addressing issues faced in resource-limited settings.

Another critical dimension explored in this research is the integration of molecular techniques in monitoring the genetic stability of hairy root lines over generations. The researchers stress that assessing the fidelity of these cultures is paramount to ensure consistent yield and quality. Genetic stability ensures that the desired traits are retained throughout successive cultures, reinforcing the reliability of the outputs generated from hairy roots.

Ultimately, the comprehensive methodology outlined by Stepanova, Gladkov, and Gladkova is a significant stride in the enhancement of hairy root technology. It not only offers a systematic framework for the creation and management of diverse hairy root collections but also advances the discussion on the sustainable applications of plant biotechnology. As the research community delves deeper into the complexities of plant cellular behavior, methodologies such as this will undoubtedly play a pivotal role in shaping the future of agricultural innovation and bioproduction.

In a world increasingly reliant on biotechnological advancements for solving pressing issues, the promise of hairy roots may usher in era-defining changes. The combination of their rapid growth, adaptability, and ability to produce valuable secondary metabolites makes them an indispensable asset in the quest for sustainable practices. As researchers continue to explore the vast potential inherent in these unique plant structures, the implications for global health and food security are profound, ensuring that the groundwork established by previous discoveries flourishes into actionable solutions.

As we celebrate this new research, we are reminded of the boundless possibilities that lie ahead. The innovative methodology for generating diverse hairy root collections signifies more than just scientific progress; it embodies the collaborative spirit of researchers committed to harnessing nature’s mechanisms for the betterment of humanity. In doing so, it paves the way for a brighter, greener future as we strive to align our agricultural practices with the ecological paradigms of our planet.

Thus, as we look toward the future, it’s imperative to recognize the significance of hairy roots in the larger landscape of plant biotechnology. These remarkable structures serve as a linchpin connecting the realms of ecology, industry, and sustainable development. The work of Stepanova and colleagues is not only a testament to the scientific inquiry but a call to action for all stakeholders to participate in leveraging the power of biotechnology in addressing the most pressing challenges of our time.

Subject of Research: Hairy root cultures and their application in biotechnology.

Article Title: A methodology for creating collections of different focus of hairy roots.

Article References:
Stepanova, A.Y., Gladkov, E.A. & Gladkova, O.V. A methodology for creating collections of different focus of hairy roots. Sci Nat 112, 40 (2025). https://doi.org/10.1007/s00114-025-01991-3

Image Credits: AI Generated

DOI: https://doi.org/10.1007/s00114-025-01991-3

Keywords: Plant biotechnology, hairy roots, Agrobacterium rhizogenes, secondary metabolites, sustainable agriculture, genetic stability, bioremediation, biopharmaceuticals.