**4. A critical look at biotechnological interventions to ginsenoside bioproduction**

The conventional approaches for ginsenoside pruning from natural populations or production using the classical agricultural systems can be time-consuming and/or not feasible, and thus it has paved the way for the development of various biotechnological approaches, which would ameliorate the productivity of ginsenosides. Plant tissue culture proved to be an important tool for the continuous production of bioactive compounds that are specialized metabolites in most of the instances. However, the notion of productivity is essential here, and it is far more significant than production. Naturally, secondary metabolites (*aka* specialized metabolites) are produced from primary metabolites (such as carbohydrates, lipids, and amino acids) that are required for plant growth and development. The key concern is that if the primary metabolites are involved too actively for the biosynthesis of a specific class of specialized metabolite, plant growth and development may deteriorate

*How Do Extraction Methods and Biotechnology Influence Our Understanding and Usages… DOI: http://dx.doi.org/10.5772/intechopen.103863*

eventually. As a result, high productivity collectively defined as "*biomass x production yield of bioactive specialized metabolite"* is significantly more desirable for efficient and continuous output of specialized metabolite at the industrial level.

For this purpose, *in vitro* systems (**Figure 3a**) for various plant species have been developed over the last decades, including undifferentiated cell cultures like a callus and cell suspension, as well as differentiated organ cultures like adventitious root and hairy root, with widely disparate results in terms of biomass production and/or ginsenoside accumulation, as recently reviewed by Gantait et al. [18].

The most notable results of these various plant biotechnology techniques are critically reviewed below, along with some perspectives:

1.During a critical evaluation of the analytical procedures developed for the extraction of ginsenosides, we observed that only a limited number of *Panax* species, as well as a small number of different ginsenosides, have been thoroughly investigated with the aid of biotechnological methods to date.

#### **Figure 3.**

*a. Flowchart depicting the main biotechnology approaches that have been developed using the various plant in vitro culture systems; b. Comparison of the total ginsenoside production (GS) obtained using various plant in vitro culture systems (callus, cell suspension, adventitious root and hairy root compared to naturally grown rhizome). \* production done in a bioreactor; DW: dry weight. Pg: P. ginseng; Pq: P. quinquefolius; Pn: P. notoginseng; Pv: P. vietnamensis [55–82].*


*How Do Extraction Methods and Biotechnology Influence Our Understanding and Usages… DOI: http://dx.doi.org/10.5772/intechopen.103863*

appealing option for redirecting ginsenoside biosynthesis [77]. It should be emphasized that elicitation has usually resulted in a reduction in growth [76].

8.Too infrequently, detailed metabolic investigation of the ginsenoside accumulation patterns has been investigated. The majority of investigations focused on total ginsenoside content or a limited number of ginsenosides. The study by Ha et al. [83] on the hairy roots of *P. vietnamensis* nicely demonstrated the benefits of in-depth LC-MS characterization for the discovery of unique accumulation patterns.

The current understanding of ginsenoside biosynthesis and regulation paves the way for metabolic engineering strategies to be developed [17]. For this purpose, in addition to the plant *in vitro* cultures, the microbial biosynthesis of ginsenosides from renewable resources may be a viable alternative technique for meeting the ever-increasing demand for ginsenosides in recent years [16]. Microbes have several benefits over plant cells, including the need for less area for growth, the ability to grow quickly with high cell density culture, the ability to regulate and describe genetics, and the ability to manipulate genetics. Yeasts, particularly *Saccharomyces cerevisiae*, are well-known as eukaryotic model organisms for the creation of highvalue compounds with complex structures. In recent years, alternative approaches for ginsenoside production have been developed using the model yeast *Saccharomyces cerevisiae* and non-conventional yeasts such as *Yarrowia lipolytica* and *Pichia pastoris* [16].

### **5. Conclusions**

**"**What you see is what you extract" remarked Y.H. Choi and R. Verpootre [84]. This is especially true for ginsenosides. Most extraction methods continue to focus only on the major bioactive ginsenosides, although more holistic approaches to extraction-based research would substantially increase our understanding of the biological activities of this family of natural products. As critically discussed in the present chapter, ginsenosides may not have provided their full potential as medicinal resources due to a global lack of effective technologies for ginsenoside extraction and/or production.

The majority of the extraction procedures involve the most common bioactive components only (i.e., PPD-type ginsenosides: Rg3, Rb1, Rb2, Rc, and Rd; and PPT-type ginsenosides: Rg1, Re, and Rg5) from a limited number of *Panax* species (*P. ginseng* and *P. quinquefolius* mainly). On the contrary, some species, like *P. sokpayensis* and *P. stipuleanatus*, have received little attention. Additional bioactive components may be found using bioactivity-oriented separation methods. Further research will be needed to understand the molecular and cellular processes, toxicity using cellular and animal models, and clinical applications of less-studied ginsenosides. This would allow for more in-depth research of the structure-activity relationships of ginsenosides, which would provide important insights into the development of a *Panax* quality control method, based on faster and more accurate analytical procedures. In addition, the development of more effective holistic strategies vis-a-vis more specific targeted extraction procedures would go a long way toward ensuring that the *Panax* species continues to reveal new secrets. It is feasible to generate richer extracts through a more precise extraction strategy (for example, using NaDES combined with ultrasonic or high-pressure extraction) and then fractionate this extract with much more specific extraction methods for certain classes of ginsenosides (e.g., with bio-imprinted polymers).

Biotechnological production of different ginsenosides using *in vitro* cultures has not been thoroughly investigated to date, nor have quantitative analyses of less common ginsenosides been undertaken. Although, there have been several publications on cell suspension cultures and bioreactors, the use of elicitors has to be investigated more often, using omics technologies (metabolomics and transcriptomics) to provide full insight, since these compounds may have a substantial influence on ginsenoside biosynthesis. Recently, the microbial cell factory has been proposed as a source of the production of main ginsenosides, for which biosynthetic genes have been isolated. In this sense, plant and microbial biotechnology approaches are complementary: plant can reveal new structures, in particular, using elicitation coupled to omics studies and allow the identification of new genes that can then be used in both plant metabolic engineering or microbial synthetic biology approaches.

*Panax* species have been widely employed in traditional medicine and are known to have pharmaceutical uses. Ginsenosides have only recently been studied, owing to advances in analytical methods since the first comprehensive phytochemical descriptions in the 1970s. The current surge in the application of advanced technologies, such as HR-MS, has enabled the discovery of an increasing number of ginsenoside structures. These unique structures have not yet been explored due to their most recent discovery, and a lack of availability of adequate quantity.
