4.2. In Vitro Propagation 

In vitro propagation, or micropropagation is a variant of the vegetative mode of propagation accomplished using plant-derived explants cultivated under aseptic in vitro conditions [87]. It offers the possibility of producing a large number of plants that can be explored for the extraction of valuable metabolites while reducing the over-exploitation of wild and endangered species [88]. The use of differentiated plantlets (micro-propagated plants) is mandatory when the bioactive molecule is exclusively produced in specialized plant organs or tissues (e.g., essential oils). Another advantage of the use of in vitro-propagated plants is associated with their stability and higher yields of secondary metabolites. The use of in vitro culture systems allows for production independent of seasonal constraints and the rapid and efficient isolation of the targeted bioactive molecule, along with the reliability and predictability of production [25].

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

Click On cistanche Chemist warehouse

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】

Some studies have pointed out the efficiency of in vitro propagation in terms of the production of bioactive compounds. Goyal et al. (2013) found that lowbush blueberry clones obtained through micropropagation displayed higher flavonoid and phenolic contents compared to those developed using the conventional method of propagation [89]. Similar findings were reported in Ziziphora senior L. by Dakah et al. (2014) [90]. The authors found that in vitro-propagated plant extracts of Ziziphora senior L. showed higher radical scavenging ability than wild plants. They also explained this noticeable difference by the stress conditions that arise through the in vitro culture establishment or the presence of plant growth regulators, which can have a stimulatory effect on polyphenol production [90]. Huperzia serrata, an important traditional herb in Chinese culture, is known to produce a valuable compound, Huperzine A (HupA). Huperzia serrata extracts obtained from micro-propagated plants displayed increased antioxidant activity. However, the production of HupA remains lower in micro-propagated plants than in wild ones. Hypericin content subsequently increased in Hypericum hookerianum micro-propagated plant extracts in comparison with the extract generated from wild plants [91]. In the in vitro culture of Salvia officinalis, abietane diterpene was only detected in shoot cultures but not in cell suspensions, calluses, or hairy roots [92]. Despite the above, in some cases, the phenolic composition and antioxidant activity can be lower in micro-propagated plants compared to wild ones, such as in Cichorium pumilum Jacq [93], Caralluma tuberculata [94], and Alocasia longiloba Miq [95].

4.3. Callogenesis and Cell Suspensions

Plants show noteworthy developmental plasticity for cell differentiation, as it is the main feature of plant cells. Due to this outstanding property, plants can form unorganized cell masses, referred to as calluses, in response to environmental constraints, most likely pathogen invasion or physical damage [96]. Callus culture establishment relies mostly on the dedifferentiation of cells. This can be defined as the process by which mature or specialized cells lose their differentiated character and become juvenile (dedifferentiated) [97].

Through their transfer to a liquid medium, callus culture clumps can desegregate into small pieces, aggregates, or even single cells, whereby cell suspension cultures are achieved. A callus is typically heterogeneous. Cell suspensions are a potential source of high-value plant-derived bioactive compounds [97,98]. Cell suspensions encompass a homogeneous cell population, which produces uniform and rapid nutrient and plant growth regulators. They also easily accommodate several biotechnological strategies such as elicitation, precursor feeding, and bioconversion or biotransformation, as well as mass production in bioreactors (scale-up) [7]. Several important plant-derived bioactive compounds have been produced using callogenesis and cell suspension technologies,  whereby the majority were obtained using cell suspensions [98]. The main PDBCs that have been produced using cell suspensions are Echinan 4 P, Acetos 10 P, Teoside 10, and Teupol 50 P [99,100].

Many studies have reported the efficacy of cell suspensions for producing desired bioactive compounds. For example, ginsenoside production was obtained through Panax quinquefolium cell suspensions developed in an MS medium in the presence of 1 mg/L of 2.4-Dichlorphenoxyacetic acid and 0.25 mg/L of Kinetin [101]. Shikonine production was assessed from cell suspensions of Onosma bublotrichum in an MS medium supplemented with 0.2 mg/L of IAA and 2.10 mg/L of Kinetin for calluses and in an SH medium for cell suspensions [102]. Glycyrrhiza uralensis cell suspensions were able to produce significant amounts of flavonoids in a Murashige and Skoog medium supplemented with a combination of 2,4-D, NAA, and BA and elicited with methyl jasmonate [103]. 20-  hydroxyecdysone was obtained from both Achtranthes bidentate and Vitex glabrata cell suspensions grown in the presence of both NAA and 0.2 mg/L of BA for Achtranthes bidentate and the presence of 2,4-D and BA for Vitex glabrata [104].

Several previous studies have underlined the great potential of callus and cell cultures in the treatment of skin disorders. Dilochos biflorus stem cell culture-derived hydrosoluble extract was characterized by Belmonte et al. (2014) for its high amount of isoflavones,  mainly daidzein, genistin, and their glucosidic derivatives. The authors found that the generated extract showed a noticeable inhibitive action of UV-induced erythema, which highlighted the protective effects of these plant-derived compounds against UV radiation,  specifically against sunburn and solar erythema [105]. Later, Imparato et al. (2016) used skin artificial models to demonstrate the UV protection capacity of Dilochos biflorus cell culture extracts on ECM components [106]. This outstanding dermo-protective activity was linked to the extract’s ability to scavenge free radicals, inhibit collagenase production on the dermis, and preserve collagen structure for up to 72 h after UVA radiation exposure [106]. Butterfly bush (Buddleja davidii) extracts obtained using cell suspension cultures produced high amounts of verbascoside, a phenylpropanoid glycoside compound known for its versatile protective properties (antioxidant, chelator, anti-inflammatory). Exploring the dermatological properties of the generated extracts showed the strong skin repair capacity and skin inflammation preventative action of this extract, attributed to the strong inhibition of collagenase activity and the repression of pro-inflammatory factors [107]. Bengal coffee (Coffea bengalensis) plant cell culture extracts are caffeine-free and displayed great potential for use in skin care. For instance, it was shown that the hydrosoluble extract derived from Coffea bengalensis cell cultures prompted collagen I and II syntheses in fibroblasts,  promoted lipase activity, and stimulated the expression of hydration-related genes in the keratinocytes [108].

5. Main Biotechnological Approaches to Increasing the Production of Plant-Derived Bioactive Compounds

Plant cell and tissue culture (PCTC) provide a promising biotechnological tool for generating a broad number of phytochemicals for pharmaceutical purposes. However,  only some successful cases are available on the market due to minimal phytochemical productivity, which is insufficient to cover the culture costs [76]. Thus, during the last decade, research has been oriented towards enhancing the production of high-value phytochemicals without increasing the production costs to scale up the use of in vitro culture techniques as “chemical factories” [109]. Several strategies, among which are elicitation,  metabolic engineering, immobilization, permeabilization, and two-phase systems, have been broadly used to increase the production of PDBCs (Figure 2) [77].

5.1. Elicitation 

Elicitation is one the most efficient procedures applied nowadays to improve the biotechnological production of PDBCs. Elicitation requires the use of specific compounds,  commonly known as elicitors, to prompt plant defense and trigger secondary metabolite biosynthesis and production [110]. Two distinct types of elicitors can be distinguished:  abiotic and biotic elicitors. Abiotic elicitors gather all non-biological substances, such as inorganic compounds, for example, metal ions or salts (calcium chloride, silver nitrate,  magnesium sulfate, mercury chloride, cobalt chloride, zinc ions, etc.), known to stimulate the production of bioactive substances through their plant secondary metabolism adjustment [43]. Unlike abiotic elicitors, biotic elicitors have a biological origin. They are either used as crude extracts or partially purified pathogen or plant-derived products. They can be either of a complex composition, such as fungus and yeast extracts, or a specific composition such as glycoproteins, purified chitosan, alginate, xanthan, polysaccharides,  etc. [111]. Several parameters, among which are the elicitor type, concentration, time of exposure, culture type, medium composition, cell line, stage, and age of the culture, are the main factors affecting the efficiency of the elicitation procedure in PDBC production [112].

Elicitation has been widely used to increase PDBC production in vitro cultures. Several reports have underlined the efficiency of this method. The elicitation of Pueraria cannoli suspension cells using salicylic acid enhanced isoflavonoid production and accumulation, more specifically, khwakhurin, daidzein, puerarin, and genistin, which are molecules that display great anti-aging properties [57]. In Solanum xanthocarpum, callus culture elicitation using blue light resulted in a peak production of methyl-caffeate, esculetin, caffeic acid, and scopoletin. These molecules are known for their great antioxidant,  anti-inflammatory, antidiabetic, and anti-aging activity [113]. The NaCl-induced salt stress application to a cultivated cardoon (Cynara cardunculus L. var altilis) callus increased the total phenolic and antioxidant content, which resulted in an increase in pro-collagen and aquaporin production in dermal cells, thereby boosting the production of bioactive compounds that can be used for cosmetic formulations [114]. Methyl jasmonate elicitation applied to Isatis indigotica hairy root cultures demonstrated outstanding results in the production of lignans. It also allowed for the discovery of AP2/ERFs TFs that have been implicated in the production of this class of bioactive compounds, as well as upregulated biosynthetic genes, which underlines the importance of eliciting in the identification of key regulatory mechanisms that can be used for metabolic engineering in vitro cultures [115]. Other examples of the efficacy of eliciting the stimulation of PDBC production are shown in Table 3.

5.2. Precursor and Nutrient Feeding

Precursor feeding is a biotechnological strategy that depends on the ability of plants and plant cell cultures to convert precursors (supplemented by the media culture) into desired products using pre-existing enzymes [135,136]. This technology has been broadly employed to trigger the production of specific compounds. For instance, numerous reports have demonstrated the efficiency of precursor feeding in the stimulation of PDBC  synthesis. Linum album hairy root cultures fed with a known lignan precursor, conifer aldehyde, resulted in a considerable increase in pinoresinol, lariciresinol, and podophyllotoxin production [137]. In Centella asiatica leaf-derived callus and cell suspensions, asiaticoside accumulation was achieved by the addition of amino acids to the culture media, more precisely, leucine [138]. Karppinen et al. (2007) reported similar findings for hyperforin production from Hypericum perforatum shoot cultures. For instance, the authors found that the administration of isoleucine and valine to the shooting culture was responsible for the production of hyperforin. By tracking the insertion of isoleucine and valine using labeled forms of these amino acids, the authors discovered that these two amino acids were incorporated into the acyl side chain of both hyperforin and hyperforin [138].

Following the same principle as precursor feeding, nutrient feeding aims to increase the yield of PDBCs by adjusting the physical and chemical factors of the culture media. This strategy was proven to be effective for biomass enhancement and the production of ginsenoside from ginseng adventitious root cultures. As reported in [139], biomass production and ginsenoside amounts increased when the culture was replenished with a  freshly prepared culture medium. Similar findings were also reported for the production of caffeic by-products from Echinacea purpurea adventitious root cultures [140] and taxol production from Taxus chinensis cell suspensions [141].

5.3. Metabolic Engineering

Metabolic engineering is defined as the production of specific substances or molecules,  such as pharmaceuticals, chemicals, fuels, and drugs, by disrupting the metabolic pathways in cells [142]. It gives a brand-new standpoint to better understand the PDBC biosynthesis routes through overexpression studies. It can also imply the repression of other pathways (competitive pathways) to enhance the metabolic flow of the specific biosynthesis route mediators to ensure elevated production [143]. The main objective of this strategy is to prompt cellular activity through the manipulation of cell functions using recombinant DNA  technology. So far, several strategies, such as the introduction of genes isolated from the same species or different organisms, promoters enhancing target gene expression (constitutive expression of targeted genes using 35S promoter, for example), or disruptive expression of targeted gene or genes (antisense, RNA interference, or CRISPR/Cas9 technologies), have been used to achieve this purpose [144]. The most common example of genetic manipulation is the use of Agrobacterium tumefacient-mediated genetic transformation, which can allow for the introduction of the desired gene.

The genetic disruption of biosynthesis pathway intermediates can also be conducted using other alternative transformation methods such as protoplast transformation, biolistics (microprojectile bombardment), liposome-mediated transformation, or pollen-tube pathways [143]. Metabolic engineering offers many advantages for the scale-up production of bioactive compounds by over-expressing genes (responsible for the production of regulatory enzymes) that are involved in their biosynthetic pathways [145]. However,  given the complexity of the regulatory process in plant cells and the presence of critical and rate-limiting enzymes responsible for the feedback regulation of the abundance of bioactive compounds, the production of PDBCs through metabolic engineering is limited. Thus, additional investigations are required to identify the rate-limiting steps and their regulation [146,147].

5.4. Immobilization 

Immobilization is one of the key strategies that can be applied to enhance the production of PDBCs in PCTC systems. It relies on the use of a gel matrix that allows cell entrapment. At the same time, cells are exposed to high concentrations of ions to neutralize the undesirable impact on cell metabolism. This strategy has attracted scientists and researchers worldwide as it allows for the increase in cell viability and stability of the bioactive compounds produced, in addition to the increase in the production of desirable molecules [148]. For cell entrapment or immobilization, several chemicals, such as agarose, alginate, agar,  and polyacrylamide combined with alginate, can be used as the gel matrix. Alginate polymers are the most common substances used for cell immobilization, as they show the best results in terms of PDBC production yields. For instance, Eurycoma longifolia cell aggregate entrapment with 2.5% of alginate polymer for three weeks resulted in a substantial rise in the production of 4H-imidazol-4-one, can thin-6-one, and strictosidine-synthase compared to non-immobilized cells [149]. For chitosanase production from Gongronella sp. cells, the highest production was achieved using cell immobilization with calcium alginate (E404)  gel combined with polyurethane foam at pH 5.5 [150]. In Juniperus chinensis, Premjet et al. (2007) found that the production of podophyllotoxin increased by 96–98% in entrapped cells using an alginate polymer [151]. Plumbago rosea-immobilized cells using E404 resulted in a threefold increase in the production of plumbagin, the important bioactive compound reported in this plant species, compared to non-entrapped cells [152,153]. The beneficial effects of cell immobilization can be explained by the fact that the gel (polymer) matrix generates an appropriate diffusion gradient over the immobilized cells, which improves biochemical communication. Polymer matrices automatically trigger the establishment of cell aggregates, thereby reducing cells’ dependence on the culture media, resulting in a  higher yield of PDBCs [148]. Although cell immobilization increases PDBC production,  bioactive compounds are often entrapped and frequently stored within cell vacuoles. Thus,  the cell immobilization and production process are economically dependent on the cell’s capacity to secrete the desirable bioactive compounds into the adjacent medium, which can occur naturally using natural (passive and active transport) or artificial (permeabilization strategy) secretion mechanisms [135].

5.5. Permeabilization 

As mentioned above, PDBCs are usually entrapped in specialized organs or cell structures, usually in cell vacuoles. Hence, PDBC release into the culture medium coupled with an appropriate purification procedure can allow for the recuperation of desired compounds. The permeabilization strategy relies on the use of chemical or physical approaches to increase the permeability of plant cell membranes. The chemical-mediated permeabilization can easily be implemented using organic solvents, such as dimethylsulfoxide [DMSO] and isopropanol, and polysaccharides such as chitosan [135]. Taxol, hexadecane, dibutyl phthalate, or decanol were used to increase Taxus chinensis cell culture permeability [141]. Other permeabilization methods, such as electric fields and sonication, can be applied to recover PDBCs from cell vacuoles [135]. Note that the accumulation of PDBCs can be altered either by the feedback regulation (inhibition) of product synthesis or by the degradation of bioactive compounds in the media. This obstacle can be avoided by using in situ product removal, which involves a direct liquid–liquid or liquid–solid separation [154], where the latter showed better results than the liquid–liquid culture system. For solid–liquid systems, XAD4, XAD7 resins, and activated charcoal are commonly used. For instance, it was previously demonstrated that the use of XAD7 improved the production of ajmalicine and serpentine in C. roseus, plumbagin in Pityriasis rosea, an alkaloid in Eschscholzia californica,  and taxuyunnanine C in Taxus chinensis [155–158]. XAD4 was successfully applied to the production of anthraquinones from Morinda elliptica [159].

6. Production of Antioxidant Substances for Cosmetic Formulations Using Biotechnology

PCTC techniques in combination with different biotechnological approaches aiming to produce high amounts of PDBCs have led to the development of several cosmetic products with anti-aging and dermo-protective activity. Some of them have been patented,  and several cosmetic products have been developed by leading companies in the cosmetic industry. Below are some examples of patents that have been registered in the last decade. They were randomly selected to show concrete applications of biotechnology, mainly plant  tissue culture techniques, to the formulation of galenic and cosmetic products:

• A patent registered in the United States by Blum et al. in 2012, related to the development of dedifferentiated plant cells from Malus domestica cv Uttwiler Spaetlauber fruits and their use in the formulation of cosmetic preparations to ensure the protection of stem cells against both internal and external stress factors, promotion of stem cell proliferation, and prevention of cell apoptosis (Patent US 8,580,320 B2). From these cell suspensions, different cosmetic preparations have been developed, among which are vanishing creams, liquid balms, intensive hair masks, and eye creams. The efficiency of the developed cosmetic preparations has been tested on stem cells originating from umbilical cords, hair follicles, and fibroblasts.

• Syringa vulgaris plant cells were successfully generated from the in vitro culture of plant tissues under aseptic conditions in growth containers supplemented with specific plant growth regulators by an Italian team (Dal Monte et al., 2006; Patent number: US 7,718,199 B2). An aqueous extraction was performed on callus-derived cell suspensions. HPLC profiling revealed the presence of significant amounts of verbascoside and verbascoside. The cell-suspension-derived extracts showed strong antioxidant and scavenging activity against free radicals. Moreover, the developed extracts displayed great anti-hair-loss properties due to their capacity to inhibit 5-alpha reductase and lipoxygenase. The generated extracts also showed strong anti-tyrosinase activity and notable skin-whitening properties.

• Undifferentiated cells of Iris plants (Iris pallida, Iris germanica, and Iris florentina) were generated by Breton and Gueniche in 2001. Galenic preparations were developed from the generated cells. Following the inventors’ claims, the developed preparations included sunscreens, with active ingredients that ensured the protection of the extra-cellular matrix proteins, such as from UV radiation, through the enzymatic inhibition of MMP proteins (Breton and Gueniche in 2001, Patent number: EP 1 174 120 B1).

• Leontopodium alpinum undifferentiated cells obtained using in vitro cell cultures were used for the formulation of cosmetic preparations by French inventors (Gracioso et al.). The discovery was published as a patent by the inventors in 2016 (Patent deposition in 2015, Patent number: WO 2016/113659 A1). The developed product was proposed as a cosmetic treatment for skin-aged cell homeostasis restoration and increasing cell metabolism and energetic activity.

• Undifferentiated cells of Marrubium vulgare were used as a raw material for the development of cosmetic preparations by Ringenbach et al. for a well-known cosmetic firm. The patent was registered in 2016. For this patent, a cosmetic composition was prepared from plant cells obtained using the in vitro cell culture process. The inventors proposed this cosmetic preparation for topical treatments to improve skin’s general condition, appearance, and appendages, more precisely, for pore tightening and skin imperfections. From the active ingredient discovered, different galenic formulations have been developed, among which are creams, serums, tissue masks, and cleansing lotions (Patent number: WO 2017/163174 A1).

• A cosmetic formulation was developed by an Italian team (Tito et al.) in 2016. The invention covered by this patent focuses on the use of the somatic embryos of three plant species: Lotus japonicus, Citrus limon, and Rosa gardenia. The generated extracts have shown great action against skin-aging imperfections and contain skin-tissue rejuvenation properties (Patent number: WO 2016/ 173867 A1).

• A cosmetic product with the ability to protect skin from drying and/or prevent UV radiation damage was developed from a Camellia sinensis var assamica dedifferentiated stem cell culture extraction by Berry et al. The developed product was patented in 2017. The invention’s efficiency was tested in human adult dermal fibroblasts. According to the inventors, the generated tea extracts displayed anti-inflammatory properties,  prevented skin cell drying, and protected skin cells from UV radiation (Patent number: WO 2017/178238 A1).

7. Conclusions 

Skin aging is one of the most frequent dermatological issues affecting human skin and its appearance, resulting in wound repair failure, wrinkle development, and loss of skin tone and elasticity. Several chemical-based products have been developed over the years to prevent skin’s anti-aging process and reduce its impact. However, with the use of chemical products, several problems have arisen, mostly linked to cell sensitivity, allergies,  and the side effects of some chemical products and substances. As an alternative, natural and plant-derived products have been proposed based on their outstanding properties. However, the development of plant-derived bioactive ingredients is highly dependent on plant material, which can be affected by both intrinsic and extrinsic factors. Plant tissue culture techniques can deliver huge quantities of homogenous plant material independent of those factors to ensure the sufficient production of bioactive compounds. In addition, PDBCs can be produced using biotechnological strategies such as elicitation, metabolic engineering, nutrient and precursor feeding, immobilization, and permeabilization. This work presented a comprehensive review of the biotechnological techniques used to produce bioactive compounds, with a focus on antioxidants displaying anti-aging properties. Some examples of plant tissue culture techniques used in the production of cosmetic products are also addressed to underline the importance of biotechnological tools for the sustainable production of PDBCs.

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】