Biotransformation of Platycosides, Saponins from Balloon Flower Root, into Bioactive Deglycosylated Platycosides

Platycosides, saponins from balloon flower root (Platycodi radix), have diverse health benefits, such as antioxidant, anti-inflammatory, anti-tussive, anti-cancer, anti-obesity, anti-diabetes, and whitening activities. Deglycosylated platycosides, which show greater biological effects than glycosylated platycosides, are produced by the hydrolysis of glycoside moieties in glycosylated platycosides. In this review, platycosides are classified according to the chemical structures of the aglycone sapogenins and also divided into natural platycosides, including major, minor, and rare platycosides, depending on the content in Platycodi radix extract and biotransformed platycosides. The biological activities of platycosides are summarized and methods for deglycosylation of saponins, including physical, chemical, and biological methods, are introduced. The biotransformation of glycosylated platycosides into deglycosylated platycosides was described based on the hydrolytic pathways of glycosides, substrate specificity of glycosidases, and specific productivities of deglycosylated platycosides. Methods for producing diverse and/or new deglycosylated platycosides are also proposed.


Introduction
Platycodon grandiflorum (Jacq.) A.DC., a perennial herbaceous flowering plant species [1], belongs to the family Campanulaceae, is commonly known as the balloon flower, and is used as a traditional herbal medicine for the treatment of cough, phlegm, sore throat, lung abscess, chest pain, dysuria, and dysentery [2]. In Northeast Asia, P. grandiflorum root (Platycodi radix), an edible vegetable, is widely used as a food supplement to make side dishes (seasoned balloon flower root), desserts (balloon flower root sweet), teas (balloon flower root tea), flavored liquor, and traditional herbal medicines for the treatment of pulmonary diseases and respiratory disorders, including sore throat, bronchitis, tonsillitis, asthma, and tuberculosis [2][3][4][5].
The absorption of glycosylated saponins in the gastrointestinal tract is poor. In contrast, deglycosylated saponins are more readily absorbed in the human gastrointestinal tract into the bloodstream and function as active compounds because of their lower molecular weight and greater hydrophobicity [48,49]. The biological activities of deglycosylated saponins are superior to those of glycosylated saponins [4,48]. Therefore, many studies have focused on the hydrolysis of the glycoside moieties in glycosylated saponins using chemical, physical, and biological transformation techniques [48,50]. Platycosides from P. grandiflorum detected by high-performance liquid chromatography (HPLC), liquid chromatographymass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) contain more than 4 glycosides; thus, platycosides with less than 4 glycosides are few or absent in Platycodi radix [4,8,[51][52][53][54][55][56][57], indicating that natural major platycosides are highly glycosylated saponins compared to natural ginseng saponins with 2~4 glycosides [48,49,58]. To acquire bioactive deglycosylated platycosides, hydrolysis of the glycoside moieties in natural glycosylated platycosides is required.
Reviews on platycosides have mainly focused on their pharmacological effects [2,3,53,59]. However, reviews specifically focusing on the biotransformation of platycosides have not been reported. The current review describes the transformation methods of platycosides by hydrolysis of their glycoside moieties for the production of bioactive deglycosylated platycosides. Furthermore, we propose the production of diverse and/or new deglycosylated platycosides using glycosidases.

Classification of Platycosides
Platycosides are structurally classified into platycodigenin (PDN)-, polygalacic acid (PGA)-, platyconic acid (PCA)-, methyl-PCA-, PCA lactone-, and other-type platycosides according to the chemical groups at C-4 of the aglycone sapogenins [2,3]. Platycosides are also divided into natural platycosides, including major, minor, and rare platycosides (depending on their content in Platycodi radix extract), and biotransformed platycosides. Platycosides determined as distinct peaks by HPLC-UV can be considered major platycosides, while platycosides detected by only HPLC-evaporative light scattering detector (ELSD) or LC-MS except for platycosides detected by HPLC-UV can be considered minor platycosides. In addition, residual platycosides as new unusual platycosides in nature can be defined as rare platycosides.
The antioxidant activities of platycosides have been estimated using a Trolox equivalent capacity assay or a total oxidant-scavenging capacity assay [6,8,9]. The antioxidant activities of platycodigenin-type platycosides using the Trolox equivalent capacity assay follow the order glc-PDN (one glycoside) > deapi-PD (four glycosides) > PD (five glycosides) > PD 3 (six glycosides) > PE (seven glycosides). The antioxidant activity determined using the total oxidant scavenging capacity assay follows the order platycodigenin (no glycosides) > deapi-PE (six glycosides) > PD (five glycosides) > PE (seven glycosides). Thus, the antioxidant activities of platycodigenin-type platycosides are increased by decreasing the number of glycoside residues.
The tyrosinase inhibitor assay has been used to determine whitening activity [9]. The whitening activities as tyrosinase inhibitory activities of the platycodigenin-type platycosides follow the order glc-PDN (one glycosides) > PD (five glycosides) > PD 3 (six glycosides) > PE (seven glycosides) [9]. The whitening activities of platycosides improve as the number of glycosides attached to the aglycone sapogenin decreases. The anti-inflammatory, antioxidant, and whitening activities of platycodigenin-type platycosides determined using assay kits increase as the number of glycosides attached to sapogenin decrease.

Physical Methods
Deglycosylated ginseng saponins have been prepared by steam heating, microwave heating, sulfur fumigation, and high hydrostatic pressure [48,50,71,75]. The deglycosylation of saponins using steam heating increases with increasing treatment temperature and time. However, the amount and production rate of deglycosylated saponins by steam heating are small and slow, respectively. Microwave heating is used as an efficient time-saving method to show a higher deglycosylation rate than steam heating [77]. Sulfur fumigation is a cheap method to decrease the drying time and generate new saponins but is environmentally harmful [78]. These physical methods cause structural changes in sapogenin structures by hydrolysis, dehydration, and decarboxylation, producing byproducts or new-type saponins [79,80] and they show low selectivity and have yet to be used in the deglycosylation of platycosides. However, the physical method using high hydrostatic pressure is used together with biological method such as enzymatic conversion to increase the productivity of deglycosylated saponins by increasing the activity and stability of glycosidases [71,75].

Chemical Methods
Under acidic and high-temperature conditions, glycosylated saponins such as natural ginsenosides are converted into deglycosylated saponins. Deglycosylation by acid hydrolysis is dependent on the pH, acid concentration, acid type, and treatment temperature and time. Deglycosylation increased with an increase in treatment temperature and time; however, the optimal pH is different for each saponin type [50]. Acids used in chemical transformation of saponins include acetic acid, formic acid, citric acid, lactic acid, tartaric acid, and hydrochloric acid. Acid hydrolysis causes epimerization, dehydration, and hydration reactions, resulting in the production of deglycosylated saponins and new saponins [81].
Deglycosylation via alkaline hydrolysis increases with increasing pH, pressure, temperature, and time. Alkaline hydrolysis requires strict treatment conditions because strong treatment conditions can cleave sapogenins [50]. Alkaline hydrolysis is an efficient method for the production of aglycone ginsenosides, such as protopanaxadiol and protopanaxatriol, because the production of sapogenins from natural ginseng saponins by alkaline hydrolysis has resulted in an 80% yield [82]. Therefore, alkaline hydrolysis can be applied to the production of aglycone platycosides such as PDN, PGA, and PCA. Alkaline hydrolysis also involves side reactions, such as epimerization, cyclization, and hydroxylation. The disadvantages of chemical methods are byproduct formation and the generation of environmental pollution.

Biological Methods
Physical and chemical methods for producing deglycosylated saponins have several limitations, such as low selectivity, generation of by-products, non-environment-friendly processes, and high energy consumption. To overcome these disadvantages, biological methods including enzyme conversion, cell conversion, and fermentation have been proposed [49]. The enzymatic conversion of saponins has been performed using recombinant enzymes [4,83], commercial enzymes [52,84], reagent enzymes [62,66,70], and native crude enzymes [8,85]. The conversion using recombinant enzymes showed the highest production of saponins compared to other enzymatic conversions. However, the application of recombinant enzymes in the food industry is limited. Enzymatic conversion exhibits high specificity, yield, and productivity for saponin deglycosylation. Moreover, this method is increasingly being recognized as a useful tool for structural modifications. However, it is less economical than fermentation because it requires enzyme purification and results in the loss of enzymes from cells during purification [86].
Cell conversion is the production of deglycosylated saponins by reactions using grown or washed microbial cells mixed with saponins, whereas fermentation involves cultivation of growing cells fed with saponins. Intestinal bacteria, yeast, fungi, and soil microorganisms have been used for the transformation of saponins [8,48,50,67,73]. Microbial transformation by intestinal bacteria requires expensive medium and exhibits low yield and poor productivity. Microorganisms isolated from soil-cultivated saponin-containing plants exhibit higher yields and productivity than intestinal bacteria [87][88][89]. However, soil microorganisms can be applied to foods only if they have been designated as generally regarded as safe (GRAS). GRAS microorganisms typically include human intestinal bacteria, lactic acid bacteria, and fungi. GRAS fungi are more suitable for the production than lactic acid bacteria because they are easier to grow in cheaper media and exhibit higher yields and productivities. Fermentation is more cost-effective than cell or enzymatic conversion because it does not require enzyme purification steps and can use both intracellular and extracellular enzymes. However, the productivity of deglycosylated saponins by fermentation is lower than that by cell conversion or enzymatic conversion. To increase the productivity and avoid the inhibition of saponins to cells during fermentation, a new strategy, such as continuous feeding of saponins in a fermenter, is required.
Hydrolytic pathways of PDN-type platycosides by glycosidases have been proposed (Figure 2). In these pathways, PE and PA are converted into PDN by the reactions A1, A2, A3, B, C, D, and E, and A3, B, C, D', and E, respectively, and 26 PDN-type platycosides are involved. The PGA-type platycoside, PGD 3 or AcPGD 3 , is converted into PGA by the reactions of A2, A3, B, C, D or D', and E, respectively, and 24 PGA-type platycosides were involved in the proposed hydrolytic pathways (Figure 3). Hydrolytic pathways of the PCA-type platycosides are proposed from PCCA to PCA via eight intermediate platycosides ( Figure 4). In total, 60 platycosides are involved in the hydrolytic pathways from glycosylated platycosides to sapogenins.
Crude enzyme from A. tubingensis has β-glucosidase, β-D-apiosidase, and β-D-xylosidase activities [72]. The multi-hydrolytic activities, which may be because the crude enzyme contains several glycosidases [85]. The crude enzyme hydrolyzes all three glucoside residues at C-3 by the reactions A1, A2, and A3 with β-glucosidase activity and the apioside and xyloside residues at C-28 by the reactions B and C with β-D-apiosidase and β-D-xylosidase activities, respectively. The crude enzyme converts PE and PGD 3 into deglc-api-xyl-PD and deglc-api-xyl-PGD, respectively. The crude enzyme can also be used to convert the unused substrates PA, AcPGD 3 , and PCAA into deglc-api-xyl-PA, deglc-api-xyl-AcPGD, and deglcapi-xyl-PCAA, respectively. The use of a combination of enzymes or cells with multiple enzymes is a good tool to produce new highly deglycosylated platycosides. If the combination of A. aculeatus pectinase and D. turgidum β-glucosidase are applied to the hydrolysis of the glycosylated platycosides PE, PA, PGD 3 , AcPGD 3 , and PCAA, all residues linked to C-3 and C-28 will be hydrolyzed into PDN, PDN, PGA, PGA, and PCA, respectively, by the reactions A1, A2, A3, B, C, D or D', and E. The human intestinal bacterium Bacteroides converts PD into ara-PDN by the reactions A1, A2, A3, B, C, and D [73]. The cells also convert deglc-api-PD into deglc-api-PGD by dihydroxylation, which is converted into api-PGD.

Conclusions and Future Perspectives
In this review, we describe the biotransformation of platycosides based on their hydrolytic pathways, glycosidases, and specific productivity of deglycosylated platycosides. However, the number of reports on the transformation of balloon flower root saponins are significantly fewer than those of ginseng saponins. Therefore, transformation methods for glycosylated ginsenosides can be applied for the transformation of glycosylated platycosides to produce diverse deglycosylated platycosides. Physical methods, including steam heating, microwave heating, and sulfur fumigation [48,50], and chemical methods, including acid and alkaline hydrolysis, which have been used in the hydrolysis of glycosylated ginsenosides, can be applied to the hydrolysis of glycosylated platycosides [50,81].
Currently, the pool of glycosidases that hydrolyze glycosylated platycosides is too small. Cloning of glycosidase genes and discovery of natural glycosidases are needed to expand the pool to produce diverse platycosides. To produce new platycosides by specific hydrolysis of the glycoside moieties in platycosides, different types of glycosidases with narrow substrate specificities are required. For instance, the discovery of β-glucosidases that hydrolyze only the outer glucoside residue at C-3, and β-D-apiosidase, β-D-xylosidase, α-L-rhamnosidase, and α-L-arabinosidase, which hydrolyze apioside, xyloside, rhamnoside, and arabinoside, respectively, but no glucosides are needed. To obtain highly deglycosylated platycosides, such as glc-ara-platycodigenin and ara-platycodigenin, glycosidases with broad substrate specificity that hydrolyze platycosides through different pathways are also required.
Microorganisms involved in saponin transformation include soil fungi and bacteria, intestinal bacteria, and GRAS fungi [48,50]. The use of GRAS microorganisms and enzymes originating from GRAS microorganisms for saponin transformation is an appropriate method for food applications. Although glycosidases from GRAS fungi such as A. aculeatus [52], A. niger [74][75][76], A. tubingensis [72], A. usamii [68], R. oryzae [8], and T. reesei [70] are used in the biotransformation of platycosides, only one human intestinal bacterium, Bacteroides [73] is applied in biotransformation. Thus, more diverse GRAS human intestinal bacteria, such as lactic acid bacteria, should be applied in the biotransformation of platycosides because they can metabolize orally ingested saponins and can be used safely in a variety of foods [48,90].
We propose methods for producing diverse and/or new deglycosylated platycosides that may contribute to the development of biotransformation processes to produce bioactive deglycosylated platycosides. The biological effects of platycosides are mainly attributed to natural platycosides. However, the effects of biotransformed platycosides using cell lines and animal models have not yet been investigated. Recently, biotransformed platycosides have increasingly been reported. Thus, biotransformed platycosides will allow for the initiation of pharmacological studies at the cell and animal levels, and platycosides with improved bioactivity are expected to be developed through pharmacological studies.