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Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration and productivity reported so far. Our results contribute to functional saponin production and the related food industries.

Platycosides are the functional saponins present in balloon flowers that exert diverse biological effects, and which can be further improved by their deglycosylation. Deapiose-xylosylated platycodin D, which is absent in balloon flowers, can be generated only by cytolase PCL5 by acting on platycoside E. To improve cytolase PCL5-catalyzed production of deapiose-xylosylated platycodin D from platycoside E, we explored the use of high hydrostatic pressure (HHP). At an HHP of 150 MPa, the optimal temperature of cytolase PCL5 activity for converting platycoside E into deapiose-xylosylated platycodin D shifted from 50 to 55 °C, and increased the activity and stability of the enzyme by 5- and 4.9-fold, respectively. Under HHP, the enzyme completely converted 1 mM platycoside E into deapiose-xylosylated platycodin D within 4 h, with a 3.75-fold higher productivity than that under atmospheric pressure. Our results suggest that the application of HHP is a potential method for the economical production of platycosides and enzyme-catalyzed biotransformation of functional saponins.

The bioactive compounds of Platycodon grandiflorum have been studied with saponin, including platycodin D (PD) and platycoside E (PE), but functional enhancement studies through bioconversion as fermentation are insufficient. In this study, we determined the potential therapeutic effects of fermented Platycodon grandiflorum extracts (FPE) on the atopic dermatitis-induced HaCaT cell line and NC/Nga mice. Atopic dermatitis was induced by TNF-α (10 ng/mL)/IFN-γ (10 ng/mL) in HaCaT cells and topical application of 0.2% 1-chloro-2,4-dinitrobenzene (DNCB) in NC/Nga mice. FPE regulated TNF-α/IFN-γ-induced the production of inflammation-related cytokines, chemokines and expression of transcription factors in HaCaT cells. Oral administration of FPE and PD/PE ameliorated DNCB-induced atopic dermatitis symptoms and reduced the number of T cells, immunoglobulin E-producing B cells, eosinophils in the dorsal skin. Taken together, our results indicate that FPE can modulate the Th1/Th2 immune balance and has potential as a functional food supplement that can prevent skin diseases.

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides, however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E &rarr, platycodin D3 &rarr, platycodin D &rarr, deapiosylated platycodin D &rarr, deapiose-xylosylated platycodin D, and polygalacin D3 &rarr, polygalacin D &rarr, deapiosylated polygalacin D &rarr, deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.

Platycodi radix (i.e., Platycodon grandiflorum root) products (e.g., tea, cosmetics, and herbal supplements) are popular in East Asian nutraceutical markets due to their reported health benefits and positive consumer perceptions. Platycosides are the key drivers of Platycodi radixes&rsquo, biofunctional effects, their nutraceutical and pharmaceutical activities are primarily related to the number and varieties of sugar side-chains. Among the various platycosides, platycodin D is a major saponin that demonstrates various nutraceutical activities. Therefore, the development of a novel technology to increase the total platycodin D content in Platycodi radix extract is important, not only for consumers&rsquo, health benefits but also producers&rsquo, commercial applications and manufacturing cost reduction. It has been reported that hydrolysis of platycoside sugar moieties significantly modifies the compound&rsquo, s biofunctionality. Platycodi radix extract naturally contains two major platycodin D precursors (platycoside E and platycodin D3) which can be enzymatically converted to platycodin D via &beta, d-glucosidase hydrolysis. Despite evidence that platycodin D precursors can be changed to platycodin D in the Platycodi radix plant, there is little research on increasing platycodin D concentrations during processing. In this work, platycodin D levels in Platycodi radix extracts were significantly increased via extracellular Aspergillus usamii &beta, d-glucosidase (n = 3, p &lt, 0.001). To increase the extracellular &beta, d-glucosidase activity, A. usamii was cultivated in a culture media containing cellobiose as its major carbon source. The optimal pH and temperature of the fungal &beta, d-glucosidase were 6.0 and 40.0 &deg, C, respectively. Extracellular A. usamii &beta, d-glucosidase successfully converted more than 99.9% (w/v, n = 3, p &lt, 0.001) of platycoside E and platycodin D3 into platycodin D within 2 h under optimal conditions. The maximum level of platycodin D was 0.4 mM. Following the biotransformation process, the platycodin D was recovered using preparatory High Performance Liquid Chromatography (HPLC) and applied to in vitro assays to evaluate its quality. Platycodin D separated from the Platycodi radix immediately following the bioconversion process showed significant anti-inflammatory effects from the Lipopolysaccharide (LPS)-induced macrophage inflammatory responses with decreased nitrite and IL-6 production (n = 3, p &lt, 0.001). Taken together, these results provide evidence that biocatalysis of Platycodi radix extracts with A. usamii may be used as an efficient method of platycodin D-enriched extract production and novel Platycodi radix products may thereby be created.

A traditional external standard method using HPLC coupled with evaporative light scattering detection has been developed for fast and accurate determination of seven platycosides in Platycodi Radix. However, inevitable difficulties in reference standards preparation process, which are quite costly and time consuming, have made its application limited. To avoid this inconvenience, a simultaneous determination of multiple components with a single reference standard strategy, which could be realized by calibrating the standard curve with internal standard and response factors, was introduced to the HPLC coupled with evaporative light scattering detection method. This is the first time that an incorporation of these two methods has been realized. Among seven ingredients, platycodin D was selected as the internal standard for its relatively easy preparation and low cost. Moreover, according to the investigation on concentration-dependent effects over response factors and robustness test, platycoside E, deapioplatycodin D, platycodin D, and polygalacin D2 were chosen to be the indicators for this novel method. The present method has not shown statistically significant differences with a traditional external standard method as verified sample analysis by the F-test (p = 95%, n = 6).

Effects of moisture content and puffing pressure on the extraction yield, crude saponin content, antioxidant activity, and platycoside content of puffed 21-year-old doraji were investigated. Sliced doraji was dried to 4.0, 6.0, and 8.0 g of water/100 g of sample, then puffed at pressures of 686, 784, 882, and 980 kPa. The extraction yield, crude saponin, and antioxidant activity of puffed doraji, compared with non-puffed doraji, increased from 35.9 to 52.7 g/100 g of dried doraji, from 34.4 to 70.5 mg/g of dried doraji, and from 34.0 to 138.1 mg of vitamin C equivalent (VCE)/g of dried doraji, respectively. Levels of the major platycosides, platycoside E, platycodin D, and D3 of puffed doraji were reduced as the puffing pressure increased. Puffing can be a favorable unit process resulting in a higher extraction yield, total crude saponin content, and antioxidant activity of doraji.

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant &beta, glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, &beta, glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

In the present work, we reported the enzymatic preparation of deapio-platycodin D (dPD) and platycodin D (PD) optimized by response surface methodology (RSM) from Radix Platycodi. During investigation of the hydrolysis of crude platycosides by various glycoside hydrolases, snailase showed a strong ability to transform deapio-platycoside E (dPE) and platycoside E (PE) into dPD and PD with 100% conversion. RSM was used to optimize the effects of the reaction temperature (35–45 °C), enzyme load (5–20%), and reaction time (4–24 h) on the conversion process. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of dPD and PD conversion yield. The optimum preparation conditions were as follows: temperature, 43 °C; enzyme load, 15%; reaction time, 22 h. The biotransformation pathways were dPE→dPD3→dPD and PE→PD3→PD, respectively. The determined method may be highly applicable for the enzymatic preparation of dPD and PD for medicinal purposes and also for commercial use.

An effective HPLC method to analyse platycosides from the balloon flower root was developed using ELSD. The optimum resolution of the platycosides was achieved on an ODS column with gradient elution of eluent A, 30mM ammonium acetate buffer (pH 4.81): methanol: acetonitrile=75:5:20 (v/v/v), and B, 69:5:26 (v/v/v). Amongst 18 platycosides, platycoside E showed the highest content, followed by polygalacin D2 and 3″-O-acetylplatyconic acid A. The sum of these three compounds was recommended for quality control of balloon flower root for medicinal purposes. The samples could be clustered into groups based on platycoside content. Group I, characterised by a high concentration of platycosides, was located near the west coast of Korea, whereas group II, characterised by a low concentration of platycosides, was located inland or in mountainous area. The method could be used to control the quality of balloon flower root.

A new oleanane-type triterpenoid saponin, named platycoside N (1), together with six known saponins, was isolated from the roots of Platycodon grandiflorum. On the basis of acid hydrolysis, comprehensive spectroscopic data analyses and comparison with the spectral data of the known compounds, its structure was elucidated as 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. The six known compounds were platycodin D (2), deapioplatycodin D (3), platycodin D3 (4), deapio- platycodin D3 (5), platycoside E (6) and deapioplatycoside E (7).

Bioassay-directed fractionation of the antiviral active fraction of the roots of Platycodon grandiflorum leads to the isolation of three new triterpenoid saponins, platycosides G1–G3 ( 1 – 3 ), as well as two known saponins, platycodin D3 ( 4 ), and platycoside E ( 5 ). The structures of the new compounds were elucidated on the basis of their spectral data and chemical evidences. The isolated saponins were tested for their antiviral activities against respiratory syncytial virus (RSV), herpes simplex type 1 virus (HSV-1) and influenza type A virus (Flu A). Compound 4 showed weak anti-RSV activity.

A new triterpenoid saponin, 3- O -β- d -glucopyranosyl 16-oxo-platycodigenin 28- O -β- d -apiofuranosyl-(1 → 3)-β- d -xylopyranosyl-(1 → 4)-α- l -rhamnopyranosyl-(1 → 2)-α- l -arabinopyra-nosyl ester, was isolated from the roots of Platycodon grandiflorum , together with three known saponins, including platycodin D, deapio platycoside E and platycoside E. The structure of the new compound, named 16-oxo-platycodin D, was elucidated on the basis of spectroscopic data.

Two new triterpenoid saponins, platycoside D [3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D- glucopyranosyl-2 beta, 3 beta, 16 alpha, 23-tetrahydroxyolean-12-ene-28-oic acid 28-O-beta-D- apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnop yranosyl- (1-->2)-alpha-L-arabinopyranoside] and platycoside E [3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl- (1-->6)-beta-D-glucopyranosyl-2 beta,3 beta,16 alpha,23,24-pentahydroxyolean-12-ene-28-oic acid 28-O-beta-D-apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alp ha-L- rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside] were isolated from the roots of Platycodon grandiflorum. Structural determination is based on spectral and chemical evidence.

Platycodi Radix, the root of Platycodon grandiflorum A. DC. (Campanulaceae), has traditionally been used as medicinal herb. Its primary constituents, platycosides (saponins), are known to have diverse functionalities, exerting anti-inflammation, anti-allergy, anti-tumor, anti-obesity and anti-hyperlipidemia effects, as well as augmenting immune responses and stimulating apoptosis in skin cells. To this date, some studies isolating the various platycosides according to their specific biological activities have been published. The present work explains how high-speed counter-current chromatography (HSCCC) coupled with ELSD was applied to the preparative separation of six platycosides (platycoside E, platycodin D, platycodin D3, and their deapiose forms) from Platycodi Radix. The HSCCC, with a two-phase solvent system composed of hexane-n-butanol-water (1:40:20, v/v) and hexane-n-butanol-water (1:10:5, v/v), in combination with effluent monitoring by ELSD, was successfully performed. Consequently, platycoside E (21mg), deapio-platycoside E (14mg), platycodin D3 (10mg), deapio-platycodin D3 (6mg), platycodin D (28mg), and deapio-platycodin D (6mg), all with purities of over 94%, were isolated from 300mg of the platycoside-enriched fraction. Additionally, their structures were characterized by electrospray ionization mass spectrometry (ESI-MS), 1H NMR, and 13C NMR. Acknowledgement: This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund KRF-2007-E0016)