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This study aimed to develop improved multi-component beads with controlled, sustained delivery of antibiotics, such as gentamicin (GM). Antibiotic-loaded beads with rapid-release and the sustained-release system can be used for bone restoration. Single and multi-component beads were prepared by gelation using various combinations of chitosan and calcium chloride as cationic components and alginate and citric acid as anions. GM release was also controlled by crosslinking using citric acid. The optimum beads were obtained using 5% or 2% sodium alginate, 3% chitosan, and 0.1 mol/L citric acid. The beads were characterized by FTIR, TG-DTG, swelling behavior, and SEM. All GM-loaded beads revealed good antimicrobial activity. The rate and kinetics of release in the phosphate buffer solution were controlled by changing the amount of chitosan in the calcium chloride solution and using citric acid as the crosslinking agent. Crosslinked beads were prepared for the release of about 80% of the loaded drug within 24 h. The study concluded that the chitosan-alginate beads provided faster GM release but crosslinking with citric acid was efficient for sustained-release beads containing gentamicin.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Urushiols are amphipathic compounds found in Rhus verniciflua Stokes that exhibit various biological activities. However, their practical use is very restricted due to their contact dermatitis-indu...

Urushiols are important active compounds found in the sap of the lacquer tree (Rhus verniciflua Stokes). Recently, various biological effects of urushiols, such as antioxidant, antimicrobial, and anticancer activities, have been reported. However, urushiols can also induce skin allergies. Nevertheless, the lacquer tree has traditionally been used in Korea as a folk medicine. In this study, we evaluated the absorption and metabolism of 3-pentadecylcatechol (PDC), a natural urushiol. PDC (48.0 mg/kg body wt.) in 1 mL propylene glycol was orally administered to rats (Sprague-Dawley, male, 6 weeks old). Blood plasma, urine, and feces were collected, separately. PDC was not detected in the extracts from rat blood plasma and urine. However, 89.4 ± 5.2% of the orally administered PDC was detected in the feces extracts, indicating that PDC was predominantly excreted and not absorbed.

Helicobacter pylori infections are a major cause of gastrointestinal disorders, including gastric ulcers, gastritis, and gastric cancer. Triple therapy, using two antibiotics and a proton pump inhibitor, is recommended for the treatment of H. pylori infections. However, antibiotic resistance in H. pylori is an emerging issue. Bamboo salt, a traditional Korean salt made by baking solar sea salt in bamboo barrels, can ameliorate the symptoms of various gastrointestinal diseases. Herein, we compared the anti-H. pylori activity of triple therapy (clarithromycin, metronidazole, and omeprazole), solar salt, and bamboo salt in vivo as a preliminary study. Four-week-old C57BL/6 male mice were inoculated for eight weeks with the H. pylori Sydney Strain 1 (SS-1) and orally administered triple therapy drugs and salts for five days. The transcript levels of the H. pylori-expressed gene CagA and inflammatory cytokines Tnfα and Il-1β significantly decreased in the bamboo salt treated mice than those in the H. pylori-infected control group. This effect was further enhanced by using triple therapy and bamboo salt together. Solar salt caused modest inhibition of H. pylori-induced inflammation. We also demonstrated the synergistic effects of bamboo salt and triple therapy against H. pylori. Thus, bamboo salt may be a potential candidate agent against the treatment of H. pylori-associated gastritis.

α-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the Δ5,6 double bond; the dehydro-type). The aglycone of α-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3βHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3βHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3βHSD1 protein revealed that Sl3βHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3βHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3βHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3βHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3βHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.

Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.

Fifteen steroidal saponins 1–15, which include 4 furostanol glycosides 1–3 and 15, and 11 spirostanol glycosides 4–14, were isolated from the tubers and leaves of lesser yam (Dioscorea esculenta, Togedokoro). Their structures were identified by nuclear magnetic resonance and liquid chromatography mass spectroscopy. Four steroidal saponins 9, 11, 14, and 15 were found to be novel compounds. Steroidal saponins from Dioscorea esculenta.

Nineteen compounds including one new flavanone were isolated from the juice of aged common sage exudate with sugar (ACSE). The isolated compounds were identified by NMR and MS analyses as levodopa methyl ester (1), 3,4-dihydroxybenzoic acid (2), (S)-8-hydroxy-4-hydroxy-phenylpropanoic acid (3), 4-hydroxybenzoic acid ethyl ester (4), cis-caffeic acid (5), trans-caffeic acid (6), esculetin (7), (S)-8-hydroxy-3,4-dihydroxy-phenylpropanoic acid ethyl ester (8), cis-rosmarinic acid (9), trans-rosmarinic acid (10), trans-rosmarinic acid methyl ester (11), 6-methoxy-7,8,3′,5′-tetrahydroxyflavanone (12), nepetin (13), trans-caffeic acid ethyl ester (14), luteolin (15), cis-caffeic acid ethyl ester (16), 6-methoxynaringenin (17), 1α-acetoxy-2-oxo-eudesman-3,7(11)-dien-8β,12-olide (18), and hispidulin (19). Compound 12 was isolated for the first time from nature and seven compounds (1, 3, 4, 7, 8, 14, and 18) were newly identified from common sage. Of them, 15 isolated phenolic compounds (1–3, 5–8, 10–15, 17, and 19) were detected in ACSE juice, while only 10 was detected in the fresh common sage.

Five proanthocyanidins, two B-type dimers and three A-type trimers, were purified and isolated from the fruit peels of Pyrus pyrifolia Nakai cv. Chuhwangbae. The isolated compounds were identified as (–)-epicatechin gallate-(4β → 8)-(–)-epicatechin (Hahashi et al. in Ann Biol Res 3:3200–3207, 2012), (–)-epicatechin-(4β → 8)-(–)-epicatechin (procyanidin B2) (Tanrioven and Eksi in Food Chem 93:89–93, 2005), (–)-epicatechin-(4β → 8, 2β → O-7)-(–)-epicatechin-(4β → 8)-(–)-epicatechin (cinnamtannins B1) (Salta et al. in J. Fun. Food 2: 153–157, 2010), (–)-epicatechin-(4β → 8)-(–)-epicatechin-(4β → 8, 2β → O-7)-(–)-epicatechin (aesculitannin A) (Challice and Westwood in Phytochemistry 11: 37–44, 1972), and (–)-epicatechin-(4β → 6)-(–)-epicatechin-(4β → 8, 2β → O→7)-(–)-epicatechin (Es-Safi et al. in J Agric Food Chem 54: 6969–6977, 2006). Their structures were determined by nuclear magnetic resonance and mass spectrometry. The three A-type proanthocyanidin trimers were identified for the first time from pear.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Eleven compounds, including a new sesquiterpene, were isolated from the culture medium of Aspergillus oryzae incubated with capsaicin. The structure of the new compound was determined to be 1,3,5a,6,7,8,9,9a-octahydro-9-hydroxy-9-(hydroxymethyl)-6-isopropyl-1-oxobenzo[c]oxepine-4- carboxylic acid, a heptelidic acid derivative. In addition, 10 known compounds were identified, namely 5-(hydroxymethyl)-3-furancarboxylic acid (flufuran), 3-hydroxypropanoic acid, 5-(hydroxymethyl)-2- furancarboxylic acid, 2-(4-hydroxyphenyl)-ethanol, 4-hydroxybenzoic acid, vanillic acid, 3,4-dihydroxybenzoic acid, 2-furanol, hydroheptelidic acid, and trichoderonic acid A, using spectroscopic data from nuclear magnetic resonance and electrospray ionization-mass spectroscopy.

A phenyl lipid alkaloid and seven phenolic compounds were isolated from the aerial part of Spergularia marina, a halophyte that grows on salt marshes and tidal flat. These compounds were identified as 2,4-di-tert-butylphenol, N-hexacosanoylanthranilic acid, tryptophan, 4-hydroxybenzyol glucopyranoside, luteolin 6-C-β-D-glucopyranoside 8-C-β-D-(2-O-feruloyl)glucopyranoside, luteolin 6-C-β-D-(2-O-feruloyl)glucopyranoside 8-C-β-D-glucopyranoside, apigenin 6-C-β-D-glucopyranoside 8-C-β-D-(2-O-feruloyl)glucopyranoside, and apigenin 6-C-β-D-(2-O-feruloyl)glucopyranoside 8-C-β-D-glucopyranoside. The structures were determined by nuclear magnetic resonance and electrospray ionization-mass spectroscopy.

This study identified metabolites of capsaicin bioconverted by Aspergillus oryzae, which is generally used for mass production of gochujang prepared by fermenting red pepper powder in Korea. A. oryzae was incubated with capsaicin in potato dextrose broth. Capsaicin decreased depending on the incubation period, but new metabolites increased. Five capsaicin metabolites purified from the ethyl acetate fraction of the capsaicin culture were identified as N-vanillylcarbamoylbutyric acid, N-vanillyl-9-hydroxy-8-methyloctanamide, ω-hydroxycapsaicin, 8-methyl-N-vanillylcarbamoyl-6(E)-octenoic acid, and 2-methyl-N-vanillylcarbamoyl-6(Z)-octenoic acid by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The capsaicin metabolites in gochujang were confirmed and quantitated by selective multiple reaction monitoring detection after liquid chromatography electrospray ionization MS using the isolated compounds as external standards. On the basis of the structures of the capsaicin metabolites, it is proposed that capsaicin metabolites were converted by A. oryzae by ω-hydroxylation, alcohol oxidation, hydrogenation, isomerization, and α- and/or β-oxidation.

Gochujang is a red pepper-soybean paste and a popular Korean fermented food. The aim of this study was to investigate the behavior of flavonoids and activity of β-glucosidase during fermentation of gochujang prepared using Aspergillus oryzae. The content of flavonoid glycosides (genistin, daidzin, apigenin 7-O-β-d-glucopyranoside, and quercetin 3-O-α-l-rhamnopyranoside) and their aglycones did not change significantly during preparation of gochujang. However, β-glucosidase was produced by A. oryzae. The polar layer of the gochujang methanol (MeOH) extract more strongly inhibited the β-glucosidase activity of the crude enzyme extracted from gochujang than that of the nonpolar layer. The red pepper extract and high salt solution of the gochujang ingredients showed high β-glucosidase inhibition activity. These results indicate that flavonoid glycosides were not hydrolyzed by the β-glucosidase produced by A. oryzae during gochujang fermentation, which may have been due to the salt and hydrophilic compound(s) present in red pepper as main ingredients of gochujang.

The changes in total phenolics, total anthocyanins, gallic acid (GA), and 3,4-dihydroxybenzoic acid (DHBA) contents during black raspberry (BR, Rubus coreanus) wine manufacturing were investigated. Total phenolic content gradually increased during BR wine manufacturing. The total anthocyanin content slightly increased, but the changes were not significant. The GA and DHBA in BR wine were detected as main compounds and their content greatly increased during manufacturing. The DPPH radical-scavenging activity was also increased during BR wine manufacturing. In particular, the antioxidant activity in the blood plasma of rats administered with BR wine concentrate was significantly higher than those administered with BR fruit extracts in inhibition of copper ion-induced cholesteryl ester hydroperoxide formation. These results indicate that an increase in antioxidant activity of BR wine may be due to low molecular phenolic compounds including GA and DHBA produced during the manufacturing.

Six triterpenes, including three caffeoyl triterpenes, were purified and isolated from pear fruit ( Pyrus pyrifolia Nakai cv. Chuwhangbae) peel extracts using various column chromatography techniques with a guided 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay. The isolated compounds were identified as betulinic aldehyde (1), lupeol (2), betulinic acid (3), 3-O-cis-caffeoylbetulinic acid (4), 3-O-trans-caffeoylbetulinic acid (5), and 3-O-trans-caffeoyloleanolic acid (6) on the basis of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Four compounds (1, 4-6) were identified from Asian pear fruit for the first time. In addition, compounds 4-6, containing a caffeic acid moiety, showed higher DPPH radical-scavenging and suppression effects against copper ion-induced oxidation of rat blood plasma than other compounds without a caffeic acid moiety.

Salvianolic acid B (Sal B) is the most abundant phenolic compound in Salvia miltiorrhiza, which has been widely used for the treatment of cardiovascular diseases in Oriental medicine. To elucidate structure of the converted compounds of Sal B by decoction in aqueous solution, Sal B (200 mg) was decocted in an aqueous solution (200 mL) at pH 4.9 and the decocted solution was purified by Sephadex LH-20 column chromatography and preparative HPLC. The 13 converted compounds were isolated and the chemical structures were determined by NMR and MS. In addition to the 4 compounds previously reported as conversion products of Sal B by decoction, 9 compounds were first reported with the complete structure of compounds isolated from decocted Sal B solution and three of the compounds were determined to be novel compounds. In addition, a conversion mechanism of compounds converted by decoction was proposed on the basis of kinetics studies, which reasonably supported the conversion mechanism of Sal B. The 13 compounds seemed to be produced by the hydrolysis of an ester bond, decarboxylation, retro oxa-Michael reaction, hydration, and radical reaction during decoction in aqueous solution.

γ-Oryzanol (steryl ferulates; SF) has been shown to be a major bioactive compound in rice. To determine the content of individual γ-oryzanols in brown rice by high performance liquid chromatography (HPLC), purification of individual SF for use as an external standard is required. Four main SF were isolated from a commercial γ-oryzanol mixture and identified as cycloartenyl ferulate (1), 24-methylenecycloartanyl ferulate (2), campesteryl ferulate (3), and sitosteryl ferulate (4) based on mass spectrometry and nuclear magnetic resonance spectroscopic data. The SF contents between conventional and organic brown rice were qualitatively determined by HPLC using SF isolated from a commercial γ-oryzanol mixture as the external standard. The total γ-oryzanol content (mg/100 g) in organic brown rice (65.6 ± 2.7) was slightly higher (P

Two acyclic diterpene glycosides were isolated from the n-butanol layer (50.84 g) of methanol extracts (1639 g) of gochujang (3 kg, wet wt.), Korean fermented red pepper paste. The chemical structures were elucidated as 6E,10E,14Z-(3S)-17-hydroxygeranyllinalool 17-O-α- l -rhamnopyranosyl(1 → 4)-[α- l -rhamnopyranosyl-(1 → 6)]-β- d -glucopyranoside (1, capsianoside XVIII, 2.3 mg, a novel compound) and capsianoside F (2, 5.3 mg) based on the spectroscopic data of MS and NMR. Compounds 1 and 2 are reported for the first time from gochujang. In addition, the origin of the compounds was determined to be red pepper (Capsicum annuum), which is one of the representative materials of gochujang.

Eight phenolic compounds were isolated from the EtOAc-soluble acidic layer of methanol extracts of Asian pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. These isolated compounds were identified to be arbutin (1), 4-(O-β-d-glucopyranosyl)-3-(3′-methyl-2′-butenyl)benzoic acid (2, malaxinic acid), 3,4-dihydroxybenzoic acid (3), trans-chlorogenic acid (4), cis-chlorogenic acid (5), isorhamnetin 3-O-β-d-glucopyranoside (6), 3,5-dicaffeoylquinic acid (7), and (-)-epicatechin (8) based on electronic spray ionization (ESI)-MS and NMR spectroscopic data. Of the isolated compounds, 2 and 5 were identified in pear for the first time.

Two novel caffeoylmalic acid methyl esters, 2-O-(trans-caffeoyl)malic acid 1-methyl ester (6) and 2-O-(trans-caffeoyl)malic acid 4-methyl ester (7), were isolated from pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. In addition, 5 known hydroxycinnamoylmalic acids and their methyl esters were identified: 2-O-(trans-coumaroyl)malic acid (1), 2-O-(cis-coumaroyl)malic acid (2), 2-O-(cis-coumaroyl)malic acid 1-methyl ester (3), 2-O-(trans-coumaroyl)malic acid 1-methyl ester (4), and 2-O-(trans-caffeoyl)malic acid (phaselic acid, 5). The chemical structures of these compounds were determined by spectroscopic data from ESI MS and NMR. Of all the isolated compounds, five hydroxycinnamoylmalic acids and their methyl esters (2-4, 6, 7) were identified in the pear for the first time.

Two novel antioxidant compounds, isoquercitrin 6″-O-methyloxalate (6) and methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate, 7), were isolated from Salicornia herbacea L.. Six known compounds were also identified as 3,5-dicaffeoylquinic acid (1), quercetin 3-O-β- d -glucopyranoside (2), 3-caffeoyl-4-dihydrocaffeoylquinic acid (3), methyl 3,5-dicaffeoyl quinate (4), 3,4-dicaffeoylquinic acid (5), and isorhamnetin 3-O-β- d -glucopyranoside (8). Their chemical structures were determined by spectroscopic data from ESI–MS and NMR. The isolated dicaffeoylquinic acid derivatives (1, 3, 4, 5, and 7) showed similar activities for scavenging 1,1-diphenyl-2-picrylhydrazyl radicals and inhibiting formation of cholesteryl ester hydroperoxide during copper ion-induced rat blood plasma oxidation. The two flavonol glucosides (2 and 6), which have no substitutions in the B ring of their aglycones, also had similar activity. However, compound 8, which has the same structure as 2 except for the presence of a methoxyl group in the C-3′ position of the B ring, showed predominantly lower antioxidant activity than the other isolated compounds.