Efficient regioselective enzymatic acylation of troxerutin: difference characterization of in vitro cellular uptake and cytotoxicity.

In this study, we developed a multi-site acylation strategy to improve the lipophilicity and cellular uptake of troxerutin, a natural flavonoid with many health-promoting bioactivities. By clarifying the acylation properties of troxerutin catalyzed by lipases from different sources, a series of troxerutin ester derivatives acylated at different sites was synthesized, including troxerutin dipropyl (TDP), tripropyl (TTP), tetrapropyl (TEP), dibutyl (TDB), monohexyl (TMH), monooctyl (TMO) and monodecyl (TMD) esters. Interestingly, the troxerutin esters acylated at multiple sites with shorter fatty chains (TDP, TTP and TEP) had similar lipophilicity to the mono-acylated esters bearing longer fatty chains (TMH, TMO and TMD, respectively) and meanwhile demonstrated surprisingly lower cytotoxicity than that of the long fatty-chain mono-esters. In particular, the multi-acylated esters with shorter fatty chains showed remarkably higher cellular uptake than the mono-esters with long fatty chains. In vitro gastrointestinal digestion suggested that the multi-acylated esters of troxerutin were more resistant to gastrointestinal degradation than the mono-esters. These results indicated that multi-site acylation with short fatty chains could be an effective alternative to introducing one-site mono-acylation for the modification of troxerutin and other flavonoid compounds.

Biocatalytical Acyl-Modification of Puerarin: Shape Gut Microbiota Profile and Improve Short Chain Fatty Acids Production in Rats.

Gut microbiota balance and metabolites have become a potentially mechanism in maintaining health. The specific aim of this study was to compare the modulation of puerarin and puerarin acid esters on gut microbial composition and metabolites. Male mice were fed a control diet or diets supplemented with puerarin, puerarin propanoate ester, puerarin hexanoate ester, puerarin myristate ester for 24 h, respectively. The result revealed that puerarin acid esters with different chain lengths showed different activities to create more own impacted bacterial. Puerarin propanoate and puerarin hexanoate ester significantly improved the diversity of microbiota and promoted the relative abundance of beneficial gut microbiota such as Lactobacillus, Barnesiella, Clostridium IV, Prevotella. Additionally, the puerarin propanoate ester group showed the capacity to deliver specific propionic acid to the colon. But esters with medium-long chain lengths had more opportunity to alter gut microbiota for enhancing the short chain fatty acids production. As a whole, puerarin acid esters with different chain lengths supplements shaped different gut microbial and short chain fatty acids metabolism, which could improve human health.

Evaluation of the digestion and transport profiles and potential immunocompetence of puerarin and its acylated derivatives.

Acylation has become one of the most widely used methods to improve the lipid solubility and bioavailability of flavonoids. In this study, puerarin acid esters (PAES) with different chain lengths were synthesized via biocatalytic acylation. This was the first study to evaluate the digestion and transport profiles and immunocompetence of PAES. The relationship between the digestion and transport profiles and potential immunocompetence of the acylated derivatives in Caco-2 cell monolayers was also explored. Puerarin and PAES remained stable in gastric phases, whereas different degrees of hydrolysis of PAES were found in the intestine. PAES with less than 12 carbon chains were positively correlated with the degree of hydrolysis, while those with more than 12 carbon chains showed higher resistance to hydrolysis by the artificial human digestive juice. The apparent permeability coefficients of puerarin, puerarin acetate, puerarin propanoate, puerarin butyrate, puerarin hexanoate, puerarin octanate and puerarin laurate were 1.62 ± 0.09, 1.70 ± 0.15, 1.89 ± 0.19, 1.86 ± 0.18, 2.29 ± 0.12, 4.06 ± 1.01 and 2.32 ± 0.88 × 10-6 cm s-1, respectively, in Caco-2 cell monolayers. The results of the immune factor assays indicated that puerarin propanoate, puerarin hexanoate and puerarin myristate could significantly promote the secretion of IL-6, TNF-α and IL-10. These findings suggested that a better absorption could be predicted after oral intake using PAES. Meanwhile, the concentration of esters and their metabolites (puerarin) found in the digestion and transport profiles directly affected their potential immunocompetence.

A modified anterior drawer test for anterior cruciate ligament ruptures.

OBJECTIVE: This study was aimed to utilize a modified anterior drawer test (MADT) to detect the anterior cruciate ligament (ACL) ruptures and investigate its accuracy compares with three traditional tests. METHODS: Four hundred patients were prospectively enrolled between January 2015 and September 2017 preoperatively to undergo knee arthroscopic surgeries. The MADT, anterior drawer test, Lachman test, and pivot shift test were used in the outpatient clinical setting and were compared statistically for their accuracy in terms of ACL ruptures, with arthroscopic findings as the gold standard. RESULTS: The prevalence of ACL ruptures in this study was 37.0%. The MADT demonstrated the highest sensitivity (0.89) and accuracy (0.92) among the four tests and had comparable specificity (0.94) and a positive predictive value (0.90) compared with the anterior drawer test, Lachman test, and pivot shift test. The diagnostic odds ratio (DOR) of MADT was 122.92, with other test values of no more than 55.45. The area under the receiver operating characteristic curve (AUC) for the MADT was 0.92 ± 0.01, with a significant difference compared with that for the anterior drawer test (z = 17.00, p < 0.001), Lachman test (z = 9.66, p = 0.002), and pivot shift test (z = 16.39, p < 0.001). The interobserver reproducibility of the MADT was good, with a kappa coefficient of 0.86. When diagnosing partial tears of ACL, the MADT was significantly more sensitive than the anterior drawer test (p < 0.001), Lachman test (p = 0.026), and pivot shift test (p = 0.013). The MADT showed similar sensitivity in detecting anteromedial and posterolateral bundle tears (p = 0.113) and no difference in diagnosing acute and chronic ACL ruptures (χ2 = 1.682, p = 0.195). CONCLUSIONS: The MADT is also an alternative diagnostic test to detect ACL tear, which is equally superior to the anterior drawer test, Lachman test, and pivot shifting test. It could improve the diagnosis of ACL ruptures combined with other clinical information including injury history, clinical examination, and radiological findings. LEVELS OF EVIDENCE: Level II/observational diagnostic studies TRIAL REGISTRATION: Chinese Clinical Trial Registry. ChiCTR1900022945 /retrospectively registered.

An Alternative Intraoperative Radiographic Method for Optimizing Cup Inclination during Total Hip Arthroplasty.

OBJECTIVE: During total hip arthroplasty (THA), the location of the acetabular prosthesis is very important for guaranteeing successful surgery. However, this remains a challenge for many surgeons. This study aimed to investigate the feasibility of using the intraoperative fluoroscopic iliopubic tangential angle (IPTA) to determine the valgus angles of acetabular prostheses. METHODS: In the first stage, the IPTA and valgus angles of native acetabula were defined and measured in 102 THAs obtained from the picture archiving and communication systems of our hospital. Obturator foramen morphology was also measured and divided into groups according to the axial length ratio. Correlations between obturator foramen morphology and IPTA and valgus angles of native acetabula were then determined. In the second stage, angular differences (angle θ) between the IPTA and valgus angles of native acetabula were measured during unilateral THA in 136 patients to determine whether this is a stable value that could be used as a reference for placement of acetabular prostheses. Postoperative data such as Harris Hip Score (HHS) scores and complications were collected and evaluated. RESULTS: The average IPTA at the apex of the true acetabulum was 58.8° ± 4.1°. The average valgus angle of native acetabula was 43.1° ± 3.9° and angle θ was 15.7° ± 1.3°. Obturator foramen morphology was divided into five groups according to the axial length ratio. IPTA was closely related to obturator foramen morphology (Pearson r = 0.489, P ≈ 0.000). Angle θ was stable and independent of obturator foramen morphology. When the IPTA and angle θ were used as references for placing acetabular prostheses in 136 THAs, the average postoperative valgus angles of acetabular prostheses was 45.13° ± 4.07° and the good-to-excellent rate was 97.05%. There were no short-term complications such as fracture, dislocation or infection. The average HHS score 6 months after surgery was 37.2 higher than the preoperative score, this difference being significant (P < 0.01). Neither infection nor dislocation was found 6 months after surgery. CONCLUSIONS: The IPTA (at the apex of the true acetabulum) and angle θ (obtained by intraoperative fluoroscopy) are consistent in determining the postoperative valgus angle of acetabular prosthesis during THA. Thus, IPTA and angle θ are of value in guiding placement of acetabular prostheses; in particular, this method can facilitate the learning of young surgeons.

Regioselective synthesis of cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium.

The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was successfully performed to achieve efficient acylation modification of a polar nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the catalytic activity and regioselectivity of the whole-cells clearly increased with increasing the polarity of the hydrophobic organic solvents used. The effects of some crucial factors on the reaction were further examined. The best reaction medium, hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90, 30 °C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired 3'-O-propional derivative of ara-C was synthesized with the yields of 88.3% and regioselectivity (>70%). The resulting biocatalytic system appears to be an effective alternative, and can thus be employed for application in highly regioselective modification of nucleoside analogues.

Immobilization of pectinase on oxidized pulp fiber and its application in whitewater treatment.

Modified pulp fiber was originally used as a new type of carrier for pectinase immobilization. Pulp fiber was oxidized by sodium periodate to produce aldehyde groups for covalently binding with amino groups of pectinase. Results showed that the enzymatic activity of immobilized pectinase on pulp fiber reached 65 µgg(-1)min(-1) when immobilization pH value, temperature and time were of 7.0, 20 °C and 15 min, respectively. The immobilized pectinase showed higher thermo stability in a wider temperature range of 40-70 °C than its free type and its optimal pH shifted from 8.0 to 8.8. Furthermore, the immobilized pectinase exhibited good operational stability. When employed in whitewater treatment of papermaking industry, it still efficiently decreased the cationic demand after operating repeatedly for six batches. The results obtained demonstrate a promising route to prepare available, cheap and biodegradable carrier for immobilizing enzymes with potential application in wastewater treatment in papermaking industry.

A facile whole-cell biocatalytic approach to regioselective synthesis of monoacylated 1-ß-D-arabinofuranosylcytosine: influence of organic solvents.

The lyophilized Pseudomonas fluorescens cell was an efficient alternative catalyst to enzymes for highly regioselective acylation of a polar nucleoside, 1-ß-D-arabinofuranosylcytosine (ara-C). The cells showed an evident solvent dependence in the reaction. Among the tested solvents except for acetonitrile-pyridine, catalytic activity of the cells clearly increased with increasing the polarity of the organic solvents used. Among all the tested solvents both pure and binary, the best results were observed in isopropyl ether-pyridine system, in which the catalyst also showed good thermal and operational stabilities. For the biocataylsis in isopropyl ether-pyridine, the optimal isopropyl ether concentration, water content, acyl donor/ara-C ratio, biocatalyst dosage and reaction temperature were 30% (v/v), 4%, 45, 50mg/mL and 30 °C, respectively, under which the initial rate, yield and 5'-regioselectivity were 2.93 mM/h, 77.1% and 97.3%, respectively. The bacterial cells exhibited comparable 5'-regioselectivity to the expensive immobilized enzyme, which could also have environmental and cost advantages.

A novel biocatalytic approach to acetylation of 1-ß-D-arabinofuranosylcytosine by Aspergillus oryzae whole cell in organic solvents.

Biocatalytic acylation of 1-ß-D-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. (13)C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3'-hydroxyl group than 5'-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3'-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3'-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.

Efficient synthesis of 5'-O-laurate of 1-beta-D-arabinofuranosylcytosine via highly regioselective enzymatic acylation in binary solvent mixtures.

Regioselective enzymatic acylations of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl laurate (VL) in binary organic solvents were explored for the preparation of 5'-O-laurate of ara-C. Among the nine kinds of enzymes, Novozym 435 showed the highest regioselectivity (>99.9%) towards the 5'-OH of ara-C. This lipase showed higher catalytic activity in hexane-pyridine than in other tested solvent mixtures. The most suitable VL to ara-C molar ratio, initial water activity, and reaction temperature were shown to be 15:1, 0.07, and 50 degrees C, respectively, under which the initial reaction rate and the maximum substrate conversion were as high as 84.0 mmol L(-1) h(-1) and 98.1%, respectively. The product of Novozym 435-catalyzed acylation was characterized by (13)C NMR and confirmed to be 5'-O-laurate of ara-C.

Highly regioselective enzymatic synthesis of 5'-O-stearate of 1-beta-D-arabinofuranosylcytosine in binary organic solvent mixtures.

In this paper, highly regioselective enzymatic acylations of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5'-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5'-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS-ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50 degrees C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol.L(-1)h(-1), 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13((13)C) NMR and confirmed to be 5'-O-stearate of ara-C.