Blends of hydrophobic and swelling agents in the swelling layer in the preparation of delayed-release pellets of a hydrophilic drug with low MW: Physicochemical characterizations and in-vivo evaluations

In this study,a hydrophobic material,ethylcellulose,which was used as its aqueous suspension Surelease^(®),was combined with a swelling agent as the swelling layer to prepare delayed-release pellets for Danshensu,which is a hydrophilic drug with low MW.A rupturable,delayed-release pellet consists of a drug core,a swelling layer containing a swelling agent(cross-linked sodium carboxymethyl cellulose)with a hydrophobic agent(Surelease^(®)),and a controlled layer composed by an insoluble,water-permeable polymeric coating(aqueous ethylcellulose dispersions)was developed in a fluidised bed.Results showed that blending Surelease^(®)into the swelling layer could effectively extend the release of Danshensu from the pellets,which may be attributed to the slowed swelling rate by reduction of water penetration and improvement of mechanical integrity of the swelling layer.Drug in the delayed pellets showed sustained release in beagle dogs after oral administration with comparable in-vivo exposure to the uncoated drug pellets.In conclusion,blends of hydrophobic and swelling agents in the swelling layer in doublemembrane pellets could achieve a delayed drug-release profile in vitro,as well as delayed and sustained absorption in vivo for highly soluble,low-MW drug.The present study highlighted the potential use of a delayed-release system for other hydrophilic,low-MW drugs to meet the formulation requirements for chronopharmacological diseases.

Novel semicarbazide-sensitive amine oxidase inhibitor screening from anti-obesity drugs using HPLC-MS based technique

Semicarbazide-sensitive the metabolism of glucose and fat, amine oxidase （SSAO） has been considered to be associated with and elevated SSAO activity was observed in obese patients. In the present study, an in vitro SSAO activity-based method was developed to screen inhibitors from 15 an- ti-obesity drugs. Among the fifteen anti-obesity drugs, four drugs including caffeine, fenfluramine, bumetanide and amfebutamone inhibited SSAO activity, and caffeine was the most effective one. When the concentration of caffeine was 1.4 mmol/L, the inhibition ratio was 31.9% and 18.8% in rabbit serum and rat adipose tissue, respectively. Inhibition of SSAO activity by caffeine was also confirmed in the in vivo study, showing the inhibition ratio of 15.6% on serum SSAO. Caffeine pro- vides a natural source of inhibition of SSAO activity and may be a promising inhibitor for the study of SSAO.

HPLC combined with chemometrics for quality control of Huamoyan Granules or Capsules

Objective:Huamaoyan Granules(HMYG)and Huamaoyan Capsules(HMYC)are Chinese patent medicines with different dosage forms of the same prescription.Due to the different preparation process,the chemical composition of these Chinese patent medicines varies greatly among different forms,but there were few studies on the difference comparison and quality control of them.In order to improve the effectiveness and safety in its clinical application,an idea combining high performance liquid chromatography(HPLC)and chemometrics was put forward to study the quality control of Chinese patent medicines in different dosage forms of the same prescription.Methods:The differential markers of HMYG and HMYC were explored based on HPLC fingerprint and chemometrics including orthogonal projections to latent structures-discriminant analysis(OPLS-DA),principal component analysis(PCA),and hierarchical cluster analysis(HCA).Finally,the quantitative analysis method of related components was established by HPLC.Results:A quality control method for HMYG and HMYC was established.Firstly,the chemical components of HMYG and HMYC were systematically analyzed by HPLC fingerprinting.Further exploration showed that there were 20 characteristic peaks and 57 common peaks.Then,the potential differential markers between HMYG and HMYC were explored by chemometrics,and the differential markers were screened after intersection with the 20 characteristic peaks.Finally,HPLC quantitative analysis methods for nine components were established,including seven differential markers(neochlorogenic acid,protocatechualdehyde,chlorogenic acid,cryptochlorogenic acid,caffeic acid,rosmarinic acid and salvianolic acid A).The results of HPLC quantitative analysis showed that the contents of eight components in HMYG and HMYC samples were significantly different.According to the above results,the differential markers between HMYG and HMYC screened based on HPLC fingerprint and chemometrics can effectively characterize the differences between the two dosage forms.Conclusion:The present work provides a rapid and effective method for routine quality evaluation and control of HMYG and HMYC.This work also provides feasible methods for the quality evaluation and control of Chinese patent medicines with different dosage forms of the same prescription.

A review of clinical practice guidelines on the management of preeclampsia and nursing inspiration

Objectives:To review,evaluate,and synthesize the recommendations of guidelines on preeclampsia(PE)from a nursing perspective.Methods:This is a systematic review of international and national guidelines on PE.Electronic databases and related guideline websites were searched from 2013 to 2023.After systematic retrieval and screening,we used the AGREE II tool to appraise the methodological quality of guidelines that met the eligibility criteria.Then,we analyzed and summarized the recommendations using descriptive analysis and the framework method.Furthermore,we rated the quality of evidence and the strength of the recommendations using the GRADE approach.Results:Ten guidelines were included,among which eight were deemed“clinically useful”and records were extracted.In total,31 recommendations,including 46 items on the nursing management of PE,were summarized from three aspects:1)antenatal care(18 recommendations),including the assessment and regular monitoring of PE,standardized blood pressure(BP)measurement,prevention education for PE,antihypertensive agent monitoring,and magnesium sulfate(MgSO_(4))use;2)intrapartum care(4 recommendations),including childbirth care;3)postpartum care(9 recommendations),including regular nursing monitoring,breastfeeding care and longer-term health counseling.Most of the evidence was rated as“very low”(19/46)or“moderate”(15/46)quality.For the strength of the recommendations,30 items were graded as“strong”and 16 items were rated as“weak”.Conclusions:This study provides professional,evidence-based nursing care resources both for preservice education for nursing staff and health care education for women with PE to help them detect and treat PE in a timely manner.

马兜铃属中药材中马兜铃酸成分的快速定量检测方法研究

Aristolochic acid (AA) composed of AA-Ⅰ and AA-Ⅱ (Fig.1) is the major toxic component extracted from Aristolochia plants[1]. Recently, it has been proved by several researches that high doses of aristolochic acids (AA) can result in a severe kidney disease named Chinese herbs nephropathy (CHN)[2]. It is characterized by a progressive interstitial fibrosis leading to a severe atrophy of the proximal tubules[3]. In the past several years, CHN has been reported in many countries including Belgium, France, Spain, UK and Japan[1]. Owing to its high toxicity, several health institutions, including the US Food and Drug Administration (FDA), and Medicines Control Agency (MCA), have warned people to pay attention to the safety information about botanical products containing AA. Many countries including UK, Canada, Australia, and Germany have announced banning to restrict the importation, sale and use of AA-containing medicines. So it is of great significance to develop effective and rapid methods to quantify the content of AA in regular herbal medicines.

Determination of Eight Sudan Dyes in Chili Powder by UPLC-MS/MS

Sudan dyes are synthetic azo dyes which are widely used in industry. Although they are not allowed in foodstuffs, they have been found contaminating in different food products and their presence is regularly reported. It is assumed that these appearances are due to cross-contamination or adulteration. In this paper, we present a newly developed fast and sensitive method for the quantification of eight Sudan dyes, using liquid-liquid extraction and UPLC-MS/MS analysis. The calibration curves were linear over the range of 0.1-25 mg/kg. Mean recovery for the eight Sudan dyes ranged from 80.7% to 104.4%, and with the inter-day and intra-day precisions ranged from 2.24% to 12.2%. The method was successfully applied in the determination of Sudan dyes in chili powder of 10 samples.

Improving the accumulation of 18α-and 18β-glycyrrhizins by over-expressing GuHMGR, GuSQS1, and GuBAS genes in Glycyrrhiza uralensis

Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.

Preparation and evaluation of a phospholipid-based injectable gel for the long term delivery of leuprolide acetaterrh

A phospholipid-based injectable gel was developed for the sustained delivery of leuprolide acetate(LA). The gel system was prepared using biocompatible materials(SPME), including soya phosphatidyl choline(SPC), medium chain triglyceride(MCT) and ethanol. The system displayed a sol state with low viscosity in vitro and underwent in situ gelation in vivo after subcutaneous injection. An in vitro release study was performed using a dialysis setup with different release media containing different percentages of ethanol. The stability of LA in the SPME system was investigated under different temperatures and in the presence of various antioxidants. In vivo studies in male rats were performed to elucidate the pharmacokinetic profiles and pharmacodynamic efficacy. A sustained release of LA for 28 days was observed without obvious initial burst in vivo. The pharmacodynamic study showed that once-amonth injection of LA-loaded SPME(SPME-LA) led to comparable suppression effects on the serum testosterone level as observed in LA solution except for the onset time. These findings demonstrate excellent potential for this novel SPME system as a sustained release delivery system for LA.

A combined strategy for the overproduction of complex ergot alkaloid agroclavine

Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis

The present study was designed to determine the effects of copy number variations(CNVs) of squalene synthase 1(SQS1) gene on the mevalonate(MVA) pathway.SQS1 gene from G.uralensis(Gu SQS1) was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy number of Gu SQS1 were constructed.HPLC was used to assay the level of ergosterol in all transgenic P.pastoris strains containing Gu SQS1.HPLC analysis showed that the contents of ergosterol in all of the transgenic P.pastoris containing Gu SQS1 were higher than that in the negative control.And with the increase of copy number of Gu SQS1,the content of ergosterol showed an increasing-decreasing-increasing pattern.The contents of ergosterol in 10-copy-Gu SQS1 P.pastoris and 47-copy-Gu SQS1 P.pastoris were significantly higher than that in the rest recombinant P.pastoris strains.In conclusion,the CNVs of Gu SQS1 influence the content of secondary metabolites in the MVA pathway.The present study provides a basis for over-expressing Gu SQS1 and increasing the content of glycyrrhizin in G.uralensis cultivars.

Comparative study on main compounds in Ginkgo biloba L. base on mathematical statistics

In this paper,an HPLC-DAD-ELSD method was developed to determine main 20 components of Ginkgo biloba L.leaves from different ages and sources,including six flavonol glycosides,five terpene lactones and nine organic acids.Using statistics method and establishing relevant mathematics models,the measured data has proceeded correlation analysis,principal component analysis,and regression statistics and the results showed generality and specific characteristics.We defined p-hydroxybenzoic acid,catechinic,KRcG and ginkgolide A as characteristic indexes representing commonness and speciality of Ginkgo biloba L.leaf.The four characteristic indexes can reflect the quality of Ginkgo biloba L.leaf,and the intermal relations between them are significant.The contents of other compounds could define the quantity relation with characteristic markers.It simplified the approach of quality control,and provided a basis for quality control of Ginkgo biloba L.

Determination of inulin-type fructo-oligosaccharides in inulin by HPLC-ELSD

To develop an HPLC-ELSD method for the determination of nine inulin-type fructo-oligosaccharides in inulin,the HPLC-ELSD system consisted of Waters XBridge■ Amide column(4.6 mm×250 mm,5μm)with a gradient elution mobile phase consisting of acetonitrile and water at a flow rate of 1.2 mL/min at 30°C.The detector was an Agilent Technologies 380-ELSD.The drift tube temperature for the ELSD was set at 55°C with a nitrogen flow rate of 1.8 L/min.The injection volume was 15μL.The results showed that the detection range for the nine inulin-type fructo-oligosaccharides was 3.81–30.60μg R^(2)=0.99969 for kestose,3.73–29.97μg R^(2)=0.99981 for nystose,3.82–30.69μg R^(2)=0.99993 for fructosylnystose,3.80–30.48μg R^(2)=0.99995 for GF5,3.73–29.96μg R^(2)=0.99993 for GF6,3.78–30.30μg R^(2)=0.99983 for GF7,3.82–30μg R^(2)=0.99989 for GF8,3.71–29.80μg R^(2)=0.99974 for GF9,3.61–29.00μg R^(2)=0.99970 for GF10,respectively.The recovery of the nine oligosaccharides ranged between 96.48%–100.84%(n=6).The method was simple,accurate,and reproducible that it could be used as an analytical method for evaluating the quality of inulin effectively.

Sphingolipids Profiling of Plasma in Patients with Diabetes Mellitus Associated with Atherosclerosis by a Novel Normal-Phase UPLC-QToF MS Method

A novel normal-phase UPLC-QToF MS method was developed for sphingolipids analysis,which was then applied to separate and identify seven classes of sphingolipids in plasma.The time-consuming of this novel method was much shorter than previous 2D LC–MS method,while the sensitivity,repeatability and resolution for sphingolipids analysis were better or comparable.Besides,the differences of lipids in human plasma among diabetes mellitus patients,diabetes mellitus associated with atherosclerosis patients and control subjects were also compared.Finally,12 sphingolipids species were identified as the amounts of which in diabetes mellitus associated with atherosclerosis patients were significantly higher than them in diabetes mellitus patients(VIP>1.0,p value<0.05,fold change>1.5).As the isomers of GalCs and GluCs were successfully separated,it was found that five GluCs,rather than GalCs,were included in these 12 potential biomarkers.These results suggest the need to separate GalCs from GluCs,and also indicated that there may be some extra activation pathways of diabetes mellitus associated with atherosclerosis,different from the atherosclerosis pathway and the diabetes mellitus pathway,causing cardiovascular disease.