Puerarin, an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae), modulates sclera remodeling caused by extremely low frequency electromagnetic fields

OBJECTIVE: To evaluate the protective effect of puerarin [an isoflavone compound extracted from Gegen(Radix Puerariae Lobatae)] in scleral remodeling induced by extremely low frequency electromagnetic fields(ELF-EMFs).METHODS: Human fetal scleral fibroblasts(HFSFs)were divided into 5 groups:(a) untreated controls;(b) cells treated with ELF-EMFs;(c) cells treated with ELF-EMFs and puerarin 0.1 μM;(d) cells treated with ELF-EMFs and puerarin 1 μM;(e) cells treated with ELF-EMFs and puerarin 10 μM. Cell proliferation activity was measured by the cell-counting kit-8 assay. Matrix metalloproteinase-2(MMP-2) activity was measured by gelatin enzymography.MMP-2 and collagenⅠ(COL1A1) m RNA, protein expression were measured by Real-Time polymerase chain reaction, Western blot analysis, respectively.RESULTS: Puerarin reduced the inhibition in cell proliferation, MMP-2 activity, m RNA, protein expression of HFSFs exposed to ELF-EMFs and enhanced the COL1A1 m RNA and protein expression.CONCLUSION: Puerarin was found to participate in the matrix remodeling process. It might be a potential agent for the treatment of extracellular matrix degradation of sclera associated with ocular conditions.

Preparation and Performance of Pueraria lobata Root Powder/Polylactic Acid Composite Films

Petroleum-based materials,such as plastic,are characterized by adverse environmental pollution;as a result,researchers have sought alternative degradable plastics that are environmentally friendly,such as polylactic acid(PLA).PLA has shown great potential to replace petroleum-based plastics.In this study,seven different samples of unmodified Pueraria lobata root powder(PRP)with different contents(i.e.,0,5,10,15,20,25,and 30 wt%)and three different modified PRPs(i.e.,treated with NaOH,NaOH-KH-550,and Formic)were used to reinforce polylactic acid(PLA)via solution casting process.These prepared PRP/PLA composite films were characterized using SEM,FTIR,UV-visible spectra analysis,TG,DSC,weight loss measurement(wt%),and mechanical measurements.The results showed that the PRP modified with KH-550(PRPK)intensified the interaction in the interface region between the PRP and the PLA matrix,thus increasing the tensile strength(54.5 MPa),elongation at break(2.8%),and Young’s modulus(3310 MPa)of the PRPK/PLA biofilms.Contact angle measurement showed that the PRP treatments contributed to the hydrophobicity of films.The transparency of PRP-10/PLA film atλ_(800)was 11.09%,and its UVA and UVB transmittance were 3.28 and 1.16,respectively.After blending PLA with PRP,the PRP/PLA composite films exhibited excellent biodegradability.In summary,PRPK improved the mechanical properties of PLA and prevented the films from ultraviolet light,suggesting that PRPK-5/PLA film could be used as packaging materials.

Removal of competitive native species combined with tree planting can accelerate the initial afforestation process: an experiment in an old field in Japan invaded by dwarf bamboo and kudzu

Restoring natural forests after field abandon- ment is a land management objective that fosters the re- covery of forest biodiversity. We performed seeding and transplanting of native tree species 40 years after the abandonment of an arable field that became dominated by a dwarf bamboo （Pleioblastus chino （Franch. et Sav.） Makino） and by kudzu （Pueraria lobata （Willd.） Ohwi）. By permutation tests, the removal of competing vegetation （gap creation） significantly increased the survival of three seeded species of Fagaceae and of eight transplanted spe- cies. In contrast, intact vegetation prevented most indi- viduals of all species from surviving for 1 year after planting. The lack of natural recruitment of Fagaceae in the nonseeded subplots indicated that seed limitation was a cause of the slow afforestation. Although litter accumula- tion in gaps at the time of seeding slightly increased sur- vival for late-germinating Quercus myrsinifolia Blume andCastanopsis sieboldii （Makino） Hatus. ex T. Yamaz. et Mashiba, the effect was not consistent among plots and was not statistically significant. Our results suggest that for successful afforestation using native trees in abandoned fields, it will be necessary to remove competitive native species to avoid severe limitations on microsite availability and that simultaneous tree establishment by seeding or transplanting should be implemented to accelerate the establishment of native tree species.

Effects of Rare Earth Elements on Promoting Isoflavonoid Production and Release of Pueraria Lobata Hairy Roots in Vitro

The effects of various rare earth elements on growth and isoflavonoid production in hairy root cultures of Pueraria lobata (Willd.) Ohwi (strain TR2) cultured in 500 ml flasks were studied. After 32 days of culture, the biomass of hairy roots increase 15 times and reach 3.2 g dry weight. Hairy root growth was inhibited by Y_2O_3, NaSeO_3 and Sm^(3+) because of brown formation. But significant promoting effect on root growth due to callus formation was observed in La^(3+) treatments. It is the most noteworthy that the production of total isoflavonoids and puerarin was enhanced greatly by La^(3+) treatment. A major portion of increased total isoflavonoids and puerarin was released into medium in La^(3+) treatment while the hairy root viabilities were preserved. Some specific secondary metabolite release processes could be induced by La^(3+) and their possible mechanism is discussed.

Comparative molecular docking studies of lupeol and lupenone isolated from Pueraria lobata that inhibits BACE1: Probable remedies for Alzheimer's disease

Objective: To discover lead lupane triterpenoid's potential isolated from Pueraria lobata roots against b-site amyloid precursor protein cleaving enzyme 1(BACE1), which serve as a rate limiting step in amyloid beta(Aβ) production altering the course of Alzheimer's disease. In addition, enzyme kinetics study and molecular docking were conducted to establish the inhibition type and structure activity relationship.Methods: A systematic study of 70% ethanolic P. lobata root extract was employed to identify its BACE1 inhibitory potential. Further, BACE1 inhibitory potential of two lupane terpenoids, yielded from ethanolic extract, was assessed. In order to determine their inhibition mode, Lineweaver–Burk plots and Michaelis–Menten model for BACE1 was performed. Auto Dock 4.2 program in addition determined the molecular interaction of BACE1 with isolated terpenoids.Results: Considering the inhibitory potential of 70% ethanolic extract of P. lobata against BACE1(IC_(50)= 80.35 mg/mL), lupeol and lupenone were subsequently isolated and exhibited notable or moderate BACE1 inhibitory activity with IC_(50) values of 5.12 and 62.98 mmol/L, respectively, as compared to the positive control quercetin(IC_(50)= 21.28 mmol/L). The enzyme kinetics study enabled us to identify both compounds as competitive inhibitors, where lupeol displayed a very potent inhibition against BACE1 with low inhibition constant(Ki) value of 1.43 mmol/L, signifying greater binding affinity.In order to understand the binding mechanism and structure–activity relationship of two triterpene-based BACE1 inhibitors, we employed computer aided docking studies which evidently revealed that hydroxyl group of lupeol formed two hydrogen bonds with the ASP32(catalytic aspartic residue) and SER35 residues of BACE1 with the binding energy of(-8.2 kcal/mol), while the ketone group of lupenone did not form any hydrogen bonds with BACE1 giving evidence for less binding affinity. These results in turn have predicted the dependence of the inhibitory activity in the presence of hydroxyl group which has provided a new basis for BACE1 blockade.Conclusions: Our results have successfully explored the molecular mechanism of lupane triterpenoids via BACE1 inhibition, suggesting that lupeol in particular could be utilized as a useful therapeutic and preventive agent to mitigate Alzheimer's disease.

Kudzu Forage Quality Evaluation as an Animal Feed Source

The nutritional value of kudzu (Pueraria lobata) was evaluated as a potential animal feed. Randomly selected samples from natural habitats with Jacksonville area (Alabama, USA) were utilized in the study. Kudzu leaves showed significantly higher dry matter and crude protein content than the stems;whereas, ADF fiber was significantly lower in the leaves. However, NDF and total digestible nutrients (TDN) were similar in the leaves and the stems. The leaf part of kudzu was especially rich in CP (24.46%) and met the requirements and recommendations for most ruminants. The TDN value for kudzu stem is 55.99%, which falls below but within the recommended range. Macronutrients concentration between the stem and leaf of kudzu, nitrogen, calcium, and magnesium showed a significantly higher in the leaf than the stem. However, phosphorous was similar in concentration in the stem and the leaf, and potassium was higher in the stem. In regard to the micronutrients, the only significant difference between the leaf and the stem was iron. However, iron, manganese, zinc, and copper were all above the recommended concentrations by the National Research Council. In general, kudzu can be considered a good source for animal feed, especially the leaf, which shows a higher value in most of the determined criteria in comparison to what was recommended by the National Research Council. This should provide a new outlook to kudzu, which has been considered a noxious weed and help in the utilization of kudzu in a positive way in the southern region of the US.

Diurnal changes in net photosynthetic rate of Pueraria lobataand its impact factors

In order to identify the relationship between diurnal changes in the net photosynthetic rate（Pn） of Pueraria lobata and environmental factors,diurnal changes in the Pn of leaves of two P.lobata cultivars were measured using a CIRAS-1 portable photosynthesis measurement system（PP-Systems,UK）.The results show that diurnal changes in Pn of both cultivars could be interpreted as double-peak curves,indicating the occurrence of an obvious midday depression.Further analyses indicate that the correlation between Pn and stomatal conductance was positive and extremely significant（p 〈 0.01）.The correlations of Pn with intercellular CO2 concentration and transpiration rate were positive and significant（p 〈 0.05）,while the correlations of Pn with air and leaf temperatures were negative and significant（p 〈 0.05）.The results indicate that among the factors affecting photosynthetic properties,some can be grouped as stomatal limitations while others are non-stomatal limitations.

Study on the mechanism of Salvia miltiorrhiza-Pueraria lobata in the treatment of type 2 diabetes mellitus based on network pharmacology

Objective:To explore the synergistic mechanism of salvia miltiorrhiza and Pueraria lobata of multi-component,multi-target and multi-pathway in the treatment of type 2 diabetes mellitus(T2DM).Methods:The chemical components of Salvia miltiorrhiza and Pueraria lobata were queried and screened through the pharmacological database and analysis platform of traditional Chinese medicine system,and the chemical components were predicted by SwissTargetPrediction database.At the same time,the related targets of T2DM were searched and screened from GeneCards,TTD,DrugBank and Disgenet databases.The chemical composition targets and disease targets are intersected,and the PPI network of intersection targets is constructed by using STRING11.0 database,and the PPI network nodes are screened to get the key targets.The GO function and KEGG pathway enrichment analysis of the key targets are carried out.Results:A total of 70 chemical constituents and 51 key targets for the interaction between chemical components and diseases were obtained through retrieval and screening.After enrichment and analysis of 51 key targets,a total of 71 cellular components,85 molecular functions,559 biological processes and 137 signal pathways were obtained.The treatment of T2DM with Salvia miltiorrhiza and Pueraria lobata may be related to AGE-RAGE,Pl3K-Akt,ErbB,insulin resistance,HIF-1 and other signal pathways.Conclusion:This study preliminarily reveals the action mechanism of Salvia miltiorrhiza and Pueraria lobata on the treatment of T2DM with multi-components,multi-targets and multi-pathways,which provides a certain basis for the study of the molecular mechanism of Salvia miltiorrhiza and Pueraria lobata pairs.

Active compounds in RenShenJian decoction ameliorate insulin resistance in vitro

Background:RenShenJian decoction,a combination of Pueraria lobata(Willd.)Ohwi and Panax ginseng C.A.Mey,has been used in China since the Song Dynasty(960-1279 C.E.)to relieve symptoms of diabetes mellitus.However,the key compounds in RenShenJian that ameliorate insulin resistance remain unclear.This study identified the anti-diabetic compounds in RenShenJian by rescuing the decreased function of adenosine 5’-monophosphate-activated protein kinase(AMPK),sirtuin 3(SIRT3),or glucose transporter isoform 4(GLUT4).Methods:After streptozotocin-induced diabetic mice were treated with RenShenJian,fasting blood glucose levels and protein expression of SIRT3,p-AMPK,and AMPK were determined.Compounds from RenShenJian in plasma were monitored using multiple responses by liquid chromatography-mass spectrometry.Additionally,two insulin-resistant cell models were incubated with compounds identified in RenShenJian in the blood.Glucose uptake was determined using the fluorescent analog 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino)-2-deoxyglucose.Protein expression levels of p-AMPK,AMPK,SIRT3,and GLUT4 were detected by western blotting.Results:RenShenJian decreased FBG levels and upregulated SIRT3 expression and AMPK phosphorylation in diabetic mice.Thirteen RenShenJian extracts were identified in the blood,11 of which increased the ratios of 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol 4-yl)amino)-2-deoxyglucose uptake in two insulin-resistant cell models.Nine extracts increased AMPK phosphorylation,nine increased SIRT3 expression,and six elevated GLUT4 expression in palmitate-induced HepG2 cells.Five extracts-puerarin,puerarin 6″-O-xyloside,genistein,ginsenoside Rb1,and ginsenoside Rd-simultaneously activated AMPK,SIRT3 and GLUT4.Conclusion:A series of compounds in RenShenJian that target AMPK,SIRT3,and/or GLUT4 was confirmed and indicate the chemical material basis of amelioration of insulin resistance by RenShenJian.

Comparative Study on Natural Selenium-enriching Ability of 7 Main Cultivars of Pueraria lobata

[Objectives]To screen out the selenium-enriched Pueraria lobata varieties suitable for cultivation in Guangxi according to the natural selenium-enriching ability of 7 main cultivars.[Methods]Taking 7 main cultivars of P.lobata in different places as experimental materials,field cultivation was carried out on the selenium-enriched yellow soil in Shanglin County,Nanning City of Guangxi,to study the difference in selenium-enriching ability of different main cultivars and the comparison of selenium content in different parts,as well as the transport mechanism.[Results]Under the condition of soil selenium content of 0.78 mg/kg,the selenium content of the root tubers of the 7 main cultivars all reached the selenium-enrichment standard.The selenium content of a single root tuber of Heping Variety in Teng County,Guangxi was the highest,at 0.061 mg/kg;Guifenge No.1 was the second at 0.055 mg/kg,and the total selenium accumulation per unit area was the largest.The ranking of selenium content in different parts of the same variety was:leaf>vine>head>root tuber.[Conclusions]Guifenge No.1 has the highest yield and the largest total selenium accumulation,so it is the most suitable natural selenium-enriched variety for cultivation.

Comprehensive Metabolic Profiling of Modified Gegen Qinlian Decoction by Ultra-high-Performance Liquid Chromatography-Diode Array Detection-Q-Exactive-Orbitrap-Electrospray Ionization-Mass Spectrometry/Mass Spectrometry and Application of High-Performance

Objective:Gegen Qinlian decoction(GQD)is a classical traditional Chinese medicine formulation which has been used for almost 2000 years.At Guang'anmen Hospital,Beijing,a modified GQD version(m GQD)with seven instead of four herbal ingredients has been applied to treat Type 2 diabetes.Quality control is a crucial prerequisite for the therapeutic application of herbal medicines.For the identification of products derived from classical GQD,the Chinese Pharmacopeia requires the analysis of only three marker compounds.Because m GQD is a more complex mixture containing seven herbs and hundreds of constituents,the pharmacopoeia method for GQD is inadequate.Materials and Methods:A more comprehensive characterization of the formula's constituents has been developed using ultra-high-performance liquid chromatography-diode array detection(UHPLC-DAD)-Q-Exactive-mass spectrometry(MS)in electrospray ionization positive and negative mode.Moreover,a new method for the fingerprint analysis of m GQD via high-performance thin-layer chromatography(HPTLC)has been established.Results:Altogether,91 compounds have been assigned to their originating plants and 84 substances were identified either by comparison with authentic references or with data from the literature.The HPTLC method is based on the application of two different mobile phases and is able to detect both lipophilic and hydrophilic constituents of m GQD.Conclusions:The modified GQD was extensively characterized by UHPLC combined with DAD and Q-Exactive Orbitrap high-resolution MS detection,leading to the assignment and identification of compounds present in the decoction.In addition,a new method for the fingerprint analysis of the m GQD using HPTLC was established,which allows fast and simple identification of the herbal ingredients in the mixture.

氯吡脲对葛根Ptexp1基因表达的影响及其与产量品质形成的关系

为研究氯吡脲对葛根Ptexp1基因表达影响及其与产量和品质的相互关系,揭示氯吡脲对葛根产量和品质影响的分子机制,本研究以‘桂葛1号’粉葛幼叶为材料,采用PCR同源克隆和RACE技术,克隆得到葛根expansin基因,命名为Ptexp1(GenBank登录号:MK169414)。该基因cDNA全长1063 bp,开放阅读框为91~846 bp,编码251个氨基酸,预测其分子量和等电点分别为26.78 kD和8.37,在64~149 aa和160~237 aa有DPBB_1和Pollen_allerg_1两个保守的蛋白质结构域。该基因与其他植物的expansin-A8基因核苷酸序列和氨基酸序列高度同源,其中与大豆、绿豆和豇豆expansin-A8基因的核苷酸序列同源性分别高达94%、92%和91%,与鸡血藤、大豆、豇豆和相思子expansin-A8基因的氨基酸序列同源性分别高达97%、95%、94%和93%。以葛根18S作为内参基因,Ptexp1基因在葛根膨大期不同组织中的表达规律为块根>根蔸>叶>茎,与不同部位发育表型吻合。1 mg/L氯吡脲处理显著提高该基因在块根、根蔸和茎的表达,但对叶片表达的影响不显著,与其处理显著提高块根、根蔸和茎的形态指标相吻合。本研究为氯吡脲调控葛根膨大的作用机理、产量和品质形成的分子机制及其新品种选育提供参考。