Design,Preparation,andCharacterizationofDioscin ... · 2019. 11. 14. · Table...

Research ArticleDesign Preparation and Characterization of DioscinNanosuspensionsandEvaluationofTheirProtectiveEffect againstCarbon Tetrachloride-Induced Acute Liver Injury in Mice

Hong-Ye JU Kun-Xia Hu Guo-Wang Zhao Zhi-Shu Tang and Xiao Song

School of Pharmacy Shaanxi University of Chinese Medicine Xianyang 712046 China

Correspondence should be addressed to Xiao Song song-xiaoyao163com

Received 11 October 2018 Revised 20 December 2018 Accepted 17 July 2019 Published 14 November 2019

Guest Editor Maria Rosaria Lauro

Copyright copy 2019 Hong-Ye Ju et al 1is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

1e purpose of this study was to prepare a dioscin nanosuspension (Dio-NS) that has a better distance and high solubility for oraladministration and to evaluate its hepatoprotective effects Optimal primary manufacture parameters including shear time shearspeed emulation temperature pressure and cycles of homogenization were determined by single-factor experiments 1econcentrations of dioscin SDS and soybean lecithin were optimized using the central composite design-response surface methodand their effects on the mean particle size (MPS) and particle size distribution of Dio-NS were investigated Characterization of theDio-NS formulations included examinations of the surface morphology and physical status of dioscin in Dio-NS the stability ofDio-NS at different temperatures in vitro solubility and liver protective effect in vivo Under optimal conditions Dio-NS had anMPS of 10672 nm polydispersity index of 0221 and zeta potential of minus 3427mV Furthermore the proportion of dioscin in Dio-NS was approximately 2126 1e observation of particles with a spherical shape and the disappearance of crystalline peaksindicated that the physical and chemical properties of Dio-NS were altered Furthermore we observed that the dissolution of Dio-NS was superior to that of a physical mixture and Dio-GZF Moreover Dio-NS was demonstrated to have a protective effectagainst CCl4-induced acute liver damage inmice that was equivalent to that of silymarin (a positive control drug) at the same dose1e good hepatoprotective effect of our Dio-NS preparation can provide a theoretical basis for investigating its absorptionmechanisms in the body

1 Introduction

Dioscin (Figure 1) also known as Paris polyphylla saponinIII is a steroidal saponin [1]that can be extracted from theChinese yam (Dioscorea paniculata) and other plants in theDioscoreaceae family [2] Dioscin has several importantpharmacological activities including antitumor [3] anti-inflammatory [4] antiliver damnification [5] antihepaticfibrosis [6 7] antihyperlipidemic and antioxidative prop-erties Furthermore the compound has therapeutic potentialin metabolic diseases and can also be decomposed intodiosgenin which has been an important basic raw materialfor the production of steroid hormone drugs [8]Meanwhileit has also been established that there is potential to developthe pharmaceutic value of dioscin In addition some reportshave been found on dioscin toxicology It has no adverse

effect on the acute toxicological studies at a dose of 5625mgkgd in mice [9 10] However on the subchronic toxico-logical assessment it was verified as the NOAEL (no-ob-served-adverse-effect level) at a dose of 300mgkgday infemale rats and in male rats was marked as the LOAEL(lowest-observed-adverse-effect level) at the same dose [11]So rational drug use is the key to lower ADR

In fact dioscin has a notable disadvantage in that whenthe valid route fails the probability of a new establishedroute occurring via secondary chain scission is large Its poorsolubility and slow dissolution rate limit its absorption thusaffecting its efficacy

Poor solubilities and dissolution rates are key factorsaffecting the absorption of drugs in the body 1us severaltechniques have been devised in order to overcome theseundesirable properties including micronization [12]

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 3907915 16 pageshttpsdoiorg10115520193907915

solubilization and salt formation [13] However thesetechniques are only partially effective 1e advent ofnanotechnology which can produce nanoscale particles withnovel functional properties offers a potential solution to theaforementioned drawbacks Nanotechnology has for ex-ample been used to enhance the bioavailability of certainantibiotics [14] and has also been applied to produce dif-ferent targeted drugs for the treatment of different diseases[15ndash17] and various nanosuspensions have been success-fully applied [18 19] Nanoparticles can also be preparedusing a variety of different processes including reversesolvent homogenization pressure milling and the pro-duction of self-nanoemulsifying self-nanosuspensions [20]

Diseases of the liver including hepatitis hepatapostemahepatocirrhosis and liver cancer are common and oftenlife-threatening Carbon tetrachloride (CCl4) [21 22] par-acetamol [23 24] lipopolysaccharide [25 26] and certainother chemicals can also cause acute liver damage andconsequently a large number of drugs are used for thetreatment of hepatic disorders [27] It was confirmed thatdioscin has significant hepatoprotective effect against CCl4-induced liver injury in mice and the cellular mechanisms ofthis effect are likely to be associated with inhibition of lipidperoxidation inflammatory cytokines necrosis and apo-ptosis 1us in the present study we formulated a dioscinnanosuspension (Dio-NS) using reverse solvent pre-cipitation combined with high-pressure homogenizationand compared its efficacy with that of the commercial drugsilymarin in protecting mice from acute hepatic damageinduced by CCl4 We assumed that the Dio-NS formulationwould enhance the solubility of dioscin and thus increase theefficacy of this compound in protecting against liver injury

2 Methods

21 Materials Dioscin was purchased from Nanjing Springand Autumn Biological Engineering (Nanjing China) withthe purity of gt99 Soybean lecithin mannitol poloxamer-188 Tween-80 PVP K30 sodium cholate sodium alkylsulfate twelve (SDS) anhydrous ethanol lactose glucosesucrose D-sorbitol silymarin 4 paraformaldehyde solu-tion methanol and acetonitrile were all of chromatographicgrade and the other reagents were all of analytical gradeAST and ALT kits were purchased from Nanjing Jiancheng

Institute of Biotechnology (Nanjing China) TNF-α IL-1βIL-6 MDA SOD and GSH-Px kits were purchased fromShanghai Elisa Biotech Co Ltd

22 Preparation ofDio-NS Dioscin was dissolved in ethanolas a solvent and dispersed for 15min in a KQ-3-DE ul-trasonic bath (Kunshan China) In the next emulsificationprocess ethanol was used as the organic phase of theemulsion 1e solution was rapidly added dropwise todouble-distilled water-saturated ethanol together with so-dium alkyl sulfate twelve and soybean lecithin as excipientsthat can be used to improve the stability of emulsionscommixing using magnetic stirrer (HJ-6B ChangzhouChina) A microemulsion was obtained by high-speedshearing (FJ-200 ShenZhen China) 1e microemulsionpreparation was then homogenized to produce a nano-emulsion via high-pressure homogenization (AH-BASICIATS Engineering Inc Canada) An EYELA N-1300 rotaryevaporator was used to remove the ethanol (LangYiShanghai China) at an evaporation temperature of 40degCthereby yielding the Dio-NS preparation In this study theoptimal process conditions were determined on the basis ofsingle-factor experimental results All the preparation stepsare shown in Figure 2

23 Preparation of a PhysicalMixture 1e desired dosage ofDio-NS was obtained by dissolving in water and freeze-drying for 12 h to yield an NS-A physical mixture

24 Screening of Excipients Eleven groups were examined toselect the best accessories that have a minimum size andpowder dispersion index

25 Single-Factor Experiment In the single-factor experi-ment particle size and multiple dispersion index were takenas indicators to determine optimal preparation parametersincluding shear time shear speed emulation temperaturepressure and cycles of homogenization

26 Optimization of the Response Surface of the Star DesignOn the basis of the results of the single-factor experimentand the feasibility of preparing nanosuspensions at thehighest or lowest level we selected the level of each factorand the star response surface design software and de-termined the best experimental results

27 Lyophilization 1eDio-NS preparation was lyophilizedusing a VaCo-5 freeze-drying apparatus (Zirbus Germany)to obtain dry powder with good physicochemical stability Inthis study a single-factor method was used to select optimalconditions and the best freeze-drying protectant was se-lected 1e results are shown in Table 1 1e content offreeze-drying protectant ranged from 2 to 10 1e levelsare shown in Table 2

OO

CH3

H3C

H3C

H3C

HH

O

HO

OHO

O

OH

H

H

CH3

HO

HOOH

H

O

CH3

HO

HOOH

OH

H

Figure 1 1e chemical structure of dioscin

2 Evidence-Based Complementary and Alternative Medicine

28 Particle Size and Zeta Potential 1e mean particle size(MPS) particle size distribution (PSD) and zeta potential ofthe formulation were determined using a Malvern particlesize meter Samples were prepared by dissolution of Dio-NSin deionized water and each property was measured 3 times

29 Morphological Observations (SEM) 1e morphology ofparticles was determined by scanning electron microscopy(SEM) (MERLIN Compact German) Samples were affixedto aluminum stubs using a double-sided carbon tape andsputter-coated with gold under an argon atmosphere

210 Transmission ElectronMicroscopy (TEM) After 4 typesof samples had been vortexed for 2min they were drippedonto a copper sheet of a transmission electron microscope(JEOL Japan) and the excess moisture was dried using afilter paper Images of the samples were observed on theperspective electron microscope

211 X-Ray Powder Diffraction 1e crystalline state ofsubstances was typically determined by X-ray powder dif-fraction Diffraction patterns of the Dio-NS and otherauxiliary material were determined using a Bruker D8

Magnetic stirring

High-speed shearing

High-pressure homogenization

Evaporation

Dissolving dioscinin ethanol

Dissolving SDS and soybean lecithinin water

Microemulsion

Dio-NS

Freeze-dried Dio-NS powder

Nanoemulsion

Figure 2 A flow diagram showing the steps involved in the preparation of Dio-NS

Table 1 Screening of freeze-drying protectants

Type Sensory evaluation Size (nm) PDI Zeta (mv)

Lactose White powder shows collapse and shrinkage unevencolor and poor dispersion 1235plusmn 061 0278plusmn 0003 minus 232plusmn 225

Glucose White powder shows collapse and shrinkage unevencolor and poor dispersion 2200plusmn 52185 1plusmn 0001 minus 423plusmn 186

Sucrose White powder shows collapse and shrinkage unevencolor and poor dispersion 11957plusmn 38227 0586plusmn 0075 minus 514plusmn 541

D-sorbitol White powder shows collapse and shrinkage unevencolor and poor dispersion 1724plusmn 699 0271plusmn 0023 minus 526plusmn 268

Mannitol White powder shows no collapse or shrinkageuniform color and good dispersion 223plusmn 156 0273plusmn 0055 minus 591plusmn 038

(n 3 Meanplusmn SEM)

Evidence-Based Complementary and Alternative Medicine 3

advance X-ray diffractometer (Bruker Germany) A Cu linewas used as the source of radiation and standard runs wereperformed at a voltage of 40 kV step length of 001deg currentof 40mA and a scanning rate of 01 sstep over a 2 thetarange of 5ndash90deg

212ermal Analysis Individual samples were placed indifferent aluminum differential scanning calorimeter(DSC) pans (214 Polyma Netzsch Germany) whichwere heated to 200degC and 150degC for approximately15 min 1ereafter the samples were quench-cooled inliquid nitrogen externally to the DSC instrument DSCwas implemented by returning the samples to a prere-frigerating room (minus 50degC) and thermograms wererecorded

213 In Vitro Dissolution of Dio-NS 1e Dio-NS powderand physical mixture were loaded into dialysis mem-branes which were placed in constant-speed oscillatorsat constant temperature and oscillated for differenttimes Samples of Dio-NS obtained at different timepoints were analyzed by HPLC (Waters e2695 USA) 1ecumulative release rate was determined using the fol-lowing equation

Q(cumulative release rate) Qt1 + Qt2 + middot middot middot + Qtn( 1113857

Qtimes 100

(1)

where Qt1Qt2 Qtn is the content of dioscin dissolved inDio-NS between time t0 and t12 n

214 Stability Study 1e stability of the dioscin nanoscalesuspension at temperatures of 4degC and 25degC was investigatedover a period of 1month and the results are shown inTable 3

215 e Effects of Dio-NS on Liver Preservation

2151 Animals and Treatment Kunming male mice (18ndash22 g) were purchased from Chengdu Dashuo BiotechnologyCo Ltd (Chendu China) 1e mice were acclimated for 1week in a controlled environment at 25plusmn 2degC under a 12 hdarklight photoperiod 1ey were subsequently randomlydivided into the following 6 groups each containing 8 an-imals group 1 (normal control) and group 2 (model control)were orally administered water for 7 days group 3 (positivecontrol) received silymarin (50mgkg) for 7 days andgroups 4 to 6 (low- medium- and high-dose groups re-spectively) were administered Dio-NS (25 50 and 100mgkg respectively) intragastrically Two hours after the finaladministration the animals in group 1 were injected in-traperitoneally with pure olive oil whereas mice in the othergroups were treated with 02 (vv) CCl4 (10mLkg bodyweight ip dissolved in olive oil) [28] 1ereafter the micewere maintained with free access to water and were sacri-ficed 24 h later at which time blood was collected for thepreparation of serum After killing the animals the liverswere immediately isolated and weighed to determine theliver index (liver index liver weightbody weighttimes 100)A portion of each liver was fixed in 4 paraformaldehydesolution for pathological study and the remainder alongwith the acquired serum samples were stored at minus 80degC forsubsequent studies1e entire process was performed on ice

2152 Serum Biochemistry Assays 1e activities of ALTAST TNF-α IL-1β and IL-6 in the serum were measuredusing commercial kits following the manufacturerrsquosinstructions

2153 Liver Lipid Peroxidation Assay After saline wasadded to the appropriate liver tissue the sample was placedinto an automated tissue homogenizer (SCIENTZ-48Ningbo China) to prepare a 10 hepatic homogenateSamples were processed using commercial kits according themanufacturerrsquos instructions and the levels of MDA GSH-Px and SOD in the liver homogenate were evaluated using amicroplate reader

2154 Histopathological Examination Fixed liver tissueswere embedded in paraffin from which 5-microm sections werecut and placed on slides 1e samples were stained withhematoxylin and eosin (HampE) and then observed at times400magnification

Table 2 Screening for the dosage of freeze-drying protectants

Ratio () Size (nm) PDI Zeta (mv)

24334 0620 minus 4084632 0553 minus 4184413 0540 minus 428






Table 3 Stability of Dio-NS freeze-dried powder over a period of2months (n 3)

Time (d) Size (nm) PDI Zeta (mV)0 1732plusmn 056 0235plusmn 001 minus 563plusmn 23615 1737plusmn 063 0237plusmn 021 minus 5654plusmn 26630 1739plusmn 075 0237plusmn 056 minus 5673plusmn 28660 1749plusmn 085 0239plusmn 046 minus 6343plusmn 126


2155 Statistical Analysis SPSS version 190 statisticalsoftware was used to analyze all the data using a one-wayANOVA Data are represented as meanplusmn SEM Differencesbetween groups were considered significant at plt 001 andplt 005 levels

3 Results

31 Evaluation of the Dio-NS Preparation 1e results ofstabilizer screening showed that different stabilizers had aconsiderable influence on the average particle size poly-dispersity index and system storage stability of drug sus-pensions [29](Table 4) On the premise of maintaining thesame total amount the combined use of different types ofstabilizers effectively reduced the 3 qualitative indexes ofdrug suspension In this study we found that when thecombined application of soybean lecithin and SDS was usedas a stabilizer for the suspension system the average size ofthe drug particles was small the distribution was narrowand the physical stability of the system was good 1ereforewe used soybean lecithin and SDS as stabilizers for Dio-NSAn SDS lecithin ratio of 1 1 proved to be the optimalcombination

As shown in Figure 3 it is clear that temperature haslittle effect on emulsification We also established that theminimum particle size and polydispersity index are obtainedunder the following conditions a shear speed of 19500 rpma shear time of 2min a homogeneous pressure of 800 barand 7 cycles of homogenization We thus obtained theoptimal operation parameters (Table 5) Table 6 shows theresults of 20-run experiments designed on the basis of 3factors and 4 levels using Design-Expert 806 software 1eresults of a regression analysis are shown in equations (2)and (3)Y +066 minus 0095A minus 0023B minus 0065C + 010AB minus 0033AC

+ 2375E minus 003BC minus 013A2

+ 0030B2

+ 0047C2

R 09411 Plt 00001

(2)

OD (d1d2 dk)1K whereK is the number of indices

dmin Ymax minus Yi( 1113857 Ymax minus Ymin( 1113857

dmax Yi minus Ymin( 1113857 Ymax minus Ymin( 1113857(3)

Figure 4 shows the results of the interaction between 2groups For Dio-NS preparation with a content of 2667(ww) dioscin 20 (ww) SDS and 5333 (ww) soybeanlecithin we obtained a maximum OD of 0868 which wasclose to that predicted As shown in Table 1 the diameter ofDio-NS dry powder particles polydispersity index and zetapotential were less than those obtained using other freeze-drying protection agents Furthermore the powder showedno collapse or shrinkage the color and luster were moreuniform and the preparation had good dispersion andtherefore soybean lecithin and SDS were selected as theprotectants for the dioscin freeze-dried powder

It can be seen from Table 2 that the particle size obtainedusing mannitol in the aqueous phase was 7 and values for

the polydispersity index and the zeta potential were thesmallest recorded

1e size zeta potential and polydispersity index of theDio-NS formulations are shown in Table 7 A particle sizediagram and zeta potential are shown in Figures 5(a) and 5(b)respectively A sharp peak of Dio-NS which represents itssize was identified at approximately 10672plusmn 890 Further-more the zeta potential of Dio-NS was minus 3427plusmn 291mVindicating the reasonable stability of the preparation

32 Characterization of Dio-NS

321 Particle Size and Zeta Potential 1e particle size andzeta potential of the optimum Dio-NS are given in Table 7

322 Surface Morphology and Particle Size of Dio-NSTransmission and scanning electron microscopy were ap-plied to analyze the morphology and structure of the Dio-NSformulations Dioscin has a tubular crystal structure(Figure 6(a)) Figure 6 shows that Dio-NS has a near-spheroid particle structure with a particle size of approxi-mately 106 nm Figure 6(b) shows that the redispersibility ofDio-NS powder in purified water was also nearly globoidand that the particle size is somewhat larger at approximately173 nm

1e scanning electron microscopy images shown inFigure 7 show that dioscin raw material (A) is acicular witha larger particle size whereas that with mannitol B is anirregular prism that with soybean lecithin (C) shows ir-regular lumps and that with twelve alkyl sodium sulfate(D) is irregular spherical and loose with pores Dio-NSnanoparticles (E) show the same shape as seen undertransmission electron microscopy and the morphologychanges As described above these features indicate thatthe freeze-drying process has significant effect on particlesize [30]

323 Physical Status of Dioscin in the Dio-NS DSC ther-mograms of pure dioscin SDS soybean lecithin mannitolphysical mixture and freeze-dried and dry powder of Dio-NS are shown in Figure 8 1e DSC thermograms of rawdioscin mannitol and SDS show a sharp endothermic peakat approximately 294ndash296degC 166degC and 204ndash207degC re-spectively A peak similar to that of pure dioscin was alsoobserved in the thermogram of the physical mixture in-dicating a weak or negligible influence However this peakwas not present in the freeze-dried and dry Dio-NS prep-arations which indicates that dioscin occurs in an amor-phous form in Dio-NS

1e diffraction intensity and distribution of each crystalwill have a special rule [31] By using X-ray diffraction wecan analyze whether the crystalline form has changed in thepreparation process 1e diffraction peak of dioscin rawpowder shown in Figure 9 indicates that it has a strongcrystalline structure whereas the diffraction peak of theexcipient soybean lecithin indicates that its structure isessentially amorphous 1e peaks of dioscin in the physical


Table 4 Screening of stabilizers

Type Content MPS PDI Macrostability of the system

F-68 05 14517 02271e suspension is white and opaque producing

flocculent precipitates and deepening the color of thesystem

SDS 05 12547 01961e suspension is white and opaque and easy toprecipitate It can be dispersed into an original

emulsion

PVP K30 05 91 03011e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

Tween-80 05 1088 03161e suspension is white and opaque It is easy toprecipitate It cannot be dispersed into an original

emulsion

Soybean lecithin 05 201 02691e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

Sodium cholate 05 8943 02981e suspension is white and opaque It is easy toprecipitate It cannot be dispersed into an original

emulsion

F-68 + SDS 025+ 025 17523 02941e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

PVP K30 + SDS 025+ 025 12857 02651e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

Tween+ SDS 025+ 025 1605 05301e suspension is white and opaque producing


Lecithin + SDS 025+ 025 9984 02281e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

F-68 + PVP K30 025+ 025 1242 02471e suspension is white and opaque and it producesmicroprecipitates which can be dispersed into an

original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)

5 10 15 20 250Temperature (degC)

00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)

4 6 8 10 122Shear time (min)

00

01

02

03

04

PDI

(b)

Figure 3 Continued


mixture and dry powder are both in the crystal form of thecarrier 1e structure did not change and the crystallinepeak of the drug in the Dio-NS freeze-dried powder es-sentially disappeared indicating that the drug occurs in anamorphous state and the freeze-drying process has an effecton the crystalline structure of the drug

324 In Vitro Dissolution and Stability As shown in Fig-ure 10 the dissolution rate of dioscin can be significantlyimproved after the preparation of nanosuspensions In PBS

(pH 74) the dissolution rate of Dio-NS during the initial 2 hwas close to 30 whereas the release rate at 12 h was close to90 1e dissolution rate of the physical mixture group wasslightly faster than that of the raw material group which canbe attributed to the solubilization effect of the twelve alkylsulfate and soybean lecithin used in the formulationHowever the dissolution rate was considerably less than thatof Dio-NS It is suggested that the dissolution rate ofnanosuspensions is not only caused by the solubilization ofexcipients but is also mainly due to the reduction of particlesize after the formation of nanoscale suspensions

MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)

500 600 700 800 900400Homoginization pressure (bar)

000

005

010

015

020

025

PDI

(e)

Figure 3 1e influences of temperature (a) number of homogenization cycles (b) shear speed (c) shear time (d) and homogenizationpressure (e) on the particle size and polydispersity index of Dio-NS preparations

Table 5 Variables and levels in the star design

VariablesLevels

minus 168 minus 1 0 +1 +168Dio (A) 002 003 004 005 006Sph (B) 003 005 008 010 012SDS (C) 003 005 008 010 012


Table 6 Response value of the formulation using a central composite design (n 3)

Run A B C MPS PDI OD1 0 0 0 9798 0250 06612 +1 minus 1 +1 13145 0203 0393 0 0 0 9798 0250 06614 0 0 0 9799 0250 06615 +1 +1 +1 1049 0267 0556 minus 1 minus 1 minus 1 8549 0226 08417 0 +168 0 969 025 0678 minus 1 minus 1 +1 9029 0235 07729 minus 1 +1 +1 1064 027 05310 +1 minus 1 minus 1 1024 0261 05911 0 0 +168 9823 0253 06512 0 0 0 9798 0250 066113 0 0 0 9798 0250 066114 +168 0 0 1416 0345 015 +1 +1 minus 1 9293 024 07416 minus 168 0 0 10824 0274 0517 0 minus 168 0 9156 0239 07518 0 0 0 9897 0250 066119 0 0 minus 168 7488 0238 086820 minus 1 +1 minus 1 10157 0263 059

03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )

Figure 41e response surfacemodel of the reciprocal action among the concentrations of dioscin (A) soybean lecithin (B) and SDS (C) onOD

Size distribution by intensity

1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)

Zeta potential distribution

0200000400000600000800000

100000012000001400000

Tota

l cou

nts

0 100 200ndash100Apparent zeta potential (mV)

(b)

Figure 5 Particle size and zeta potential distribution of Dio-NS formulations

(a) (b) (c)

Figure 6 Transmission electron micrographs of samples dispersed in water (a) Dioscin (b) Dio-NS (c) Dio-NS powder

Table 7 Particle size and zeta potential of the optimum Dio-NS (n 3 xplusmn s)

Batch no Particle size (nm) PDI ZP (mV)VAL1001 1065plusmn 050 0177plusmn 002 minus 3213plusmn 235VAL1002 11573plusmn 076 0231plusmn 0002 minus 331plusmn 526VAL1003 9793plusmn 126 0254plusmn 002 minus 376plusmn 253Mean value 10672plusmn 890 0221plusmn 004 minus 3427plusmn 291


(a) (b) (c)

(d) (e)

Figure 7 Scanning electron micrographs of (a) dioscin raw material (b) with mannitol (c) with soybean lecithin (d) with twelve alkylsulfates and (e) Dio-NS lyophilized powder

Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)

Soybean lecithin166degC

166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)

50 100 150 200 250 3000Temperature (degC)

Figure 8 DSC patterns of freeze-dried Dio-NS (a) physical mixture (b) soybean lecithin (c) mannitol (d) raw dioscin (e) SDS (f) and dryDio-NS (g)


1e results showed that although the non freeze-driedDio-NS had poor stability at room temperature (Figures 11and 12) the freeze-dried powder remained stable for2months (Table 3)


331 Serum Biochemistry and Hepatic Lipid PeroxidationAssays In order to assess the influence of Dio-NS on CCl4-induced liver damage the activities of ALT AST TNF-α IL-1β and IL-6 in the serum were monitored (Figure 13) 1e

results indicate that our model is successful given that themodel groups show significant difference compared with thecontrol (plt 001) Compared with the model groups therewere significant differences in groups with different Dio-NSand silymarin doses (plt 001 and plt 005)

1e results of our analysis of liver lipid peroxidationshowed that the activity of GSH-Px and SOD in differentdose groups of Dio-NS were markedly increased comparedwith those of the model control (plt 001 and plt 005)(Figure 13) In contrast the level of MDA was significantlyreduced (plt 001 and plt 005) In the different drug groupsthe liver index was reduced to some extent Consequently

(a) Mannitol

(b) Dioscin

(c) SDS

(d) Soybean lecithin

(e) DIO-DGF

(f) DIO-GZF

(g) Physical mixture

20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta

Figure 9 X-ray diffraction patterns of mannitol (a) raw dioscin (b) SDS (c) soybean lecithin (d) freeze-dried Dio-NS (e) dry Dio-NS (f)and physical mixture (g)

Dio-NSPhysical mixtureDioscin

0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)

Figure 10 In vitro dissolution of dioscin nanosuspensions


there was an equivalent or even better effect from the high-dose group (100mgkg) of Dio-NS compared with that ofsilymarin (the positive control drug)

HE staining showed that the liver cells in the blankcontrol group were arranged normally the cytoplasm wasuniform the nucleus and nucleolus were obvious thecentral vein was clearly visible and the cells had local

edema (Figure 14) In the model group a large number ofnecrotic hepatocytes were observed in the tissue as in-dicated by a more intense staining or disintegration ofliver cells Furthermore cytoplasmic eosinophilia wasenhanced accompanied by infiltration of inflammatorycells and a small amount of white blood cells were seen inthe veins In the silymarin group and the high-dose group

Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)

Figure 11 Particle size polydispersity index and zeta potential of dioscin nanoscale suspension NS-B at 25degC

Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028



of Dio-NS the structure of hepatocyte was more completewith few inflammatory cells and little necrosis and thepathological damage was clearly ameliorated Comparedwith the model group the low- and medium-dose groupsof Dio-NS decreased the infiltration of inflammatory cellsand reduced the degree of hepatocyte damage to a certainextent however the effect was not as conspicuous as in thesilymarin and high-dose Dio-NS groups

4 Discussion

It is well known that nanoparticle formulations improve thesolubility of drugs to some extent because of their small sizeHowever in the process of drug preparation excessive heatand solvent residues can adversely affect the production ofgood formulations However dioscin dissolves in ethanolwhich has beneficial effects with regards to our preparation

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)

Figure 13 Effect of Dio-NS on CCl4-induced hepatotoxicity (a) 1e activities of ALTin the serum (b) 1e activities of ASTin the serum(c) 1e liver index (d) 1e activities of TNF-a in the serum (e) 1e activities of IL-1β in the serum (f ) 1e activities of IL-6 in the serum(g)1e levels of MDA in the liver (h)1e levels of GSH-px in the liver (i)1e levels of SOD in the liver lowastlowastP lt 001 vs control +P lt 005 and++P lt 001 vs model


as the rapid volatility of ethanol ensures low amounts ofsolvent residue

In previous experiments we used the reverse solventpreparation method and this method combined with high-pressure homogenization to prepare nanoemulsionsHowever when using high-pressure homogenization wefound that the heat generated caused the production ofexcessive amounts of foam which we believe may be at-tributable to the thermal decomposition of surfactants ordioscin Moreover using the reverse solvent preparationmethod alone we were not able to obtain particles of therequisite size 1erefore in the present study we examinedthe applicability of using magnetic stirring and high-speedshearing combined with high-pressure homogenization toprepare the Dio-NS Using this approach the excessive heatgenerated by homogenization can be dissipated therebyminimizing or eliminating foaming or degradation of thedrug preparation

Furthermore we adopted a star design-response sur-face optimization method in order to determine the op-timal formulation ratio 1is enabled us to clearly assesseach level of interaction We were surprised to discover thatthe ratio of dioscin in the formulation was as high as2126

1rough an evaluation of the physical and chemicalproperties and morphology of Dio-NS we discovered thatthe nature of dioscin in the formulation had undergonechanges Observations indicated that the dioscin no longerretained a crystalline form but instead occurred as sphericalparticles as indicated by the disappearance of peaks onthermal analysis

On the basis of an in vitro solubility test we found thatthe solubility of dioscin was significantly increased in the

Dio-NS formulations 1is was confirmed by pharmaco-dynamic experiments 1e possible reasons for these ob-served properties are as follows Firstly the particle sizedecreases the adsorption surface is enlarged the saturationsolubility increases the dissolution rate increases the per-meability of the biofilm increases at the same time theretention time of the gastrointestinal tract is prolonged theconcentration of drug in the blood is enhanced and thus theefficacy of the drug is enhanced Secondly when drugparticles are less than 1 μm in size the internal dissolutionrate will suddenly increase

However we did not evaluate the bioavailability of Dio-NS In this study we examined the use of ultrahigh liquidphase and liquid mass in combination but were unable todetect any dioscin On the one hand dioscin exists in thebody in the form of glycogen On the other hand there wereimpertinent methods on handling samples or the type ofinstruments 1ese problems will be difficult to solve insubsequent experiments

5 Conclusion

We have demonstrated that nanosuspensions can be suc-cessfully prepared using an emulsification method in whichhigh-speed homogenization is used in combination withhigh-pressure homogenization which is better than pre-viously prepared drug formulations [32 33] Dioscin is aneffective component extracted from plants that belong to thefamily Dioscoreaceae 1e present study shows that dioscinhas protective effect against acute hepatic injury in miceinduced by intraperitoneal injection of CCL4 Howeverdioscin is difficult to dissolve in water although it can bedissolved in the organic solvent ethanol [34] 1erefore in

(a) (b) (c)

(d) (e) (f )

Figure 14 HE staining of liver cells (a) control (b) model (c) silymarin (d) Dio-NS (high) (e) Dio-NS (medium) and (f) Dio-NS (low)


this study we adopted the methods of reverse solventprecipitation and high-pressure homogenization [35] toprepare nanosuspensions 1e particle size distribution ofthe resulting suspensions is between 70 and 120 nm 1esuspensions also have a high specific surface area and in-creased solubility which may promote their bioavailability

1erefore we performed various pharmacodynamicanalyses to evaluate the bioavailability of our newly preparednanosuspensions (Dio-NS) We accordingly found that at ahigh dose Dio-NS reduces the levels of ALT and ASTenhances SOD activity reduces MDA content and ame-liorates liver pathology to some extent compared withsilymarin (the positive control drug)

1us the results of the present study will provide areference for the development and clinical application ofdioscin formulations Finally in further studies we in-tend to evaluate the bioavailability of our dioscin for-mulations and investigate the precise mechanismsunderlying the hepatoprotection properties of thesepreparations

Abbreviations

Dio DioscinDSC Differential scanning calorimetryDio-NS Dioscin nanosuspensionE t Emulsification timeHP Homogenization pressureHPH High-pressure homogenizationHPLC High-performance liquid chromatographyMPS Mean particle sizeOD Overall desirabilityPSD Particle size distributionP s PassesSEM Scanning electron microscopeSDS Sodium alkyl sulfate twelveTem Transmission electron microscopeXRPD X-ray powder diffractionZP Zeta potential

Data Availability

All relevant data are within the paper and its SupportingInformation files

Conflicts of Interest

1ere are no conflicts of interest

Authorsrsquo Contributions

Xiao Song and Hongye Ju contributed equally to thiswork Xiao Song conceived and designed the experi-ments Hongye Ju Kunxia Hu Li-Zhu Han and Guo-Wang Zhao performed the experiments 1e rest of theauthors were also involved in the different processes ofthe experiments Xiao Song Hongye Ju Guo-WangZhao analyzed the data and wrote the paper Xiao Songand Zhi-Shu Tang provided funding for scientificresearch

Acknowledgments

1e authors would like to acknowledge the financial supportfrom the National Natural Science Foundation of China(nos 81373944 and 81373978) the Department of Scienceand Technology of Shaanxi Province (2012KTCQ03-13) theKey Laboratory of the Basic and New Medicine Research ofthe Traditional Chinese Medicine in Shaanxi Province theSpecial Subsidization of the Key Subjects of the TraditionalChinese Medicine Pharmacy in Shaanxi Province and the1ree Level Laboratory of the Chinese Medicine Preparationof the State Administration of Traditional Chinese Medicine(TCM)

Supplementary Materials

Supplementary Table S1-3 three experimental data duringthe preparation of nanosuspensions which describe theeffects of shear speed shear time temperature homog-enization times and homogenization pressure on theparticle size and polydispersity index of nanosuspensionsand the evaluation results in vitro and in vivo 1e ex-perimental results were plotted by origin 85 andGraphPad Prism 5 Supplementary Material 1 AnimalEthics Society approval document (SupplementaryMaterials)

References

[1] C Zhu N Bao S Chen and J Zhao ldquoDioscin enhancesosteoblastic cell differentiation and proliferation by inhibitingcell autophagy via the ASPP2NF-κβ pathwayrdquo MolecularMedicine Reports vol 16 no 4 pp 4922ndash4926 2017

[2] H Zeng L Yang X Zhang Y Chen and J Cai ldquoDioscinprevents LPS-induced acute lung injury through inhibitingthe TLR4MyD88 signaling pathway via upregulation ofHSP70rdquo Molecular Medicine Reports vol 17 no 5pp 6752ndash6758 2018

[3] Y S Zhang Y L Ma K1akur et al ldquoMolecular mechanismand inhibitory targets of dioscin in HepG2 cellsrdquo Food andChemical Toxicology vol 120 pp 143ndash154 2018

[4] S Zhu S Tang and F Su ldquoDioscin inhibits ischemic strokeinduced inflammation through inhibition of the TLR4MyD88NFκB signaling pathway in a rat modelrdquo MolecularMedicine Reports vol 17 no 1 pp 660ndash666 2018

[5] L Zheng L Yin L Xu et al ldquoProtective effect of dioscinagainst thioacetamide-induced acute liver injury via FXRAMPK signaling pathway in vivordquo Biomedicine amp Pharma-cotherapy vol 97 pp 481ndash488 2018

[6] C Li Y Lu S Du et al ldquoDioscin exerts protective effectsagainst crystalline silica-induced pulmonary fibrosis in micerdquoeranostics vol 7 no 17 pp 4255ndash4275 2017

[7] L Yin Y Qi Y Xu et al ldquoDioscin inhibits HSC-T6 cellmigration via adjusting SDC-4 expression insights fromiTRAQ-based quantitative proteomicsrdquo Frontiers in Phar-macology vol 8 p 665 2017

[8] X Tao L Yin L Xu and J Peng ldquoDioscin a diverse actingnatural compound with therapeutic potential in metabolicdiseases cancer inflammation and infectionsrdquo Pharmaco-logical Research vol 137 pp 259ndash269 2018

[9] Y Qin X Wu W Huang et al ldquoAcute toxicity and sub-chronic toxicity of steroidal saponins from Dioscorea


zingiberensis CHWright in rodentsrdquo Journal of Ethno-pharmacology vol 126 no 3 pp 543ndash550 2009

[10] X J Liang Y Z Shang H R Song and W J Dong ldquoAcutetoxicity study of whole saponin of Dioscorea nipponicaMakino in micerdquo Journal of Chengde Medical College vol 27pp 117-118 2010

[11] T Xu S Zhang L Zheng L Yin L Xu and J Peng ldquoA 90-daysubchronic toxicological assessment of dioscin a naturalsteroid saponin in SpraguendashDawley ratsrdquo Food and ChemicalToxicology vol 50 no 5 pp 1279ndash1287 2012

[12] L Padrela M A Rodrigues A Duarte A M A DiasM E M Braga and H C de Sousa ldquoSupercritical carbondioxide-based technologies for the production of drugnanoparticlesnanocrystalsmdasha comprehensive reviewrdquo Ad-vanced Drug Delivery Reviews vol 131 pp 22ndash78 2018

[13] L R Zawar and S B Bari ldquoPreparation characterization andin vivo assessment of repaglinide nanosuspension for oralbioavailability improvementrdquo Recent Patents on Drug De-livery and Formulation vol 12 no 3 pp 162ndash169 2018

[14] A G1ombre J C Shah K Sagawa andW B Caldwell ldquoInvitro and in vivo characterization of amorphous nano-crystalline and crystalline ziprasidone formulationsrdquo In-ternational Journal of Pharmaceutics vol 428 no 1-2pp 8ndash17 2012

[15] M H He L Chen T Zheng et al ldquoPotential applications ofnanotechnology in urological cancerrdquo Frontiers in Pharma-cology vol 9 p 745 2018

[16] N Kamaly J C He D A Ausiello and O C FarokhzadldquoNanomedicines for renal disease current status and futureapplicationsrdquo Nature Reviews Nephrology vol 12 no 12pp 738ndash753 2016

[17] D C Snow-Lisy M K Samplaski V Labhasetwar andE S Sabanegh Jr ldquoDrug delivery to the testis current statusand potential pathways for the development of novel thera-peuticsrdquo Drug Delivery and Translational Research vol 1no 5 pp 351ndash360 2011

[18] P J Patel B Y Gajera and R H Dave ldquoA quality-by-designstudy to develop Nifedipine nanosuspension examining therelative impact of formulation variables wet media millingprocess parameters and excipient variability on drug productquality attributesrdquo Drug Development and Industrial Phar-macy vol 44 no 12 pp 1942ndash1925 2018

[19] A N Oktay A Karakucuk S Ilbasmis-Tamer and N CelebildquoDermal flurbiprofen nanosuspensions optimization withdesign of experiment approach and in vitro evaluationrdquoEuropean Journal of Pharmaceutical Sciences vol 15 no 122pp 254ndash263 2018

[20] E B Basalious and M A Ahmed ldquoPhospholipid based self-nanoemulsifying self-nanosuspension (p-SNESNS) as a dualsolubilization approach for development of formulation withdiminished food effect fastfed in vivo pharmacokineticsstudy in humanrdquo European Journal of Pharmaceutical Sci-ences vol 109 pp 244ndash252 2017

[21] H Liu Z Zhang H Hu et al ldquoProtective effects of Liu-weiwuling tablets on carbon tetrachloride-induced hepaticfibrosis in ratsrdquo BMC Complementary and AlternativeMedicine vol 18 no 1 p 212 2018

[22] X Jiang H Zhang K Mehmood et al ldquoProtective effects ofHerpetospermum caudigerum extracts against liver injuryinduced by carbon tetrachloride in mouserdquo Journal of Bi-ological Regulators amp Homeostatic Agents vol 32 no 3pp 699ndash704 2018

[23] J Leng Z Wang C-L Fu et al ldquoNF-kappaB and AMPKPI3KAkt signaling pathways are involved in the protective

effects of Platycodon grandiflorum saponins against acet-aminophen-induced acute hepatotoxicity in micerdquo Phyto-therapy Research vol 32 no 11 pp 2235ndash2246 2018

[24] C Ning X Gao C Wang et al ldquoGinsenoside Rg1 protectsagainst acetaminophen-induced liver injury via activatingNrf2 signaling pathway in vivo and in vitrordquo RegulatoryToxicology and Pharmacology vol 98 pp 58ndash68 2018

[25] H Zhao Q Han N Lu D Xu Z Tian and J ZhangldquoHMBOX1 in hepatocytes attenuates LPSD-GalN-inducedliver injury by inhibiting macrophage infiltration and acti-vationrdquo Molecular Immunology vol 101 pp 303ndash311 2018

[26] H Lv X Fan L Wang H Feng and X Ci ldquoDaphnetinalleviates lipopolysaccharided-galactosamine-induced acuteliver failure via the inhibition of NLRP3 MAPK and NF-κBand the induction of autophagyrdquo International Journal ofBiological Macromolecules vol 119 pp 240ndash248 2018

[27] P Kanmani and H Kim ldquoProtective effects of lactic acidbacteria against TLR4 induced inflammatory response inhepatoma HepG2 cells through modulation of toll-like re-ceptor negative regulators of mitogen-activated protein kinaseand NF-κB signalingrdquo Frontiers in Immunology vol 9p 1537 2018

[28] B Lu Y Xu L Xu et al ldquoMechanism investigation of dioscinagainst CCl4-induced acute liver damage in micerdquo Environ-mental Toxicology and Pharmacology vol 34 no 2pp 127ndash135 2012

[29] Y-J Son P W Longest G Tian and M Hindle ldquoEvaluationand modification of commercial dry powder inhalers for theaerosolization of a submicrometer excipient enhanced growth(EEG) formulationrdquo European Journal of PharmaceuticalSciences vol 49 no 3 pp 390ndash399 2013

[30] G Pilcer T Sebti and K Amighi ldquoFormulation and char-acterization of lipid-coated tobramycin particles for drypowder inhalationrdquo Pharmaceutical Research vol 23 no 5pp 931ndash940 2006

[31] W Yang J Tam D A Miller et al ldquoHigh bioavailability fromnebulized itraconazole nanoparticle dispersions with bio-compatible stabilizersrdquo International Journal of Pharmaceu-tics vol 361 no 1-2 pp 177ndash188 2008

[32] Y Li X Zhao Y Zu and Y Zhang ldquoPreparation andcharacterization of paclitaxel nanosuspension using novelemulsification method by combining high speed homogenizerand high pressure homogenizationrdquo International Journal ofPharmaceutics vol 490 no 1-2 pp 324ndash333 2015

[33] S E Lee S F Bairstow J O Werling et al ldquoPaclitaxelnanosuspensions for targeted chemotherapymdashnanosuspensionpreparation characterization and userdquo Pharmaceutical De-velopment and Technology vol 19 no 4 pp 438ndash453 2014

[34] Y Zhang L Jin J Liu et al ldquoEffect and mechanism of dioscinfrom Dioscorea spongiosa on uric acid excretion in animalmodel of hyperuricemiardquo Journal of Ethnopharmacologyvol 214 pp 29ndash36 2018

[35] G Pilcer F Vanderbist and K Amighi ldquoPreparation andcharacterization of spray-dried tobramycin powders con-taining nanoparticles for pulmonary deliveryrdquo InternationalJournal of Pharmaceutics vol 365 no 1-2 pp 162ndash169 2009


Stem Cells International

Hindawiwwwhindawicom Volume 2018


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Disease Markers


BioMed Research International

OncologyJournal of



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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

solubilization and salt formation [13] However thesetechniques are only partially effective 1e advent ofnanotechnology which can produce nanoscale particles withnovel functional properties offers a potential solution to theaforementioned drawbacks Nanotechnology has for ex-ample been used to enhance the bioavailability of certainantibiotics [14] and has also been applied to produce dif-ferent targeted drugs for the treatment of different diseases[15ndash17] and various nanosuspensions have been success-fully applied [18 19] Nanoparticles can also be preparedusing a variety of different processes including reversesolvent homogenization pressure milling and the pro-duction of self-nanoemulsifying self-nanosuspensions [20]

Diseases of the liver including hepatitis hepatapostemahepatocirrhosis and liver cancer are common and oftenlife-threatening Carbon tetrachloride (CCl4) [21 22] par-acetamol [23 24] lipopolysaccharide [25 26] and certainother chemicals can also cause acute liver damage andconsequently a large number of drugs are used for thetreatment of hepatic disorders [27] It was confirmed thatdioscin has significant hepatoprotective effect against CCl4-induced liver injury in mice and the cellular mechanisms ofthis effect are likely to be associated with inhibition of lipidperoxidation inflammatory cytokines necrosis and apo-ptosis 1us in the present study we formulated a dioscinnanosuspension (Dio-NS) using reverse solvent pre-cipitation combined with high-pressure homogenizationand compared its efficacy with that of the commercial drugsilymarin in protecting mice from acute hepatic damageinduced by CCl4 We assumed that the Dio-NS formulationwould enhance the solubility of dioscin and thus increase theefficacy of this compound in protecting against liver injury

2 Methods

21 Materials Dioscin was purchased from Nanjing Springand Autumn Biological Engineering (Nanjing China) withthe purity of gt99 Soybean lecithin mannitol poloxamer-188 Tween-80 PVP K30 sodium cholate sodium alkylsulfate twelve (SDS) anhydrous ethanol lactose glucosesucrose D-sorbitol silymarin 4 paraformaldehyde solu-tion methanol and acetonitrile were all of chromatographicgrade and the other reagents were all of analytical gradeAST and ALT kits were purchased from Nanjing Jiancheng

Institute of Biotechnology (Nanjing China) TNF-α IL-1βIL-6 MDA SOD and GSH-Px kits were purchased fromShanghai Elisa Biotech Co Ltd

22 Preparation ofDio-NS Dioscin was dissolved in ethanolas a solvent and dispersed for 15min in a KQ-3-DE ul-trasonic bath (Kunshan China) In the next emulsificationprocess ethanol was used as the organic phase of theemulsion 1e solution was rapidly added dropwise todouble-distilled water-saturated ethanol together with so-dium alkyl sulfate twelve and soybean lecithin as excipientsthat can be used to improve the stability of emulsionscommixing using magnetic stirrer (HJ-6B ChangzhouChina) A microemulsion was obtained by high-speedshearing (FJ-200 ShenZhen China) 1e microemulsionpreparation was then homogenized to produce a nano-emulsion via high-pressure homogenization (AH-BASICIATS Engineering Inc Canada) An EYELA N-1300 rotaryevaporator was used to remove the ethanol (LangYiShanghai China) at an evaporation temperature of 40degCthereby yielding the Dio-NS preparation In this study theoptimal process conditions were determined on the basis ofsingle-factor experimental results All the preparation stepsare shown in Figure 2

23 Preparation of a PhysicalMixture 1e desired dosage ofDio-NS was obtained by dissolving in water and freeze-drying for 12 h to yield an NS-A physical mixture

24 Screening of Excipients Eleven groups were examined toselect the best accessories that have a minimum size andpowder dispersion index

25 Single-Factor Experiment In the single-factor experi-ment particle size and multiple dispersion index were takenas indicators to determine optimal preparation parametersincluding shear time shear speed emulation temperaturepressure and cycles of homogenization

26 Optimization of the Response Surface of the Star DesignOn the basis of the results of the single-factor experimentand the feasibility of preparing nanosuspensions at thehighest or lowest level we selected the level of each factorand the star response surface design software and de-termined the best experimental results

27 Lyophilization 1eDio-NS preparation was lyophilizedusing a VaCo-5 freeze-drying apparatus (Zirbus Germany)to obtain dry powder with good physicochemical stability Inthis study a single-factor method was used to select optimalconditions and the best freeze-drying protectant was se-lected 1e results are shown in Table 1 1e content offreeze-drying protectant ranged from 2 to 10 1e levelsare shown in Table 2

OO

CH3

H3C

H3C

H3C

HH

O

HO

OHO

O

OH

H

H

CH3

HO

HOOH

H

O

CH3

HO

HOOH

OH

H

Figure 1 1e chemical structure of dioscin






Magnetic stirring

High-speed shearing


Evaporation



Microemulsion

Dio-NS


Nanoemulsion















Qtimes 100

(1)




















3 Results




+ 0030B2

+ 0047C2

R 09411 Plt 00001

(2)




















emulsion


original emulsion


emulsion


original emulsion


emulsion


original emulsion


original emulsion




original emulsion


original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)


00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)


00

01

02

03

04

PDI

(b)

Figure 3 Continued





MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























Magnetic stirring

High-speed shearing


Evaporation



Microemulsion

Dio-NS


Nanoemulsion















Qtimes 100

(1)




















3 Results




+ 0030B2

+ 0047C2

R 09411 Plt 00001

(2)




















emulsion


original emulsion


emulsion


original emulsion


emulsion


original emulsion


original emulsion




original emulsion


original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)


00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)


00

01

02

03

04

PDI

(b)

Figure 3 Continued





MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























Qtimes 100

(1)




















3 Results




+ 0030B2

+ 0047C2

R 09411 Plt 00001

(2)




















emulsion


original emulsion


emulsion


original emulsion


emulsion


original emulsion


original emulsion




original emulsion


original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)


00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)


00

01

02

03

04

PDI

(b)

Figure 3 Continued





MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















3 Results




+ 0030B2

+ 0047C2

R 09411 Plt 00001

(2)




















emulsion


original emulsion


emulsion


original emulsion


emulsion


original emulsion


original emulsion




original emulsion


original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)


00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)


00

01

02

03

04

PDI

(b)

Figure 3 Continued





MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























emulsion


original emulsion


emulsion


original emulsion


emulsion


original emulsion


original emulsion




original emulsion


original emulsion

MPSPDI

0

20

40

60

80

100

120

MPS

(nm

)


00

01

02

03

04

PDI

(a)

MPSPDI

020406080

100120140160180200220240

MPS

(nm

)


00

01

02

03

04

PDI

(b)

Figure 3 Continued





MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















MPSPDI

106

108

110

112

114

116

118

120

122

124

126

128M

PS (n

m)

17500 18000 18500 19000 1950017000Shear speed (rpm)

000

005

010

015

020

025

030

035

PDI

(c)

MPSPDI

0102030405060708090

100110120130140

MPS

(nm

)

000

005

010

015

020

025

030

PDI

5 6 7 8 94Ps

(d)

MPSPDI

100102104106108110112114116118120122124126128130

MPS

(nm

)


000

005

010

015

020

025

PDI

(e)



VariablesLevels





03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















03

04

05

06

07

08

09

Od

005 006 006 007 007 008 008 009 009 010 010

003003

004004

005005

B sph A dio

(a)

005 006 006 007 007 008 008 009 009 010

010

005006 006

007 007 008 008 009 009 010010

03

04

05

06

07

08

09

Od

B sphC sds

(b)

Od

005006006007007008008009009010

010

003003

004004

005005

03

04

05

06

07

08

09

A dioC sds

(c)

Od

05

06

06

07

6

005

006

006

007

007

008

008

009

009

010

010

B sp

h

003 004 004 005 005003A dio

(d)

Figure 4 Continued


Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Od

065

07

07508

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

006 006 007 007 008 008 009 009 010 010005B sph

(e)

Od

04

05

06

07

6

005

006

006

007

007

008

008

009

009

010

010

C sd

s

003 004 004 005 005003A dio

(f )



1 10 100 1000 1000001Size (dnm)

0

2

4

6

8

10

Inte

nsity

(per

cent

)

(a)


0200000400000600000800000

100000012000001400000

Tota

l cou

nts


(b)


(a) (b) (c)





(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















(a) (b) (c)

(d) (e)


Dioscin

294~296degC

SDC

Mannitol

Physical mixture

Dio-DGF(a)

(b)

(c)

(d)

(e)

(f)

(g)


166degC

204-207degC 294-296degC

204-207degC

Dio-GZF

ndash8

ndash6

ndash4

ndash2

0

2

4

6

8

10

12

Hea

t flow

(wg

)









(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























(a) Mannitol

(b) Dioscin

(c) SDS


(e) DIO-DGF

(f) DIO-GZF


20000

40000

60000

80000

100000

120000

140000

Coun

ts

20 40 60 80 10002-theta



0

20

40

60

80

100

Cum

ulat

ive r

elea

se d

egre

e Q (

)

10 20 300t (h)






Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

022

023

024

025

026

027

028

ndash90

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

2 5 7 15 300Time (d)


Size (nm)PDIZeta

100

110

120

130

140

150

160

170

180

190

2 5 7 15 300Time (d)

ndash80

ndash70

ndash60

ndash50

ndash40

ndash30

022

023

024

025

026

027

028




4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















4 Discussion


Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

AST

0

100

200

300

400

UL

++

++

++

++

lowastlowast

(a)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

ALT

0

50

100

150

UL

++

++

++

+

lowastlowast

(b)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

Liver index

0

2

4

6

8

Live

r wei

ghtb

ody

wei

ght 1

00

++ ++ ++ ++

lowastlowast

(c)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

TNF-α

0

200

400

600

800

1000

pgmiddotm

lndash1

++

++

++++

lowastlowast

(d)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-1β

0

50

100

150

pgmiddotm

lndash1

++ ++

+

lowastlowast

(e)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

IL-6

0

50

100

150

200

250

pgmiddotm

lndash1

++

++

++

lowastlowast

(f )

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

MDA

0

2

4

6

umol

middotgpr

ondash1

++ +++

lowastlowast

(g)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

GSH-Px

0

50

100

150

umiddotm

gpro

ndash1 ++

++++

++

lowastlowast

(h)

Cont

rol

Mod

el

Sily

mar

in

Dio

-NS

(low

)

Dio

-NS

(mid

dle)

Dio

-NS

(hig

h)

SOD

0

10

20

30

40

50

um

gpro

ndash1 ++

+++

lowastlowast

(i)










5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























5 Conclusion


(a) (b) (c)

(d) (e) (f )






Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Abbreviations


Data Availability






Acknowledgments




References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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