determination of ascorbic acid, epinephrine, and uric acid by differential pulse voltammetry
Sensors and Actuators B 150 (2010) 321–329
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Sensors and Actuators B:
Chemical
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s n
b
Simultaneous determination of ascorbic acid,epinephrine,and uric acid by differential pulse voltammetry using
poly(3,3 -bis[N,N -bis(carboxymethyl)aminomethyl]-o -cresolsulfonephthalein)modi?ed glassy carbon electrode
Ali A.Ensa??,B.Rezaei,S.Z.Mirahmadi Zare,M.Taei
Department of Chemistry,Isfahan University of Technology,Isfahan 84156-83111,Iran
a r t i c l e i n f o Article history:
Received 23April 2010
Received in revised form 17June 2010Accepted 29June 2010
Available online 7 July 2010
Keywords:
Simultaneous determination Ascorbic acid Epinephrine Uric acid
Voltammetry
Poly(3,3 -bis[N,N -bis(carboxymethyl)aminomethyl]-o -cresolsulfonephthalein)
a b s t r a c t
A selective and stable poly(3,3 -bis[N,N -bis(carboxymethyl)aminomethyl]-o -cresolsulfonephthalein)modi?ed glassy carbon electrode was prepared using the electrochemical polymerization technique for the simultaneous determination of ascorbic acid (AA),epinephrine (EP),and uric acid (UA).The modi?ed electrode showed an excellent electrocatalytic activity for AA,EP,and UA oxidations and for accelerated electron transfer between the electrode and the substances.The separation potentials of the oxidation peak potentials for EP–AA and EP–UA were about 180mV and 130mV,respectively.Calibration curves in the ranges 1.0–2000?mol L ?1,0.20–175?mol L ?1,and 0.020–2000?mol L ?1were obtained for AA,EP,and UA,respectively.The lowest detection limits (S /N =3)were 0.4?mol L ?1,0.03?mol L ?1,and 0.009?mol L ?1for AA,EP,and UA,respectively.The proposed procedure was also successfully applied for the simultaneous detection of AA,EP,and UA in injectable medicine,blood plasma,and urine samples.
? 2010 Elsevier B.V. All rights reserved.
1.Introduction
The development of voltammetric sensors for the determina-tion of epinephrine (EP),uric acid (UA),and ascorbic acid (AA)has received considerable attention during the last few years.EP is an important catecholamine neurotransmitter in the mam-malian central nervous system.It has been used for the treatment of myocardial infarction,hypertension,bronchial asthma,cardiac arrest,and cardiac surgery in clinics.Many physiological phenom-ena are correlative to EP’s level in the body ?uid.Therefore,a simple,fast,and sensitive method is required for the determination of EP in both biological ?uids and pharmaceutical preparations.
UA and AA also coexist in biological ?uids such as blood and urine.Extreme abnormalities of UA levels are symptoms of sev-eral diseases [1].Thus,the determination of EP concentration in human blood or urine is very important to warn the presence of cer-tain diseases,such as Lesch–Nyhan syndrome,immunode?ciency,gout,and gouty nephropathy.However,a major obstacle usually encountered in the detection of EP is the interference of UA and
?Corresponding author.Tel.:+983113913269;fax:+983113912350.E-mail address:ensa?@cc.iut.ac.ir (A.A.Ensa?).AA [10],which are usually present at high concentrations and can be oxidized at a potential close to that of EP.Thus,simultaneous determination of the three has always been considered as a serious challenge.
Several methods including gas chromatography [2],high per-formance liquid chromatography [3–5],and spectrophotometry [6]have been used for the determination of neurotransmitters compounds.Among these techniques,HPLC coupled with electro-chemical detection [7,8]has been widely used to detect a large number of interesting and important compounds.
Electrochemical methods are selective and sensitive methods used for the determination of inorganic and organic compounds.Electrochemical detection of EP at the surface of bare electrodes has a high overpotential,which results in weak electrochemical responses.In addition,the oxidation peak potential of many elec-trodes to EP is commonly very close to those of AA and UA and,hence,their signals usually overlap [9].As a result,the accuracy of the determinations is remarkably low in mixture samples.Several papers have reported new,modi?ed electrodes for the determi-nation of EP and/or EP plus AA [10–14].Carbon paste electrode (CPE)modi?ed with iron(II)phthalocyanine [15]has been used for the determination of EP in the presence of AA and UA.A pre-treated glassy carbon electrode (GCE)by electrochemical activation
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322 A.A.Ensa?et al./Sensors and Actuators B150 (2010) 321–329
Table1
Comparison of some characteristics of the different modi?ed electrodes for the determination of AA,EN,and UA.
Slope(?A(?mol L?1))?1Limit of detection(?mol L?1)Linear dynamic range(?mol L?1)Interferences Reference AA EN UA AA EN UA AA EN UA
7.450.070.077.00.20.620–1000 2.0–805–300l-Lysine,glucose,NADH,asparagine,glutamic
acid,glycin and chitosan l-cystine
10
0.480.740.60130.42950–5000.1–70020–500Not reported12
––––0.0030.1–0.04–45–200Not reported13
––––0.5––1–30–Ascorbic acid,uric acid15
– 3.58 1.81–0.030.01–0.3–210.05–28Cysteine,glycin,l-cystine,citric acid and
glutamic acid
16
0.11–0.35 1.4–0.025–240–0.1–18Cysteine,copper(II),tyrosine,oxalate,citric
acid
17
0.0260.5130.1240.40.030.0091–1000.2–3.00.02–70No interference This work 0.0100.1300.021100–2000 3.0–17570–2000
has been proposed for simultaneous determination of EP and UA using differential pulse voltammetry[16].The presence of a?vefold AA concentration has been found to cause a serious interference in the voltammetric determination of EP.Recently,organic dyes have been applied to modify electrode surfaces for voltammetric determination of EP in the presence of AA and UA[17,18].Function-alized carbon nanotubes form another example of the modi?cation of electrode surfaces and voltammetric determination of EP in biological compounds[19,20].However,their simultaneous deter-mination is rarely reported.Table1shows the comparison of the proposed method with other reported electrochemical methods for the determination of EP,AA,and UA.
Treatment of the surface of solid electrodes has been extensively used for improving the electrochemical performance of electrodes. Especially,electrochemical pretreatment of the electrode is used to activate its surface for the analysis of special compounds [13,21–24].For this purpose,such various groups as quinoidal,car-boxyl,and phenolic functionalities can be added onto the surface of carbon and/or metal electrodes using a variety of methods includ-ing cyclic voltammetry,differential pulse polarography,scanning electron microscope,and scanning probe microscopy[25,26].These active groups catalyze many electrochemical reactions.Modi?-cation of the surface of electrodes improves their selectivity, decreases their kinetic overpotential,increases their electrocataly-sis effect,and frees them from surface fouling[27,28].
For the purposes of this study,GCE was modi?ed with a?lm of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)in a0.3mol L?1NaOH solution by cyclic voltammetry(CV).The polymer was found to be electrocatalyt-ically active for the oxidation of AA,EP,and UA.The oxidation potential of EP was well separated from those of AA and UA with potential differences of200mV,150mV,and350mV for EP–AA,UA–EP,and UA–AA,respectively.The modi?ed GCE was used for the simultaneous determination of EP,AA,and UA in both biological?uids and pharmaceutical samples with satisfactory results.
2.Experimental
2.1.Apparatus
Electrochemical measurements,differential pulse voltammetry (DPV),chornoamperometry,and cyclic voltammetry(CV),were performed in a conventional three electrode cell with a working electrode poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)modi?ed glassy carbon electrode (PBCACPMGCE),a platinum wire counter electrode,and an Ag/AgCl(3.0mol L?1KCl)as the reference electrode.All the elec-trochemical measurements were carried out with a Metrohm instrument,Model797VA processor.A glassy carbon electrode (GCE)with a formal surface area of0.0314cm2was used.All potentials reported are vs.Ag/AgCl.
Impedance spectroscopic measurements were performed with a conventional three-electrode cell,powered by an electrochemical system comprising Autolab system(PGSTAT12and FRA2boards, Eco Chemie B.V.,Utrecht,and The Netherlands).The system was run on a PC using GPES and FRA4.9software.For impedance mea-surements,a frequency range of100kHz–10Hz was employed.The AC voltage amplitude used was5mV,and the equilibrium time was 120s.The PBCACPMGCE and GCE as working electrodes,a graphite electrode and an Ag/AgCl(3.0mol L?1KCl)reference electrode was employed as an auxiliary and reference electrode,respectively. 2.2.Reagents
All chemicals were of analytical grades and double distillated water was used throughout.All the reagents including AA,EP,and UA were purchased from Sigma–Aldrich.
Stock solutions of EP and AA(0.010mol L?1)were prepared daily by dissolving suitable amounts of them in water in a10-mL volu-metric?ask.UA stock solution(0.010mol L?1)was prepared daily by dissolving enough amount of UA in alkaline water in a10-mL vol-umetric?ask.Working solutions of the substances were prepared daily by appropriate dilution of the stock solutions with water.
Phosphate buffer solutions(0.25mol L?1PBS)with different pH levels were prepared by mixing different volumes of0.125mol L?1 Na2HPO4and0.125mol L?1NaH2PO4solutions.pH of the sam-ple solutions were adjusted using the buffer solution and/or 0.10mol L?1NaOH solution.
Patient and healthy human urine as real samples were pre-pared from the laboratory of Isfahan hospital.Epinephrine injection ampoules were purchase from Daropakhsh Company(Iran)in two different batch number and volume.Vitamin C tablet was prepared from three different companies by different contents of ascorbic acid.
2.3.Preparation of PBCACPMGCE
Prior to each experiment,a glassy carbon electrode(GCE) was polished with0.05-?m alumina in a water surrey using the polishing cloth.Then,GCE was sonicated in a solution of water–ethanol(90%(v/v))after each polishing step fol-lowed by electrochemical pretreatment of the GCE by cycling the potential between?0.10and+1.30at a scan rate of 100mV s?1for?ve times in a0.10mol L?1H2SO4solution to get a stable background current.The electrode was subse-quently placed in a solution containing0.20mol L?1NaOH and 0.0010mol L?13,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein.Then,a cyclic potential sweep was applied in the range of?0.20V to+1.40V at a scan rate of100mV s?1
A.A.Ensa?et al./Sensors and Actuators B150 (2010) 321–329323
for30times(the anodic peak potential and the current tended to be stable after25scans).To enhance surface reproducibil-ity,the modi?ed electrode was washed after polymerization with distilled water and scan-cycled at pH4.0between?0.20V and0.80V for eight times to eliminate untreated3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein.
2.4.Real sample preparation
To analyze injection solutions,10–20?L of each ampoule (1.0mg/mL EP)was added into a5-mL buffer solution(0.1mol L?1 of pH4.0).The test solution was transferred into the electrochemi-cal cell to determine epinephrine levels using the standard addition method.
For tablet analysis,3tablets of vitamin C(labeled1000mg, 500mg,and180mg vitamin C per tablet)were each completely ground and homogenized.Then,978mg,352mg,and176mg weights(equal to0.01mol ascorbic acid in25mL)from each tablets powder were accurately weighed and dissolved in25mL water by sonication for4min.10?L of each solution plus5mL of the buffer(0.10mol L?1pH4.0)was transferred into the electrochemi-cal cell and the analysis was carried out using the standard addition method.
Urine samples from?ve people(suffering from the same dis-ease)were collected,mixed and centrifuged(4000rpm)to remove the solid particles.Then,1.0mL of the mixed sample solution plus 10mL of the0.25mol L?1buffer(pH5.0)was transferred into a 25-mL volumetric?ask and diluted with water.5mL of this test solution was subsequently transferred into the electrochemical cell and the analysis was performed using the standard addition method.
3.Results and discussion
3.1.Formation of PBCACPMGCE and its electrochemical
properties
The modi?cation consisted in the formation of electroactive poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)by oxidation of3,3 -bis[N,N-bis(carbo-xymethyl)aminomethyl]-o-cresolsulfonephthalein on the glassy carbon electrode surface.The poly(3,3 -bis[N,N-bis(carboxy-methyl)aminomethyl]-o-cresolsulfonephthalein)thin?lm at the surface of GCE acts as a suitable electrocatalyst for the oxidation of AA,EP,and UA.In addition,the oxidation peak potentials separated well from each other.Fig.1displays the cyclic voltammogram of electro-polymerization of3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein over the range of?0.20to+1.30V at a scan rate of100mV s?1for25 cycles.The growth of the polymer?lm on the electrode surface was observed with all the monomers in the form of decreas-ing anodic peak currents.Our experiments showed that after 20cycles,both the anodic peak potential and current tended to be stable and the relative standard deviation(RSD%)of the peak current was less than5%.After25cycles the peak current was nearly constant and the RSD%of the peak current was less than1%.Thus,we selected25cycles for further study.These observations suggest that the initially formed poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm had a leaching process with the scan cycle increasing up to25 times,which may imply a self–adjustment of the polymer?lm thickness at the GCE.
The electro-deposited behavior of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)
at the surface of GCE was similar to that reported
elsewhere Fig. 1.Cyclic voltammogram of electro-polymerization of3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein over the range of?0.20to+1.30V at scan rate of100mV s?1for25cycles in a solution containing0.20mol L?1NaOH and0.0010mol L?13,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein.
[29,30].The voltammogram of the PBCACPMGCE in the range of0.20–0.80V at various sweep rates in the buffer solution (pH 4.0)was investigated(not shown here).The anodic peak current(I pa)depended linearly on the scan rate( )with a regression equation of I(?A)=4.00 (V s?1)+0.190(r2=0.998). The peak potential was0.337V at a low scan rate(10mV s?1) and did not change with increasing scan rate.The fact that E p?E p/2was equal to0.034V suggests that the poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm reaction could have been a double-electron transfer process(n=2). In addition,the above results suggest that the electrode reaction was a reversible electron transfer process[17].
The electrochemical response of PBCACPMGCE depends on the pH of the supporting electrolyte solution.The results showed that the anodic peak potential(E pa)depended lin-early on pH level with an equation of E p(V)=0.426?0.032 pH(r2=0.9280).This result con?rms that the oxidation and reduction of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm were both involved in the pro-ton transfer.These imply that the ratio of the participating protons to the electrons transferred through poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm must be1:2(Scheme1)including that,the redox process must have been con?ned to the polymer-modi?ed surface of the electrode, which con?rms the immobilized state of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm.
Total re?ectance Fourier transform IR spectroscopy(FT-IR) was employed to check the formation of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)
?lm at the surface of GCE.FT-IR spectra of3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein
and poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm were recorded.The results showed that a wide and strong absorption band at around3423cm?1in3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephth-alein spectra possibly resulted from the stretching vibration of a –OH(aryl group),whereas this band was disappeared for the poly-mer?lm.In addition,another band at1725cm?1was appeared for the polymer?lm,possibly caused by the stretching vibration of C O group,whereas this band did not observed for the monomer. Those observation con?rmed the formation of poly(3,3 -bis[N,N-
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150 (2010) 321–329
Scheme 1.Electrooxidation of 3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o -cresolsulfonephthalein at GCE.
bis(carboxymethyl)aminomethyl]-o -cresolsulfonephthalein)?lm at the surface of GCE by electropolymerization,and this is match with previous report [31].
3.2.Electrooxidation of AA,EP,and UA at PBCACPMGCE
Differential pulse voltammograms of the oxidation of AA,EP,and UA mixture on the surfaces of the bare GCE and PBCACP-
MGCE are shown in Fig.2(curve b)showed differential pulse of a mixture of AA,EP,and UA.This voltammograms shows a broad peak potential for AA at about 0.25V and a peak poten-tial at about 0.40V vs.Ag/AgCl for the mixture of EP and UA together at a bare GCE.Moreover,Fig.2(curve d)shows that all the three compounds were oxidized with well-de?ned and distin-guishable sharp peak potentials at about 0.12V,0.30V,and 0.43V vs.Ag/AgCl for AA,EP,and UA,respectively when using PBCACP-
A.A.Ensa?et al./Sensors and Actuators B 150 (2010) 321–329
325
Fig.2.Differential pulse voltammogram of the electrodes on the buffer solution (a and c),and mixture of 200?mol L ?1of AA,20?mol L ?1of EP,and 20?mol L ?1of UA (b and d)at the surface of bare GCE (dash line)and at PBCACPMGCE (solid line).
MGCE as a working electrode.It was also found that the oxidation peak potentials are separated completely into three well-de?ned peaks AA,EP,and UA,respectively.AA and EP exhibited nega-tive potential shifts,which together with their enhanced currents with PBCACPMGCE indicate that the modi?ed electrode must has played a catalytic effect on the oxidation of AA,and EP.The sep-arated oxidation peak potentials were about 0.200V and 0.150V,respectively.
3.3.Electrochemical impedance spectroscopy
The oxidation of ascorbic acid on both GCE and PBCACPMGCE was investigated using impedance spectroscopy.Fig.3shows the Nyquist plot of the impedance surface densities ( cm ?2)and admittance surface densities (mS cm ?2)with GCE (a)and with PBCACPMGCE (b)recorded at +0.25V as DC offset for 500?mol L ?1AA at pH 4.0.Impedance and admittance quantities depend on the microscopic areas of the electrodes.The surface areas of the bare GCE and the modi?ed electrodes were different;therefore,the impedance elements were normalized post-experimentally during data analysis with respect to electrode surface area.The ratio of the microscopic surface of a bare GCE to the modi?ed electrode was determined by recording the CVs of 1.0mmol L ?1K 4Fe(CN)6in 0.1mol L ?1KNO 3in different scan rates,using the same size GCE.For a reversible process,the Randles–Sevcik equation was used [32]:
I pa =2.69n 3/2AC 0D R 1/2 1/2
(1)
where I pa (A)refers to the anodic peak current,n is the number of transfered electron,A (cm 2)is the surface area of the elec-trode,D R (cm 2s ?1)is the diffusion coef?cient,C 0(mol cm ?3)is the concentration of K 3Fe(CN)6,and (V s ?1)is the scan https://www.sodocs.net/doc/3a3435309.html,ing 1.0mmol L ?1K 3Fe(CN)6in the presence of 0.10mol L ?1KCl elec-trolyte with n =1and D R =7.6×10?6cm 2s ?1,the microscopic areas of the electrodes can be calculated from the slope of the I pa versus 1/2.
In addition,the oxidation of EP and UA at PBCACPMGCE were investigated too.Table 2shows electrochemical impedance spec-troscopic data ?t for the oxidation of AA at GCE and PBCACPMGCE surfaces,plus oxidation of EP and UA at PBCACPMGCE.These elec-trodes have a charge transfer resistance (R ct ),a mass transfer resistance (R S ),and a capacitor (C Id )showing the adsorption of the analyte at the surface of GCE.Due to the inhomogeneity of the PBCACPMGCE electrode surface,R S and C Id appeared as Q contain-ing the capacitor and the resistance in parallel.When PBCACPMGCE was used (Fig.3),two counteracting parameters affected the charge transfer resistance (R ct ):one,the polymer coating that caused the charge transfer resistance to increase and,the other,the electrocat-alyst effect of poly(3,3 -bis[N,N
-bis(carboxymethyl)aminomethyl]-
Fig.3.Nyquist plot of the impedance surface densities (k cm ?2)and admittance surface densities (mS cm ?2)with (a)GCE;and (b)PBCACPMGCE.Conditions:recorded at +0.25V as DC offset for 500?mol L ?1AA at pH 4.0.
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Fig.4.Chronoamperograms were recorded for different EP concentrations as(a)0;
(b)50;(c)75;and(d)100?mol L?1of EP with one potential step,potential of0.20V, and sampling current of0.05with duration of10s.
o-cresolsulfonephthalein)for the oxidation of AA which caused it to decrease.Therefore,the two different parameters balanced each other’s effect,?nally reducing charge transfer resistance.
To study the electrocatalytic system,we compared the values of the charge transfer coef?cient from the modi?ed electrode in the absence and presence of500?mol L?1AA,EP,and https://www.sodocs.net/doc/3a3435309.html,ing?tting process,the value of R ct for the modi?ed electrode was obtained as 510±0.5 in the absence of the analytes(blank solution).On the other hand,this amount was equal to449±4.5 ,453±11 ,and 480±5 in the presence of500?mol L?1AA,EP,and UA,respec-tively.Bigger difference between the R ct values in the absence and presence of the analytes con?rms higher electrocatalytic activity of the modi?ed electrode.Thus,according to the above R ct values,the modi?ed electrode showed better electrocatalytic activity for the oxidation of the analytes as AA>EP>UA.In addition,the decreasing in the diameter of the semicircles were as AA>EP>UA,con?rm-ing the electrocatalytic capability of the modi?ed electrode for the oxidation of AA and EP.
3.4.Chornoamperometric studies
The oxidation of EP at PBCACPMGCE was studied using chornoamperometry for different EP concentrations(Fig.4).The experimental plots of I vs.t?1/2were employed with the best ?ts for different concentrations of EP.The slopes of the resulting straight lines were then plotted vs.EP concentration.We calcu-lated the diffusion coef?cient as1.15×10?4cm2s?1for EP.The chornoamperograms of AA and UA were also studied using the same procedure described above for EP.Then,the diffusion coef-?cients of AA and UA were calculated as5.6×10?6cm2s?1and
2.2×10?6cm2s?1,respectively.
3.5.In?uence of solution variables
The effects of pH on the anodic peak potentials and peak currents of AA,EP,and UA were investigated by DPV in a solution containing 20.0?mol L?1AA,EP,and UA(Fig.5).The results showed that the peak potentials of AA,EP,and UA depended on the pH of the solu-tion at slopes values of?48mV pH?1,?58mV pH?1and?60mV pH?1,respectively.This con?rms previous reports indicating that the redox of AA,EP,and UA include1:1proton–electron transfer processes.
The results of the present study indicate that AA,EP,and UA obtain their maximum peak currents at pH values of5.0,4.0,and 5.0,respectively.This is because AA and UA are weak acids with p K a values of4.17and3.89,respectively.Thus,the negatively anionic charge is predominant at pH levels higher than4.17for AA and 3.89for UA.Hence,AA and UA were adsorbed on the electrode surface by a high hydrophobic group.On the other hand,the p K a value of R–SO3H(R=aryl group)is usually about four;hence,the R–SO3Na of poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm favorably dissociated into a nega-tively charged group(–SO3?)under this condition,and the alkaline –NH group of EP(p K a=8.55)obtained one proton to form a positive ion of EP,which has a great af?nity toward the poly(3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein)?lm. Therefore,for the simultaneous determination of these com-pounds,a pH of4.0(PBS,0.10mol L?1)was selected for further study.
The DPV parameters including pulse amplitude,pulse time,and voltage step time were optimized for solutions containing AA, EP,and UA at concentrations of200?mol L?1,20?mol L?1,and 20?mol L?1,respectively.Maximum peak current was observed when the pulse amplitude was50mV,pulse time was0.005s,and voltage step time and value were0.1s and5mV,respectively.These variables were selected for further study.
The in?uence of scan rate on the anodic peak current of EP was studied by cyclic voltammetry.The results showed that increasing scan rate increased the peak current.The good linear relation-ship holding between 1/2and I pa for a scan rate in the range of10–100mV s?1con?rms a diffusion-controlled process on the modi?ed electrode(r2=0.997).
4.Simultaneous determination of AA,EP,and UA
As shown above,3,3 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein possesses an excellent electrocatalytic activity regarding AA,EP,and UA oxidation.Since they have similar oxidation potentials at most solid electrodes,individual
Table2
Electrochemical impedance spectroscopy data that?t to oxidation of500?mol L?1AA at the surface of GCE(curve(a))and PXGCE(curve(b)).
R1(Q[R2W])Electrolyte at PXGCE AA at GCE AA at PXGCE EP at PXGCE UA at PXGCE Value Error%Value Error%Value Error%Value Error%Value Error%
R1220 1.03500 0.010209 2.30211 0.29207 1.98 C–– 1.000?F0.008––––––
Q–––––
y00.10×10?5 1.0––0.18×10?5 5.790.11×10?5 4.120.08×10?5 3.27 n 1.00–––0.938 1.050.9430.870.961 1.42 R2510 0.10500 0.013449 1.0453 2.35480 1.07 W0.65×10?30.190.100×10?30.0110.72×10?5 2.990.86×10?5 1.780.93×10?5 1.20
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327
Fig.5.Effects of pH on the peak current (a),and on the peak potential (b)for AA,EP,and UA in solutions containing a mixture of 20.0?mol L ?1AA,20.0?mol L ?1EP,and 20.0?mol L ?1UA.
determination of these species poses a great problem due to their overlapping signals.In order to establish a sensitive and selective method for the quanti?cation of AA,EP,and UA,the ability of the modi?ed electrode to promote the voltammetric resolution of AA,EP,and UA was investigated.The differences in the oxidation peak potentials for AA–EP and EP–UA were 0.18V and 0.13V,respectively,which were large enough separations to allow for the simultaneous determination of AA,EP,and UA in one mixture.Two linear segments with different slopes were observed for AA concentration;namely,for 1.0–100.0?mol L ?1of AA,the regression equation was I p (?A)=0.026C +1.794(r 2=0.9970,n =9),while for 100–2000?mol L ?1of AA,the regression equa-tion was I p (?A)=0.010C +3.026(r 2=0.991,n =7).Two linear segments for EP were found as:0.2–3.0?mol L ?1of EP,the regres-sion equation was I p (?A)=0.513C +0.132(r 2=0.9971,n =9),while 3.0–175.0?mol L ?1for EP,the regression equation was I p (?A)=0.130C +2.840(r 2=0.970,n =7).In addition,two linear segments for UA were found as:0.02–70.0?mol L ?1or UA,the regression equation was I p (?A)=0.124C +1.405(r 2=0.9910,n =9),while for 70–2000?mol L ?1of UA,the regression equation was
I p (?A)=0.021C +10.070(r 2=0.9920,n =7),where C is ?mol L ?1concentration of the analytes and I p is the peak current.
The lowest detection limits (S /N =3)were 0.4?mol L ?1,0.03?mol L ?1,and 0.009?mol L ?1for AA,EP,and UA,respectively.In order to check the intermolecular effects between AA,EP,and UA,three different experiments were carried out under the opti-mum conditions at pH 4.0at a scan rate of 100mV s ?1,using DPV.In each experiment,the concentration of one of the three com-pounds was changed while the concentrations of the other two species were kept constant.The results are shown in Fig.6.The peak currents for AA,EP,and UA increased linearly with an increase in their respective concentration.Based on the results obtained,no obvious change was observed in EP or UA oxidation currents (2.0?mol L ?1EP and 10.0?mol L ?1UA)with varying AA concentra-tion but the peak current of AA increased linearly with a correlation coef?cient of 0.993when AA concentration was increased (Fig.6a).Various concentrations of EP in the presence of 10.0?mol L ?1AA and 10.0?mol L ?1UA exhibited excellent DPV responses to AA,EP,and UA without any obvious intermolecular effects.The peak current of EP increased linearly by increasing EP
concentration
Fig.6.Voltammograms of (a)different concentrations of AA (0.0,6.0,10,30,50,70,100,200,300,and 400?mol L ?1)in the presence of 2.0?mol L ?1EP,and 10.0?mol L ?1UA;(b)different concentrations of EP (0.0,5.0,10,30,50,70,90,110,130,160,and 220?mol L ?1)in the presence of 10.0?mol L ?1AA and 10.0?mol L ?1UA;and (c)different concentrations of UA (0.0,3.0,7.0.10,30,50,and 70?mol L ?1)in the presence of 2.0?mol L ?1EP,and 10.0?mol L ?1AA.
328 A.A.Ensa?et al./Sensors and Actuators B150 (2010) 321–329
with a correlation coef?cient of0.997(Fig.6b).We also examined the in?uence of AA and EP on UA oxidation under the optimum conditions at pH4.0using PBCACPMGCE.No obvious change was observed in the EP and AA peak currents(10.0?mol L?1AA and 2.0?mol L?1EP)in response to varying UA concentration.However, the oxidation peak current of UA increased linearly with a corre-lation coef?cient of0.996when UA concentration was increased (Fig.6c).The variation in these peak currents is due to the error caused by sample addition and extraction of their voltammograms. These results indicate that the modi?ed electrode is suitable for the simultaneous determination of these three compounds without interference posed by any of them.
5.Interference study
In order to investigate the selectivity of the modi?ed glassy carbon electrode for simultaneous determination of AA,EP,and UA,several compounds were checked as potential interfering sub-stances in the analysis of the AA,EP,and UA.The tolerance limit was taken as the maximum concentration of foreign substances that caused a relative error of approximately+5%for the determi-nation of10.0?mol L?1AA,10.0?mol L?1EP,and10.0?mol L?1UA plus the potential interfering substances at pH4.0.The interference study was conducted by placing the PBCACPMGCE to a solution con-taining AA,EP,and UA at pH4.0.The DPV responses resulting from the presence of interfering substances were compared with those Table3
Interference studied of some foreign substances for20.0?mol L?1EN, 100.0?mol L?1AA,and20.0?mol L?1UA.
Species AA EN UA
Starch500500500 Citric acid400500500 Sucrose500500500 Glucose100100100 Urea400500500 Thiourea200300300 Fructose500500500 Valine100100100 Leucine300300200 Glycine100300300 Histidine200300300 Cystine10010050 Mg2+,Ca2+,K+,Na+,NH4+,CO32?,
SO42?,HCO3?,Br?,Cl?,ClO?4,NO3?
1000a1000a1000a a Maximum concentration tested.
obtained for AA and EP plus UA.The results are reported in Table3.It is clear that no interference occurred due to common substances or due to the ions Ca2+,Mg2+,glucose,fructose,carbonate and starch.
6.Analysis of real samples
In order to evaluate the applicability of the proposed method for the determination of AA,EP,and UA in real samples,its utility
Table4
Simultaneous determination of EP,AA,and UA as mixture in synthetic samples.
Sample Added(?mol L?1)Found(?mol L?1)Recovery(%) AA EP UA AA EP UA AA EP UA
150.010.010.049.3(±0.6)9.8(±0.3)10.1(±0.3)98.698.0101.0 2100.020.015.099.8(±0.9)19.7(±0.5)15.2(±0.5)99.898.5101.3 3200.035.025.0201.0(±2.0)34.6(±0.8)24.9(±0.6)100.598.999.6 4350.050.040.0347.0(±4.0)49.4(±1.0)39.8(±0.9)99.198.899.5 5500.075.060.0505.2(±7.0)74.5(±0.9)60.0(±0.8)101.099.3100.0
Table5
Determination of AA,EN,and UA in real samples.
Sample Target Added(?mol L?1)Found(?mol L?1)Recovery(%)RSD(%)HPLC(?mol L?1)
Tablet a AA20.019.5±0.597.4 2.619.2±0.7 Tablet a AA40.039.6±0.499.10.939.8±0.5 Tablet b AA20.019.7±0.298.6 1.119.6±0.6 Tablet c AA20.019.6±0.498.1 1.919.4±0.8
Ampoule d EP20.020.4±0.4101.9 2.120.0±0.5 Ampoule d EP40.039.6±0.499.0 1.139.2±0.8 Ampoule e EP20.09.7±0.548.6 5.39.6±0.45
Urine f UA–130.1±2.699.9* 2.0131.3±3.8 Urine g UA–121.6±3.0100.8* 2.5120.6±2.3 Urine h UA–68.4±1.2100.8* 1.870.0±1.3 Urine h UA–71.3±1.5101.8* 2.170.0±1.3 EP20.019.3±0.696.5 3.120.0±0.5
Plasma i AA– 5.1±0.2102.0 3.9 5.0±0.3 EP20.019.7±0.498.5 2.019.5±0.6
Plasma i AA– 5.3±0.2106.0 3.8 4.8±0.6 UA10.09.6±0.396.0 3.19.7±0.4
EP20.019.6±0.598.0 2.620.3±0.6
*Recovery was calculated based on the of?cial method.
a Vitamin C(18mg AA per each4g tablet,Vitalia Company,Germany).
b Vitamin C(500mg AA per each4g tablet,Osveh Pharmaceutical Company,Iran).
c Vitamin C(500mg AA per each2g tablet,Sunkist Company,Germany).
d 1.0mL of Epinephrin
e injection ampoule(0.1mg mL?1,Darou Pakhsh Company,Iran).
e10.0mL of Epinephrine injection ampoule(0.1mg mL?1,Darou Pakhsh Company,Iran).
f Diabetic urine sample(female,64years old).
g Urinary tract infection(UTI)urine sample(female,25years old).
h Healthy urine sample(male,25years old).
i Healthy plasma sample(male,25years old).
A.A.Ensa?et al./Sensors and Actuators B150 (2010) 321–329329
was tested by determining these compounds in several synthetic samples(Table4).The good recoveries of the mixture samples indi-cate the applicability of the proposed method for the simultaneous determination of AA,EP,and UA.In addition,we tested the applica-bility of PBCACPMGCE for the determination of AA in commercially available vitamin C tablets,EP in dopamine hydrochloride injec-tion,and UA in different urine samples.The results are given in Table5.The recovery ratios indicate that the modi?ed electrode is effective for determining AA,EP,and UA and that it can be applied for their detection in real https://www.sodocs.net/doc/3a3435309.html,parisons of the proposed method with high performance liquid chromatography[33,34]also con?rmed the accuracy of the results obtained by our proposed method,showing no signi?cant differences between those meth-ods.
7.Conclusions
We have shown that poly(3,3 -bis[N,N-bis(carboxymethyl) aminomethyl]-o-cresolsulfonephthalein)?lm modi?ed glassy car-bon electrode exhibits highly electrocatalytic activity for the oxidation of AA,EP,and UA.The modi?ed electrode not only improved the electrochemical catalytic oxidation of AA,EP,and UA, but also resolved the overlapping anodic https://www.sodocs.net/doc/3a3435309.html,ing the modi?ed electrode with the DPV method,AA,EP,and UA were measured simultaneously at as low levels as0.4?mol L?1,0.03?mol L?1,and 0.009?mol L?1,respectively.Moreover,the results obtained from the application of the proposed method for determining AA,EP, and UA in real samples such as urine and pharmaceutical samples con?rmed its satisfactory accuracy and precision.
Acknowledgements
The authors wish to thank Isfahan University of Technology (IUT)Research Council,Center of Excellence in Sensor and Green Chemistry,and Iranian Nanotechnology Initiative Council for their support.
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Biographies
Ali A.Ensa?was graduated in Chemistry(MSc)from Shiraz University(Iran)in1988 and he received his PhD in1991in Analytical Chemistry in the same University.Then, he joined the Department of Chemistry at Isfahan University of Technology(Iran). He became full professor in2001.His research interest comprises the development of new electrochemical or chromogenic chemosensors and molecular probes for anions,cations and pharmaceutical compounds.
B.Rezaei is a Professor of Chemistry,Isfahan University of Technology(IUT),Isfahan, Iran.BS from Esfahan University,1988;MS from Esfahan University in Analytical Chemistry,1991;PhD from Isfahan University of Technology in Analytical Chem-istry,1999.His research interests cover electrochemical,?ow injection method of analysis and electrochemical sensors.
Z.Mirahmadi Zare is currently a PhD student at Isfahan University of Technology (Iran).She has received her BS(2003)from Isfahan University of Technology and MS (2005)from Isfahan University of Technology(IUT).Her research activities concern on nanotubes and biomaterial and electrochemical sensors.
M.Taei received his BSc degree in2002and MSc degree in2004from Department of Chemistry,Yazd University(Iran).She is PhD student in Analytical Chemistry in Isfahan University of Technology(Iran)now.
药学毕业论文齐墩果酸和熊果酸保护神经的药理作用综述
齐墩果酸和熊果酸保护神经的药理作用综述 齐墩果酸和熊果酸是通过抗氧化、抗炎而产生神经保护 作用，以下是搜集整理的一篇相关论文范文，欢迎阅读参考。 齐墩果酸(oleanolicacid)和熊果酸(ursolicacid)同属 五环三萜酸类化合物，它们又是同分异构体，因此药理作用几乎 相同。现已证实齐墩果酸和熊果酸都具有抗炎、降糖、调脂、抗 肥胖、抗动脉粥样硬化药理作用[1-4],有望成为抗代谢综合征新药。代谢综合征可诱发神经系统方面的并发症，如脑中风、疼痛、糖尿病脑病、痴呆等。我国正在步入老龄社会，尤其是很多大城 市已经进入老龄社会，老年性神经精神疾病如老年痴呆、帕金森 病等发病率快速增长，而目前又缺乏安全有效、毒副作用低的防 治老年性神经精神疾病药物。齐墩果酸和熊果酸除了通过抗代谢 综合征阻滞神经系统并发症外，已有大量实验研究发现齐墩果酸 和熊果酸具有神经保护作用。本文综述齐墩果酸和熊果酸保护神 经细胞、镇痛、抗精神失常、改善学习记忆等神经精神药理方面 的研究进展，为齐墩果酸和熊果酸防治老年痴呆、帕金森病和抑 郁症的研发提供依据。 1、对离体神经细胞的保护 β淀粉样肽、过氧化氢、谷氨酸、6-羟基多巴胺等都具 有神经毒性作用，能直接损伤神经细胞引起的神经元凋亡和神经 功能障碍。齐墩果酸和熊果酸对这些神经毒素引起的离体神经细 胞损伤都有保护作用。 1.1对抗β淀粉样肽的神经毒性
PC12细胞源自大鼠髓质嗜铬细胞瘤，为神经源性细胞，具有多巴胺能神经特性，被广泛用作神经细胞体外模型，常被用来筛选神经保护药物。齐墩果酸(20、40、80mg/L)预处理PC12细胞，可浓度相关地对抗β淀粉样肽诱导乳酸脱氢酶渗漏，提高PC12细胞存活率，降低细胞凋亡率。这种神经保护作用可能与齐墩果酸促进抗凋亡基因bcl-2表达，抑制促凋亡基因bax表达有关[5].Hong等[6]报道熊果酸0.5~125μmol/L浓度相关的抑制β淀粉样肽诱导PC12细胞产生活性氧，从而减少DNA断裂和细胞凋亡。也可通过抑制上游激酶ERK1/2、p38和JNK磷酸化，阻滞核因子-κB(NF-κB)的p65亚单位核易位，抑制β淀粉样肽诱导PC12细胞表达诱生型一氧化氮合酶和环氧化酶-2,阻止神经炎症反应发生[7].Wilkinson等[8]报道熊果酸阻滞β淀粉样肽与小神经胶质细胞结合并抑制活性氧生成。用表达人CD36的中国仓鼠卵巢细胞实验，发现熊果酸浓度相关地阻滞β淀粉样肽与其受体CD36结合，浓度增至20μmol/L时作用达到峰值，最大抑制率为64%,认为熊果酸通过阻滞β淀粉样肽与CD36结合，抑制前炎性细胞因子和神经毒活性氧产生，从而阻断神经变性发生。 1.2对抗过氧化氢的神经毒性 Yu等[9]报道熊果酸0.05~2mg/L通过诱生抗氧化酶过氧化氢酶、谷胱甘肽过氧化物酶和抑制脂质过氧化反应，对抗过氧化氢损伤HEI-OC1听觉细胞。Rong等[10]报道齐墩果酸1、 10μmol/L可对抗过氧化氢损伤PC12细胞，表现为显着降低乳酸脱氢酶活性和丙二醛水平，逆转低下的线粒体膜电位、琥珀酸脱氢酶和超氧化物歧化酶活性、还原型谷胱甘肽水平，从而提高 PC12细胞存活率。Cho等[11]报道齐墩果酸通过对抗过氧化氢升高细胞内钙离子浓度和活性氧生成，抑制过氧化氢诱导大鼠皮质神经元凋亡。
熊果酸含量测定方法研究进展
熊果酸含量测定方法研究进展 （作者:___________单位: ___________邮编: ___________） 【关键词】熊果酸；含量测定方法 熊果酸（ursolie acid，UA）又名乌索酸、乌苏酸，属于三萜类化合物，广泛存在于熊果、白花蛇舌草、女贞子、乌梅等天然植物和草药中，具有抗炎、护肝、降血脂等多种生物学活性［1～4］。目前许多中药材及其制剂常选用熊果酸的含量作为其质量控制指标。本文对熊果酸含量测定方法的研究进展综述如下。 1 分光光度法 李国章等［5］建立了湘产3种苦丁茶中熊果酸含量测定的分光光度法，具体操作是取苦丁茶经索氏提取器提取后，加入5%香草醛－冰醋酸、高氯酸显色，在波长548 nm处测定吸光度，结果熊果酸在4～20 μg·ml-1浓度范围内线性关系良好，加样回收率为98.96%。罗启剑等［6］采用同样方法测定了连钱草中熊果酸含量，结果熊果酸在0～18 μg·ml-1浓度范围内线性关系良好，加样回收率为100.64%。 2 薄层扫描法
白洁等［7］采用双波长薄层扫描法测定了夏枯草中熊果酸的含量，具体操作为取夏枯草药材粗粉经95％乙醇超声提取两次，滤液用70℃水浴蒸干，残渣用石油醚浸泡两次，挥干溶剂，用95％乙醇溶解后测定。采用硅胶G板，以环己烷－氯仿－醋酸乙酯（20∶5∶8）为展开剂，用10％硫酸乙醇溶液显色，选用λS=540 nm，λR=700 nm进行双波长反射式锯齿扫描，结果熊果酸在0.314～1.570 μg范围内线性关系良好，加样回收率为99.3％。张军等［8］建立了狼疮静颗粒中熊果酸的薄层扫描法测定方法，具体操作为取狼疮静颗粒用乙醚萃取3次，合并乙醚萃取液水浴蒸干乙醚，残留物加无水乙醇－氯仿（3∶2）混合液溶解后测定，以环己烷－氯仿－醋酸乙酯－甲酸（20∶5∶8）为展开剂，用10％的硫酸乙醇液显色，以λS=520 nm，λR=700 nm进行双波长薄层扫描，结果熊果酸在0.498～3.084 μg范围内线性关系良好，加样回收率为97.91％。 3 高效液相色谱法 3.1 HPLC－UV法戚志华等［9］采用HPLC法测定了陕西女贞子中熊果酸的含量，其方法为取女贞子粉末经超声提取后采用HPLC法，选用Lichrospher C18（ 4.6 mm×250 mm，5 μm）色谱柱，流动相为乙腈－甲醇－水－磷酸－三乙胺（50∶30∶20∶0.02∶0．04），流速1 ml·min-1，测定波长为205 nm，结果熊果酸在20.48～102.4 μg范围内线性关系良好，加样回收率为100.6％。梁洁等［10］采用HPLC法测定了广西产美味猕猴桃根中熊果酸的含量，具体操作为取
乌梅药材中齐墩果酸和熊果酸的高效液相色谱含量测定(一)讲解
乌梅药材中齐墩果酸和熊果酸的高效液相色谱含量 测定(一) 作者：范成杰，刘友平，陈鸿平，石宇华 【摘要】目的应用高效液相色谱（HPLC）法测定乌梅药材中齐墩果酸和熊果酸的含量，并以齐墩果酸和熊果酸为指标成分建立乌梅肉的质量标准。方法采用Hypersil ODS C18(150 mm×4.6 mm, 5 μm)；甲醇－0.2%乙酸铵水溶液（83∶17）为流动相；检测波长210 nm，流速0.8 ml/min，柱温25℃。结果 齐墩果酸的线性范围为0.168～1.512 μg，r＝0.999 6，回收率为98.00%（RSD=0.53%）；熊果酸的线性范围为0.452～4.068 μg，r=0.999 9，回收率为97.13%（RSD=1.17%）。结论该方法简便、可靠、准确，可用于乌梅药材中 齐墩果酸和熊果酸的含量比较和乌梅肉的质量控制。 【关键词】高效液相色谱法乌梅肉齐墩果酸熊果酸 Abstract：ObjectiveTo determine the ursolic acid and oleanolic acid in the pulp of Fructus Mume by HPLC. MethodsThe ursolic acid and oleanolic acid were separated on C18 column.Methanol-0.2% Ammonium acetate solution (83∶17) was used as mobile phase and the detection wavelength was 254nm.ResultsThe linearity of ursolic acid and oleanolic acid was in the range of 0.168～1.512μg, 0.452～4.068μg, respectively; the average recoveries of ursolic acid and oleanolic acid were 98.00%(RSD=0.53%,n=5) and 97.13%(RSD=1.17%,n=5). ConclusionThe method is simple, quick and reproducible，and it can be used for the quality control of the pulp of Fructus Mume. Key words：HPLC; Pulp of Fructus Mume; Ursolic acid; Oleanolic acid 乌梅，别名酸梅、黑梅，由蔷薇科植物梅Prunus mume (Sieb.) Sieb. et Zucc.（Armeniaca mume Sieb.）的干燥近成熟果实加工而成。关于乌梅的入药方式，历代本草记载有去核和连核使用两种，现代研究证明乌梅中有效成分有机酸及水浸出物大多集中在果肉中，核含量甚少〔1〕，且核仁含大量脂肪油成分，具滑泄作用，与乌梅的固涩作用相驳；同时也有报道认为乌梅核特征明显，有利于乌梅的鉴定，而且乌梅核还含有约5％的有机酸，且去核繁琐费时 费工〔2〕，所以乌梅是否应分部位药用仍存在争议。《中国药典》Ⅰ部（2005年版）中，净乌梅和乌梅肉都有收载〔3〕。因此明确乌梅的入药方式，是建立乌梅规范的质量标准的前提。本课题组前期对乌梅各部位的化学成分进行了初步比较研究，结果表明乌梅各部位的药理作用不同，代表乌梅涩肠止泻作用的主要药用部位为乌梅果肉。本实验以齐墩果酸和熊果酸为对照品，建立了乌梅
熊果酸及五环三萜同类物的研究进展
湖南工业大学学报Journal of Hunan University of Technology Vol.23 No.5Sep.2009 第23卷 第5期2009年9月熊果酸及五环三萜同类物的研究进展 李宏杨１，刘国民１，刘 飞２，张凤琴２，李小龙２ （1. 海南大学 农学院，海南海口570228；2. 湖南工业大学包装与材料工程学院，湖南株洲412007） 摘要：五环三萜类化合物种类繁多，广泛分布在植物体中，且大多具有重要的药理活性，临床应用前景 十分诱人。随着研究的不断深入，有关五环三萜结构与活性的研究取得了大量进展，新的同类化合物不断的被发现。就熊果酸及五环三萜同类物结构与分类、在植物中的分布情况和药理作用的研究进展进行了综述。 关键词：熊果酸；五环三萜；抗肿瘤 中图分类号：Q541 文献标志码：A 文章编号：１６７３－９８３３（２００９）０５－００１８－０４ Research of Ursolic Acid and Similar Pentacyclic Triterpenoid Li Hongyang 1，Liu Guomin 1，Liu Fei 2，Zhang Fengqing 2，Li Xiaolong 2 （1. School of Agriculture ，Hainan University ，Haikou 570228，China ； 2. School of Packaging and Material Engineering ，Hunan University of Technology ，Zhuzhou Hunan 412007，China ） Abstract ：Various pentacyclic triterpenoids, widely distributing in plants, have excellent pharmacological activities. The clinical application of such compounds has attracted much attentions. The research of the structure and classification of Ursolic Acid and similar pentacyclic triterpenoids and their distribution and pharmacological functions are summarized. Keywords ：ursolic Acid ；pentacyclic triterpenoid ；antitumor 收稿日期：2009－08－17 基金项目：国家科技支撑计划子课题（2007BAD76B05－02）作者简介：李宏杨（1983－），男，河南信阳人，海南大学硕士研究生，主要研究方向为作物种质资源的创新与利用，E-mail ：hyang896@https://www.sodocs.net/doc/3a3435309.html, ；刘国民（1955－），男，湖南祁东人，海南大学教授，博士生导师，主要研究方向为作物种质资源的创新与利用，E-mail ：kudingcha_no1@https://www.sodocs.net/doc/3a3435309.html, 五环三萜类化合物是一类重要的天然产物，大多以游离形式或者与糖结合成苷的形式广泛存在于自然界中。熊果酸（ursolic acid ）又名乌索酸、乌苏酸，属于α－香树脂烷（α－amyrin ）型五环三萜类化合物。1990年，日本将熊果酸列为最有希望的癌化学预防药物之一[1]。大量研究表明，熊果酸及五环三萜同类物具有抗肿瘤、抗HIV 、抗糖尿病、抗菌、抗病毒、增强免疫功能和降血脂等多种生物学活性。近年来，国内外学者围绕熊果酸及五环三萜同类物的药理学作用、以及新五环三萜结构的发现做了大量的研究工作，取得了丰硕的成果，这些成果亦展示了五环三萜广泛的应用前景，为五环三萜的综合开发利用提供了可靠的实验依据。 １熊果酸及五环三萜同类物的结构 目前已发现的三萜类化合物多数为四环三萜和五 环三萜。五环三萜类成分在药用植物中较为常见，主要的结构类型有乌苏烷型、齐墩果烷型、羽扇豆烷型和木栓烷型等[2]，见图1。 乌苏烷（ursane ）型又称α－香树脂烷（α－amyrane ）型，如熊果酸、积雪草酸[3]、蔷薇酸[4]、坡模酸[5]、2α－羟基乌苏酸[6]；齐墩果烷（oleanane ）型又称(β－香树脂烷（β－amyrane ）型，如齐墩果酸、甘草酸、甘 草次酸[7]、丝石竹皂苷元[8]、蒲公英萜醇[9]、刺囊酸[10]等；羽扇豆烷（lupane ）型如白桦脂醇、白桦脂酸[11]、 与分类
熊果酸抗肿瘤作用机制的研究进展
Studies in Synthetic Chemistry 合成化学研究, 2016, 4(3), 19-27 Published Online September 2016 in Hans. https://www.sodocs.net/doc/3a3435309.html,/journal/ssc https://www.sodocs.net/doc/3a3435309.html,/10.12677/ssc.2016.43003 文章引用: 孟艳秋, 杨丽娜, 潘洪双, 于婷婷, 张伟晨, 宁梓廷. 熊果酸抗肿瘤作用机制的研究进展[J]. 合成化学研究, Research Progress on Antitumor Action Mechanism of Ursolic Acid Yanqiu Meng, Lina Yang, Hongshuang Pan, Tingting Yu, Weichen Zhang, Ziting Ning Shenyang University of Chemical Technology, Shenyang Liaoning Received: Oct. 1st , 2016; accepted: Oct. 16th , 2016; published: Oct. 21st , 2016 Copyright ? 2016 by authors and Hans Publishers Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). https://www.sodocs.net/doc/3a3435309.html,/licenses/by/4.0/ Abstract Ursolic Acid is a type of pentacyclic triterpene compounds with many kinds of pharmacological ac-tivities, especially its antitumor activity. The antitumor mechanism of ursolic acid is multifaceted. In this paper, the research progress of anti-tumor mechanism of ursolic acid has been reviewed and forecasted. Keywords Ursolic Acid, Antitumor, Action Mechanism 熊果酸抗肿瘤作用机制的研究进展 孟艳秋，杨丽娜，潘洪双，于婷婷，张伟晨，宁梓廷 沈阳化工大学，辽宁 沈阳 收稿日期：2016年10月1日；录用日期：2016年10月16日；发布日期：2016年10月21日 摘 要 熊果酸是一种具有多种药理活性的五环三萜类化合物，其抗肿瘤活性尤为显著。熊果酸的抗肿瘤机制是多方面的。本文对熊果酸的抗肿瘤作用机制的研究进展进行综述并进行展望。 Open Access
熊果酸药理作用研究进展
熊果酸药理作用研究进展 ，相对分子量456 科植物毛子草的地上部分熊果酸具有广泛的 之 对致癌、促癌物有抵抗作用 多研究认为，熊果酸能通过化学预防、抗突变、细胞生长抑制和细胞毒等作用来抑制 到预防恶性肿瘤的目的。癌的发生、发展一般要经历始发突变、促癌和演变三阶段，干扰三个阶段即可达到延缓或阻止
显增加，部分细胞在
熊果酸在自然界中分布广泛，资源丰富，具有化学预防，保肝、抗肝炎，抗肿瘤、抗菌、抗病毒等多种药理活性，熊果酸药用开发景已被众多研究机构所重视，有望成为一种高效低毒的多用途新药。 【参考文献】 1 李开泉，陈武，熊筱娟，等.乌索酸的化学、药理及临床应用进展.中成药，2002， 2 4（9）:709-711. 2 Muto Y，Ninomiya M，Fujiki H.Present status of research on cancer chemopre vention in Japan.Jpn J Clin Oncol，1990，20（3）:219-221. 3 黄镜，孙燕.熊果酸的抗肿瘤活性.中国新药杂志，1997，6（2）:101-104. 4 Niikawa M，Hayashi H，Sato T，et al.Isolation of substances from glossy prive t（Ligustrum lucidum Ait）inhibiting the mutagenicity of benzo（α）preene in bacteri a.Mutat Res，1993，319（1）:1-4. 5 Young HS，Chung HY，Lee CK，et al.Ursolic acid inhibits aflatoxin B1-induced mutagenicity in a Salmonella assay system.Biol Pharm Bull，1994，17（7）:990-99 3. 6 Huang MT，Ho CT，Wang ZY，et al.Inhibition of skin tumorigenesis by rosema ry and its constituents carnosol and ursolic acid.Cancer Res，1994，54（3）:701-70 5. 7 Ohigashi H，Takamura H，Koshimizu K，et al.Search for possible an-titumor p romoters by inhibition of12-O-tetrade-canoylphorbol-13-acetate-induced Epstein-Barr v irus activation;ursolic acid and oleannolic acid from an anti-inflammatory Chinese m edicnal plant，Glechoma hederaceae L.Cancer Lett，1986，30（2）:143-148. 8 Ames BN.Dietary carcinogens and anticarcinogens.Science，1983，221:1256-12 58. 9 Balanehru S，Nagarajan B.Protective effect of oleanolic acid and urso-lic acid against lipid peroxidation.Biochem Int，1991，24（5）:981-984.
不同产地及外观形态夏枯草中齐墩果酸和熊果酸的含量比较
不同产地及外观形态夏枯草中齐墩果酸 和熊果酸的含量比较 （作者:___________单位: ___________邮编: ___________） 【摘要】目的比较不同产地、长度、色泽的夏枯草中齐墩果酸和熊果酸的含量，为更全面地控制夏枯草的质量提供依据。方法采用高效液相色谱法，以Shim Pack C18为色谱柱，乙腈甲醇水乙酸铵(体积比68∶16∶16∶0.5)为流动相，检测波长为215 nm，流速为0.8 mL/min。结果与结论不同产地夏枯草中齐墩果酸和熊果酸的含量差异较大，果穗短者两者含量高于果穗长者，紫红色果穗者两者含量高于棕色果穗。 【关键词】夏枯草产地果穗齐墩果酸熊果酸 Abstract:Objective To compare the contents of oleanolic acid and ursolic acid in Prunella vulgaris with different appearances and from different provenances,in order to control the quality of Prunella vulgaris.Methods Samples were analyzed on a Shim Pack C18 column. The mobile phase consisted of acetonitrile methol water ammonium acetate (68∶16∶16∶0.5)
under a flow rate of 0.8 mL·min-1. The detection wavelength was set at 215 nm.Results and Conclusions There were large differences in contents of oleanolic acid and ursolic acid among Prunella vulgaris with different provenances and appearances, higher in Prunella vulgaris with short and mauve ears. This method was suitable for the quality control of Prunella vulgaris. Key words:Prunella vulgaris;oleanolic acid;ursolic acid 夏枯草Prunella vulgaris为常用中药，以干燥果穗或全草入药，主要用于治疗目赤肿痛、头痛眩晕、瘰疬瘿瘤、乳痈肿痛、高血压等[1]。夏枯草中含有三萜及苷类、苯丙素类、黄酮类等多种化学成分，其中齐墩果酸和熊果酸是主要活性成分[2,3]。目前文献报道有采用衍生化气相色谱法、毛细管胶束电动色谱法、高效液相色谱法等方法评价夏枯草的质量[4-6]，但对于不同产地、长度、色泽的夏枯草中这两种三萜酸的含量比较还未见报道。本文以高效液相色谱法测定并比较不同产地、长度、色泽夏枯草样品中齐墩果酸及熊果酸的含量，旨在为更全面控制夏枯草的内在质量提供依据。 1 仪器与试药 Agilent 1100高效液相色谱仪：包括G1313A自动进样器，Agilent ChemStations数据处理软件，美国安捷伦科技有限公司;齐墩果酸对照品、熊果酸对照品：中国药品生物制品检定所，批号分别为110709200304、110742200516;乙腈：色谱纯，美国TEDIA公司;甲醇：分析纯及色谱纯，江苏汉邦科技有限公司;石油醚、乙酸铵：
熊果酸的功效与作用
熊果酸是由天然植物中的一种三萜类化合物而来，具特殊的气味，是存在于天然植物中的一种五环三萜类化合物。那么熊果酸具体有哪些的作用与功效呢？下边不妨一起来简单了解一下吧。 一、熊果酸的作用及功效 据临床医学表示，熊果酸中有的有益分子，非常显著而迅速的可以降低谷丙转氨酶、血清转氨酶等，对于消退小儿黄疽以及增进食欲等方面都有很好的促进，而对于抗纤维化和恢复肝功能症状，效果也非常的明显，而且还特别的稳定。熊果酸还具备有非常明显的抗氧化功效，这也使得它成为医药及化妆品方面的常备原料，例如医药方面对于肝部的治疗非常有益，能够保护并稳定稳定肝细胞膜、细胞器，从而使输出、运输等功能相继慢慢的恢复，在化妆品方面，由于熊果酸有很好的抗氧化作用，能使肌肤具有抵抗力，与此同时还有效淡化皱纹、修肌肤等皮肤难题。且熊果酸其实还是一味天然的保湿剂，大病可以调理，小病可以医治，又给人们带来不少的惊喜。
二、含量测定标准 1、色谱条件： 硅胶G薄层板;环己烷-氯仿-乙酸乙酯(20:5:8)为展开剂，上行展开;展距12～18cm;5%硫酸乙醇溶液，110℃加热5min显色。 2、样品溶液的制备： 精密称取栀子粉碎样品20g (炒栀子按得率折合后称取)，置索氏提取器内，加乙醚300ml回流提取至无色，回收溶剂至干，残留物加石油醚浸泡2次，每次15ml，约浸泡2min，倾去石油醚，用无水乙醇-乙醚(2：3)混合液微热使溶解并定容于5ml量瓶中，作为样品溶液。 3、对照品溶液的配制： 精密称取熊果酸对照品适量，加无水乙醇：乙醇(3：2)的混合溶液制成每毫升含1mg的溶液，作为对照品溶液。 4、测定： 准确吸取样品溶液及对照品溶液2μl，点于同一薄层板上。按上述色谱条件展开，显色。照薄层扫描法扫描，λS=520nm，λR=700nm;双波长反射法锯齿
齐墩果酸与熊果酸结构修饰物的药理活性和构效关系研究进展_刘丹
齐墩果酸与熊果酸结构修饰物的药理活性和构效关系研究进展 刘丹1,2 孟艳秋23 赵娟2 (1天津大学药物科学与技术学院 天津 300072; 2沈阳化工学院制药工程教研室 沈阳 110142) 刘丹 女,35岁,副教授,主要从事天然活性成分的结构修饰和医药中间体的合成工作。 3联系人,E 2mail :myq6581@https://www.sodocs.net/doc/3a3435309.html, 辽宁省自然科学基金资助项目(20042009) 2005-12-12收稿,2006-08-15收稿 摘 要 齐墩果酸(OA )和熊果酸(UA )均属于五环三萜类化合物,广泛存在于自然界中,具有多种显著的 生物活性。本文综述了近年来齐墩果酸及熊果酸结构修饰物的药理活性和构效关系的研究进展。 关键词 齐墩果酸 熊果酸 结构修饰 药理活性 R ecent Advance in the Study on Derivatives of Oleanolic Acid and U rsolic Acid Liu Dan 1,2,Meng Y anqiu 23,Zhao Juan 2(1C ollege of Pharmaceuticals &Biotechnology ,T ianjin University ,T ianjin 300072;2Faculty of Pharmaceutical Engineering ,Shenyang Institute of Chemical T echnology ,Shenyang 110142) Abstract Oleanolic acid and urs olic acid belong to triterpene acids which having numerous pharmacological activities and widely presenting in food ,medicinal herbs and other plants.Here a brief introduction of the recent progresses on pharmacological activities and the structure-activity relationship of derivatives of olean olic acid and urs olic acid are given out. K ey w ords Oleanolic acid ,Urs olic acid ,M odification ,Pharmacological activities 齐墩果酸(oleanolic acid ,OA ,1)和熊果酸(urs olic acid ,UA ,2)为结构类似物,均属于五环三萜类化合物,在自然界分布十分广泛,且具有多种生物活性。为了寻找高效低毒的衍生物,需对齐墩果酸和熊果酸的作用机制进行深入的研究;通过对32位羟基,C 122C 13位双键和282位羧基等官能团的结构修饰合成了一系列衍生物,进行了相关的药理活性测试,并与母体进行比较,获得了相应的构效关系。本文将就齐墩果酸及熊果酸结构修饰物的药理活性和构效关系研究进展进行综述。 1 齐墩果酸及熊果酸化学及药理活性简介 齐墩果酸(C 30H 48O 3)属于β-香树脂醇型五环三萜类化合物,据不完全统计,它以游离形式或与糖结合的形式存在于大约60个科190种植物中[1],具有保肝[2,3]、消炎[4]、降糖[5]、抗HI V [6,7]和抗肿瘤[8～10]等药理作用。 熊果酸(C 30H 48O 3)为α-香树脂醇型五环三萜类化合物,以游离形式或与糖结合的形式存在于大约
熊果酸
打开的谱图文件：D:\10\熊果酸标准品3(00001).hw ───────────────────────────序号 保留时间 名称 峰面积% 峰面积 ─────────────────────────── 1 0.20 2 0.00464 3 118 2 0.23 3 0.001742 44 3 0.278 0.002292 58 4 0.444 0.009194 233 5 0.652 0.006404 163 6 0.922 0.006128 156 7 1.150 0.005266 134 8 1.199 0.002263 57 9 1.236 0.002975 76 10 1.453 0.009436 240 11 1.652 0.0075 190 12 1.803 0.001693 43 13 2.070 1.414 35901 14 2.659 0.09665 2454 15 2.777 0.1092 2773 16 3.084 1.454 36908 17 3.726 1.346 34185 18 4.277 0.2177 5529 19 4.675 0.4557 11571 20 4.886 0.322 8176 21 5.498 93.84 2382895 22 7.284 0.02985 758 23 7.588 0.006965 177 24 7.853 0.003855 98 25 8.058 0.01011 257 26 8.294 0.0007985 20 27 8.404 0.002138 54 28 8.594 0.00232 59 29 8.920 0.006411 163 30 9.097 0.003013 77 31 9.357 0.005584 142
熊果酸及五环三萜同类物的研究进展
熊果酸及五环三萜同类物的研究进展 李宏杨1，刘国民1，刘飞2，张凤琴2，李小龙2 (1. 海南大学农学院，海南海口570228 ；2. 湖南工业大学包装与材料工程学院，湖 南株洲412007) 摘要：五环三萜类化合物种类繁多，广泛分布在植物体中，且大多具有重要的药理活性，临床应用前景十分诱人。随着研究的不断深入，有关五环三萜结构与活性的研究取得了大量进展，新的同类化合物不断的被发现。就熊果酸及五环三萜同类物结构与分类、在植物中的分布情况和药理作用的研究进展进行了综述。 关键词：熊果酸；五环三萜；抗肿瘤 五环三萜类化合物是一类重要的天然产物，大多以游离形式或者与糖结合成苷的形式广泛存在于自然界中。熊果酸(ursolic acid) 又名乌索酸、乌苏酸，属于oc 一香树脂烷(r,-amyrin) 型五环三萜类化合物。1990 年，日本将熊果酸列为最有希望的癌化学预防药物之一。大量研究表明，熊果酸及五环三萜同类物具有抗肿瘤、抗HIV、抗糖尿病、抗菌、抗病毒、增强 免疫功能和降血脂等多种生物学活性。近年来，国内外学者围绕熊果酸及五环三萜同类物的药理学作用、以及新五环三萜结构的发现做了大量的研究工作，取得了丰硕的成果，这些成果亦展示了五环三萜广泛的应用前景，为五环三萜的综合开发利用提供了可靠的实验依据。 1 熊果酸及五环三萜同类物的结构与分类 目前已发现的三萜类化合物多数为四环三萜和五环三萜。五环三萜类成分在药用植物中较为常见，主要的结构类型有乌苏烷型、齐墩果烷型、羽扇豆烷型和木栓烷型等，见图1。 乌苏烷(Ill'Sane) 型又称一香树脂烷(r,-amyrane) 型，如熊果酸、积雪草酸、蔷薇酸引、坡模酸I 、2 一羟基乌苏酸：齐墩果烷(oleanane) 型又称( 口一香树脂烷(B-amyrane) 型，如齐墩果酸、甘草酸、甘草次酸、丝石竹皂苷元引、蒲公英萜醇、刺囊酸等；羽扇豆烷(1upane)型如白桦脂醇、白桦脂酸n”羽扇豆醇、乙酸羽扇豆醇酯ml等；木栓烷(friedeiane) 型如木栓酮㈣、雷公藤红素、demethylzeylasteral 、salaspermic acid 、2,3 — dihydroxy-friedel-6 ，9(1 1) 一en 一29-oic acid 等。 2 熊果酸及五环三萜同类物在植物中的分布 据不完全统计，在自然界已有34科108种植物中能分离得到熊果酸，主要分布在女贞子、山楂、珍珠菜、夏枯草、车前草、甘草、连翘和苦丁茶等药用植物中。齐墩果酸是一种齐墩果烷型五环三萜类化合物，广泛分布于约60 科190种植物中，如：青叶胆全草、白花蛇舌草、女贞果实等，以游离形式或与糖结合成苷存在。就冬青科苦丁茶而言，目前发现含量 最丰富的五环三萜类化合物是熊果酸和齐墩果酸，主要存在于苦丁茶冬青、大叶冬青的枸骨嫩芽和功能叶之中。除此之外，冬青科苦丁茶中尚含有若干种含量较低的其它五环三萜类化合物。人们在大叶冬青中分离鉴定了 1 3 种新型三萜皂苷和7 种三萜苷元；在苦丁茶冬青
熊果酸的生物活性及其研究热点
2010年第卷第期 137[收稿日期]2010-05-14 [作者简介]刘柯彤（1988-），陕西榆林人，陕西理工学院化学工程与工艺专业2008级本科生。摘要：综述了近年来熊果酸生物活性的研究进展，重点讨论了其研究热点，展望了其今后的发展趋势。 关键词：熊果酸；生物活性；研究热点 中图分类号：O624 文献标识码：A doi ：10.3969/j.issn.1007-7871.2010.07.002 熊果酸的生物活性及其研究热点 刘柯彤，陶亮亮，马雄，刘军海 （陕西理工学院化学与环境科学学院，陕西汉中723001） 0前言熊果酸（Ursolic Acid ，UA ），又名乌索酸、乌苏酸，属α-香树脂醇型五环三萜类化合物。纯净的熊果酸为白色针状晶体，熔点为285~287℃，易溶于二氧六环吡啶，可溶于甲醇、乙醇，微溶于丙酮、苯、氯仿和乙醚。在自然界中熊果酸主要分布在女贞子、山楂、夏枯草、车前草和苦丁茶等多种植物中[1，2]。近年来，有关熊果酸的提取已经成为天然产物加工领域的研究热点之一；随着对熊果酸生物活性研究的深入，其在医药、食品、保健和化妆品等领域获得了广泛的应用。本文综述了熊果酸的提取工艺及其生物活性的研究进展，重点讨论了研究的热点问题及发展趋势，以期为熊果酸进一步的开发利用提供参考。1熊果酸的生物活性熊果酸具有广泛的生物活性，尤其在抗肿瘤、抗氧化、抗菌抗病毒、保肝和美容护肤等方面的作用显著，值得重视。1.1抗肿瘤作用研究发现，熊果酸具有抗致癌、抗促癌、诱导F9畸胎瘤细胞分化和抗肿瘤血管形成的作用。其主要通过以下几方面进行作用：熊果酸能抗突变、抑制癌变的启动、能直接杀伤肿瘤细胞，具有肿瘤逆转作用及抗侵袭性、诱导肿瘤细胞凋亡和抑制肿瘤血管形成，从而抑制癌变的形成；并且对多种肿瘤细胞体内、外均有抑制作用，并可 以减少放疗、化疗之后对造血系统的损害[3]。熊果酸对结肠癌、黑色素瘤和胃癌等多种细胞系具有抑制作用，并诱导其凋亡，其机制可能是通过影响细胞周期及癌相关基因的表达而实现的[4]。于丽波等研究发现熊果酸可明显抑制卵巢癌细胞生长，并诱导细胞凋亡，其主要机制与调节Bcl-2和Bax 的蛋白表达有关[5]。刘琼等研究表明， 熊果酸具有抑制恶性胶质瘤裸鼠移植瘤生长的作用，其机制可能与下调ERK1、C -Jun 、C -Myc 、CyclinD1表达有关[6]。 1.2抗氧化作用 氧化作用是造成人类衰老现象的重要原因之一，在防止人体内不良胆固醇（LDL ）的氧化，保持血管的年轻化方面，熊果酸扮演着重要角色。 熊果酸具有明显的抗氧化功能，因而被广泛 地用作医药和化妆品原料。抗氧化作用对人体抗 衰老和皮肤祛斑、祛色素、美容都有积极作用。 对于熊果酸的抗氧化作用有人曾作过研究，如卢静等采用邻苯三酚自氧化法，Feton 体系法测定熊果酸对超氧阴离子和羟自由基的清除能力，从而 探究熊果酸的抗氧化性能。结果表明熊果酸对超 氧阴离子和羟自由基有明显的清除作用，对超氧 阴离子的最高清除率可达88.42%，但是清除作用弱于同浓度下的VC 。熊果酸对羟自由基的清除作用最高可达86.35%，并且强于同浓度下的甘露醇，具有明显的抗氧化性[7]。王建梅等研究发现，熊果酸有保护糖尿病大鼠血管损伤的作用，机制可能 Surveys &Reviews 综述与述评 11
熊果酸的研究进展
作者：肖坤福郑云法刘成左张春牛 【关键词】熊果酸；,,,检测方法；,,,提取方法；,,,动物实验；,,,临床研究熊果酸是广泛存在于白花蛇舌草、女贞子、乌梅、夏枯草等天然植物中的一种五环三萜类化合物，有研究表明熊果酸具有镇静、抗炎、抗菌、抗糖尿病、降血糖等多种生物学效应，早在1996年，日本学者神藏美枝子等人从杜鹃花科越桔属植物越桔叶中提取了具有较高熊果酸浓度的熊果酸产品，产品中除含有25%的熊果酸外，尚含有大量的熊果酸衍生物、熊果苷和其它三萜类化合物等，具有多种活性成分，用于增强机体免疫力、预防心血管系统疾病、稳定肝功能等。现在人们对其研究越来越多，并在很多方面取得了可喜的研究进展。 1 提取方法 对三萜类化合物的分离提纯是其研究起点，没有较好的提取工艺，就更谈不上对其进一步研究。近年来，许多科研工作者在这方面做出了突出的贡献。有些科研工作者［1～5］，通过正交实验确定85%～95%的乙醇提取熊果酸效果最佳。崔星明等［6］采用超临界流体萃取得到的芦笋提取物，用甲醇溶解，采用液相色谱-质谱联用仪检测，得到了56个组分。发现有保留时间和熊果酸基本一致的峰。其质谱分子离子峰和特征碎片峰都与熊果酸的一致，确定该化合物为熊果酸。 2 检测方法 在检测方法方面，有不少科研工作者对其进行了较深入的研究。熊慧敏［7］全面地论证了薄层层析-双波长扫描法测定大山楂咀嚼片中熊果酸含量的检测限的线性范围可行性，并考查了此方法的测定熊果酸的稳定性和重复性，从而证明薄层层析-双波长扫描法测定熊果酸精密度、稳定性、重现性、回收率均都达有关规定的要求，本方法操作简单方便，结果可靠，可作为检验产品质量的一个快速而实用的定量方法。朱玉琴等［8］在采用薄层扫描检测保驾胶囊中熊果酸的含量时首次对显色剂进行了改进，并验证了醋酸-浓硫酸 (9∶1)比10%硫酸乙醇显色要好，检测限低等优点。与此同时，杨云等［9］在采用薄层层析检测牙周康泰胶囊熊果酸的含量验证了10%磷钼酸乙醇液比10%硫酸乙醇显色要好。 罗启剑等［10］采用分光光度法测定连钱草中熊果酸含量。郭艳玲等［11］和陈志强［12］采用薄层扫描法分别测定了补肾颗粒和消食贴中熊果酸含量，方法简便、快速、准确，可作为该产品的质量控制方法。 周静等［13］探求用毛细管气相色谱法测定女贞子中齐墩果酸和熊果酸含量。取女贞子氯仿提取液自然挥发干，甲醇溶解经甲酯化后，进样分析。色谱条件：hp－1毛细管柱（25 cm×0.32 mm，0.52 μm），柱温280℃，氮气为载气，氢火焰检测器。分析结果表明熊果酸的线性良好，回收率为99.8%±2.1%。所建立的气相色谱法可以作为女贞子药材的质量检测方法。 施慧君等［14］采用hplc法测定知柏地黄冲剂中熊果酸的含量。选用ods-c：色谱柱，流动相：甲醇-水(99∶1)，磷酸调ph 2.5，检测波长：206 nm，流速：1.2 ml/min，柱温25℃的条件下，熊果酸线性关系良好(r=0.999 8) 平均回收率达98.7%(rsd=0.66%)。罗晓清等［15］采用rp-hplc法测定石精叶中熊果酸含量，色谱柱为zorbax ods柱，用甲醇（1%），醋酸水溶液(88∶12)为流动相。检测波长为215nm。结果熊果酸和齐墩果酸的平均收率分别为98.5%、97.6%(n=3)。rsd分别为1.62%，1.89%(n=3)。方法准确、灵教、快速，可作为石抽叶药材的质量检测方法之一。 杨书良等［16］采用高效液相色谱法从影响熊果酸测定的各个方面(例如：粒度、煎煮次数、溶媒量、提取时间等)进行测定和和正交分析，从而论证了它比薄层层析法更精确、更可靠、灵敏度更高，重现性更好。鞠建华等［17］，也采用高效液相色谱测定熊果酸含量，但他在前人的基础上调节流动相的比例和加入改性剂乙酸胺，验证了此改进方法可以使熊果酸和它的同分异构体齐墩果酸达到基线分离，并使基线平稳、峰型和分离效果较佳。鄢立新等
熊果酸
枇杷叶提取物 药材来源及工艺： 熊果酸以游离形式或与糖结合成苷的形式分布于约7 个科46个属62种植 物 。主要为木犀科植物女贞(Ligustrum lucidum Ait.)叶，杜鹃药科植物熊果 Actostaphylos uva-ursi(L.)Spreng ，蔷薇科植物枇杷Eriobotrya japonica(Thunb.)Lindl. 的叶，玄参科植物毛泡桐（Paulownia tomentosa(Thunb.)Steud.）叶，唇形科植物夏枯草(Prunella vulgaris L.)的全 草，冬青科冬青属铁冬青(Ilex rotunda Thunb.)的叶等。 性状： 本品为类白色粉末。 可供规格： 熊果酸：25%—95%（HPLC ） 功能： 熊果酸是存在于天然植物中的一种三萜类化合物，具有镇静、抗炎、抗菌、抗糖尿病、抗溃疡、降低血糖等多种生物学效应，熊果酸还具有明显的抗氧化功能，因而被广泛地用作医药和化妆品原料。 1 保肝，抗肝炎作用 熊果酸临床表现有显著而迅速降低谷丙转氨酶、血清转氨酶、消退黄疽、增进食欲、抗纤维化和恢复肝功能的作用，具有见效快、疗程短、效果稳定的特点。 印度学者Saraswat 等 发现熊果酸(5～20 mg?kg ) 对CC1 引起的大鼠肝毒性有保护作用。用熊果酸作预处理可明显提高大鼠肝细胞的成活率，并在实验中观察到熊果酸具有抗胆汁淤积的作用，胆汁流量及其内容物均有增加。由CC1 的作用机制推测熊果酸对损伤肝细胞的治疗作用机制可能与其同分异构体齐墩果酸相似，在于保护和稳定肝细胞膜及细胞器的生物膜系统，使其变动通透及主动运输功能恢复正常，细胞内外离子和水的移动及分布亦随之复原，再生能力相继恢复，促进肝小叶中央区坏死肝细胞修复。 “抗肝炎中药一类新药乌索酸及其制剂的研究开发”课题取得重大进展，被列入2002年国家“863”计划，获国家科研资助经费100万元，成为国家级重大研究课题（2002AA2Z3217）。 2 抗肿瘤作用 熊果酸还对多种致癌、促癌物有抵抗作用，研究发现熊果酸能明显抑制HL －60细胞增殖，并诱导其凋亡；能使小鼠的巨噬细胞吞噬功能显着提高，能抑制人舌鳞癌细胞株TSCCa 细胞增殖，对TSCCa 细胞的半数生长的抑制剂量约为12.5 μmol?L －1，在24h 内表现为一定的量效关系；原位杂交显示熊果酸对TSCCa 细胞的抑制作用与抑制核转录因子的原位表达有关。。体内试验证明熊果酸可以明显增强机体免疫功能。说明熊果酸的抗肿瘤作用广泛，熊果酸极有可能成为低毒有效的新型抗癌药物。 熊果酸对肿瘤形成生长各阶段具有预防和抑制作用，抑制肿瘤形成生长以及诱导癌细胞分化作用。Li J 等通过实验及临床试验证明熊果酸能抗DNA 突变、抑制癌变的启动, 并且熊果酸能对抗致癌物如苯并芘，黄曲霉毒素Bl 诱发基因突变。王鹏试验对早期抗体(EBV-EA)活化试验型来筛选皮肤癌促癌物的抑制剂，发现熊果酸对TPA 诱导Raji 细胞EBV-EA 活化具有几乎同维甲酸相同的抑制作用，并使Raji 细胞的存活率更高；利用同位素标记的佛酯化合物3H-PDB2证明熊果酸不影响TPA 与受体的结合，不是通过竞争TPA 位点，而是有效地阻断致癌物与受体结合后的环节发挥作用。Guevara 等研究显示熊果酸能使诱变剂所致的多染色质的微核红细胞数减少76％ 。 Ohigachi 等研究结果显示熊果酸对佛波酯(TPA)诱导 Raji 细胞EBA —EA 活化有抑制作用，其程度与维甲酸几乎相同，Raji 细胞存