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*• •''"""'•'of Medicmal Materials, 2016, VoL 21, No. 5 (pp. 315-320) SIMULTANEOUS QUANTITATIVE DETERMINATION OF LIMONIN,
^ t VODIAMDNE AND RUTAECARPINE IN EVODIAE FRUCTUS l^ BY HPLC/DAD
,,' Nguyen Thi Ngoc Van *, Ta Chieu Phung
^. Can Tho University of Medicine and Pharmacy. Vielnam ,'. 'Corresponding author: ntnvangctump.edu.vn
(Received September, 0 5 , 2 0 1 6 )
'• -. , Sumrairy ,1 !..n.ult,n,o„, Quantilaliv, D.termin.tion of Limonin, Evodiamin. and Rutaecarpim , A.im t irnir- *ti Enodlae fructus hy m-hCliiAB
, .nd ™,Xi™ in C : ' Z'^'T ': f""""""''"™'""°" "'""" - J " ' " ' " " • ^ '•"«•"'"• "»•'•"»'
''• .de„d,„tne, „ „ , , ™ o r e c . „ . ™ ,„.,3.f„Mi»n,„,?0,.;::;"^^^^
Keyword,; D„e™™„„„, £vorf„„„, Ruiaecarpine. Limonin. E,odia, fruclus 1. Background c- t , • .
Evodiaf fr.„.f„= I. .1, J • J • , . beveral analytical assays have been reported Evodtae fructus ts the dr.ed, unr.pen fru.t of for the detennination of evodiamine and Evodla rutaecarpa (Juss.) Benth., which
' belonging to the family Rutaceae. It has been
• widely used as one o f a tradilional Vietnamese . medicine for treatment of gastrointestinal
disorders, postpartum hemorrhage and amenorrhea [I], [2], The diree major components in this herbal drug are limonin, evodiamine and ruiaecarpine (Fig. 1). Modem pharmacological studies have proved Iheir various activities, such as inhibition of corticosterone production, anti-inflammation, anti-obesity, anticancer, cardiotonic, vasodilatoty and antithrombotic acliviljes [3],[4], [5]. In Vietnam, Evodla rutaecarpa (Juss.) Benth is widely distributed in Ha Giang province.
".'.••• Ul ...uuiiiiiijiie ano lulaecarpine which included liquid chromatography, liquid cliromatography-tandem mass spcctromeffv (LC/MS/MS) [6,7]. Although the two methods were highly sensitive and selective, their popularities were limited because of the high cost of instrumentation. Until no%v, no study on simultaneous quantitative determination of limonin evodiamme and ruiaecarpine from Evodiae fniclus had been carried out in Vietnam.
Therefore, the aim of this sludy are lo develop a simple rapid, sensitive, and robust analytical method for quantification of biologically
•mportant components namely limonin, evodiamine and rutaecaTine in Evodiae fniclus for the quality control of this herbal dmg.
(1) (2) Fig.!. Smicnnes of ovodimiinc (I), mi.ecapine (2) and Imonin (3) *^' Journal of Medicinal Materials, 2016, VoL 21, No. 5
315
2. Materials and Methods Chemical and materials
Evodiae Fructus, the dried fniit of Evodia rutaecarpa, was purchased from traditional drug stores in Can Tho market. Voucher specimens were deposited at the Laborator>' of Drug Control and Toxicolog>', Can Tho University of Medicine and Pharmacy. The collected samples were stored in sealed packages to avoid moisture and light.
Evodiamine (99% purit>0; Rutaecarpine {99%
purity); Limonin (98.47% purity) was purchased from Sigma-Aldrich.
Instrumentation
Hitachi HPLC L-2000 system (Hitachi, Japan), with an L-2130 pump, an L-2300 temperature control system and an L- 2455 diode- array detector was used for the analysis.
Preparation of standard solutions Standard stock solutions of limonin (Img/ml), evodiamine (0.2 mg/ml) and rutaecarpine (0.2 mg/m!) were prepared in acetonitnle. Working standard solutions containing each of the three compounds were prepared by diluting the stock solutions with mobile phase to suitable concentrations for limonin, evodiamine, and rutaecarpine
Preparalion of sample solutions Three batches of Evodiae fructus pulverized into powder, passed through a 0.3 mm sieve (30 meshes) and stored in a desiccator until taken for determination of three marker compounds. An accurately weighed amount (0.10 g) of each powder sample was repeatedly extracted with 50 ml of ethanol 80% (3 times) in an ultrasonic bath for 20 min. The extracted solution was combined, filtrated through analytical filter paper and then evaporated to dryness by rotary vaporization under reduced pressure. The dried residue was dissolved in 10 ml of mobile phase and filtered through a 0.45 jim membrane. The filtrate was then transferred to a vial and injected into the HPLC system for quantitative analysis.
Optimization of chromatographic conditions Different compositions of mobile phases and now rate were investigated. The chromatographic separation was achieved using a Germini C,g
column (150 mm x 4.6 mm; 5fim) at matt temperature; detection wavelength was 210 na The mobile phase consisted of acetonitrile (Al methanol (B), water (C). The gradient progna was as followed: 0-18 min, linear gradient 4(Bi A, 5% B with flow rate of 1 ml/min; 18-30 min, linear gradient 100% A with flow rate U ml/min. The injection volume was 10 pi.
Method validation
For validation of the analytical method, guidelines of the Intemational Conference on Harmonization of Technical Requirements forthe Registration of Pharmaceuticals for Human Use were followed [8]. The requirements for the validation included: system suitability, specificih|
linearity, limits of detection (LOD) and quantification (LOQ), accuracy and precision.
3. Results a n d Discussion
Optimization of chromatographic conditions Limonin, evodiamine and rutaecarpine were identified by their retention times (tn) and by co- injection with standards. The detection wavelength for limonin, evodiamine and rutaecarpine in E. rutaecarpa (Juss.) Benth, wm selected by using photodiode-array detection (DAD). The detection wavelength was set at 21(1 nm because both the total peak areas and tbe numbers of detectable peaks reached a maxiraani in the HPLC chromatographic diagram. Theped:
purity of three compounds in the sample WBS 99.9% obtained from spectrum overlaying grajAs of three-point purity detection. The optimizatiffli of the chromatographic conditions was perfonnrf by using Phenomenex Gemini RP-C|g column with different compositions of mobile phases (1}
methanol-water system, (2) acetonitrile-water and (3) acetonitrile-methanol- water system and different ratio of solvents in isocratic mode. Tbe result showed that limonin eluted very fast (2-3 minutes), evodiamine and rutaecarpine eliflfll very slow {50 minutes) with system (I). TTf three components have good resolution bH analysis time was too short (under 8 minutes) with system (2). This proves inconvenient ftf herbal matrix. Good resolution, baseline, shsfp and symmetrical peaks, suitable retention tu«
316 Journal of Medicinal Materiab, 2016, VoL 21, NoJ
^\ ™ °^^'"^d by using system (3). The mobile sg^P"^^ "'as acetonitrile-methanol-water in the p^^^^ratio of 40:5:55 (v/v/v), retention time of limonin,
^^^ evodiamine and mtaecarpine were 7.93; 12.80 [,^^^^and 15.85 minutes, respectively. However, due to
polar impurity compounds in the extract solution,
we shorten analysis time by using gradient mode to elute them. The representative chromatogram of the sample and standard (Fig. 2) could be concluded that limonin, evodiamine and rutaecarpine were eluted with highly symmetrical peaks under this condition. Analysis time was 30 minutes.
F,g. 2. Repre enm,,.e HPLC cl„.n,.l„|,.n,s of mixed s,»d„ds .nd dteexMci of£ rutaecarpa .1210 nn, (1) d.»lv,ng solvent (m=lhin,ol). (2) mobile ph„., ,3, „ „ . d ,i.„d„ds of the diree „ . , t o c o m p l d s (4) exlr.c,
of £ rntaecarpa. (5) extr„l of £ rutaecarpa spiked w.O, limonin, e.odi™inc and ^l.ecrpme
•^^ Zhuo et al. developed an LC method for the
^ detennination of dehydroevodiamine, j^^ wuchuyuamide-I, 5-hydroxyrutaecarpine, 14.
Tormyldihydrorutaecarpine, evodiamine and - rutaecarpine, but it took a long analysis time of
^70 min and complicated mobile phase, which (•consisted of methanol, acetonitrile and
•^phosphoric acid-trielhylamine-buffer solution
?[91. Tang et al. published repon on .1 simultaneous determination of six bioactive
^components, rutaevine, limonin, evodiamine, : rutaecarpine, A'-formyldihydrorutaecarpine and jdihydroevocarpine, using a complex mobile : phase consisted of tetrahydrofuran-acid
phosphoric 0.02%-acetonihile) [6]. In this study, we conducted i simple chromatographic condition using very popular solvent in laboratory and in shon time (within 30 minutes) can be used for the simultaneous quantification of 3 bioactive compounds in Evodiae fructus.
Method validation
System suitability
System suitability was tested by performing six replicate injections and detemiining theoretical plate number (N), resolution (Rs), symmetrical factor (As) and repeatability (RSD of retention times and peak areas) for the analytes of interest. The %RSD values of peak area and retention time for all peaks were less than 2%
mdicating the precise analysis of limonin, evodiamine and mtaecarpine by this system. All' the results showed that the proposed method met the requirements
Specijlclly
The selectivity was tested by applying ,he HfLC method to analyze methanol extracts of Evodtae fmclos It was evaluated by comparing the retention time of each standard compound wn 1 that of the respective peaks obtained by analyzmg samples. The HPLC method was able to discriminate limonin, evodiamine and rutaecarpine of Evodiae fructus from the other conslituenls of the plant material (flavonoid qu.nolone alkaloids, etc.). There was n '
Journal of Medicinal Materials, 2016, VoL 21, No. 5317
mterference with the peaks of limonin, three compounds in the sample was 99.9^
evodiamine and rutaecarpine in Evodiae fructus obtained from spectrum overlaying graphs of (shown in Fig. 2), Therefore, the peak purity of three-point purity detection {Fig. 3)
1 .
(A) limonm
-
'^t^ - '^z:'''^:^:-^ - - —
:
(C) rataecaipine
- = - ' • ? - •= : - -
(B) evodiamine
!
:s^:::^^tzzz '
-
I ( D ) 3D spectrum
• - > ^ ^ - < ' ' ' '
" ''
Fig. 3. UV-VIS speclnim compmson bctwctn sumdinds md respective peaks in Ihe chrom.logram of Evodi.t fractus alno:
limonin (A), evodi.mine (B). rulaeciirpine (C), 3 D spectrum of Evodiae fruclus extract (D) Linearity, limits of detection and quanllficallon LOD was 0.0625 pg/ml for three components.
The result for regression equation and coirelation The LOQ were 0.125 pg/ml for limonin Had coefllcients (r=) are summarized in Table i. The mtaecarpine and 0.2 pg/ml for evodiamine.
Table 1. Linear regression data, LOD and LOQ of the HPLC meihod Parameter
Regression equalion R' Linear range (ue/ml)
LOD (ua/m!1 LOQ(ua/mU
Limonin V = 5l262x
0.9977 5-200 0.0625
0 125 0.2
1 Rmaecar ine—1
0.125 Regression equations for six calibration points
IS y = ax + b, where y is the peak area of analjles, x is concentration, a is slope, b is mtereept, and r' is the squared conelation coefficient
Precision and Accuracy
The RSDs of intra-day and inter-day were 4.81 ~ 6.07%, 2.15 - 6.79% and 6.01 - 6.13% for limonin, evodiamine and rutaecarpine, respectively (data were shown in Table 2).
for Ihe assay of the investigaied compounds in Evodiae fruclus Ham ltr9Tigifigjma
Rutaecarpine
Journal of Medicinal Materiab, 2016, VoL 21,N&5
^^ able 3 showed a summary of extraction -recovery m E.odiae fructus sample. The developed method had good accuracv uith
^overall recover, ua^ in7 7-?o/ r r
• ^^3s lUZ./iVo tor iimonm, ' _____ Table 3 Recoveries for the asia\ of im
10158% for evodiamine and 103 34% for rutaecarpine wilh RSD less than 5% for the anahies. Considering Ihe results of the recovery test, the method was pro\ed to be accurate.
;\odiamme and rutaecarpine
Sample Concentration (jig/ml) Mean RSD
recovery | (%)
102 73 ! 4 9
! 19-87
• 1987 : Rutaecarpini I 20.64 ! 20 64
46-05 3601 46 96
103 34 3.78 105 30
Recovery (%) = ((found-original)/added) MOO The samples were spiked with known amounts of standards at low level (80% of the known amounts).
The samples were spiked with known
• amounts of standards at medium level (same as the known amounts).
The samples were spiked with known amounts of standards at high level (120% of the known amounts)
Table 4. Conienl o f limonm evodianii
Quality evaluation of Evodia rutaecarpa (Juss.) Beath in the market
The contents of major compounds in 3 commercial samples of Evodiae fructus were summarized in Table 4. Data are expressed as % (grams per 100 gram) of dry weight. The quantification data of marker compounds in Evodiae fructus were 0,32-0.54 g/l OOg for hmonin, 0.2 - 1.02 g/lOOg for evodiamine and 0 21 - 0.74 g/l OOg for rutaecarpine.
le and ruiaecarpine in Evodiae fruclus
Fig. 4. Represenlative HPLC chrDmatogmms of three ba.ches ofEvodiae fructus
exiraci in the marliel Journal of Medicinal Materiab, 2016, VoL 21, No. 5
319
4. CoDclusioD analysis of limonin, evodiamine and rutaecaipmea In this work, the HPLC-DAD for analysis of complex matrices such as Evodiae fructus extracts major components in Evodiae fructus was Acknowledgements: This stuefy was financl^
developed and frilly validated. The method was supported by Can Tho University of Medicine oti simple, precise, and economical in terms of time Pharmacy, Can Tho. Vietnam. The authors thai and solvent usage. The validation procedure Pharmaceutical Analytical Laboratory ofCTUMpJjg confirmed that this technique afforded reliable theuseof analytical instruments
References
1. Loi D T- (2004). TTie medicinal plants and tradilional medicines in Vielnam, Medical Publishing House, 378-379.2.1^
Slate Pharmacopoeia Commission of China (2005), Pharmacopoeia of the People's Republic of China. 1, 93. 3. Jiang J„ Hi C. (2009), Evodiamine. A novel anti-cancer alkaloid from Evodia rutaecarpa. Molecules. 14(5), 1852-1859. 4, MatsudaR, Yoshika«-a M.. Linuma M . Kubo M. (1998). Antinociceptive and anti-inflammatory activities of limonin isolated frotn tte fruits of Evodia rutaecarpa var bodinieri, Planta Medica, 64(4) 339-342 5. Sheu J R. (1999), Phannacological effeclsof Rutaecarpine, An alkaloid isolated from Evodia rutaecarpa". Cardiovascular Drug Reviews, 17(3), 237-245. 6. TaneX, Huang Z, Chen Y.. Liu Z, Liu Y , Zhao J., Yi J. (2013), Simultaneous detemiination of six bioactive compounds in Evodiie fructus by high-performance liquid chromatography with diode array detection. Journal of Chromatographic Science. 32(2), 149-156. 7. Zhao Y.. Zhao Y., Zhou X, Gong X., (2014), Development and validation of an UPLCMS/MS method for determination of dehydroevodiamin, limonm, evodiamin and rutaecarpine in Evodiae fructus. Pharmacognosy Magasm, 10(39), 374-383. 8. ICH (2005), Validation of analytical procedures: Text and Methodology Q2(RI). 9. Zhao M. V. and Yang X. W (2008), Optimization of the extraction conditions and simultaneous quantification by RP-LC of six alkaloids n Evodiae fruclus. Chromatographia. 67(7-8), 543-550.
Journal of Medicinal Materials, 2016, VoL 21, No. 5 (pp. 320 - 325) IMMUNOSTIMULATING EFFECTS OF A PREPARATION COMBINED
FROM THE CULTIVATED CORDYCEPS SINENSIS, GANODERMA LUCIDUM ANT) ANGELICA SINENSIS IN CYCLOPHOSPHAMIDE-
INDUCED IMMUNOSUPPRESSED MICE
Trinli Thi Diep'' * Truong Binh Nguyen', Pham Thi Van Anh^Dalat University, Vietnam; 'Hanoi Medical Umversity. Vietnam
* Corresponding author: dieptt(gd lu.edu. vn (Received July, 29^ 2016)
Summary
I..mun»„l„„|.,i„g Efcis of. Pr.p.r.lio. Combir.d fr.n, tb. Cultivated Cortlyoep, sinensis, Ganoderma Ittdd..
.nd Angelica sinensis in Cyclopliospli.mid.-lnditced Immnnosiippre.i.d Mic.
.k culwlT r"'!"" """"'*"'°""""" """"™" "'""'•""s =«•«' '<"" »1"=»"' »•""» CSGL m^a ft.
i l i o Z r """""• ° ° " " ' ' ™ ° '""''"'" " " " " « ' " " ' " » " " ™ 1>= cyclophosph™id..i«ii«J
g t r c s G ^ r.w° ? r T ! " • ""' " " " " " ^ ™ " "•""" '" CSGL-t„.t.d groups when comp J d to »™l sensitized m,„ r ^ r ^ ',• '"'"'•'"= l-yPTsensilivity reaction by f.ciiilating the footpad thicltness response to OA •
histopatb.,:;:, sid z rir™"''"'""" "••' ™'""-'" """"^ ""»•'»"'•™"""'"" "**
cjclophospliamide " " "™""'«S"« "Iso showed that CSGL clearly helped to recover the damages cased » Keywords, Cordyceps sloeosis. Gaooderioa liicdum, Aogelica slneiisls. Immuooslimttlatlng eMict
Journal of Medicinal Malerials, 2016, VoL 21, Na.i
