全 文 ：Open Access开放获取 Related Articles推荐阅读
DOI:10.3736/ jcim20110117
http:// ww w.jcimjournal.com
Mohan Maruga Raja MK , Patel RS , Mishra SH.
Identifica tion and quantification of adrenaline
from the leaves of Clerodendrum phlomidis
using thin- laye r chromatog raphy.J Chin Integr
Med.2011;9(1):105-108.
Mohan Maruga Raja MK , Patel RS , Mishra SH.
薄层色谱法检测海州常山桐树叶中肾上腺素的
含量.中西医结合学报.2011;9(1):105-108.
Received Septembe r 21 , 2010;accepted Octobe r
25 , 2010;published online January 15 , 2011.
Full-tex t LinkOut at PubMed.Journal title in
PubMed:Zhong X i Y i J ie He X ue Bao.
Correspondence:Muthu Kumarado ss Mohan Maruga
Raja , PhD;Tel:+ 91-265-2794051;E-mail:
mohanma rugar aja@gmail.com
郭春燕 , 白雪梅 , 张万明 , 王治宝 , 付煜荣.高效液相色谱法测定生地
黄和桃红四物汤中梓醇的含量.中西医结合学报.2004;2(2):135-
137.
Guo CY , Bai XM , Zhang WM , Wang ZB , Fu YR.Determination o f
catalpol in dried rehmannia roo t and Taohong Siw u Decoction with high
perfo rmance liquid chroma to g raphy.J Chin Integr Med .2004;2(2):
135-137.
Full text availableat http://ww w.jcimjournal.com/ articles/ publishAr ticles/
pdf/ 2006322864238910.pdf
陈建伟 , 李祥 , 袁铸人.顶空气相色谱法快速鉴定牡丹皮及其它类似
的芳香类中药.中西医结合学报.2003;1(3):215-217.
Chen JW , Li X , Yuan ZR.Rapid identification of Corte x moutan radicis
and similar aromatic Chinese he rbs by headspace gas chromatog raphy.
J Chin Integr Med.2003;1(3):215-217.
Full text availableat http://ww w.jcimjournal.com/ articles/ publishAr ticles/
pdf/ 2006322734854546.pdf
More related articles at http:// w ww .jcimjournal.com/ FullTex t2.aspx?
ar ticleID=jcim20110117
Short Report　短篇报道　
Ident ification and quantification of adrenaline from the
leaves of Clerodendrum phlomidis using thin-layer
chromatography
Muthu Kumaradoss Mohan Maruga Raja , Ruchi Sureshbhai Patel , Shri Hari Mishra
Herbal Drug Techno log y Labo rato ry , Pharmacy Depar tment , The Maha raja Sayajirao Univ ersity o f Baroda ,
Vadodara 390002 , Gujar at , India
Keywords:Clerodendrum , epinephrine;chromatography , thin-layer;plant extracts
　In the Ayurvedic , Siddh a and other medical
systems of India , pr oducts der ived fr om herbs ,
used either as active ingr edients or as adjuvants ,
hold par amount importance as alter nat ive medi-
cines.However , their standardization poses a great
ch al lenge.Corr ect ident ificat ion of these drugs is
of ten problemat ic in both their complete form as
well as in powder form , since these medicinal
plants are known by a varie ty of vernacular names
and frequent ly many medicinal plants share the
same name.Comparat ive thin-layer chromatography(co-TLC) with chemical or biological marker
compounds can be used to standardize raw materials.
Moreover , due to its simplicity , accuracy , cost
effectiveness and rapidity , TLC is often used as
an al ter native to other chromatographic tech-
niques for quant ifying plant pr oducts.　Clerodendrum phlomidis Linn.f.(syn.Clero-
dendrum mult if lorum (Burm.f) O.Kuntze ,
abbr.C .phlomidis)of the Lamiaceae f amily is
an important and well-known medicinal plant in
the Ayurvedic and Siddha systems of medicine.I t
is commonly known as Thalludhalai , Agnimantha ,
or Arani and is a const ituent of more than 50 indige-
nous medicinal formulat ions.Popular uses include
the t reatment of inflammat ion , diabetes , nervous
disorders , asthma , rheumat ism , digestive disorders ,
and urinary disorders and also as a bi tter tonic
[ 1] .
Non-clinical investigat ions have revealed anti-
inflammatory , hypoglycemic , immunomodulatory ,
ant idiarrhoeal and antiplasmodial pr opert ies
[ 1] .　Pr elimin ar y phytochemical screening and co-
TLC studies of C .phlomidis leaves in this study
revealed the presence of adr enal ine f or the first
t ime.Chemical substances that play an essent ial
role in the per ipher al and centr al neur ot ransmis-
sion of animals , such as acetylcholine and biogenic
monoamines , have been demonstr ated in the
plant kingdom
[ 2 , 3] .Adrenaline or 4-[ 1-hydroxy-
2-(methylamino)ethyl ] benzene-1 , 2-diol , is a
biogenic monoamine belonging to the catecholamine
gr oup.Adrenaline , noradrenaline , dopamine and
·105·中西医结合学报 2011年 1月第 9卷第 1期　J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
their der ivat ives have been de tected in many plant
families
[ 4] .In contrast to the considerable knowl-
edge concerning the r ole and act ion of adrenaline
in mammals , very lit t le is known of the role of
adr enaline in plants.Sever al repor ts suggest th at
adr enaline is a precursor for alkaloids
[ 5]
while others
suggest that it may interact with plant hormones
[ 6 , 7] .
At present , high-performance l iquid chromatog-
r aphy (HPLC)methods are typically used for
identification and quant ification of catecholamines
using fluorescence or electrochemical detection
[ 8-10] .
Considering the wide physiological signif icance of
adr enaline and also its potent ial as a mar ker for
standardizat ion of raw mater ial , a simple , sensi-
tive and accurate TLC method was developed for
the quantificat ion of adrenaline in C .phlomidis leaves.
1　Materials and methods
1.1　Reagents and chemicals　Pure adrenaline was
pr ocured f rom Himedia Laborator ies Pvt.Ltd.(Mumbai , India), other solvents and chemicals
wer e of analytical gr ade.Silica gel 60 F 254 TLC
plates were purchased from Merck (Darmstadt ,
Germany).A Camag TLC system equipped with
Camag Linomat V and a computerized Camag
TLC scanner (f or densitometr ic scanning and
UV-Visible spect ra)was used.
1.2 　Plant materials 　Leaves of C .phlomidis
wer e collected from mult iple plants in the out-
skir ts of Tr ichy city , Tamilnadu , India and were
authenticated by the Botanical Survey of India ,
Souther n Circle , Coimbatore , Tamilnadu , India.
A voucher specimen (Pharmacy/HDT/CP/08-09/
15/MKM)h as been deposited in the Pharmacy
Department of The M.S.University of Bar oda ,
Vadodar a , Gujara t , India.
1.3　Preparation of sample and standard solution　
Accur ately weighed 2.5 g of coarse powder of C .
phlomidi s leaves was extracted separa tely with
1%glacial acet ic acid in water (4×50 mL)under
reflux(30 min each t ime).The combined extracts
wer e fil tered , concentrated and tr ansfer red into a
25 mL volume tr ic flask and the volume was made
up with the same solvent.A standar d stock solu-
tion of adr enaline (100 μg/mL)was prepared in
1% glacial acet ic acid in water .Working solu-
tions wer e pr epared by appr opria te dilution of the
stock solution (10 μL each)to obtain 100 , 200 ,
300 , 400 and 500 ng/spot in order to prepare a
five-point calibrat ion curve of peak area versus
concentra tion.
1.4　Thin-layer chromatography　A Camag TLC
system equipped with a Camag Linomat V , an
automat ic TLC sample spot ter and a Camag glass
twin tr ough chamber (20 cm ×10 cm)was used
for analysis.Chromatogr aphy was per formed by
using preact ivated (60 ℃ for 5 min) silica gel
60 F254 TLC plates(20 cm×10 cm;layer thickness
250 μm).Samples and standards were applied to
the plate as 8 mm wide bands by using an automatic
TLC sampler under a flow of nit rogen;10 mm
fr om the bottom of the plate , 10 mm from the
side and leaving a space between the spots of
15 mm.The linear ascending development was
carr ied out in a Camag twin trough chamber
satur ated with 20 mL mobile phase (acetone ︰
chloroform︰n-butanol ︰ glacial acet ic acid ︰
water (60︰40︰ 40︰40︰35 , v/v/v/v/v))for
20 min at room tempe rature((25±2)℃and 40%
relative humidity).The plates were developed up
to 8 cm under chamber satur ation condi tions.
Subsequent to the development , TLC pla tes we re
dried with the help of a hair dryer.Adrenaline
oxidizes r apidly hence post chroma tographic
derivatiza tion was carried out in potassium
hexacyanofer rate (Ⅲ)-e thylenediamine reagent
followed by heat ing a t 80 ℃ for 15 min[ 11] .
Qualitative identification of adrenaline was carried
out by compar ing the retardat ion f actor (R f )
value and the UV-Visible spect ra of a standard
adrenaline spot and an identical spot in the le af
extr act.Quantif ication of adrenaline in the le af
extr act was performed by the external standard
method.Quant itative evaluat ions of the plates
were performed with Camag scanner 3(CATS 4.0
integra tion software).Densi tometric scanning
was performed in the absorption-re flection mode
a t 407 nm by using a slit width of 6 mm ×
0.45 mm and data r esolution 100 μm step and a
scanning speed 20 mm/s wi th a computerized
Camag TLC scanner.
1.5　Quantification of adrenaline in test sample　A
total of 10 μL sample solut ion was appl ied to a
TLC plate , developed and scanned as above.
Peak ar eas were re corded and the amount of
adrenaline was calculated by using the cal ibr ation
cur ve.
1.6　Specificity　Specif ici ty of the me thod was
determined by analyzing the adrenaline standard
and the leaf extr act samples.The spot for adr ena-
line in the leaf ext ract was confirmed by compa-
ring its R f and UV-Visible spect ra with tha t of
the standard adrenal ine spot.The peak pur ity of
adrenaline was assessed by compar ing the spectr a
a t the peak star t , peak apex and peak end posi-
t ions of the spot.
1.7　Method validation　The method was validated
for precision , accur acy and r epeatability[ 12] .
Inst rumental precision was checked by r epeated
scanning of the same standard spot 100 and 500 ng
three t imes and was expressed as the coef ficient of
variance (rela tive standard deviat ion , %RSD).
Method precision was studied by analyzing the
standards 100 and 500 ng/spot under the same
analyt ical pr ocedure and labor atory condi tions ,
on the same day and on different days (inter day
precision), the results wer e expressed as %RSD.
Accuracy of the method was tested by performing
recovery studies of pre-analyzed samples with the
adrenaline standar d at three levels (23.6 , 26.3
·106· 中西医结合学报 2011年 1月第 9卷第 1期　J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
and 29.0μg/mL), and the pe rcentage of recovery
was calcula ted.
2　Results and discussion
2.1　TLC separation optimization　Diff erent com-
posit ions of the mobile phase wer e tested and the
desired resolution of adrenaline with a symmetrical
and reproducible spot was achieved by using
ace tone ︰chloroform ︰ n-butanol ︰ glacial acetic
acid︰ water (60 ︰ 40︰ 40 ︰ 40 ︰ 35 , v/v/v/
v/v)with 20 min of chamber sa tur at ion.The
standard peak f or adrenaline was seen at R f =
0.45.The leaf ext ract of C .phlomidis , when
subjected to TLC , as per the methodology
described above , showed the presence of adrenaline.
A comparison of the spectr al character ist ics of the
peaks for the adrenal ine standard and th at of the
sample peak confirmed the presence of adrenaline.
Peak pur ity was assessed by comparing UV-Visible
spect ra of both the adr enal ine standard and the
adr enaline spot in leaf ext ract t rack.
2.2　Linearity and detection limit　Lineari ty was
confirmed by applying standard solutions of
adr enaline at five different concentrat ion levels.
A cal ibr ation cur ve was dr awn in the concentra-
tion r ange of 100 to 500 ng/spot.The equat ion
for the calibrat ion curve of adr enal ine is y =
311.08+7.41x and the correla tion coefficient of
the calibra tion cur ve is 0.998 , indicating good
linearity.Detection and quantif ication limits
wer e dete rmined as per ICH Guidelines Q2(R1)(1996/2005)[ 12] .Resul ts of regression analysis on
the calibrat ion curve and detection limits are
pr esented in Table 1.
2.3 　Precision studies 　I nst rumental precision
was checked by repeated scanning of the same
spots(100 and 500 ng/spot)of standard adrenaline
three times and the %RSD values were 0.94 and
0.80 respect ively.To determine the precision of
the developed assay me thod , 100 and 500 ng/
spots of adr enal ine standard were analyzed three
t imes within the same day to determine the intraday
variability.The %RSD values were 0.96 and 0.78
for the 100 and 500 ng/spot spots , r espect ively.
Similarly , the interday precision was tested on the
same concentr at ion levels on two consecutive days
and the %RSD values were 1.28 and 0.87 ,
respect ively(Table 2).
2.4 　Quantitative analysis and recovery studies　
This developed TLC me thod was subsequently
applied for the quantitat ive analysis of adrenaline
in the leaf extr act of C .phlomidis .The adr ena-
line content of the leaves by this proposed method
collected from multiple plants were f ound to be
0.013% w/w.For the examination of r ecovery
r ates , 10.60 , 13.30 and 16.00 μg/mL of pure
adrenaline were added to pre-an alyzed samples
and quantitat ive analysis was performed.The
recoveries were between 97.03% and 99.19%(Table 3).
Table 1　Linearity regression data for quantif ication of
adrenaline using proposed thin-layer chromatography
densitometric method
Parameter Result s
R f 0.45
Dyn amic range(ng/spot) 100 to 500
Equation y =311.08+7.41 x
Slope 7.41
Intercept 311.08
Limit of detect ion(ng) 18.33
Limit of quanti ficat ion(ng) 61.11
Lineari ty (correlation coeff icient) 0.998
Table 2　Precision data for quantification of adrenaline using proposed thin- layer chromatography densitometric method
Concent ration
(ng/spot)
Ins trum en tal
precision(%RSD)
Method p recision(%RSD)
Int raday In terday
100 0.94 0.96 1.28
500 0.80 0.78 0.87
　　%RSD:relative standard deviation.
Table 3　Recovery data for quantification of adrenaline using proposed thin-layer chromatography densitometric method
Am ount of adrenaline in the sample (μg) Amount of adrenaline added(μg) Amoun t of ad renaline found(μg) Recovery (%)
13.00 10.60 23.41 99.19
13.00 13.30 25.52 97.03
13.00 16.00 28.37 97.82
3　Conclusion
　Thin-layer chr omatogr aphy is a useful alterna-
tive under cir cumstances where the othe r slower
and more cost ly chr omatogr aphic methods are not
appr opria te.I t is a suitable method to standardize
raw her bs , act ive const ituent-enriched extracts
and their formulations.The TLC method developed
for the quant ificat ion of adrenaline in C .phlomidis
leaves is simple , specific , precise , accurate , rapid
and cost-effect ive.This TLC procedure may be
used e ffectively for the qual ita tive and quant ita-
t ive dete rmina tion of adr enal ine , allowing for the
standardiza tion of adrenaline in this plant and its
derived pr oducts.
·107·中西医结合学报 2011年 1月第 9卷第 1期　J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
REFERENCES
1　Mohan Maruga Raja MK , Mishra SH.Com prehensive
review of Clerodend rum phlomidis:a tr aditionally used
bitter.J Chin Integr Med.2010;8(6):510-524.Eng-
lish with abstrac t in Chinese.
　　Mohan Maruga Raja MK , Mishra S H.传统苦味药用
植物苦郎树.中西医结合学报.2010;8(6):510-
524.
2　T re tyn A , Kendrick RE.Ace ty lcho line in plants:pres-
ence , me tabo lism and mechanism o f action.Bo t Rev.
1991;57(1):33-73.
3　Kuklin A I , Conger BV.Ca techolamines in plants.J
Plant G rowth Regul.1995;14(2):91-97.
4　Smith TA.Phenethy lamine and related compounds in
plants.Phy to chemistry.1977;16(1):9-18.
5　Lundstr om J.Simple isoquino line a lkaloids.In:Brossi
A.The alkaloids:chemistry and bio lo gy.Vol 21.New
Yo rk:Academic P ress.1983:255-327.
6　Dai YR , Michaels PJ , Flo res HE.Stimulation of ethylene
production by catecho lamines and pheny lethylamine in
po tato cell suspension cultur es.P lant G row th Regul.
1993;12(3):219-222.
7　Pro tacio CM , Dai YR , Lew is EF , F lo res H E.Grow th
stimula tion by catecho lamines in plant tissue/org an
cultures.Plant Phy siol.1992;98(1):89-96.
8　Gro ssi G , Bargossi AM , Lucarelli C , Pa radisi R ,
Sprov ieri C , Sprov ieri G.Improvements in automated
analysis of catecholamine and related metabolites in biological
samples by co lumn-sw itching high-perfo rmance liquid
chromato graphy.J Chromatogr.1991;541(1-2):273-284.
9　Sarzanini C , Menta sti E , Nerva M.Determination o f
catecholamines by ion chromatog raphy and electrochemical
detec tion.J Chromato g r A.1994;671(1-2):259-264.
10　H ansen AM , Kristiansen J , Nielsen JL , By rialsen K ,
Christensen JM.Valida tion o f a high perfo rmance liquid
chroma tog raphy analy sis fo r the de te rmination of no ra-
drenaline and adrenaline in human urine with an on-line
sample purification.Talanta.1999;50(2):367-379.
11　Geissler HE , Mutschler E.Die direkte quantitativ e
ausw er tung von dǜ nnschichtchromatog rammen durch
remissions- und f luore szenzmessungen:6.Mitteilung
bestimmung von adrena lin , nor adrenalin und dopamin
aus dem ha rn.J Chromatog r A.1971;56:271-279.
German w ith abstract in English.
12　ICHH T Guideline.Validation o f analy tical procedure s:
tex t and methodology Q2(R1).http://ww w .ich.or g/
LOB/media/M EDIA417.pdf , 2005.
薄层色谱法检测海州常山桐树叶中肾上腺素的含量
Muthu Kumaradoss Mohan Maruga Raja , Ruchi Sureshbhai Patel , Shri Hari Mishra
Herbal Drug Technology Labo rator y , Pha rmacy Depa rtment , The Maharaja Sayajirao Unive rsity o f Baroda , Vadoda ra 390002 ,
Gujar at , India
关键词:海州常山属;肾上腺素;色谱法 , 薄层;植物提取物
·108· 中西医结合学报 2011年 1月第 9卷第 1期　J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1