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DOI:10.3736/jcim20120116
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Mohan Maruga Raja MK,Mishra SH.Isolation,
characterization and thin-layer chromatography method
development of clerosterol palmityl ester:a chemical
marker for standardization of leaves of Clerodendrum
phlomidis.J Chin Integr Med.2012;10(1):109-113.
Mohan Maruga Raja MK,Mishra SH.薄层色谱法提取
和鉴定海州常山桐树叶中的化学标记物赤桐甾醇十六
烷基酯.中西医结合学报.2012;10(1):109-113.
Received September 28,2011;accepted November 9,
2011;published online January 15,2012.
Ful-text LinkOut at PubMed.Journal title in PubMed:
Zhong Xi Yi Jie He Xue Bao.
Correspondence:Muthu Kumaradoss Mohan Maruga Raja,
PhD;Tel:+91-80-25747191;E-mail:mohanmarugaraja
@gmail.com
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Original Experimental Research 实验论著
Isolation,characterization and thin-layer chromatography
method development of clerosterol palmityl ester:a chemical
marker for standardization of leaves of Clerodendrum phlomidis
Muthu Kumaradoss Mohan Maruga Raja1,Shri Hari Mishra2
1.Department of Pharmacognosy and Phytochemistry,Karnataka Kaveri Educational and Cultural Society
Colege of Pharmacy,Bangalore 560103,Karnataka,India
2.Department of Pharmacy,The Maharaja Sayajirao University of Baroda,Vadodara 390002,Gujarat,India
OBJECTIVE:Clerosterol palmityl ester(CPE)is a unique clerosterol derivative isolated and
characterized from the leaves of Clerodendrum phlomidis.Considering the uniqueness of this
compound,the present study was planned to use CPE as a specific chemical marker and
develop a new validated thin-layer chromatography (TLC)method for standardisation of
C.phlomidis.
METHODS:Separation and quantification of CPE were achieved by TLC using a mobile phase
of petroleum ether(60 to 80 ℃)and ethyl acetate (95∶5,volume ratio)(Rf0.64)on
precoated silica gel 60F254aluminium plates.Densitometric determination was carried out after
derivatization with anisaldehyde sulphuric acid reagent in absorption mode at 527 nm.
RESULTS:The calibration curve was linear in the concentration range of 100 to 500 ng/spot.
The method was validated for precision,repeatability and accuracy.The proposed method was
found to be simple,specific,precise,accurate,rapid and cost-efective.
CONCLUSION:This TLC procedure may be used efectively for quantitative determination of
CPE,identification of the plant and standardization of this plant or its derived products.
KEYWORDS:chromatography,thin-layer;Clerodendrum;plant extracts
In India,Ayurvedic,Siddha and other herb-derived
products which are used either as active ingredients or
as adjuvants hold paramount importance as alter-
native medicines but their standardization poses a
great chalenge.An accurate identification of
these drugs both in their entire form or in powder
form has lead to quandary,since these medicinal
plants are known by a variety of vernacular names
while frequently numerous medicinal plants are
known by one vernacular name.Comparative
·901·中西医结合学报2012年1月第10卷第1期 Journal of Chinese Integrative Medicine,January 2012,Vol.10,No.1
thin-layer chromatography (TLC)with chemical
or biological marker compounds can be used to
standardize the raw materials.Besides,TLC is
often used as an alternative to other chromato-
graphic techniques for quantification of plant
products due to its simplicity,accuracy,cost
effectiveness and time efficiency.
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Clerodendrum phlomidis Linn.f.(syn.Clerodendrum
multiflorum (Burm.f)O.Kuntze)of the family
Lamiaceae is an important and wel-known medicinal
plant in the Ayurveda and Siddha systems of medicine.
It is commonly known as Thaludhalai,Agnimantha
or Arani and as a constituent of more than50indigenous
drug formulations.The popular uses include treatment
of inflammation,diabetes,nervous disorders,
asthma,rheumatism,digestive disorders,urinary
disorders and also as a bitter tonic[1-6].Non-clinical
investigations have revealed the anti-inflammatory,
hypoglycemic,immunomodulatory,antidiarrheal and
antiplasmodial activities[7].The genus Clerodendrum
is known to be a source of rare sterols[8-10].
C.phlomidis like many other species is a rich
source of sterols.Clerosterol palmityl ester(CPE),a
unique clerosterol derivative,has been isolated
and characterized from the leaves of C.phlomi-
dis.CPE is not yet reported in higher plants and
thereby provides the specificity as a chemical
marker.Standardization of C.phlomidis extracts by
TLC with24β-ethylcholesta-5,22E,25-triene-3β-ol as
a chemical marker has been reported[11].Since
this sterol is considered as a chemotaxonomic
marker of the entire Clerodendrum genus,the
present study was planned to use a specific chemical
marker(CPE)and develop a new validated TLC
method for standardisation of C.phlomidis
extracts.
1 Materials and methods
1.1 Plant materials Leaves of C.phlomidis were
colected from the suburbs of Trichy city,Tamilnadu,
India and were authenticated by the Botanical
Survey of India (southern circle),Coimbatore,
Tamilnadu,India.A voucher specimen(Pharmacy/
HDT/CP/08-09/15/MKM )was deposited in the
Pharmacy Department of The Maharaja Sayajirao
University of Baroda,Vadodara,Gujarat,India.
1.2 Chemicals and reagents The solvents and
chemicals used were of analytical grade.Silica gel
60F254TLC plates were purchased from Merck
(Darmstadt,Germany).The marker compound
CPE was isolated and characterized by spectral
analysis in the authorslaboratory.
1.3 Extraction and isolation of CPE fromC.phlomidis
leaves Coarsely powdered leaves of C.phlomidis
were extracted with methanol in Soxhlet apparatus
until exhaustion;the extract was concentrated in
vacuo by a rotary evaporator and dried in a desiccator.
The methanol extract was fractioned with petroleum
ether(60 to 80 ℃).The dried petroleum ether
fraction of methanol extract was saponified to
obtain the unsaponifiable matter.A total of 2.5 g
of the unsaponified petroleum ether fraction of
methanol extract (UPFMCP )was subjected to
column chromatography on silica gel 60 to
120 mesh and eluted with petroleum ether(60 to
80℃ )∶ethyl acetate in varying proportions.A
total of 100 fractions of 25 mL each were colected.
The fractions obtained by elution with petroleum
ether(60 to 80℃ )∶ethyl acetate(95∶5)yielded
80 mg of a yelow oily substance(CPE).
1.4 Preparation of sample and standard solution
Totaly 7.5 g of accurately weighed coarse powder
of C.phlomidis leaves was extracted with methanol
(4×50 mL)under reflux(30 min each time)on a
water bath.The combined extracts were filtered,
concentrated and transferred to a 50 mL volumetric
flask and the volume was made up with methanol.
A standard stock solution of CPE (100μg/mL)
was prepared in methanol. Working solutions
were prepared by appropriate dilution of the stock
solution to achieve 100,200,300,400 and
500 ng/spot for preparing a five-point calibration
curve of peak area versus concentration.
1.5 Chromatography A Camag TLC system
equipped with Camag Linomat V,an automatic
TLC sample spotter,Camag glass twin trough
chamber(20 cm×10 cm)was used for the analysis.
Chromatography was performed using pre-activated
·011· 中西医结合学报2012年1月第10卷第1期 Journal of Chinese Integrative Medicine,January 2012,Vol.10,No.1
(60 ℃ for 5 min)silica gel 60F254 TLC plates
(20 cm×10 cm;layer thickness 250μm).Samples
and standards were applied to the plate as 8 mm
wide bands with an automatic TLC sampler under
a flow of nitrogen gas,10 mm from the bottom
and 10 mm from the side and the space between
the two spots was 15 mm of the plate.The linear
ascending development was carried out in a
Camag twin trough chamber saturated with20 mL
mobile phase petroleum ether (60 to 80 ℃ )∶
ethyl acetate(95∶5,volume ratio)for 20 min at
room temperature of(25±2)℃and 40%relative
humidity.The plates were developed up to 8 cm
under chamber saturation conditions.Subsequent
to the development,TLC plates were dried in
current air with the help of a hair dryer.The post
chromatographic derivatization was carried out in
anisaldehyde sulphuric acid reagent folowed by
heating at 80℃for 15 min
[12].Quantitative eval-
uations of the plates were performed with Camag
scanner 3 (WinCATS 4.0integration software).
Densiometric scanning was performed in the
absorption mode at 527 nm,using a slit width of
6 mm×0.45 mm and data resolution 100μm step
and scanning speed 20 mm/s with a computerized
Camag TLC scanner.Quantification of CPE in
the leaf extract was performed by external standard
method.
1.6 Quantification of CPE in test sample A total
of 10 mL of methanol extract was applied to a
TLC plate,developed and scanned as above.
Peak areas were recorded and the amount of CPE
was calculated using the calibration curve.
1.7 Specificity Specificity of the method was
determined by analyzing the standard and the
unknown sample.The spot for CPE in the sample
was confirmed by comparing the Rfand spectra of
the spot with that of the standard.The peak purity
of CPE was assessed by comparing the spectra at
three different levels,namely,peak start,peak
apex and peak end positions of the spot.
1.8 Method validation The method was validated
for precision,accuracy and repeatability
[13].
Instrumental precision was checked by repeated
scanning of the same standard spot 100 and500 ng
three times and was expressed as coefficient of
variance(%relative standard deviation(RSD)).
Method precision was studied by analyzing the
standards 100 and 500 ng/spot under the same
analytical procedure and laboratory condition on
the same day and on different days (inter-day
precision);the results were expressed as%RSD.
Accuracy of the method was tested by performing
the recovery studies of the pre-analyzed sample
with standard at three levels(76.62,95.80 and
114.9μg/mL),and percentage of recovery was
calculated.
2 Results
2.1 Characterization of CPE Infrared potassium
bromide(KBr)νmax:2 933,2 851,1 726,1 643,
1 460,1 379,1 190,1 132,1 058,960,889,800/cm;
electro spray ionisation-mass spectrometry m/z:
393.4[M-palmitic acid]+,255.2[palmitoxyl]+,
202.2[C15H24]+,137.2 [C10H16]+;1 H nuclear
magnetic resonance (NMR)(400 MHz,CDCl3)
δ:5.35(1H,-CH),4.69(2H,-CH2),2.42
2.41(4H,-CH2),2.28 2.17(1H,-CH),
2.04 1.97 (2H,-CH),1.85 1.83 (4H,
-CH2),1.67 1.64(1H,-CH),1.56(26H,
-CH2),1.48 1.38(3H,-CH),1.25(12H,
-CH2),1.21 1.11(4H,-CH2),1.07 1.00
(9H,-CH3),0.96 0.93(6H,-CH3),0.89
0.85(3H,-CH3).The structure of the isolated
compound CPE (C45H78O2;molecular weight 650)
(Figure 1 )was unambiguously elucidated by
analysis of IR,1 H NMR and mass spectral data
and confirmed as reported[14].
Figure 1 Structure of clerosterol palmityl ester
2.2 TLC separation optimization The desired
resolution of CPE with symmetrical and reproducible
peak was achieved by using the mobile phase of
petroleum ether (60 to 80 ℃ )︰ ethyl acetate
(95∶5,volume ratio)with 20 min of chamber
saturation.The peak for CPE was seen at Rf
0.64.The leaf extract of C.phlomidis,when
subjected to TLC as per the methodology described
above showed the presence of CPE peak (Figure
2).An excelent separation was achieved by the
conditions described above.A comparison of the
spectral characteristics of the peaks for isolated
CPE and that of the sample peak confirmed the
presence of CPE (Figure 3).Peak purity of CPE
was assessed by comparing its ultraviolet-visible
spectra in standard and sample track.
Figure 2 Densitogram of the ST and CPE
ST:sample track;CPE:clerosterol palmityl ester.
·111·中西医结合学报2012年1月第10卷第1期 Journal of Chinese Integrative Medicine,January 2012,Vol.10,No.1
Figure 3 Spectral comparison for the peaks of ICPE
and CPE in C.phlomidis leaf extract
ICPE:isolated clerosterol palmityl ester;CPE:clerosterol palmityl
ester.
2.3 System suitability test
2.3.1 Linearity and detection limit Linearity
was checked by applying standard solutions of
CPE at five different concentration levels.A
calibration curve was drawn in the concentration
range of 100 to 500 ng/spot.The equation for the
calibration curve of CPE is Y=480.006+10.106 X
and the correlation coefficient of the calibration
curve was 0.999,indicating good linearity.Limit
of detection (LOD )and limit of quantification
(LOQ)were calculated by using the formulas as
folowing:LOD = (3.3×standard deviation of
the response)/slope of the calibration curve and
LOQ= (10×standard deviation of the response)/
slope of the calibration curve.Results of regres-
sion analysis on the calibration curve and detec-
tion limits are presented in Table 1.
Table 1 Linearity regression data for quantification of
clerosterol palmityl ester using proposed thin-layer
chromatography densitometric method
Parameter Result
Rf 0.64
Dynamic range(ng/spot ) 100to 500
Equation Y=480.006+10.106 X
Slope 10.106
Intercept 480.006
Limit of detection 15.49ng
Limit of quantification 46.95ng
Linearity(correlation coefficient ) 0.999
2.3.2 Precision studies Instrumental precision
was checked by repeated scanning of the same
spots (100 and 500 ng/spot)of standard CPE
three times and the%RSD values were 0.58 and
0.17,respectively.To determine the precision of
the developed assay method,100 and 500 ng/spot
of CPE standard was analyzed three times within
the same day to determine the intra-day variability.
The%RSD values were 0.79 and0.22 for 100 and
500 ng/spot,respectively.Similarly,the inter-day
precision was tested on the same concentration
levels on two days and the%RSD values were 1.
31 and 0.37,respectively(Table 2).
Table 2 Precision studies data for quantification of
clerosterol palmityl ester using proposed thin-layer
chromatography densitometric method
Concentration
(ng/spot)
Instrumental precision
(%RSD)
Method precision(%RSD)
Intra-day Inter-day
100 0.58 0.79 1.31
500 0.17 0.22 0.37
RSD:relative standard deviation.
2.3.3 Sample analysis and recovery studies This
developed TLC method was subsequently applied
to the analysis of CPE in methanolic leaf extract
of C.phlomidis.The CPE content of the leaves
measured by this proposed method was found to
be 0.012% (plant dry weight basis).For the
examination of recovery rates,80%,100% and
120% of pure CPE were added to pre-analyzed
sample and quantitative analysis was performed.
The recoveries were between 95.84%and99.50%
(Table 3).
Table 3 Recovery studies data for quantification of CPE using
proposed thin-layer chromatography densitometric method
Amount of CPE
in the sample(μg)
Amount of CPE
added(μg)
Amount of
CPE found(μg)
Recovery
(%)
95.79 76.62 165.24 95.84
95.79 95.80 184.71 96.41
95.79 114.90 209.63 99.50
CPE:clerosterol palmityl ester.
3 Conclusion
TLC is a useful alternative under circumstances
where the other slower and more costly chromato-
graphic methods are not appropriate.It is a suitable
method to standardize raw herbs,active constituent-
enriched extracts and their formulations.The
TLC method developed for the quantification of
CPE in C.phlomidis leaves is simple,specific,
precise,accurate,rapid and cost-effective.This
TLC procedure may be used effectively for quan-
titative determination of CPE,identity of the
plant and standardization of this plant or its
derived products.
4 Competing interests
The authors declare that they have no competing
interests.
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薄层色谱法提取和鉴定海州常山桐树叶中
的化学标记物赤桐甾醇十六烷基酯
Muthu Kumaradoss Mohan Maruga Raja1,Shri Hari Mishra2
1.Department of Pharmacognosy and Phytochemistry,Karnataka Kaveri Educational and Cultural Society Colege of Pharmacy,
Bangalore 560103,Karnataka,India
2.Department of Pharmacy,The Maharaja Sayajirao University of Baroda,Vadodara 390002,Gujarat,India
目的:赤桐甾醇十六烷基酯(clerosterol palmityl ester,CPE)是一种从海州常山桐(Clerodendrum phlomidis)
树叶中提取的特殊的赤桐甾醇衍生物。鉴于这种物质的特殊性,本研究以CPE作为化学标志物,采用一种
新的薄层色谱法鉴定海州常山桐树叶。
方法:CPE的分离和量化采用薄层色谱法,流动相石油醚(60~80 ℃)与乙酸乙酯的体积比为95∶5
(Rf0.64),硅胶60F254薄板层析检测。茴香醛浓硫酸试剂显色后使用光密度仪检测样品在527nm处的吸
光度。
结果:CPE在浓度范围100~500ng/孔的校正曲线呈线性分布。通过系统适用性检测,这种鉴定方法被证
实具有很好的准确性和可重复性,特异性强并且具有较高的成本效益。
结论:这种薄层色谱法能够有效地量化CPE的含量从而鉴定海州常山桐树叶及其衍生物。
关键词:色谱法,薄层;海州常山属;植物提取物
·311·中西医结合学报2012年1月第10卷第1期 Journal of Chinese Integrative Medicine,January 2012,Vol.10,No.1