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 Analytical & Bioanalytical Taralkar and Chattopadhyay, J Anal Bioanal Techniques 2012, 3:3
http://dx.doi.org/10.4172/2155-9872.1000134
Techniques

Research Article Open Access

A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from
their Methanolic Extracts of Vitex Negundo Linn
S. V. Taralkar1*and S. Chattopadhyay2
1
Chemical Engineering Department, MAEER’s Maharashtra Academy of Engineering, Alandi (D), Pune-412105, Maharashtra, India
2
Polymer Engineering Department, Indian Institute of Technology, Roorkee-247667, Uttarakhand, India

Abstract
Ursolic and betulinic acids are very useful nutraceuticals available in herbs. Their quantitative estimation
from the solvent extracts of the herbs is a great challenge. So far all the chromatographic methods available for
identification and quantification of these acids are distinctly different, meaning thereby, each acid would require a
separate method. Reverse phase (RP-HPLC) method is developed for determination of ursolic acid and betulinic
acid from their methanol extract of Vitex negundo Linn leaves. Analysis was carried out using Waters’ symmetry
C-18 column with acetonitrile: methanol (80:20) as isocratic elution mode with UV detection (λ=210 nm). The method
is pretty linear for ursolic acid in the range of 0.01-0.1 mg/ml (R2 = 0.9961) and for betulinic acid in the range of
0.003-0.018 mg/ml (R2 = 0.999). The peaks of ursolic acid and betulinic acid were confirmed by LC-MS. The method
was validated by mixing these acids standards in methanol and found that it is accurate, sensitive and has a good
reproducibility.

Keywords: Vitex negundo linn; Ursolic acid; Betulinic acid; HPLC; weight 456.68 and melting point is 283-285°C. The structure of betu-
LC-MS linic acid is as shown in Figure 2.

Introduction Determination of ursolic acid and betulinic acid content in the

leaves of Vitex negundo linn can be obtained by various methods.
Vitex negundo linn a member of Verbenaceae family, an important HPLC is the one of the best and accurate method for determination of
medicinal plant is found throughout India. In Hindi, it is called as “Nir- ursolic acid and betulinic acid in extract of Vitex negundo linn leaves.
gudi” or “Lagundi”. The extract from the leaves is the most important Many researchers have investigated and used different HPLC meth-
in the field of medicine [1]. The essential oils [2], β-sitosterol [3], Fla- ods for determination of ursolic acid and betulinic acid separately in
vonoids [4] and triterpenoids- ursolic acid and betulinic acid [5] were plant extracts. Simone et al. [9] used HPLC method with acetonitrile:
isolated from the leaves and seeds of Vitex negundo linn. Ursolic acid is water (70:30) as mobile phase, λ=203 nm wavelength with Novapack
known to have anti-inflammatory, antitumor, antimicrobial, antibacte- C-18 column to identify/estimate ursolic acid from Ilex paraguariensis
rial and antifungal activity [6]. Ursolic acid (3β-hydroxy-urs-12-en-28- aqueous extract. Hypersil C-18 column with methanol: water (95:5) as
oic acid) is a pentacyclic triterpenoids. The physical properties of urso- mobile phase and LC-MS detector was used by Liao et al. [10] to deter-
lic acid are: molecular formula C30H48O3, molecular weight 456.68 and mine ursolic acid from Rat plasma. Ursolic acid from Perilla frutescens
melting point is 283-285°C. The structure of ursolic acid is as shown in
Figure 1 [6].
Betulinic acid (3β-hydroxylup-20-(29)-en-28-oic acid) is a penta-
cyclic triterpenoids [7]. Betulinic acid has been found to selectively kill
human melanoma cells while leaving healthy cells alive. Due to its ap-
parent specificity for melanoma cells, betulinic acid seems to be a more
promising anti-cancer substance. Betulinic acid has also been found
to retard the progression of HIV 1 infection, which eventually leads
to AIDS, by preventing the formation of syncytia (cellular aggregates).
In addition, betulinic acid has antibacterial properties and inhibits the
growth of both Staphylococcus aureus and Escherichia coli [8]. Betu-
Figure 2: Structure of betulinic acid.
linic acid is not very poisonous, is relatively inexpensive. The physical
properties of betulinic acid are: molecular formula C30H48O3, molecular

*Corresponding author: S. V. Taralkar, Chemical Engineering Department,

MAEER’s Maharashtra Academy of Engineering, Alandi (D), Pune-412105, Ma-
harashtra, India, Tel: +912030253621; +919011332500; Fax No: +912030253799;
E-mail: suyogkumartaralkar@yahoo.co.in

Received April 23, 2012; Accepted June 01, 2012; Published June 07, 2012

Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination

of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of Vitex Negundo
Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134

Copyright: © 2012 Taralkar SV, et al. This is an open-access article distributed

under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
Figure 1: Structure of ursolic acid.
original author and source are credited.

J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 3 • 1000134
Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of
Vitex Negundo Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134
was estimated by Chen et al. [11] using RP-HPLC method with Sper-
isob Octadecylsilil Silica (ODS) column with acetonitrile and aqueous
H3PO4 as mobile phase and UV detector (λ=206nm). Novotny et al.
[12] analyzed methanol extract of Staphylea holocorpa Hemsl for ur-
solic acid content using LC-MS and Shim Pack CLC-CN, C-18 column
and a mobile phase of methanol: 1% acetic acid. The content of ursolic
acid in the ethanol extract of Eriobotrya was determined by Zuo et al.
[13] using HPLC with Methanol: water (88:12) as the mobile phase and
λ=210nm wavelength with Symmetry shield RP C-18 column.
Zhao et al. [14] reported RP-HPLC method for simultaneous de-
termination of betulin and betulinic acid in white birch bark using
Diamonsil C18 column and acetonitrile:water (88:16, v/v) as mobile
phase and UV detector of λ=210nm wavelength. Quantification of
betulinic acid in leaf extract of Orthosiphon stamineus was carried out
by Akowuah et al. [15] using HPLC with C-18 column and acetoni-
trile: water acidified to pH 2.08 with phosphoric acid (80:20) at λ=210
nm wavelength. Puder et al. [16] developed process for extraction of
betulinic acid from ground plane bark and used HPLC and NMR for
determination and quantification of betulinic acid. Markus [17] inves-
tigated process for obtaining betulinic acid from methanolic extract of
ground plane tree cortex and used HPLC for quantification of betulinic
acid. HPLC method was used by Abreu et al. [18] for determination of
betulinic acid from extract of Hypericum brasiliense with C18 micro-
sorb-MV column with acetonitrile: water (9:1) pH 3.0 adjusted with
phosphoric acid at λ=210 nm wavelength.
With the above discussion and from the literature available, num-
ber of medicinal plants consists of ursolic acid and betulinic acid, which
can be extracted simultaneously due to their wide medicinal applica-
tions. All the existing methods separately determine either ursolic or
betulinic acid from their extracts and each of them was tried to apply
for a mixture of both these acids and it was found that none of them
is suitable to apply for simultaneous determination of ursolic acid or
betulinic acids from their mixtures.
To the best of our knowledge there is no report available that sepa-
rates and estimates both these acids from their mixtures. The objective
of this work is to determine the ursolic acid and betulinic acid content
simultaneously by using one method from their methanol extract of
Vitex Negundo Linn leaves, because none of the earlier method really
does it.
Materials and Methods
The leaves of VN were collected from nearby area of Raigad dis-
trict, Maharashtra, India. The leaves were washed with water and shade
dried (at 30°C for 168 hrs) and pulverized. The powdered mass was
then sorted into various sizes by screening technique and 0.42-0.60 mm
size was taken for further experiments.
Ursolic acid (90%) and betulinic acid (90%) standard were obtained
from Fluka, USA and used as standards for calibration of the HPLC
system. Acetonitrile and methanol of HPLC grade were obtained from
S.D. Fine, India.
Apparatus and chromatographic conditions
Standards of ursolic acid and betulinic acid dissolved in metha-
nol and all unknown samples of methanolic extract were analyzed by
HPLC system of Waters’ (USA). The system consists of Waters HPLC
600 pump, a Rheodyne 7725i injector with 5 µl sample loop, on-line de-
gasser AF (Waters, USA), Waters 2487 Dual λ absorbance UV detector
(at λ=210 nm, for ursolic acid and betulinic acid) with 0.05 aufs sensi-
tivity. Ursolic acid and betulinic acid were analyzed using a Symmetry
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
Page 2 of 6
C-18 (4.6 × 250 mm, 5 µm) column equipped with automatic tempera-
ture (± 0.1°C) controller module. An isocratic mobile phase of aceto-
nitrile: methanol (80:20 v/v) with an elution volume of 0.5 ml/min was
selected for identification of both acids. The column temperature was
maintained at 35°C ( ± 0.1°C).
Preparation and analysis of extract of VN leaves
5 gm of shed dry and crashed leaves (particle size 0.42 to 0.60 mm)
was subjected to fully baffled 150 ml stirred borosilicate glass vessel (5
cm ID, and 9.5 cm height) along with methanol (100 ml) as solvent. A
four blade turbine type agitator (2 cm diameter) was used for stirring at
500 rpm to ensure all particles remain in suspended condition. Extrac-
tion of ursolic and betulinic acids were carried out at 28°C ( ± 1°C) for
2 hrs. The schematic experimental setup is as shown in Figure 3. The
samples were collected at fixed interval of time. The collected samples
were filtered through 0.2 µm membrane filter and analyzed in HPLC
system for determination of recoverable ursolic acid and betulinic acid
from their mixtures.
LC-MS analysis of ursolic acid and betulinic acid standard
and methanolic extract of Vitex Negundo Linn leaves
The methanolic solutions of standards of ursolic acid and betulinic
acid were analyzed with LC-MS for confirmation of the peaks and its
positions. The methanolic extract of Vitex Negundo linn leaves also
analyzed with LC-MS for identification of ursolic acid and betulinic
acid in extract. Waters Aquity LC-MS system, Waters TQD Mass detec-
tor and Waters Masslynx 4.1 software was used along with Symmetry
C-18 column (4.6 × 250 mm, 5 µm) with same mobile phase mentioned
earlier.
Results and Discussion
Determination of ursolic acid and betulinic acid from metha-
nolic extract
Series of HPLC methods available in literature were tried to sepa-
rate ursolic acid and betulinic acid content from the methanolic extract,
but none of them was found suitable for separation of both acids by
any single method. To overcome this problem, we tried different pos-
sibilities of solvent compositions to change the polarity of mobile phase
for ursolic acid and betulinic acid separation from the methanol ex-
tract. The reported HPLC method was found suitable for ursolic acid
and betulinic acid separation and quantification simultaneously. In this
Figure 3: Schematic of experimental setup.
Volume 3 • Issue 3 • 1000134
Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of
Vitex Negundo Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134

Page 3 of 6

method, acetonitrile:methanol (80:20) was used as mobile phase with for betulinic acid is 10.92 min ( ± 0.2 min). The method developed
0.5 ml/min elution flow rate, λ=210 nm UV wavelength with 0.05 aufs certainly indicates improved resolution of peaks and better selectivity.
sensitivity. Symmetry C-18 (4.6 × 250 mm, 5 µm) column was used for The chromatogram for methanol extract of leaves is shown in Figure
separation and isolation of ursolic and betulinic acids. The HPLC chro- 6. A distinct peak appearing at 12.36 min indicates ursolic acid while
matograms of ursolic and betulinic acid using the developed method the peak at 10.92 min matches with betulinic acid retention time. The
are shown in Figure 4 and Figure 5. peak fractions appearing at 12.36 mins and 10.92 mins were separately
The retention time for ursolic acid is 12.36 min ( ± 0.2 min) while collected in separate sample vowels after injecting the extracts several

1400.00
1200.00
1000.00
Ursolic acid
800.00
mV

600.00
400.00
200.00
0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Minutes
Figure 4: HPLC Chromatogram of ursolic acid (Residence time = 12.36 min) with Chromatographic conditions: Symmetry
C-18 (4.6×250mm, 5µm) column, acetonitrile:methanol (80:20), λ=210 nm, 0.5 ml/min flowrate.

1200.00

1000.00 Betulinic acid

800.00

600.00
mV

400.00

200.00

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Minutes

Figure 5: HPLC Chromatogram of betulinic acid (Residence time = 10.92 min) with Chromatographic conditions:
Symmetry C-18 (4.6×250mm, 5µm) column, acetonitrile:methanol (80:20), λ=210 nm, 0.5 ml/min flowrate.

2200.00

2000.00

1800.00

1600.00

1400.00

1200.00
mV

1000.00
Betulinic acid
800.00

600.00
Ursolic acid
400.00

200.00

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00
Minutes

Figure 6: HPLC Chromatogram of methanolic extract of Vitex Negundo Linn leaves with Chromatographic conditions:
Symmetry C-18 (4.6×250mm, 5µm) column, acetonitrile:methanol (80:20), λ=210 nm, 0.5 ml/min flow rate.

J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 3 • 1000134
Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of
Vitex Negundo Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134
Figure 7: HPLC calibration of ursolic acid.
Figure 8: HPLC calibration of betulinic acid.
Figure 9: LC-MS analysis of ursolic acid standard with Symmetry C-18 col-
umn (4.6×250 mm, 5 µm), acetonitrile:methanol (80:20 V/V), 210 nm wave-
length.
times. These two fractions were then purified and MS analysis was car-
ried out to confirm the collected fractions are separately of ursolic and
betulinic acids respectively.
Ursolic acid calibration
Standard solutions of ursolic acid (0.01, 0.02, 0.03, 0.04 0.05 0.06
and 0.1 mg/ml) were prepared in methanol. The solutions were filtered
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
Page 4 of 6
Figure 10: LC-MS analysis of betulinic acid standard with Symmetry C-18
column (4.6×250 mm, 5 µm), acetonitrile:methanol (80:20 V/V), 210 nm.
Figure 11 (a): LC-MS chromatogram of methanolic extract of Vitex Ne-
gundo Linn leaves with Symmetry C-18 column (4.6×250 mm, 5 µm),
acetonitrile:methanol (80:20 V/V), 210 nm wavelength.
through 0.2 µm (PALL Ultipor N66) membrane filter and analyzed in
HPLC. Evaluation of each point was repeated thrice. The area under
peak of ursolic acid vs. concentration plot shows a linear fit with cor-
relation coefficient of 0.9961 (Figure 7). The same calibration chart was
subsequently used for quantification of unknown samples.
Betulinic acid calibration with HPLC
Standard solutions of betulinic acid (0.003, 0.006, 0.009, 0.012,
0.015, 0.018, and 0.06 mg/ml) were prepared in methanol. All stan-
dard as well as samples were filtered through 0.2 µm (PALL Ultipor
N66) membrane filter and injected in the HPLC system through an-
other syringe filter 0.2 µm. The area under the peak of betulinic acid
was calculated using Waters Empower-II software and that corresponds
to the concentration of betulinic acid present. The plot of area against
concentration of betulinic acid standard injected shows a linear fit with
correlation coefficient 0.9991 (Figure 8). The same calibration chart was
subsequently used for quantification of unknown samples.
Volume 3 • Issue 3 • 1000134
Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of
Vitex Negundo Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134
Figure 11 (b): LC-MS spectra of methanolic extract of Vitex Negundo Linn
leaves with Symmetry C-18 column (4.6×250 mm, 5 µm), acetonitrile:methanol
(80:20 V/V), 210 nm wavelength.
Figure 12: HPLC method validation plot for ursolic acid solution.
Figure 13: HPLC method validation plot for betulinic acid solution.
LC-MS analysis of ursolic acid and betulinic acid standard
and methanolic extract of Vitex Negundo Linn leaves
Figure 9, Figure 10 and Figure 11 shows the chromatogram and
spectra of the ursolic acid, betulinic acid and methanolic extract of Vitex
Negundo Linn leaves respectively by LC-MS. The top chromatograms
in each figure shows that the position of ursolic acid and betulinic acid
at 210 nm wavelength of UV Detector and the bottom chromatograms
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
Page 5 of 6
Figure 14: Batch extraction of ursolic acid and betulinic acid from Vitex Ne-
gundo Linn leaves with methanol as solvent, 0.42-0.60mm particle size and
at 28°C.
shows that the position of ursolic acid and betulinic acid with MS de-
tector and spectra shows the mass of that peak fraction which matches
with standards of ursolic acid and betulinic acid. These chromatograms
are matching with each other at that position and show the exact mass
of pure compounds (ursolic acid and betulinic acid). Figure 11 (a) rep-
resents the chromatograms with UV and MS detector while Figure 11
(b) represents the spectra of extracted sample of identified peaks of ur-
solic acid and betulinic acid. From these figures it is clear that the HPLC
method used for analysis of ursolic acid and betulinic acid is valid. The
results obtained by UV detector at 210 nm wavelength are matching
with MS detector for analysis of ursolic acid and betulinic acid from
methanolic extract of Vitex Negundo Linn Leaves.
Method validation
The HPLC method developed was validated with different propor-
tions of ursolic acid and betulinic acid standards (ursolic acid: betulinic
acid; 0:100, 10:90, 25:75, 40:60, 50:50, 60:40, 75:25, 90:10, 100:0 (v/v))
solutions in methanol. From Figure 12 and Figure 13, it is observed that
the method is linear for the ursolic acid and betulinic acid concentra-
tion for the range 0.099 to 0.10 mg/ml concentration. Above 0.1 mg/ml
concentration of both acids the linearity lost. The validation plot shows
the R2 value more than 0.97 for both acids (Figure 12 and Figure 13).
This value of standard deviation and the linearity equation shows the
method is valid for the given range of concentrations.
Batch extraction of ursolic acid and betulinic acid from leaves
The batch extraction experiment was carried out with methanol as
solvent and at 28°C to extract the ursolic acid and betulinic acid from
leaves of Vitex Negundo Linn. The collected samples were analyzed
with proposed HPLC method to determine the ursolic acid and betu-
linic acid content. Figure 14 shows, how the concentration of ursolic
acid and betulinic acid increases with increase in extraction time. This
increase in concentration of both acids is due to diffusion mechanism
of solutes (ursolic acid and betulinic acid) in solvent (methanol).
This experiment was carried out to verify the validity of the pro-
posed method. It is found that the proposed HPLC method is valid for
simultaneous determination of ursolic acid and betulinic acid from
leaves of Vitex Negundo Linn.
Conclusion
The HPLC methods available in the literature are tried with the
given conditions and with Symmetry C-18 column. These methods did
Volume 3 • Issue 3 • 1000134
Citation: Taralkar SV, Chattopadhyay S (2012) A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of
Vitex Negundo Linn. J Anal Bioanal Techniques 3:134. doi:10.4172/2155-9872.1000134
not show any results in case of ursolic acid and betulinic acid for extract
of Vitex Negundo Linn. The developed method of HPLC is suitable for
simultaneous determination of ursolic acid and betulinic acid from
leaves of Vitex Negundo Linn. The method showed linearity for ursolic
acid and betulinic acid different concentrations. LC-MS analysis shows
the validation of method. From the results of batch extraction experi-
ments, the method is accurate, sensitive and has a good reproducibility.
Acknowledgements
The financial support from AICTE Project (Grant no. 8023/RTD/RPS52/2004-
05) and TEQIP (World-Bank project) for this research work is gratefully acknowl-
edged. Authors are thankful to Waters India Ltd for providing LC-MS facility.
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