Volume 3 Issue 3 1000134

J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal
Open Access Research Article
Analytical & Bioanalytical
Techniques
Taralkar and Chattopadhyay, J Anal Bioanal Techniques 2012, 3:3
http://dx.doi.org/10.4172/2155-9872.1000134
Keywords: Vitex negundo linn; Ursolic acid; Betulinic acid; HPLC;
LC-MS
Introduction
Vitex negundo linn a member of Verbenaceae family, an important
medicinal plant is found throughout India. In Hindi, it is called as Nir-
gudi or Lagundi. Te extract from the leaves is the most important
in the feld of medicine [1]. Te essential oils [2], -sitosterol [3], Fla-
vonoids [4] and triterpenoids- ursolic acid and betulinic acid [5] were
isolated from the leaves and seeds of Vitex negundo linn. Ursolic acid is
known to have anti-infammatory, antitumor, antimicrobial, antibacte-
rial and antifungal activity [6]. Ursolic acid (3-hydroxy-urs-12-en-28-
oic acid) is a pentacyclic triterpenoids. Te physical properties of urso-
lic acid are: molecular formula C
30
H
48
O
3
, molecular weight 456.68 and
melting point is 283-285C. Te structure of ursolic acid is as shown in
Figure 1 [6].
Betulinic acid (3-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 specifcity 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-
linic acid is not very poisonous, is relatively inexpensive. Te physical
properties of betulinic acid are: molecular formula C
30
H
48
O
3
, molecular
weight 456.68 and melting point is 283-285C. Te structure of betu-
linic acid is as shown in Figure 2.
Determination of ursolic acid and betulinic acid content in the
leaves of Vitex negundo linn can be obtained by various methods.
HPLC is the one of the best and accurate method for determination of
ursolic acid and betulinic acid in extract of Vitex negundo linn leaves.
Many researchers have investigated and used diferent HPLC meth-
ods for determination of ursolic acid and betulinic acid separately in
plant extracts. Simone et al. [9] used HPLC method with acetonitrile:
water (70:30) as mobile phase, =203 nm wavelength with Novapack
C-18 column to identify/estimate ursolic acid from Ilex paraguariensis
aqueous extract. Hypersil C-18 column with methanol: water (95:5) as
mobile phase and LC-MS detector was used by Liao et al. [10] to deter-
mine ursolic acid from Rat plasma. Ursolic acid from Perilla frutescens
*Corresponding author: S. V. Taralkar, Chemical Engineering Department,
MAEERs 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
original author and source are credited.
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
identifcation and quantifcation 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 (=210 nm). The method
is pretty linear for ursolic acid in the range of 0.01-0.1 mg/ml (R
2
= 0.9961) and for betulinic acid in the range of
0.003-0.018 mg/ml (R
2
= 0.999). The peaks of ursolic acid and betulinic acid were confrmed 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.
A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from
their Methanolic Extracts of Vitex Negundo Linn
S. V. Taralkar
1
*and S. Chattopadhyay
2
1
Chemical Engineering Department, MAEERs Maharashtra Academy of Engineering, Alandi (D), Pune-412105, Maharashtra, India
2
Polymer Engineering Department, Indian Institute of Technology, Roorkee-247667, Uttarakhand, India
Figure 1: Structure of ursolic acid.
Figure 2: Structure of betulinic acid.
Volume 3 Issue 3 1000134
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
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 2 of 6
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 (=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. Te 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
=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 =210nm wavelength. Quantifcation 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 acidifed to pH 2.08 with phosphoric acid (80:20) at =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 quantifcation of betulinic acid. Markus [17] inves-
tigated process for obtaining betulinic acid from methanolic extract of
ground plane tree cortex and used HPLC for quantifcation of betulinic
acid. HPLC method was used by Abreu et al. [18] for determination of
betulinic acid from extract of Hypericum brasiliense with C
18
micro-
sorb-MV column with acetonitrile: water (9:1) pH 3.0 adjusted with
phosphoric acid at =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. Te 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
Te leaves of VN were collected from nearby area of Raigad dis-
trict, Maharashtra, India. Te leaves were washed with water and shade
dried (at 30C for 168 hrs) and pulverized. Te 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). Te system consists of Waters HPLC
600 pump, a Rheodyne 7725i injector with 5 l sample loop, on-line de-
gasser AF (Waters, USA), Waters 2487 Dual absorbance UV detector
(at =210 nm, for ursolic acid and betulinic acid) with 0.05 aufs sensi-
tivity. Ursolic acid and betulinic acid were analyzed using a Symmetry

C-18 (4.6 250 mm, 5 m) column equipped with automatic tempera-
ture ( 0.1C) 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 identifcation of both acids. Te column temperature was
maintained at 35C ( 0.1C).
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 bafed 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 28C ( 1C) for
2 hrs. Te schematic experimental setup is as shown in Figure 3. Te
samples were collected at fxed interval of time. Te collected samples
were fltered through 0.2 m membrane flter 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
Te methanolic solutions of standards of ursolic acid and betulinic
acid were analyzed with LC-MS for confrmation of the peaks and its
positions. Te methanolic extract of Vitex Negundo linn leaves also
analyzed with LC-MS for identifcation of ursolic acid and betulinic
acid in extract. Waters Aquity LC-MS system, Waters TQD Mass detec-
tor and Waters Masslynx 4.1 sofware was used along with Symmetry

C-18 column (4.6 250 mm, 5 m) 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 diferent pos-
sibilities of solvent compositions to change the polarity of mobile phase
for ursolic acid and betulinic acid separation from the methanol ex-
tract. Te reported HPLC method was found suitable for ursolic acid
and betulinic acid separation and quantifcation simultaneously. In this
Figure 3: Schematic of experimental setup.
Volume 3 Issue 3 1000134
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
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
for betulinic acid is 10.92 min ( 0.2 min). Te method developed
certainly indicates improved resolution of peaks and better selectivity.
Te chromatogram for methanol extract of leaves is shown in Figure
6. A distinct peak appearing at 12.36 min indicates ursolic acid while
the peak at 10.92 min matches with betulinic acid retention time. Te
peak fractions appearing at 12.36 mins and 10.92 mins were separately
collected in separate sample vowels afer injecting the extracts several
method, acetonitrile:methanol (80:20) was used as mobile phase with
0.5 ml/min elution fow rate, =210 nm UV wavelength with 0.05 aufs
sensitivity. Symmetry

C-18 (4.6 250 mm, 5 m) column was used for

separation and isolation of ursolic and betulinic acids. Te HPLC chro-
matograms of ursolic and betulinic acid using the developed method
are shown in Figure 4 and Figure 5.
Te retention time for ursolic acid is 12.36 min ( 0.2 min) while
m
V
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
Minutes
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
Ursolic acid
Figure 4: HPLC Chromatogram of ursolic acid (Residence time = 12.36 min) with Chromatographic conditions: Symmetry

C-18 (4.6250mm, 5m) column, acetonitrile:methanol (80:20), =210 nm, 0.5 ml/min fowrate.
m
V
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
Minutes
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
Betulinic acid
Figure 5: HPLC Chromatogram of betulinic acid (Residence time = 10.92 min) with Chromatographic conditions:
Symmetry

C-18 (4.6250mm, 5m) column, acetonitrile:methanol (80:20), =210 nm, 0.5 ml/min fowrate.
m
V
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
Minutes
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
Betulinic acid
Ursolic acid
Figure 6: HPLC Chromatogram of methanolic extract of Vitex Negundo Linn leaves with Chromatographic conditions:
Symmetry

C-18 (4.6250mm, 5m) column, acetonitrile:methanol (80:20), =210 nm, 0.5 ml/min fow rate.
Volume 3 Issue 3 1000134
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
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 4 of 6
times. Tese two fractions were then purifed and MS analysis was car-
ried out to confrm 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. Te solutions were fltered
through 0.2 m (PALL Ultipor N
66
) membrane flter and analyzed in
HPLC. Evaluation of each point was repeated thrice. Te area under
peak of ursolic acid vs. concentration plot shows a linear ft with cor-
relation coefcient of 0.9961 (Figure 7). Te same calibration chart was
subsequently used for quantifcation 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 fltered through 0.2 m (PALL Ultipor
N
66
) membrane flter and injected in the HPLC system through an-
other syringe flter 0.2 m. Te area under the peak of betulinic acid
was calculated using Waters Empower-II sofware and that corresponds
to the concentration of betulinic acid present. Te plot of area against
concentration of betulinic acid standard injected shows a linear ft with
correlation coefcient 0.9991 (Figure 8). Te same calibration chart was
subsequently used for quantifcation of unknown samples.
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 (4.6250 mm, 5 m), acetonitrile:methanol (80:20 V/V), 210 nm wave-
length.
Figure 10: LC-MS analysis of betulinic acid standard with Symmetry

C-18
column (4.6250 mm, 5 m), 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 (4.6250 mm, 5 m),

acetonitrile:methanol (80:20 V/V), 210 nm wavelength.
Volume 3 Issue 3 1000134
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
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 5 of 6
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. Te top chromatograms
in each fgure shows that the position of ursolic acid and betulinic acid
at 210 nm wavelength of UV Detector and the bottom chromatograms
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. Tese 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 identifed peaks of ur-
solic acid and betulinic acid. From these fgures it is clear that the HPLC
method used for analysis of ursolic acid and betulinic acid is valid. Te
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
Te HPLC method developed was validated with diferent 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. Te validation plot shows
the R
2
value more than 0.97 for both acids (Figure 12 and Figure 13).
Tis 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
Te batch extraction experiment was carried out with methanol as
solvent and at 28C to extract the ursolic acid and betulinic acid from
leaves of Vitex Negundo Linn. Te 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. Tis
increase in concentration of both acids is due to difusion mechanism
of solutes (ursolic acid and betulinic acid) in solvent (methanol).
Tis 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
Te HPLC methods available in the literature are tried with the
given conditions and with Symmetry C-18 column. Tese methods did
Figure 11 (b): LC-MS spectra of methanolic extract of Vitex Negundo Linn
leaves with Symmetry

C-18 column (4.6250 mm, 5 m), 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.
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 28C.
Volume 3 Issue 3 1000134
J Anal Bioanal Techniques
ISSN:2155-9872 JABT, an open access journal
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 6 of 6
not show any results in case of ursolic acid and betulinic acid for extract
of Vitex Negundo Linn. Te developed method of HPLC is suitable for
simultaneous determination of ursolic acid and betulinic acid from
leaves of Vitex Negundo Linn. Te method showed linearity for ursolic
acid and betulinic acid diferent 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 fnancial 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.
References
1. Chadha YR (1976) The wealth of India: A dictionary of Indian raw material and
industrial products. CSIR (Council of Scientifc & Industrial Research), New
Delhi, India.
2. Taneja SC, Gupta RK, Dhar KL, Atal CK (1979) The essential oil of Vitex ne-
gundo linn. Indian Perfumer, India.
3. Dutta KP, Chowdary S, Chakravarty AK, Achari B, Pakrashi CS (1993) Studies
of Indian medicinal plants part LXXV; Nishindaside, a novel iridoid glycoside
from Vitex negundo. Tetrahadran 39: 3067-3072.
4. Prema MS, Misra GS (1978) Leucoanthocynidins of Vitex Negundo. Ind J
Chem, India.
5. Chawla AS, Sharma AK, Ha SS, Dhar KL (1992) Chemical investigations and
anti-infammatory activity of Vitex negundo seeds. J Nat Prod 55: 163-167.
6. Liu J (1995) Pharmacology of oleanolic and ursolic acid. J Ethnopharmacol
49: 57-68.
7. Chandramu C, Manohar RD, Krupadanam DG, Dashavantha RV (2003) Isola-
tion, characterization and biological activity of betulinic acid and ursolic acid
from Vitex Negundo L. Phytother Res 17: 129-134.
8. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, et al. (1995) Discov-
ery of betulinic acid as a selective inhibitor of human melanoma that functions
by induction of apoptosis. Nat Med 10: 1046-1051.
9. Simone CB, Eloir PS, Valquiria LB (2005) HPLC method to assay total saponins
in Ilex paraguariensis aqueous extract. J Braz Chem Soc 16: 723-726.
10. Liao Q, Yang W, Jia Y, Chen X, Gao Q, et al. (2005) LC-MS determination and
pharmacokinetic studies of ursolic acid in rat plasma after administration of
the traditional chinese medicinal preparation Lu-Ying extract. Yakugaku Zasshi
125: 509-515.
11. Chen JH, Xia ZH, Tan RX (2003) High perfrmance liquid chromatographic
analysis of bioactive triterpenes in Perilla frutescens. J Pharm Biomed Ana 32:
1175-1179.
12. Novotny L, Abdel-Hamid M, Hamza H, Masterova I, Grancai D (2003) Devel-
opment of LC-MS method for determination of ursolic acid: application to the
analysis of ursolic acid in Staphylea holocarpa Hemsl. J Pharm Biomed Anal
31: 961-968.
13. Zou S, Chen W, Li K (2004) Preparation and HPLC analysis of ursolic acid from
Eriobotrya japonica Lindi. Acta Agri Univer Jiangxiensis 26: 808-810.
14. Zhao G, Yan W, Cao D (2007) Simultaneous determination of betulin and betu-
linic acid in white birch bark using RP-HPLC. J Pharm Biomed Anal 43: 959-
962.
15. Akowuah GA, Zhari I, Norhayati I, Sadikun A, Sundram K, et al. (2003) Quanti-
fcation of betulinic acid in leaf extracts. J Tropical Medicinal Plants 4: 225-228.
16. Puder CH, Graef H, Thumerer MJ, Heitzmann M (2007) Process for the extrac-
tion of betulinic acid. United State Patent, USA.
17. Markus S (2005) Process for obtaining betulinic acid. United State Patent, USA.
18. Abreu AN, Porto ALM, Marsaioli AJ, Mazzafera P (2004) Distribution of bioac-
tive substances from Hypericum brasiliense during plant growth. Plant Science
167: 949-954.
Submit your next manuscript and get advantages of OMICS
Group submissions
Unique features:
User friendly/feasible website-translation of your paper to 50 worlds leading languages
Audio Version of published paper
Digital articles to share and explore
Special features:
200 Open Access Journals
15,000 editorial team
21 days rapid review process
Quality and quick editorial, review and publication processing
Indexing at PubMed (partial), Scopus, DOAJ, EBSCO, Index Copernicus and Google Scholar etc
Sharing Option: Social Networking Enabled
Authors, Reviewers and Editors rewarded with online Scientifc Credits
Better discount for your subsequent articles
Submit your manuscript at: http://www.omicsonline.org/submission