Acta Chromatographica 25(2013)1, 181-199

DOI: 10.1556/AChrom.25.2013.1.12

Rapid Extraction, Isolation, and Quantification

of Oleanolic Acid from Lantana camara L. Roots
Using Microwave and HPLCPDA Techniques
S.C. VERMA1,2,*, C.L. JAIN2, S. NIGAM1, AND M.M. PADHI1
1

Central Council for Research in Ayurvedic Sciences, 61-65, Institutional Area, Opp.-D-Block, Janakpuri, New Delhi-110058, India
2Department of Chemistry, M. M. H. College, Ghaziabad, U.P., India
*E-mail: scvpharma@gmail.com

Summary. An ecofriendly solvent polarity based microwave-assisted extraction (MAE)

technique was developed for the rapid extraction and isolation of bioactive oleanolic
acid from roots of Lantana camara L. Several different influential extraction parameters
such as microwave power, extraction time, solvent type, and volume were studied in a
systematic fashion for the determination of optimum extraction conditions. Simply
modified and rapid high-performance liquid chromatographydiode array detector
(HPLCDAD) method was also developed and validated for quantitative determination
of oleanolic acid from roots of L. camara. Under optimum conditions, using a mixture of
CHCl3:MeOH (60:40, v/v, 15 mL) as a solvent, 600 W microwave powers, and 50 C temperature for 6 min of MAE produced a maximum yield of 1.23% (dry weight of roots).
No degradation of the target analyte was observed at the optimum conditions as evidenced from the recovery studies performed with standard oleanolic acid. The proposed
method also showed high degree of reproducibility; hence, it may be useful for maximum extraction and isolation of biologically active oleanolic acid.
Key Words: Lantana camara L. roots, microwave-assisted extraction and isolation,
oleanolic acid, HPLCPDA method

Introduction
Lantana camara L., commonly known as wild or red sage, is the most widespread species of the Lantana genus, growing luxuriantly at elevations up
to 2000 m in tropical, sub-tropical, and temperate regions [1]. It is regarded
both as a notorious weed and a popular ornamental garden plant and has
found various uses in folk medicine in many parts of the world [2]. L.
camara is poisonous to stocks and humans [3]. Traditionally, the plant is
used as diaphoretic, carminative, antispasmodic, tonic, and useful in the
treatment of tetanus, vitiated conditions of vata, epilepsy, and gastropathy.
A decoction of fresh roots is a good gargle for odontalgia, and this is used
by hill tribes for all types of dysentery. Powdered leaves are used for cuts,
02312522 2012 Akadmiai Kiad, Budapest

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wounds, ulcers, and swellings. An infusion of the leaves is good for bilious
fever, eczema, and eruptions. The fruits are useful in fistula, pustules, tumors, and rheumatism [1, 35].
Different parts of the plant are reported for pharmacological activities
like antilymphocytic and immunosuppressive, hepatoprotective, thrombin
inhibitory, termiticidal, antimotility, antifilarial, in vitro cytotoxic, and antimicrobial activity and promising anti-hyperglycemic activity against alloxan-induced diabetic rats [613]. The root has established uses in malarial
control, rheumatism, skin rashes as well as dermatitis, eczema, and related
mycotic infections as well as in the management of respiratory tract infections including influenza and tuberculosis [1415].
Major products investigated in Lantana plants belong to mono- and
sesquiterpenes (bisabolene derivatives, (E)-nuciferal and (Z)-nuciferol, curcumene, -curcumene), triterpenes (lantadene A, B, C, and D; lantanolic,
lantanilic, and lantic acid; icterogenin; oleanolic acid (OA) (Fig. 1); camarinic
and camaric acid; pomolic acid), iridoid glycosides (theveside, geniposide,
and lamiridoside), furanonaphthoquinones (diodantunezone), flavonoids
(3-methoxy-, 3,7-dimethoxy-, and 3,7,49-trimethoxyquercetin; hispidulin;
flavone glycoside camaraside), phenylethanoid glycosides (verbascoside
and isoverbascoside), oligosaccharides (ajugose, stachyose, verbascotetraose, verbascose, and lantanose A and B), and other groups [2, 17].
The profile of triterpenoids in the roots of L. camara is different from that in
the leaves. Roots are rich source of OA [16], and it is found in the form of
free acid or aglycones. Oleanolic acid (pentacyclic triterpenoid) is the focus
of attention of our research due to its wide applicability similar to use for
anti-cancer, anti-AIDS, anti-inflammatory, antimicrobial activity, and hepatoprotective [23] etc., and, in fact, the rootlets and root bark of L. camara
provide a plentiful (2%) supply of OA [18].
30

12

29

19

26

3
HO

23

5
H

28

13
14

8
7

OH

16
15

1
10

22
17

9
2

21

18

11
25

20

27

24

Fig. 1. Structure of oleanolic acid (3-hydroxyolean-12-en-28-oic acid),

[M+] = m/z 456, corresponding to C30H48O3

Oleanolic Acid from Lantana camara L. Roots

183

Optimum extraction is the pivotal step in the recovery of active ingredients from plant matrix. Conventional extraction methods like solvent extraction, soxhlet extraction, and heat reflux extraction are time and solvent
consuming, thermally unsafe, and involve risk of thermal decomposition of
bioactive compounds. Soxhlet extraction and other conventional methods
operate through cell permeation followed by solubilizing the active constituents by the extracting solvent [19]. In the last decade, there has been an
increasing demand for new extraction techniques, amenable to automation,
with shortened extraction times, and reduced organic solvent consumptionpreventing pollution in analytical laboratories and reducing sample
preparation costs [20, 21]. Microwave assisted extraction (MAE), supercritical fluid extraction (SFE), and pressurized liquid extraction (PLE) are some
of the alternatives to conventional techniques [22]. Extraction of OA from L.
camara roots has been reported by various methods like maceration [23],
soxhlet extraction [24], cold percolation [13], and response surface methodology [25]. These techniques are often time consuming and require large
volume of organic solvent whose subsequent disposal creates severe environmental hazards. Due to the consumption of huge energy resources, it
also adds up to the huge carbon load which is a severe problem affecting
the entire mankind. In contrast, MAE is known as one of the best green
technologies with advantages like high extraction efficiency, good reproducibility, and low consumption of organic solvents. MAE is based upon
the selective and rapid localized heating of moisture in the sample by microwaves. Several applications of MAE for biologically active compounds
have appeared in the literatures, such as extraction of four phenolics from
Cynodon dactylon Linn. Whole Plant [26], extraction of gallic acid in leaves of
Eucalyptus hybrida Maiden [27], extraction of vanillin from Vanilla planifolia
pods [28], and extraction of curcuminoids from Curcuma longa rhizome [29],
and so on.
The objective of the present work is to evaluate the applicability of microwave energy for the performance evaluation of a rapid, reliable, and reproducible extraction and isolation method for the large scale production of
OA from roots of L. camara Linn. Such attempt with the support of this
study has not been earlier reported for the extraction and isolation of
oleanolic acid from L. camara by MAE. In the proposed method, effects of
various experimental conditions on the extraction yield as well as quantitative analysis of OA by high-performance liquid chromatographydiode array detector (HPLCDAD) method are studied in order to determine the
optimum extraction and isolation of OA from the roots of L. camara. Furthermore, the contents of OA in different polarity microwave-assisted extracts of L. camara were determined with the developed MAEHPLC techniques.

S.C. Verma et al.

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Experimental
Plant Material and Chemicals
Fresh roots of L. camara L. were collected from village Jallapur, District
Sitapur, Uttar Pradesh, India, in the month of April, 2010 and washed it
thoroughly with clean water. Some cleaned roots were sent to National Institute of Sciences Communication and Information Resources (NISCAIR),
New Delhi for identification, and specimen voucher (NISCAIR/RHMD/
Consult/2010-2011/1632/230) has been procured. After authentication, the
roots were dried under a gentle stream of air in the laboratory till no loss in
weight (temperature 30 2 C and relative humidity 50 5%) and powdered in an electric grinder. Solvents and chemicals used were of analytical
grade (E. Merck), and those used for HPLC were of HPLC grade. Oleanolic
acid (2 mg, purity 97%) was obtained as a gift sample. Isolated oleanolic
acid (purity 95%) was used as standard OA for study. For sample and solvent filtration, 0.45-m membrane filters (Millipore, Germany) were used,
and solvents were degassed prior to use.

Equipment and Operating Conditions

The extraction system comprised of a microwave oven (Domestic) manufactured by KENSTAR (Ahmednagar, Maharashtra, India) equipped with a
magnetron of 2450 MHz with a nominal maximum power of 900 W, 10
power levels, time controller, and 10 convection temperature sensor; exhaust system was used for extraction; and nuclear magnetic resonance
(NMR) spectra were recorded on BRUKER DRX-300 MHz (Bruker BioSpin,
Switzerland). HPLC analysis of OA was carried out on a ZORBAX SB-AQ
RRHT (Agilent Technologies, U.S.A.) C18 column (100 mm 4.6 mm,
1.8 m) using an Agilent-1290 HPLC system (U.S.A.) equipped with a degasser, an auto-sampler, a diode array detector (DAD), and 20-L injector
loop. Gradient mixture of buffer solution (Solvent A) prepared by dissolving 1.36 g of potassium dihydrogen phosphate in 900 mL Milli-Q water, adjusted pH to 2.8 with dilute phosphoric acid, and diluting it to 1000 mL with
Milli-Q water and methanol (Solvent B) was used as the mobile phase with
a gradient elution as per Table I with a flow rate of 1.2 mL min1 for 16 min
to elute out OA at 11.5 min. Column was equilibrated under the starting
conditions for 5 min. Chromatogram was monitored at 210 nm wavelengths
and analyzed using Agilent 2D and 3D Chemstation software.

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Table I. Mobile phase program for gradient elution of OA

Time (min)

Solvent A (%)

Solvent B (%)

90

10

70

30

15

85

15

15

85

16

90

10

Flow rate and max

1.2 mL min1 and

210 nm, equilibration
time (5 min)

Solvent A = prepared by dissolving 1.36 g of potassium dihydrogen phosphate in

900 mL Milli-Q water, adjusting pH to 2.8 with dilute phosphoric acid, and diluting it to
1000 mL with Milli-Q water, and Solvent B = methanol.

Microwave-assisted Extraction (MAE) of Roots of L. camara

Roots of L. camara were extracted with variety of solvents ranging from nonpolar to polar as per Table II. After allowing a preleaching time of 10 min,
each suspension was irradiated with microwave at different experimental
conditions for selection of suitable solvents. Approximately 4.0 g roots
powder were taken in four different 100-mL conical flasks; 10, 15, 20, and
25 mL of a mixture of CHCl3:MeOH (60:40, v/v) were added separately in
each flask as solvents; and microwave irradiation powers 500, 600, 700, and
Table II. Preparation of MAE sample for oleanolic acid analysis

Solvent

Powdered
roots
(g)

Extract
wt. (g)

% Yield
of extract

Sample conc. for HPLC

analysis

n-Hexane

4.1122

0.0098

0.238

5.5 mg/5 mL in EtOAc

Dichloromethane

4.1356

0.0352

0.851

5.15 mg/5 mL in EtOAc

Ethyl acetate

3.4402

0.0624

1.813

4.41 mg/5 mL in MeOH

CHCl3:MeOH
(60:40, v/v)

3.8904

0.1982

5.094

10.12 mg/10 mL in MeOH

Ethanol

8.8255

0.258

2.923

5.85 mg/5 mL in MeOH

MeOH:water
(60:40, v/v)

3.8786

0.3541

9.129

7.27 mg/5 mL in MeOH

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S.C. Verma et al.

800 W were applied for 4, 6, 8, and 10 min, respectively, for maximum extraction of OA. The sample was treated under microwave irradiation in an
intermittent way, i.e., irradiation coolingirradiation. The irradiation time
was kept for 1 min, and 1 min was taken to cool the sample solution between two irradiations. Each extract was filtered by using Whatman filter
paper no. 1 and the solvents were removed under vacuum at 50 C, separately and lyophilized till each extract was free from solvents. The concentrated extracts were re-dissolved separately in HPLC grade solvents as per
Table II, passed through 0.45-m membrane filter before injecting in to the
HPLC system.

Isolation of Oleanolic Acid from the Roots of L. camara

The powdered roots of L. camara (100 g) were defatted thrice with n-hexane
and then microwave-assisted extracted with a mixture of CHCl3:MeOH
(60:40, v/v, 500 mL) three times, applied 600 W microwave powers for
6 min, and kept overnight at room temperature. The solvent was removed
under vacuum at 50 C, and the crude was dissolved in CHCl3 and left
overnight for precipitation. The precipitate so obtained was crystallized

Fig. 2. TLC of OA (std. and isolated) vs. MAE extract (chloroform:methanol) of

L. camara roots, after derivatization with anisaldehydesulfuric acid reagent

Oleanolic Acid from Lantana camara L. Roots

187

with ethanol (99%), which showed single spot on thin-layer chromatography (TLC) as evident in Fig. 2. Precipitation and crystallization process were
repeated three times, which gave OA in 0.86 g with recovery of 70% and
purity of 95% by HPLC.

Spectral Analysis (OA)

White amorphous powder, m.m.p.: 306307 C, 1H-NMR (300 MHz, C5D5N)
H: 0.81, 0.87, 0.93, 0.94, 1.10, 1.15 (each 3H, s, CH3 6), 1.20 (3H, s, H-27),
3.20 (1H, dd, J = 3.6, 10 Hz, 18-H), 3.37 (1H, t, J = 8.2 Hz, 3-H), 5.43 (1H,
brs, H-12) (Fig. 3).

Fig. 3. 1H-NMR spectra (300 MHz, pyridine-d5) of isolated oleanolic acid

from roots of L. camara L.
13C-NMR

(75 MHz, C5D5N): C (from C-1 to C-30) 40.70t, 29.59t, 79.92d,

39.07s, 57.57d, 20.47t, 32.57t, 40.98s, 49.84d, 39.07s, 25.45t, 124.22d, 146.47s,
41.48s, 27.82t, 25.45t, 48.39s, 43.71d, 48.39t, 31.56s, 34.90t, 32.57t, 30.41q,
18.09q, 17.18q, 19.10q, 29.99q, 181.86s, 34.99q, 25.45q (Fig. 4). The structure
of OA (Fig. 1) was determined by NMR data and confirmed by comparison
of 1H and 13C-chemical shifts with the reported data [30], and also crosschecked by Co-TLC and MMP with an authentic sample of oleanolic acid.

S.C. Verma et al.

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Fig. 4. 13C-NMR spectra (75 MHz, pyridine-d5) of isolated oleanolic acid

from roots of L. camara L.

Preparation of Standard Stock Solution

A stock solution (1 mg mL1) of OA was prepared by accurately weighing
10 mg of OA and transferred in to 10 mL of volumetric flask. Approximately 10 mL of methanol was added and dissolved to obtain final standard
solution of 1 mg mL1 of OA. Standard solutions were prepared by diluting
the stock solution with methanol to obtain concentrations of 0.04, 0.08, 0.16,
0.32, 0.4, and 0.5 mg mL1 of OA for method suitability studies. Each solution was filtered through 0.45-m membrane filter paper prior to inject in to
HPLC system.

Method Validation Parameters

Validation of the analytical method was done according to the International
Conference on Harmonization guideline [31]. The method was validated for
its linearity, specificity, precision, accuracy, limit of detection (LOD), and
limit of quantitation (LOQ) [2629].

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Linearity and Range

The linearity of the method was determined at six concentration levels ranging from 0.04 to 0.5 mg mL1 for OA. For preparing a calibration curve of
OA, each working standards solution was separately injected in to the
HPLC system in triplicate. Six-point calibration curve of OA was obtained
by plotting the concentration of OA versus peak area to check the linearity of
response as evident in Table III.
Table III. Summary of validation parameters of oleanolic acid
Parameters

Results

Linearity:
Range (mg mL1)
Linear equation
Slope (m)
Intercept (b)
Correlation coefficient (r)
r-Square (r2)
Standard deviation (SD)

0.0400.50
y = 2932.24x + 21.985
2932.24
21.985
0.9999
0.999
7.86

Precision (%RSD):
Intra-day (n = 6)
Repeatability of peak area of standard
Repeatability of retention time (Rt)
Inter-day (n = 6)
Repeatability of peak area of sample
Repeatability of Rt

0.4409
0.0119
0.2203
0.0135

Limit of detection (LOD) (g mL1)

8.8

Limit of quantification (LOQ) (g mL1)

26.8

Specificity

Specific

Recovery (%)

95.75103.42

Specificity
The specificity of the method was ascertained by analyzing the standard
and the samples. The peak of OA in sample was confirmed by comparing
the retention time and UV spectra of the standard OA (Figs. 57).

S.C. Verma et al.

190
DAD1 A, Sig=210,4 Ref=off (F:\AYUSH-LC\AYUSH210311 2011-03-22 00-02-01\AYUSH000050.D)
1 1 .5 0 7

mAU

600

500

400

300

200

100

0
0

10

12

14

16

min

16

min

Fig. 5. HPLCPDA chromatogram of isolated oleanolic acid at 210 nm

DAD1 A, Sig=210,4 Ref=off (F:\AYUSH-LC\AYUSH210311 2011-03-21 05-51-55\AYUSH000023.D)
1 1 .5 0 8 - O A

mAU
800
700
600
500
400
300
200
100
0
0

10

12

14

Fig. 6. HPLCPDA chromatogram of MAE extract obtained from a mixture of

chloroform:MeOH (60:40, v/v) at 210 nm

Oleanolic Acid from Lantana camara L. Roots

191

*DAD1, 11.508 (441 mAU, ) Ref = 11.445 & 11.602 of OA, below line
*DAD1, 11.505 (368 mAU, ) Ref = 11.441 & 11.581 of sample, upper line
Norm.
400
350
300
250
200
150
100
50
0
200

225

250

275

300

325

350

375

Fig. 7. Overlay UV spectra of MAE extract obtained from mixture of

chloroform:MeOH (60:40, v/v) with isolated OA at 210 nm

Precision
Six injections of 2 L of OA were injected separately from a single stock solution (0.4 mg mL1) in to HPLC and analyzed by the proposed method to
determine variations due to the chromatographic conditions (system precision) as shown in Table III. To determine variations due to the HPLC system, six different samples of ethyl acetate extract of the concentration
0.882 mg mL1 were injected in to HPLC and analyzed by the proposed
method (Table III).

Accuracy
The accuracy of the method was determined at three levels by calculating
the recovery of OA by the method of standard addition. Known amount of
OA was added to pre-quantified sample solutions of L. camara roots, and
the mixtures were analyzed according to the proposed method; the
amounts of OA were estimated by measuring the peak area and by fitting
these values to the straight line equation of calibration curve (Table III).

nm

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192

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ of the developed method were determined by using linear
regression equation of the calibration curve. The LOD and LOQ were calculated based on the standard deviation (SD) of the y-intercept and the slope
(S) as 3.3 SD/S and 10 SD/S, respectively.

Results and Discussion

Characterization of OA
It gave positive Liberman and Noller test and developed yellow color with
tetranitromethane (TNM) indicating triterpenoid nature of the molecule. It
did not respond Molisch's test showing non-glycosidic nature of the molecule. The 1H-NMR spectrum of compound (OA) exhibits presence of seven
tertiary methyl groups at 0.81, 0.87, 0.93, 0.94, 1.10, 1.15, and 1.20, and a
characteristic olefinic proton of C12C13 double bonded pentacyclic triterpenoid at 5.43 (1H, brs, H-12) suggesting an olea-12-ene skeleton. One methine proton at 3.37 (1H, t, J = 8.2 Hz, 3-H) (Fig. 3) showed that OA has at
least one hydroxyl group on the olean-12-ene-skeleton.
The 13C-NMR spectrum of (OA) revealed presence of signals due to an
oxygenated carbon signal at 79.92 (C-3), one tri-substituted double bond at
124.22 (C-12) and 146.47 (C-13), and one carboxyl group at 181.86 (C-28)
(Fig 4). Moreover 13C-NMR signals due to C-18 to C-22 at [43.71 (C-18),
48.39 (C-19), 31.56 (C-20), 34.90 (C-21), and 32.57 (C-22)] suggested that
(OA) was an olean-12-en derivative. It forms mono acetate with acetic anhydridepyridine suggesting the presence of one hydroxyl group in the
molecule. On the basis of above spectral and chemical evidences compound,
OA was identified as oleanolic acid (Fig. 1). The fundamental difference between the two triterpenes is at C-29 and C-30 of the ring E. In type triterpenoid (ursolic acid), both the methyl groups at ring E are secondary
whereas in type triterpene (oleanolic acid), both the methyl groups are tertiary. By comparing the 13C values of the compound against that of and
type triterpenes, the compound was found to show a closer resemblance to
the type. The interpretation is further supported by the 1H-NMR spectra
of the compound. A double doublet at 3.20 ppm with a J value of 3.6 and
10 Hz was indicated coupling between a single proton at C-18 and two protons at C-19. This double doublet only appears if the compound is of the type triterpenoid (where only two protons are attached to C-19). On the
other hand, if the compound is of -type, a single doublet will appear (be-

Oleanolic Acid from Lantana camara L. Roots

193

cause the two groups attached to C-19 are hydrogen and methyl, and the
coupling between a single proton at C-18 and at C-19 would give a doublet).
The identity of the compound was finally determined by Co-TLC and MMP
with an authentic sample (Fig. 2) and by comparison of 1H and 13C-chemical
shifts with the reported data [30].

Effect of Solvent and Volume on Extraction Yield

Quantity of root powder (100120 mesh) was extracted with 15 mL of different solvents (mentioned in Table II) for 6 min. The microwave power was
600 W, and the temperature was 50 C. The results of extraction yield are
shown in Table II. The yields of OA reached the maximum when the solvent
was a mixture (60:40, v/v) of chloroform and methanol. In conventional extraction method, the polarity of solvent is an important factor to extraction
yield. However, the dielectric constant and dissipation factor of solvent significantly influence the extraction yield in MAE [32]. Different solvent volumes (10, 15, 20 mL, and 25 mL) of a mixture (60:40, v/v) of chloroform and
methanol were taken for the extraction of OA, while other conditions (microwave power, irradiation time, and temperature) were same as above.
The yields of OA reached the maximum when the solvent volume was
15 mL as evident in Table IV. Therefore, 15 mL of a mixture (60:40, v/v) of
chloroform and methanol was chosen for optimum extraction of OA from
roots of L. camara.
Table IV. Optimization of MAE conditions for OA extraction
Solvent volume (mL)
CHCl3:MeOH (60:40, v/v)

10

15

20

25

Microwave power (W)

500

600

700

800

Irradiation time (min)

10

OA (%) Yield

1.18

1.23

1.21

1.18

Effect of Microwave Power

Table IV indicated that there was significant improvement in extraction
yield with the increase in microwave power between 500 W and 600 W. A
sharp decrease in extraction yield was obtained between 600 W microwave
power and 700 W microwave power with the increase in extraction time.
However, there was no significant increase in extraction yield at higher
power level between 700 W and 800 W for any extraction time. More elec-

S.C. Verma et al.

194

tromagnetic energy was transferred to the extraction system quickly and

improved the extraction efficiency, when the microwave power increased
from 500 W to 600 W. Based on the above observation, 600 W microwave
power was considered to be optimum.

Effect of Irradiation Time

Microwave irradiation time of 4 min, 6 min, 8 min, and 10 min at 600 W microwave power on the extraction yield of OA was calculated (Table IV).
Three intervals were observed in the process of microwave extraction. Initially, a short rise in extraction yield between 4 min and 6 min which indicates the first quantities extracted, located at the surface of root particles
representing approximately 1.18% of the OA. Extraction yield at 6 min resulted to the highest yield of OA (1.23%) due to intern warming of the natural moisture located in the plant cells. Since no significant difference in extraction yield was obtained between 6-min and 10-min extraction time, the
6 min was considered optimum for maximum extraction. MAE reached the
highest extraction yield of OA (1.23%, w/w) when irradiation time was
6 min.

Optimization of HPLCPDA Method

Selection of the HPLC conditions was guided by the requirement for good
resolution of adjacent peaks within as short a time as possible, especially
when large numbers of samples were analyzed. Due to very labile characteristics of OA, C18 columns are preferred for the HPLC analysis. Preliminary studies were performed under isocratic conditions using a ODS (Waters) column (4.6 250 mm, 5 m) with binary mobile phases comprising
MeOH or ACN and an aqueous solution containing 0.1% acetic acid to keep
the acidic compounds in neutral form. The retention behavior of the extracts
in both solvent systems was examined. Peaks were broad, and isomeric
peak was not resolved. Further, manipulation in the mobile phase like
methanol or ACN with phosphate buffer which suppresses the ionization of
triterpenes [33], sharpens peak shapes and improves analytical sensitivity
and resolution, but tR window was long. Because the separations were performed on a reversed-phase column, the most polar compound eluted earlier than OA, as expected. Resolution and retention were highly dependent
on the composition and flow rate of the mobile phase. Higher percentage of
organic solvents and higher flow markedly reduced run time, but at the expense of reducing the resolution of isomeric peaks [34]. An ACN-containing
mobile phase was too high to separate isomeric overlapping peaks with a

Oleanolic Acid from Lantana camara L. Roots

195

sufficient resolution because of its eluotropic strength. MeOH was chosen as

the optimum solvent with aqueous phase for separation of isomeric peaks.
Isocratic elution was usually used for the separation of triterpenes in plant
tissues, containing different components, difficult to separate during a short
period of the run time. The C18 column (100 mm 4.6 mm, 1.8 m) gave better resolution of OA and other constituents, but with a markedly increased
retention time. However, a flow rate of 1.2 mL min1 resulted in satisfactory
resolution in a reasonable analysis time (less than 20 min); however, the
resolution of peaks in L. camara real samples was even better (Fig 5). The UV
absorbance maximum, tested in MeOH, was found to be optimal for the
highest sensitivity at 206.19 nm for OA (Fig. 7). To optimize the detection
wavelength for the mobile phase, the best responses were observed at
200 nm but, because the baseline was very unstable at this wavelength,
210 nm was chosen as optimum for detection for oleanolic acid.
Therefore, satisfactory separation was performed using a ZORBAX SBAQ RRHT C18 column (100 mm 4.6 mm, 1.8 m), gradient mixture of
phosphate buffer solution (Solvent A) and methanol (Solvent B) as the mobile phase with a gradient elution as evident in Table I with a flow rate of
1.2 mL min1 for 16 min to elute out oleanolic acid at 11.5 min, as shown in
Figs. 56, while the detection wavelength, column temperature, and injection volume were set 210 nm, 30 C and 2 L, respectively.

Validation of HPLCPDA Method

The specificity of the method was determined by comparing the chromatographic profile, and the data obtained for the standards and samples, considering the following parameters like retention time, maximum wavelength of absorption and overlay of UV spectrum [26]. The peaks of OA in
sample was identified by matching their retention times and UV-spectra
with corresponding standards as shown in Table III and Figs. 57. Peak purity of compounds was assessed by comparing the spectra at three different
points, i.e., peak start, peak apex, and peak end positions, and found the
proposed method is specific for OA analysis.
The linear equation between the concentration of the standards injected
and the peak area can be expressed as y = mx + b, where y is the peak area
and x is the concentration of the standard, and slop (m) and intercept b are
constants [31]. The slopes, y-intercepts, and correlation coefficients (r2) obtained from regression analysis are shown in Table III. The calibration curve
was linear in the tested concentration range (0.040.5 mg mL1); good correlation coefficients (r2) were found greater than 0.999 for OA (Table III), indicating good linearity of the proposed method.

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LOD is the lowest amount of analyte in a sample that can be detected

but not necessarily quantified. LOD of OA was found 8.8 g mL1 (Table III).
LOQ is defined as the lowest concentration that can be accurately quantitated with acceptable accuracy and precision. LOQ of OA was found
26.8 g mL1 (Table III). These results indicate that the method provided satisfactory sensitivity.
Six replicate injections of same solution and six injections of different
solution of same concentration were analyzed by the proposed method to
determine the system precision and method precision, respectively. Relative
standard deviation (%RSD) value of retention time and peak area was obtained within 1%, indicating very little variation of the measured values
(Table III).
The % recovery has been studied and established within the range of 50
to 150%. Percentage recovery of OA was found within range 95.75103.42%,
representing the good accuracy of the method (Table III).
In the present study, linearity, LOD, LOQ, precision, and recovery results were in accord with the required criteria. Therefore, the proposed
method was found to be most suitable for rapid extraction and simultaneous quantitation of OA.

Quantitative Determination of OA in the Microwave Assisted

Extract
HPLCPDA method was standardized for the qualitative and quantitative
analysis of OA in extracts of L. camara roots, extracted by different solvents.
These extracts were analyzed by the proposed HPLCPDA method although maximum yield of microwave-assisted extract was obtained (9.13%)
in MeOH:H2O (60:40, v/v) mixture, while minimum yield of extract
Table V. Yield (%) of oleanolic acid in MAE of Lantana camara roots
MAE extract

% OA (w/w) in dry roots

n-Hexane

Not detected

Dichloromethane

Not detected

Ethyl acetate

0.834

CHCl3:MeOH (60:40, v/v)

1.229

Ethanol

0.817

MeOH:water (60:40, v/v)

0.041

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197

(0.238%) with n-hexane was observed. Further, the percentage of OA content was found maximum in extract obtained from a mixture of chloroform:MeOH (60:40, v/v), while the percentage of OA found minimum in extract obtained from a mixture of MeOH:water (60:40, v/v). However, OA
was not detected in extract obtained from n-hexane, and dichloromethane
and results were summarized in Table V.

Conclusion
An ecofriendly, simple, precise, and relatively cost-effective MAEHPLC
DAD method was developed for maximum extraction and isolation of OA
from roots of L. camara. Moreover, a mixture of chloroform:MeOH (60:40,
v/v) is proposed as most favorable solvent for MAE of OA to get the maximum yield. High extraction efficiency, less labor cost, minimum uses of solvent, ease, and rapidity are the advantages of performing the extraction using microwave rather than other conventional methods. The HPLCPDA
method was developed and validated in compliance with the International
Conference on Harmonization (ICH) guidelines 1997 and is found to be
suitable for the determination of the individual triterpenoid in extracts with
excellent precision, accuracy, and linearity. The method of sample preparation and assay procedure is simpler and more rapid than reported methods.
Therefore, we suggested that the proposed method may be helpful for rapid
isolation of OA with maximum yield from L. camara roots for Pharma industries, and it may also be useful for quantitative analysis of oleanolic acid in
its formulations for quality control purposes.

Acknowledgment
The authors are grateful to Dr. R.M. Johari, officiating Principal, and Dr.
Ayodhya Singh, Head of the Chemistry Department, M.M.H. College,
Ghaziabad and Director General, CCRAS, New Delhi for providing necessary facilities in completion of this project. The authors are grateful to Mr.
Ramesh N., Application Chemist, Agilent Technology, Bangalore for necessary help related to the study. The authors also appreciate the kind help extended by Dr. D. K. Aggarwal, R.O. (Botany), CCRAS and Dr. H.B. Singh,
Scientist, NISCAIR, New Delhi for plant material identification.

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Accepted by MWH