oleanolic acid of Lantana

Acta Chromatographica 25(2013)1, 181-199 DOI: 10.1556/AChrom.25.2013.1.12

02312522 2012 Akadmiai Kiad, Budapest

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

1Central Council for Research in Ayurvedic Sciences, 61-65, Institutional Area, Opp.-D-Block, Janak-

puri, New Delhi-110058, India 2Department of Chemistry, M. M. H. College, Ghaziabad, U.P., India

*E-mail: [email protected]

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 tem-perature 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 evi-denced from the recovery studies performed with standard oleanolic acid. The proposed method also showed high degree of reproducibility; hence, it may be useful for maxi-mum 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 wide-spread 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,

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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, tu-mors, 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 anti-microbial activity and promising anti-hyperglycemic activity against al-loxan-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 infec-tions 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, verbas-cotetraose, 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 hepa-toprotective [23] etc., and, in fact, the rootlets and root bark of L. camara provide a plentiful (2%) supply of OA [18].

HOH

H

O

OH

H

20

18

13

15

27

12

2611

810

25

53

1

23 24

2

46

7

9 14 16

17

19 21

22

28

30 29

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

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Optimum extraction is the pivotal step in the recovery of active ingre-dients from plant matrix. Conventional extraction methods like solvent ex-traction, 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 con-stituents 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 consump-tionpreventing pollution in analytical laboratories and reducing sample preparation costs [20, 21]. Microwave assisted extraction (MAE), supercriti-cal 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 method-ology [25]. These techniques are often time consuming and require large volume of organic solvent whose subsequent disposal creates severe envi-ronmental 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 repro-ducibility, and low consumption of organic solvents. MAE is based upon the selective and rapid localized heating of moisture in the sample by mi-crowaves. 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 mi-crowave energy for the performance evaluation of a rapid, reliable, and re-producible 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 quantita-tive analysis of OA by high-performance liquid chromatographydiode ar-ray detector (HPLCDAD) method are studied in order to determine the optimum extraction and isolation of OA from the roots of L. camara. Fur-thermore, the contents of OA in different polarity microwave-assisted ex-tracts of L. camara were determined with the developed MAEHPLC tech-niques.

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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 In-stitute 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 pow-dered 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 sol-vent 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) manufac-tured 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; ex-haust 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 de-gasser, an auto-sampler, a diode array detector (DAD), and 20-L injector loop. Gradient mixture of buffer solution (Solvent A) prepared by dissolv-ing 1.36 g of potassium dihydrogen phosphate in 900 mL Milli-Q water, ad-justed 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 (%) Flow rate and max 0 90 10

6 70 30

9 15 85

15 15 85

16 90 10

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 non-polar 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 ex-tract

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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800 W were applied for 4, 6, 8, and 10 min, respectively, for maximum ex-traction 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 be-tween two irradiations. Each extract was filtered by using Whatman filter paper no. 1 and the solvents were removed under vacuum at 50 C, sepa-rately and lyophilized till each extract was free from solvents. The concen-trated 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


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with ethanol (99%), which showed single spot on thin-layer chromatogra-phy (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 cross-checked by Co-TLC and MMP with an authentic sample of oleanolic acid.

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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. Approxi-mately 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 solu-tion 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 rang-ing 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).

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min0 2 4 6 8 10 12 14 16

mAU

0

100

200

300

400

500

600

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

11.

507

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

min0 2 4 6 8 10 12 14 16

mAU

0

100

200

300

400

500

600

700

800

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

1.50

8 -

OA

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

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


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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 so-lution (0.4 mg mL1) in to HPLC and analyzed by the proposed method to determine variations due to the chromatographic conditions (system preci-sion) as shown in Table III. To determine variations due to the HPLC sys-tem, 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).

nm200 225 250 275 300 325 350 375

Norm.

0

50

100

150

200

250

300

350

400

*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

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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 calcu-lated 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 mole-cule. 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 triter-penoid at 5.43 (1H, brs, H-12) suggesting an olea-12-ene skeleton. One me-thine 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 an-hydridepyridine 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 be-tween the two triterpenes is at C-29 and C-30 of the ring E. In type triter-penoid (ursolic acid), both the methyl groups at ring E are secondary whereas in type triterpene (oleanolic acid), both the methyl groups are ter-tiary. 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 pro-tons 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-


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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 dif-ferent 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 ex-traction method, the polarity of solvent is an important factor to extraction yield. However, the dielectric constant and dissipation factor of solvent sig-nificantly influence the extraction yield in MAE [32]. Different solvent vol-umes (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 (mi-crowave 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) 4 6 8 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-

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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 mi-crowave power on the extraction yield of OA was calculated (Table IV). Three intervals were observed in the process of microwave extraction. Ini-tially, a short rise in extraction yield between 4 min and 6 min which indi-cates the first quantities extracted, located at the surface of root particles representing approximately 1.18% of the OA. Extraction yield at 6 min re-sulted to the highest yield of OA (1.23%) due to intern warming of the natu-ral moisture located in the plant cells. Since no significant difference in ex-traction 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 charac-teristics of OA, C18 columns are preferred for the HPLC analysis. Prelimi-nary studies were performed under isocratic conditions using a ODS (Wa-ters) 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 per-formed on a reversed-phase column, the most polar compound eluted ear-lier 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 ex-pense of reducing the resolution of isomeric peaks [34]. An ACN-containing mobile phase was too high to separate isomeric overlapping peaks with a


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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 bet-ter 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 SB-AQ 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 mo-bile 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 injec-tion 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 chroma-tographic profile, and the data obtained for the standards and samples, con-sidering the following parameters like retention time, maximum wave-length 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 pu-rity 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) ob-tained from regression analysis are shown in Table III. The calibration curve was linear in the tested concentration range (0.040.5 mg mL1); good corre-lation coefficients (r2) were found greater than 0.999 for OA (Table III), indi-cating 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 quanti-tated with acceptable accuracy and precision. LOQ of OA was found 26.8 g mL1 (Table III). These results indicate that the method provided sat-isfactory 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 ob-tained 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 re-sults were in accord with the required criteria. Therefore, the proposed method was found to be most suitable for rapid extraction and simultane-ous 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 al-though 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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(0.238%) with n-hexane was observed. Further, the percentage of OA con-tent was found maximum in extract obtained from a mixture of chloro-form:MeOH (60:40, v/v), while the percentage of OA found minimum in ex-tract 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 MAEHPLCDAD 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 maxi-mum yield. High extraction efficiency, less labor cost, minimum uses of sol-vent, ease, and rapidity are the advantages of performing the extraction us-ing 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 prepara-tion 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 indus-tries, 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 neces-sary facilities in completion of this project. The authors are grateful to Mr. Ramesh N., Application Chemist, Agilent Technology, Bangalore for neces-sary help related to the study. The authors also appreciate the kind help ex-tended by Dr. D. K. Aggarwal, R.O. (Botany), CCRAS and Dr. H.B. Singh, Scientist, NISCAIR, New Delhi for plant material identification.

S.C. Verma et al.

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oleanolic acid of Lantana