High-Performance Thin-Layer Chromatographic Analysis for ... · 10/12/2019 · purpurea L. was tested and a high-performance thin-layer chromatography (HPTLC) determination of antioxidants

Journal of Planar Chromatography 29 (2016) 3 423

SummaryA high-performance thin-layer chromatography (HPTLC) meth-od for the simultaneous quantitative determination of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia leaves was developed for the first time. For achieving good separation, a mobile phase of toluene–ethyl acetate–formic acid (8:2:0.1, v/v) was used. The densitometric determination was carried out at 550 nm and 520 nm in reflection–absorption mode for lupeol and ursolic acid, respectively, which were linear in the range of 100–600 ng per band. During the analysis, lupeol (0.15%) and ursolic acid (0.11%) were found to be the highest in the leaves of B. acuminata. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the obtained data proves that the method is reproducible and selective and can be used for the routine analysis of the reported terpenoids in crude drug and extracts. The simultaneous quantification of these compounds has not yet been reported in the leaves of the studied Bauhinia spe-cies which may be utilized for the proper standardization of these species.

1 Introduction

The genus Bauhinia belonging to the family Caesalpiniaceae consists of over 250 species of trees and shrubs [1]. All Bau-hinia species have characteristic butterfly-shaped leaves and they are used in many traditional medicinal applications for the treatment of different kinds of ailments [2, 3].

Bauhinia purpurea L., Bauhinia variegata L., Bauhinia acumi-nata L., and Bauhinia tomentosa L., commonly known as

A. Gupta, S. Verma, and A.K.S. Rawat, Pharmacognosy and Ethnopharmacolo-gy Division, CSIR-National Botanical Research Institute, Lucknow, India; and A. Gupta and H. Dwivedi, School of Pharmacy, Babu Banarsi Das University, Faizabad Road, Lucknow, India.E-mail: [email protected]

Kanchanar in Hindi, are usually found in Southeast Asia [4–7]. The aerial parts of the plant are reported to contain amino acids, flavonoids, steroids, terpenoids, tannins, lactones, gly-colipids, glycosyl steroids, quinines, phenyl fatty ester, pacha-rin, bauhiniastatins, β-sitosterol, flavanones, and dihydrod-ibenzoxepins as well as bibenzyls [8–12], lutein, isoquercetin, astragalin, etc. [13].

All these four plant species have been used in traditional med-icine for the cure of body pain, rheumatism, fever [14], dropsy, skin diseases, septicemia, diarrhea, dysentery, hemorrhoids, piles, obesity, and stomatitis and as tonic, astringent, laxative, anthelmintic, antileprotic, antigoitrogenic, and carminative as well as antidote for snake poisoning, dyspepsia, and flatulence [15–18]. Several activities like antidiabetic [19], antibacterial [20, 21], wound-healing activity, antioxidant activity, antima-larial, antimycobacterial, antifungal, antimicrobial [22], and anti-cancerous activities have been reported [11].

The effect of extraction techniques on the phenolic content as well as on the antioxidant and antimicrobial activities of B. purpurea L. was tested and a high-performance thin-layer chromatography (HPTLC) determination of antioxidants was performed by Annegowda et al. [23]. Simultaneous determi-nation of the major flavonoids (apigenin, ursolic acid, rutin, luteolin, and quercitrin) in B. variegata has been performed earlier [24]. The paper chromatography of flavonoids showed the presence of kaempferol, ursolic acid, and apigenin in B. acuminata L. [25]. Phytol, sesquiterpenoids, β-caryophyl-lene, and caryophyllene oxide were identified as the major constituents in B. acuminata L. leaf oil [26]. Several chem-ical compounds including palmitic acid, three phthalic acid esters, phthalic acid, gallic acid, and ursolic acid were iden-tified from the leaves of B. acuminata [27]. Some researchers have performed the qualitative HPTLC analysis of B. tomen-tosa L. leaves and flowers [28, 29]. We have earlier performed the simultaneous estimation of four phenolic compounds in B. purpurea L., B. variegata L., and B. acuminata L. flow-ers and floral buds [30]. The simultaneous quantification with method validation of lupeol and ursolic acid has not yet been reported in these species under study which may be utilized for the proper standardization of these species.

High-Performance Thin-Layer Chromatographic Analysis for the Simultaneous Quantification of Lupeol and Ursolic Acid in the Methanolic Fraction of Four Different Species of Bauhinia

Abhishek Gupta, Shikhar Verma, Harinath Dwivedi, and Ajay Kumar Singh Rawat*

Key Words:

BauhiniaHigh-performance thin-layer chromatographyLupeolUrsolic acid

Journal of Planar Chromatography 29 (2016) 6, 423–428 DOI: 10.1556/1006.2016.29.6.40933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

HPTLC Analysis for the Simultaneous Quantification of Lupeol and Ursolic Acid

424 Journal of Planar Chromatography 29 (2016) 6

2 Experimental

2.1 Chemicals and Reagents

HPTLC analyses were performed on 20 cm × 10 cm HPTLC silica gel 60 F254 (0.25 mm) plates (Merck, Darmstadt, Ger-many). Lupeol and ursolic acid were supplied by Sigma-Aldrich (Munich, Germany). All the reagents used in the experiment were of analytical grade and were supplied by Merck.

2.2 Preparation of Standard Solutions

The stock solutions of lupeol and ursolic acid were prepared separately by dissolving them (0.1 mg mL−1) in methanol.

2.3 Plant Material

The plant material, i.e., leaves of B. purpurea L., B. variegata L., B. acuminata L., and B. tomentosa L., were collected from Lucknow, U.P., India (Figure 1). The plant was identified and authenticated by Dr. Tariq Hussain, CSIR-NBRI, and voucher specimens were submitted in LWG herbarium.

2.4 Sample Preparation

The fresh leaves of three different Bauhinia species were col-lected, thoroughly washed with water to remove all debrises. The plant materials were shade-dried and powdered by using electric grinder at 60 mesh size. Extraction was performed by the soxhletion method. Firstly, the powdered plant material was defatted under Soxhlet assembly using 250 mL of 98% petroleum ether for 6 h. This was followed by 9 h soxhletion of defatted powder by using 250 mL chloroform, followed by methanol. The final methanolic fraction obtained was passed through Whatman No. 1 filter paper. The obtained filtrate was concentrated under vacuum in a rotary evaporator at 40°C and stored at 4°C for further use. The dried extracts were dissolved in 98% methanol to obtain a stock solution of 10 mg mL−1 which was used for the application (10 µL) of bands on HPTLC plates.

2.5 Development of HPTLC Fingerprinting of Lupeol and Ursolic Acid

2.5.1 Instrumentation and Chromatographic Conditions

The following were the instruments and chromatographic con-ditions used: spotting device: Linomat V automatic sample applicator, CAMAG (Muttenz, Switzerland); syringe: 100 μL

Hamilton (Bonaduz, Switzerland); TLC chamber: glass twin-trough chamber (20 cm × 10 cm × 4 cm), CAMAG; densitom-eter: TLC Scanner 3 linked to winCATS software V. 4.06, CAMAG; HPTLC plates: 20 cm × 10 cm, 0.2 mm thickness precoated with silica gel 60 F254, E. Merck (Darmstadt, Ger-many). The following were the experimental conditions used: band length: 6 mm, distance between tracks: 11.3 mm; tem-perature, 25 ± 2°C; relative humidity, 40%. A misture of tolu-ene–ethyl acetate–formic acid (8:2:0.1) was used as the solvent system, and the HPTLC chamber was saturated for a period of 45 min. The detection wavelength was 550 nm for lupeol and 522 nm for ursolic acid. The TLC plate was dipped in anisalde-hyde–sulfuric acid reagent, then used as a visualization agent, and heated in hot-air oven at 40°C for a period of 3 min [31]. The slit dimension was 5.00 mm × 0.45 mm; scanning speed, 10 mm s−1; and source of radiation, deuterium lamp.

2.5.2 Calibration Curves of Lupeol and Ursolic Acid

The stock solutions of lupeol (94% pure) and ursolic acid (90% pure) (100 μg mL−1) were prepared in HPLC grade methanol. Different volumes of the stock solutions (0.5–5.0 μL) were applied on the TLC plate to obtain concentrations of 100–600 ng per band of lupeol and ursolic acid, respectively. The data of peak areas plotted against the corresponding concentra-tions were treated by least square regression analysis method validation.

2.6 Method Validation

The method was validated according to the International Con-ference on Harmonization (ICH) guidelines [32, 33], and the statistical analysis was done using Excel 2000 (MS Office®).

2.6.1 Precision

Repeatability studies of the sample application and measure-ment of the peak area were carried out using nine determinants (3 concentrations/3 replicates) covering the specified ranges for the procedure (200, 400, and 600 ng per band of lupeol and ursolic acid) and they were expressed in terms of relative standard deviation (RSD). Intra- and inter-day variations for the determination of lupeol and ursolic acid were examined on three different concentration levels of 200, 400, and 600 ng per band. The acceptance criteria for a procedure’s repeatability or intermediate precision are based on the intended use of the ana-lytical method.

2.6.2 Robustness of the Method

By introducing small changes in the mobile-phase composition, mobile-phase volume, duration of mobile-phase saturation, and activation of prewashed TLC plates with methanol, the effects on the results were examined. The robustness of the method was tested in triplicate at a concentration level of 200 ng per band for lupeol and ursolic acid, and the RSD and SD of peak areas were calculated.

2.6.3 Limit of Detection and Limit of Quantitation

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank methanol was applied six times and the signal-to-noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally

Figure 1

The morphologies of four different Bauhinia species.



verified by diluting the known concentrations of lupeol and ursolic acid until the average responses were approximately 3 or 10 times of the responses for six replicate determinations.

2.6.4 Recovery

The preanalyzed samples were spiked with extra 50, 100, and 150% of the standard lupeol and ursolic acid, and the mixtures were reanalyzed by the proposed method. The experiment was

conducted six times. This was done to check the recovery of the targeted analytes at different levels in the plant extracts.

2.6.5 Ruggedness

Lupeol and ursolic acid solutions of concentration 200 ng per band were prepared and analyzed on day 0 and after 6, 12, 24, 48, and 72 h. The data were treated for % RSD to assess the ruggedness of the method.

Figure 2

(A) HPTLC plate showing tracks of standard markers and samples. (B) HPTLC densitogram showing peaks of standards and samples. (C) Peaks of BPLM. (D) Peaks of BVLM. (E) Peaks of BALM. (F) Peaks of BTLM.



2.6.6 Specificity

The specificity of the method was confirmed by analyzing the standard drugs and the extract. The band for lupeol and ursolic acid in the sample was confirmed by comparing the Rf values and spectra of the band with those of the standard. The peak purities of lupeol and ursolic acid in the plant extracts were assessed by comparing the spectra at three different levels, viz., peak start (S), peak apex (M), and peak end (E) positions of the band.

3 Results and Discussion

In this study, several solvent systems used for the individual estimation of these terpenoids were investigated to evaluate the combinatorial separation of these compounds in a single sol-vent system and between different components of the extract. Among the different solvents systems investigated, the mobile phase consisting of toluene–ethyl acetate–formic acid in the ratio of 8:2:0.1 (v/v) demonstrated compact bands with typical

Figure 3

(A) Peak of standard lupeol. (B) Peak of standard ursolic acid. (C) Spectra of lupeol. (D) Spectra of ursolic acid. (E) Calibration curve of lupeol. (F) Calibration curve of ursolic acid.



Gaussian shaped peaks with good resolution between other peaks of the extract.

The procedure for the separation and determination of differ-ent compounds in the methanolic fraction of Bauhinia using HPTLC–densitometry is reported at six-point calibration curve in which lupeol and ursolic acid were observed and quantified with method validation (Table 1). HPTLC chromatogram and densitograms were obtained from standard compounds and methanolic fractions (Figure 2), and both targeted compounds were identified by retention factor (Rf), peak purity, and overlaid ultraviolet (UV) spectrum (Figure 3). Precision studies have been performed by analyzing intra- and inter-day variation for the determination of these terpenoids which was carried out at three different concentration levels of 200, 400, and 600 ng per band; the mean percentage relative standard deviation val-ues were found to be 0.31 and 0.53 for lupeol and ursolic acid, respectively, in intra-day analysis, while inter-day analysis showed mean percentage relative standard deviation values of 1.42 and 1.45 for lupeol and ursolic acid, respectively, showing a good precision (Table 2). For recovery studies, preanalyzed samples of different Bauhinia species were spiked with extra 50, 100, and 150% of the standard compounds and the mixtures were reanalyzed which shows a good recovery ranging from

97.14% to 100.05% for lupeol and 97.96% to 99.20% for ursolic acid. The experimental data are expressed as mean percentages of the recovered analytes and standard deviation, and relative standard deviation is also presented (Table 3). The concentra-tions of lupeol and ursolic acid in the methanolic fraction of B. purpurea L., B. variegata L., B. acuminata L., and B. tomen-tosa L. are represented in Table 4.

4 Conclusion

A validated HPTLC analytical method has been developed for the simultaneous determination of lupeol and ursolic acid in the leaves of B. purpurea L., B. variegata L., B. acuminata L., and B. tomentosa L. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the obtained data proves that the method is reproducible and selective and can be used for the routine analysis of the reported compounds in crude drug and extracts. The method can be used to deter-mine the purity and identity of different Bauhinia species avail-able from various sources by detecting the related impurities as well as for the quality control of herbal formulations containing Bauhinia leaves as ingredients. The HPTLC analysis has indi-cated the presence of optimum amount of lupeol and ursolic acid in the samples. This can be used in the pharmaceutical industry as a pharmacognostical tool to identify these medic-inally important plant species. In addition, it can be adopted as a chemo-taxonomical tool in plant systematics. Further, the identification of other terpenoids and their separation and char-acterization from the plants are to be evaluated and reported in the near future.

Table 1

Summary of validation parameters.

Parameters Lupeol Ursolic

Rf 0.46 ± 0.01 0.68 ± 0.01

Linearity range 100–600 100–600

Regression via area y = 18.847*x + 29.620 y = 21.577*x − 1617.168

r 0.996 0.998

Slope 18.847 21.577

Intercept 29.620 1617.168

LOD (ng) 40 35

LOQ (ng) 100 100

Scanning (nm) 550 522

Table 2

Intra-day and inter-day precisions.

Standard markers

Conc. (ng band−1)

Intra-day Inter-day

% RSD Mean RSD % RSD Mean RSD

Lupeol

200 0.53 1.69

400 0.62 0.54 1.53 1.47

600 0.47 1.19

Ursolic acid

200 0.68 1.32

400 0.42 0.51 1.73 1.37

600 0.44 1.07

Table 3

Recovery analyses of lupeol and ursolic acid.

Standards Amount added (%) Amount recovered (%) Mean SD RSD

Lupeol

50 97.62 1.61 1.65

100 99.05 1.14 1.15

150 97.80 1.30 1.32

Ursolic acid

50 99.31 1.27 1.28

100 101.07 1.15 1.14

150 97.92 1.00 1.02

Table 4

Quantification of lupeol and ursolic acid in the methanolic fraction of different Bauhinia species.

Plant sample Lupeol (%) Ursolic acid (%)

BPLM 0.035 0.06

BVLM 0.043 0.03

BALM 0.15 0.11

BTLM 0.028 0.03



Acknowledgment

The authors are thankful to Director, CSIR-NBRI, for provid-ing all the facilities to conduct this research work.

Funding

This work was supported by the Council of Science and Tech-nology, Uttar Pradesh, India.

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Ms received: May 19, 2016Accepted: September 22, 2016

High-Performance Thin-Layer Chromatographic Analysis for ... · 10/12/2019 · purpurea L. was tested and a high-performance thin-layer chromatography (HPTLC) determination of antioxidants

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