Validated Simultaneous High-Performance Thin …...2019/10/21 · green, climbing, much branched...

Journal of Planar Chromatography 29 (2016) 3 1

Summary

A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination and validation of ursolic acid, β-sitosterol, lupeol, and quercetin in the methanolic fraction of Ichnocarpus frutescens L. 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. Densitometric determination was carried out at 500 nm for ursolic acid, 550 nm for β-sitosterol, 650 nm for lupeol, and 310 nm for quercetin in re-flection–absorption mode, and the calibration curves were linear in the range of 100–600 ng per spot. During the analysis, the meth-anolic fraction of I. frutescens L. showed the presence of ursolic acid (0.34%), β-sitosterol (0.27%), lupeol (0.27%), and quercetin (0.26%). The proposed method is simple, precise, specific, and ac-curate. The obtained data can be used for routine analysis of re-ported biomarkers in crude drug and extracts. The simultaneous quantification and method validation of these biomarkers have not yet been reported in I. frutescens L., which may be utilized for the proper standardization of the plant.

J. Dwivedi and A.K.S. Rawat, Pharmacognosy & Ethnopharmacology Division, CSIR ‒ National Botanical Research Institute, Lucknow, India; A. Gupta, Pran-veer Singh Institute of Technology, Department of Pharmacy, Kanpur, India; S. Verma, Amity Institute of Pharmacy, Amity University, Lucknow, India; and S. Paliwal and J. Dwivedi, Department of Pharmacy, Banasthali Vidyapeeth, Banasthali, India.*E-mail: [email protected]

1 Introduction

Ichnocarpus frutescens L. (Apocynaceae) is a large ever-green, climbing, much branched shrub, ascending up to an altitude of 4000 ft, and is found almost throughout India [1]. The leaves of I. frutescens are used extensively in the form of decoction for its antidiabetic activity by the tribes of Karna-taka and Utter Pradesh states, for treating diabetes [2]. Some of the constituents of the plant, such as polyphenols and flavo-noids, were shown to have jaundice, antioxidant, anti-cancer, and hepatoprotective activities [3–7]. The leaves are boiled in oil and applied in headaches and fevers; they are also applied to wounds [1]. The whole plant is used as a tribal medicine in atrophy, bleeding gums, cough, and dysentery [8]. Stalk and leaves in decoction are used in the treatment of skin erup-tions [9], antiurolithiatic activity [10]. Diabetes, hyperlipi-demia, anti-inflammatory, antioxidant activity, and alpha-glu-cosidase inhibitory activity of this plant have been reported [11–16]. It is also used by some tribes in atrophy, convulsions, delirium, dysentery, measles, splenomegaly, tuberculosis, abdominal, and glandular tumors, and its roots are used as alterative, antidysenteric, and antipyretic agents [17–19]. The active constituents in I. frutescens include flavonoids, simple phenolic acids, coumarins, and pentacyclic triterpenoids [20]. The plant contains a wide range of iridoid glycosides, triter-penoids [21], and polyphenolic compounds, but it contains no alkaloids [22], α-amyrin and its acetates, lupeol and its acetates, friedelin, epifriedelinol, and β-sitosterol from its stems [23, 24]. Its leaves mainly contain flavones and glycoflavones [25], ursolic acid acetate, kaempferol, kaempferol-3-galactoside (tri-folin), and mannitol [26], and its flowers contain quercetin and quercetin-3-O-β-D-glucopyranoside [27]. Isolation of n-butyl oleate, n-octyl tetracontane, tetratriacontadiene, n-nonadeca-nyl benzoate, and benzocosanylarachidate is reported from the stems of I. frutescens [28]. Thin-layer chromatography (TLC) fingerprinting of I. frutescens was performed earlier [29–31]. The simultaneous quantification with method validation of ursolic acid, β-sitosterol, lupeol and quercetin in I. frutescens has not yet been reported, which may be utilized for the proper standardization of this important medicinal plant.

Validated Simultaneous High-Performance Thin-Layer Chromatographic Analysis of Ursolic Acid, β-Sitosterol, Lupeol, and Quercetin in the Methanolic Fraction of Ichnocarpus frutescens

Jyotsana Dwivedi, Abhishek Gupta, Shikhar Verma, Sarvesh Paliwal, and Ajay Kumar Singh Rawat*

Key Words:

β-SitosterolHigh-performance thin-layer chromatographyIchnocarpus frutescensLupeolQuercetinUrsolic acid

Journal of Planar Chromatography 32 (2019) 2, 1–6 DOI: 10.1556/1006.2019.32.2.00933-4173 © Akadémiai Kiadó, Budapest

Validated Simultaneous HPTLC Analysis of Ursolic Acid, β-Sitosterol, Lupeol, and Quercetin in I. frutescens

2 Journal of Planar Chromatography 32 (2019) 2

2 Experimental

2.1 Chemicals and Reagents

High-performance thin-layer chromatography (HPTLC) anal-yses were performed on Merck 20 cm × 10 cm HPTLC silica gel 60 F254 (0.25 mm) plates. Ursolic acid, β-sitosterol, lupeol, and quercetin were supplied by Sigma Aldrich (Darmstadt, Germany). All other reagents used in the experiment were of analytical grade and were supplied by Merck (Darmstadt, Germany).

2.2 Preparation of Standard Solutions

Stock solutions of ursolic acid, β-sitosterol, lupeol, and querce-tin were prepared separately by dissolving 0.1 mg mL−1 of these in methanol.

2.3 Plant Material

The plant material, i.e., aerial parts of I. frutescens L., was col-lected from the Council of Scientific & Industrial Research-Na-tional Botanical Research Institute (CSIR-NBRI), Lucknow, India. The plant was identified and authenticated by Dr. A.K.S. Rawat, and voucher specimens were submitted in LWG herbarium.

2.4 Sample Preparation

Fresh leaves of I. frutescens were collected and thoroughly washed with water to remove all debris. The plant materials were shade-dried and powdered by using an electric grinder with a 60 mesh size. Extraction was performed by soxhlation method. First, 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 soxhlation 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 filtrate obtained 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 is used for appli-cation of spots on the HPTLC plates.

2.5 Development of HPTLC Fingerprinting of Quercetin, Ursolic Acid, Lupeol and β-Sitosterol

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). HPTLC chamber: glass twin-trough chamber (20 cm × 10 cm × 4 cm, CAMAG). Densi-tometer: HPTLC Scanner 3 linked to winCATS software V. 4.06 (CAMAG). HPTLC plates: 20 cm × 10 cm, 0.2-mm thickness pre-coated with silica gel 60 F254 (E. Merck, Darmstadt, Ger-many). Band size: 6 mm; slit dimension: 5.00 mm × 0.45 mm; scanning speed: 10 mm s−1; and source of radiation: deuterium lamp. The extract with 10 mg mL−1 concentration was applied on HPTLC plates with 10-µL volume. Experimental condi-tions: a temperature of 25 ± 2°C and relative humidity of 40%.

Solvent system: toluene–ethyl acetate–formic acid (8:2:0.1). Detection wavelengths were 500 nm for ursolic acid, 550 nm for β- sitosterol, 650 nm for lupeol, and 310 nm for quercetin, and for post-chromatographic treatment, anisaldehyde–sulfuric acid reagent was used as the visualization agent.

2.5.2 Calibration Curve of Quercetin, Ursolic Acid, Lupeol, and β-Sitosterol

Stock solutions of ursolic acid, β-sitosterol, lupeol, and querce-tin (100 μg mL−1) were prepared in HPLC-grade methanol. Different volumes of the stock solutions were spotted on the HPTLC plate to obtain concentrations of 100–600 ng per band of ursolic acid, β-sitosterol, lupeol, and quercetin, respectively. The data of peak areas plotted against the corresponding con-centrations were treated by least squares regression analysis method validation.

2.6 Method Validation

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

2.6.1 Specificity

The specificity of the method was confirmed by analyzing the standard drugs and the extract. The band for ursolic acid, β-si-tosterol, lupeol, and quercetin in the sample was confirmed by comparing the RF values and spectra of the band with those of the standard. The peak purity of ursolic acid, β-sitosterol, lupeol, and quercetin was assessed by comparing the spectra at 3 different levels, viz., peak start (S), peak apex (M), and peak end (E) positions of the band.

2.6.2 Robustness of the Method

By introducing small changes in the mobile phase composi-tion, mobile phase volume, duration of mobile phase saturation, and activation of prewashed HPTLC plates with methanol, the effects on the results were examined. The robustness of the method was determined in triplicate at a concentration level of 200 ng per band for ursolic acid, β-sitosterol, lupeol, and quercetin and the relative standard deviation (RSD) and SD of peak areas were calculated.

2.6.3 Limit of Detection and Limit of Quantification

In order to estimate the limit of detection (LOD) and limit of quantification (LOQ), blank methanol was spotted 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 verified by diluting the known concentrations of ursolic acid, β-sitosterol, lupeol, and quercetin until the average responses were approximately 3 or 10 times of the responses for 6 repli-cate determinations.

2.6.4 Ruggedness

Ursolic acid, β-sitosterol, lupeol, and quercetin solutions of concentration 200 ng per band were prepared and analyzed on day 0 and after 6, 12, 24, 48, and 72 h. Data were treated for %RSD to assess the ruggedness of the method.



2.6.5 Accuracy

The pre-analyzed samples were spiked with extra 50, 100, and 150% of the standard ursolic acid, β-sitosterol, lupeol, and quercetin, 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 formulations.

2.6.6 Precision

The repeatability of the sample application and measurement of peak area was determined using nine determinants (3 con-centrations per 3 replicates) covering the specified range for the procedure (200, 400, and 600 ng per band of ursolic acid, β-si-tosterol, lupeol and quercetin) and it was expressed in terms of

%RSD. The intra- and inter-day variations for the determination of ursolic acid, β-sitosterol, lupeol, and quercetin were carried out at 3 different concentration levels of 200, 400, and 600 ng per band. The acceptance criteria for a procedure’s repeatabil-ity or intermediate precision are based on the intended use of the analytical method.

3 Results and Discussion

Among the different mobile phases investigated, the mobile phase consisting of toluene–ethyl acetate–formic acid in the ratio of 8:2:0.1 (v/v) demonstrated good resolution between other peaks of the extract.

Table 1

Summary of validation parameters.

Parameters Ursolic acid β-Sitosterol Lupeol Quercetin

RF 0.66 ± 0.005 0.70 ± 0.005 0.75 ± 0.005 0.42 ± 0.005

Linearity range [ng] 100–600 100–600 100–600 100–600

Regression via area y = 11.058x + 58.942 y = 3.7646x + 101.12 y = 8.523x + 399.73 y = 10.157x − 18.467

r 0.9996 0.9994 0.9991 0.9996

Slope 11.058 3.7646 8.523 10.157

Intercept 58.942 101.12 399.73 18.467

LOD [ng] 60 80 50 45

LOQ [ng] 181.80 242.40 151.5 136.35

Scanning [nm] 500 550 650 310

Figure 1

HPTLC profile of Ichnocarpus frutescens with standard marker compounds (ursolic acid, quercetin, β-sitosterol, and lupeol).



The procedure for the separation and determination of differ-ent biomarkers in methanolic fraction of I. frutescens using HPTLC–densitometry is reported at 6-point calibration curve, in which ursolic acid, β-sitosterol, lupeol, and quercetin were quantified with method validation (Table 1). The HPTLC pro-file (Figure 1) with chromatograms was obtained for standard compounds and plant extracts, and both targeted compounds were identified by retention factor (RF) as well as peak purity (Figures 2). Precision studies have been performed by ana-lyzing intra- and inter-day variations for the determination of these compounds, which were carried out at concentration lev-els of 200, 400, and 600 ng per band; mean percentage relative

standard deviation values were found to be 0.67 for ursolic acid, 0.86 for β-sitosterol, 0.83 for lupeol, and 0.59 for querce-tin, respectively, in intra-day analysis, while inter-day analy-sis showed mean percentage relative standard deviation values of 1.87 for ursolic acid, 1.81 for β-sitosterol, 1.78 for lupeol, and 1.59 for quercetin, respectively, which shows good preci-sion (Table 2). For recovery studies, pre-analyzed samples of I. frutescens were spiked with extra 50%, 100%, and 150% of the standard compounds, and the mixtures were reanalyzed which shows a good recovery ranging from 96.89% to 98.69% for ursolic acid, 97.16% to 98.61% for β-sitosterol, 97.11% to 98.81% for lupeol, and 97.67% to 100.07% for quercetin. The

Figure 2

HPTLC chromatograms of Ichnocarpus frutescens with standard marker compounds (quercetin in the UV range without derivatization and ursolic acid, Β-sitosterol, and lupeol in the visible range after derivatization).

Table 2

Intra-day and inter-day precisions.

Standard marker Conc. [ng per band]Intra-day Inter-day

%RSD Mean RSD %RSD Mean RSD

Ursolic acid

200 0.53 1.91

1.87400 0.78 0.67 1.73

600 0.69 1.97

β-Sitosterol

200 0.88 1.72

1.81400 0.78 0.86 1.79

600 0.92 1.91

Lupeol

200 0.92 1.72

1.78400 0.91 0.83 1.85

600 0.66 1.77

Quercetin

200 0.69 1.69

1.59400 0.56 0.59 1.74

600 0.52 1.35



experimental data are expressed as mean percentages of the recovered analytes, standard deviation and relative standard deviation, which are also presented in Table 3. The concentra-tions of ursolic acid, β-sitosterol, lupeol and quercetin in meth-anolic fraction of I. frutescens are represented in Table 4.

Table 3

Recovery analysis of of ursolic acid, β-sitosterol, lupeol, and quer-cetin.

Standard Amount added [%]

Amount recovered [%] Mean SD RSD

Ursolic acid

50 96.89 1.85 1.91

100 98.69 2.88 2.92

150 97.88 1.27 1.30

β-Sitosterol

50 98.52 1.60 1.63

100 97.16 2.21 2.27

150 98.61 1.71 1.77

Lupeol

50 98.06 2.36 2.40

100 97.11 1.79 1.84

150 98.81 1.77 1.79

Quercetin

50 100.07 2.28 2.28

100 99.28 1.32 1.33

150 97.67 2.01 2.06

Table 4

Quantification of ursolic acid, β-sitosterol, lupeol, and quercetin in the methanolic fraction of Ichnocarpus frutescens.

Plant sampleUrsolic acid β-Sitosterol Lupeol Quercetin

[%]

Ichnocarpus frutescens 0.34 0.27 0.27 0.26

4 Conclusion

A validated HPTLC analytical method has been developed for the simultaneous determination of ursolic acid, β-sitosterol, lupeol, and quercetin in I. frutescens. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the data obtained proves that the method is reproducible and selective, and can be used for routine analysis of the reported compounds in crude drug and extracts. The method can be used to determine the purity of I. frutescens available from various sources by detecting the related impurities as well as for the quality control of herbal formulations containing I. frutescens as an ingredient. HPTLC analysis has indicated the presence of quantifiable amount of ursolic acid, β-sitosterol, lupeol, and quercetin in the samples. This can be used in the pharma-ceutical industry as a pharmacognostical tool to identify this medicinally important plant. In addition, it can be adopted as a chemo-taxonomic tool in plant systematics. These 4 biomarkers

from I. frutescens were simultaneously quantified for the first time. Further, isolation of other compounds and their character-ization from the plant and in vivo anti-diabetic activity are to be evaluated and reported in the near future.

Acknowledgment

The authors are thankful to the Director of CSIR-NBRI for pro-viding all the facilities to conduct this research work.

Conflict of Interest

We declare that we have no conflict of interest.

Funding

This work was supported by the Department of Science & Technology, India, under the INSPIRE Fellowship Program.

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Ms received: August 10, 2018Accepted: January 16, 2019

Validated Simultaneous High-Performance Thin …...2019/10/21 · green, climbing, much branched...

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