a-modified-hptlc-method-development-and-validation-for-comparative-quantification-of-analgesic-salicin-in-industrially-important-b-64.html A Modified HPTLC Method Development and Validation for Comparative Quantification of Analgesic Salicin in Industrially Important Bergenia Species

Journal of Research Analytica

Research Article Article

A Modified HPTLC Method Development and Validation for Comparative Quantification of Analgesic Salicin in Industrially Important Bergenia Species

Nishi Srivastava*, Sharad Srivastava, Rachit, Jai Chand and Ajay Kumar Singh Rawat*

1Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, India
2Central Instrument Facility, CSIR- National Botanical Research Institute, Lucknow-226001, India

* Corresponding author: Nishi Srivastava & Ajay Kumar Singh Rawat E-mail: pharmacognosy1@rediffmail.com, nishisri13jan@gmail.com
Copyright: ©2017 OLOGY Group.

Abstract:

NA modified sensitive, selective, precise and robust high-performance thin layer chromatography (HPTLC) method for the determination and comparative quantification of salicin was developed and validated for the salicin in two species of Bergenia viz. Bergenia ciliata (BC) and Bergenia stracheyi (BS). Quantification of salicin was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. Linear ascending development was carried out in twin tough glass chamber saturated with mobile phase consisting of toluene-ethyl acetate-formic acid (4:5.5:1.5v/v) at room temperature. Camag TLC scanner III was used for densitometric scanning and analysis in visible absorbance mode at wavelength λmax 500 nm. The system was found to give compact spots for salicin (Rf value of 0.37 ± 0.02). The linear regression analysis data for the calibration plots showed good linear relationship with regression equation y = 1707.5x + 1618.1, correlation coefficient (r) 0.998 in the concentration range 2-7 μg/spot with respect to peak area. According to International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery and robustness. The limit of detection and limit of quantification were determined. Statistical analysis of the data showed that the method is reproducible and selective for the estimation of analgesic salicin. From quantitative result, higher amount of salicin is found in (BS) comparative to (BC).On industrial level, Bergenia and its species can be used as an alternative source for isolation of natural analgesic compounds salicin.

Untitled Document

Abbreviations

BC: Bergenia Ciliate; BS: Bergenia Stracheyi; HPTLC: High-performance Thin Layer Chromatography; TLC: Thin Layer Chromatography; RSD: Relative Standard Deviation

1. Introduction

The genus Bergenia (family Saxifragraceae) and its species viz. Bergenia ciliata (BC) and Bergenia stracheyi (BS) is an evergreen perennial herb, generally distributed in Central and East Asia. It is also found in temperate Himalayas from Kashmir to Bhutan at high altitude 7000-10000 feet and in khasia hill at 400 feet [1]. Previous studies on phytochemical analysis of B. ciliata have been shown the isolation of bergenin (C-glycoside of 4-O-methyl gallic acid), gallic acid (3,4,5 trihydroxybenzoic acid), (+)-catechin, leucocyanidin, (+)-catechin-3-gallate, (+) catechin-7-O-beta-D-glucopyranoside, paashaanolactone, β-sitosterol, β-sitosterol-D-glucoside, and (+)-afzelechin [2]. These phytochemicals have a range of biological activities such as antioxidant [3,4] antidiarrhoeal, anti-inflammatory, menorrhagia, excessive hemorrhage [5,6] antibacterial, antitussive [7,8] and in the treatment of pulmonary infections [9].

Phytomedicines are successfully used alone or as adjuvance therapy for the treatment of degenerative rheumatism and painful arthrosis. salicin is a glycoside, which acts as a precursor compound for the synthesis of acetyl salicylic acid. Glycoside consists of a carbohydrate molecule (sugar) and a non-sugar component (aglycone). Salicin is well known for its effect on treating rheumatic fever and sub-acute bacterial endocarditis. Also salicin has been used as a traditional analgesic in Europe [10]. Salicin is a prodrug which is gradually hydrolyzed to ligenin by intestinal bacteria, and converted into salicylic acid after absorption. Thus it produces an antipyretic action without causing gastric injury [11].

The aim of our work was the development of simple and rapid methods of quantification for salicin from hydrolyzed extracts of two Bergenia species viz. Bergenia ciliata(BC) and Bergenia stracheyi(BS). We have chosen HPTLC for determination and quantification of salicin in extracts of Bergenia species. As per previous report on HPLC method quantification of the phenolic glycosides after hydrolysis to salicin in literature [12,13] we have adopted the extraction methodology reported in our previous work and further quantification is followed by development of modified HPTLC method [14,15]. Due to several advantage over other analytical methods such as the rapidity, less amount of test sample and extremely limited solvents waste, HPTLC has gained widespread interest as most acceptable technique for the determination of pharmacologically interesting compounds in the biological matrices like plants and its different parts and even in formulations [16,17].We have also validated the developed method in terms of accuracy, precision, recovery and robustness as per ICH norms [18]. From the literature survey and to our best knowledge no previous report is available on determination and comparative quantification of salicin in Bergenia species. However report on salicin quantification using HPTLC is available in Salix sp. [19].This is the first time report on determination and comparative quantification of salicin in two different Bergenia species.

 Voucher No.

Plant 

State 

Region explored

Collection stage 

 GPS Information

Material 

262557

B.stracheyi

Uttrakhand

Juda ka talab

Pre-flowering

9400 feet, N 31003.116’ E 78011.096’

Rhizome

254013

B.ciliata

Uttrakhand

Way to Taluka

Pre-flowering

7135 feet, , N 16°46'51" E 73°44'48"

Rhizome

Table 1: Details of collection of Bergenia species.

2. Experimental

2.1. Chemicals and materials

Standard salicin (purity: 98% w/w) was procured from Sigma-Aldrich USA. All the solvents used were of analytical grade from Rankem laboratory, India.

2.2. Preparation of crude extracts

Bergenia ciliata (BC) and Bergenia stracheyi (BS) were collected from Lansdowne and Juda ka talab, Uttrakhand, India in the month of November 2012, deposited (voucher specimen no. 254013 and 262557) in repository of CSIR-National Botanical Research Institute, Lucknow (Uttar Pradesh) India(Table 1). After washing with tap water, rhizomes were chopped and dried under shade conditions at temperature 40°C. The dried rhizomes (100 g) were crushed into powdered and soaked in absolute methanol (4 × 250 mL) at room temperature (25 ± 2°C) for 5 days. The suspension was filtered and evaporated to dryness by using rotary evaporator (Buchi, USA). Methanol extract of (BC) and (BS) were further hydrolyzed in acidic medium as per reported by Srivastava et al., 2014 [14, 15].

2.3. HPTLC method

2.3.1. Apparatus

Camag Linomat V automated TLC applicator, Camag twin trough glass chamber, ascending. Camag TLC scanner model 3 equipped with Camag Wincats IV software were used during the study at temperature 27 ± 2°C, relative humidity.

2.3.2. Sample preparation

Salicin standard stock solution was prepared by dissolving pure 1 mg standard salicin into 1 ml methanol and extract of (BC) and (BS) was dissolved separately into 10 ml methanol. After dissolution both standard and samples are vortex for 10 min.

2.3.3. Chromatographic experiments

Sample solution and standards were applied on precoated silica gel 60F254 HPTLC plates with 6 mm band width using Camag 100 microlitre sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland) under a flow of N2 gas. The Linear ascending development was carried out with Toluene/ethyl acetate/formic acid (4:5.5:1.5 v/v/v) as a mobile phase in a Camag glass twin trough chamber (20 × 10 cm).The saturation time of the TLC chamber in the mobile phase was optimized to 20 min for a good resolution of the tested markers and total run time was about 25 minutes at room temperature (27 ± 2°C), 50% ± 2 % relative humidity. After run, plates were dried over hair drier and TLC image was taken on white light after derivatization (Figure 1). Scanning of TLC plate were performed by using Camag TLC Scanner 3 at λmax 500 nm in Visible absorbance mode for all tracks after derivatization, TLC plate were developed at distance of approximately 80 mm from the point of application and slit dimensions were 4 mm × 0.45 mm (Figure 2). Quantification evaluation of the plate was performed using peak area with linear regression of amount 2-7 µg/band (Table 2). Peak profiling was done in after derivatization in visible range region at λmax 500 nm (Variable wavelength was used to get best absorbance range) check the identity of the bands, Visible absorption spectrum of standard salicin was overlaid with the corresponding band in the samples track (Figure 3). Standards six point concentration chromatogram is illustrated in (Figure 4).

image


Figure 1: Image of TLC plate at white light after derivatization.

image

Figure 2: All track chromatogram at wavelength λmax = 500 nm. (BC)=Bergenia ciliata, (BS)=Bergenia stracheyi and Salicin.

2.3.4. Postchromatographic derivatization

Postchromatographic derivatization was necessary for the evaluation of the two dimensional stability. Derivatizing reagent anisaldehyde-sulphuric acid solution (anisaldehyde:methanol:acetic acid:sulphuric acid (0.5:85:10:5, v/v)) was used to visualize the spot of salicin in visible range. After air drying for 2 min the plates were heated at 120°C for 5 min (Figure 1).

 3. Results and Discussion

3.1. Method validation

3.2. Specificity

The specificity of the methods was determined by analyzing the standards and samples bands. The bands for the salicin in sample solution were confirmed by comparing the Rf and Visible spectra with the reference standards (Figure 4). A densitometer is used for providing whether the spot contains one compound or more by measuring its visible spectrum at the up slope (peak start), apex (peak apex) and down slope (peak end). The value of correlation coefficient of up slope to apex (rsm) and apex to down slope (rme) are found (≈0.99), so it can be conclude that the peak is pure (Table 3).

3.3. Calibration and quantification

The calibration curves for standard salicin was linear in the concentrations range of 2-7 µg/spot with correlation coefficient (r) 0.998 respectively. The regression data obtained showed a good linear relationship (Table 4). Plate development and spot scanning as well as quantification were performed as mentioned in chromatographic experiment section and calibration curve was constructed (Table 4).

3.4. Estimation of salicin in extracts of Bergenia species

A single spot at Rf = 0.37 Was observed in the chromatogram of the salicin in the extracts of Bergenia species. There was no interference in the analysis from the other components of extract matrix. The total salicin contents were found to be 0.88±0.02 μg/10 mg (mean ± SD) in extract of (BC) and 1.42 ± 0.04 μg/10 mg (mean ± SD) in extract of (BS) respectively (Table 2).

Sample

Extract (MeOH)

Applied sample volume

Content of Salicin in 10mg extract (Mean±SD)

10mg/ml; 10µl

Salicin μg/10mg

B.ciliata

MeOH extract

10

0.88±0.02

B.stracheyi

MeOH extract

10

1.42±0.04

 

 

 

 

Table 2: Quantification of Salicin in (BC) & (BS), (BC)= Bergenia ciliata and (BS)=Bergenia stracheyi.

image

Figure 3: Overlay spectra comparison and peak purity correlation of standard Salicin with (BC) and (BS). (BC)=Bergenia ciliata and (BS)=Bergenia stracheyi.

image

Figure 4: Calibration of standard Salicin at six concentration level.

 3.5. Accuracy

The accuracy of the methods was determined by analyzing the percentage recovery of the salicin in the both samples. To obtain it, three sets were prepared from BC.The sample was spiked with three concentrations: 50, 100 and 150 μg of salicin. The spiked sample was recovered in triplicate and then analyzed by proposed HPTLC method. The average recoveries for salicin in (BC) were found to be 99.49%, within the acceptable range. (Table 5).

Standard & Sample

 r (s, m)

 r (m, e)

Salicin

0.999546

0.999947

B.ciliata

0.999139

0.999275

B.stracheyi

0.999314

0.999512

Table 3: Peak purity test of Salicin, (BC) and (BS), (BC)=Bergenia ciliata and (BS)=Bergenia stracheyi

Parameters

Salicin

Rf value

0.37±0.02

Slope

1.7075

Intercept

1618.1

Linearity equation

y = 1707.5x + 1618.1

Linearity range

2-7 μg

Regression coefficient (R2)

0.997

Correlation Coefficient (r=√R2)

0.998

p-value

0.002

Intercept coefficient

1618.09

Standard deviation

2.87

LOD (μg)

0.9

LOQ (μg)

2.8

Parameters

Salicin

Rf value

0.37±0.02

Slope

1.7075

Intercept

1618.1

Linearity equation

y = 1707.5x + 1618.1

Linearity range

2-7 μg

Regression coefficient (R2)

0.997

Correlation Coefficient (r=√R2)

0.998

p-value

0.002

Intercept coefficient

1618.09

Standard deviation

2.87

LOD (μg)

0.9

LOQ (μg)

2.8

Table 4: Statistical analysis of calibration curves in HPTLC determination of Salicin

Compounds

 

 Intraday Interday

 

Conc.(μg/band)

%RSD

%RSD

2

0.94

0.93

Salicin

4

0.81

1.01

6

0.83

1.1


Table 5: Recovery study to evaluate accuracy of method.
%RSD= Percent Relative standard deviation

Standard

Amount of Salicin present (μg/10mg)

Amount of Salicin added (μg)

Theoretical value

Mean±SD

Observed value

Recovery (%)

Average recovery (%)

0.88

50

50.88

51.27±0.01

51.27

99.24

99.49

Salicin

0.88

100

100.88

101.5±0.14

101.5

99.39

 

0.88

150

150.88

151.1±0.05

151.1

99.85


Table 6: Inter- and Intra-day precision of Salicin.

Parameters

RSD% of peak area

Salicin

Time interval difference between spotting and plate development

0.22

Mobile phase composition

0.38

Time interval between drying and scanning

0.42

Table 7: Robustness testing of the HPTLC method.

3.6. Precision

Instrumental precision was checked by repeated scanning of the spot of standards salicin five times each. The repeatability of the sample application and measurements of peak area was expressed in terms of percent relative standard deviation (%RSD). Intra-day precision study was achieved at different concentrations levels of 2-6 µg/spot of standard salicin was spotted in three times within 24 h and expressed in terms of percent relative standard deviation %RSD (Table 6). For inter-day precision study, same concentrations levels of 2-6 µg/spot of salicin used over a period of 5 days and expressed as %RSD. The results showed no significant inter and intraday variation was observed in the analysis of the salicin.

3.7. Limit of detection (LOD) and Limit of quantification (LOQ)

In order to estimate the limit of detection (LOD) and limit of quantification (LOQ), the signal to noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. In the present study LOD for salicin estimation in samples was found to be 0.9 μg/band, respectively whereas LOQ for estimation in samples was found to be 2.8 μg/band (Table 4).

3.8. Robustness

Robustness is a measure of the method to remain unaltered by small but deliberate variations in the method conditions, and is indicate of the reliability of the method. For robustness study different mobile phase composition, developing TLC distance and different TLC plate lots were assessed (Table 7).

4. Conclusion

The present study can be further utilized to understand the chemical compounds responsible for analgesic activity of extract and fractions of Bergeniasp. Among both species (BS) was found to contain higher amount of salicin comparative to (BC).On industrial level and in herbal medicinal system Bergeniaand its species can be used as an alternative sources for analgesic compound salicin. The modified developed HPTLC method is precise, sensitive, accurate and robust for the determination of salicin in the extracts of Bergeniaspecies. Statistical interpretation of the data proves that the method is reproducible and selective for the analysis of salicin. Since the proposed mobile phase effectively resolves salicin, the method can be used for qualitative and quantitative analysis of salicin in extract of Bergeniaspecies. In future, the proposed method can be extended to study the degradation of salicin under different stress conditions, as per recommendations of I.C.H guidelines.

Acknowledgement

The authors thank to the Director Dr. C.S.Nautiyal CSIR-NBRI, Lucknow for his kind support and to make available required resources during this study. First author (NS) is thankful to CSIR (New Delhi) for the award of Senior Research fellowship. We are also thankful to CSIR-NBRI for the support of project BSC 106.

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