Quantitative analysis of benzyl isothiocyanate in Salvadora persica extract and dental care herbal formulations using reversed phase C18 high-performance liquid chromatography method
Maged Saad Abdel-Kader1, YT Kamal2, Prawez Alam2, Mohammed Hamed Alqarni2, Ahmed Ibrahim Foudah2
1 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia; Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria 21215, Egypt
2 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia
|Date of Submission||21-Mar-2017|
|Date of Acceptance||27-Apr-2017|
|Date of Web Publication||11-Oct-2017|
Maged Saad Abdel-Kader
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Benzyl isothiocyanate is the active antimicrobial agent in Salvadora persica (siwak) widely used in Islamic countries for oral hygiene. Aims: Quantification of benzyl isothiocyanate in the ethanol extract of S. persica and some dental care herbal formulations labeled to contain siwak. Settings and Design: Simple and sensitive high-performance liquid chromatography method was designed. Subjects and Methods: Separation was achieved on reverse phase C18 (250 mm × 4.6 mm, 5 μ ) column with a mobile phase comprising acetonitrile and water (1:1). The detection was carried out at 190 nm using ultra violet-visible detector. The flow rate was kept at 1 mL/min. Results: A sharp and well-defined peak was obtained at the retention time of 9.322 ± 0.3 min. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 0.5–500 μg/mL with a regression coefficient (r2) of 0.9977. The method was validated for accuracy, precision, robustness, and sensitivity. All the parameters examined met the current recommendations for the International Conference on Harmonization guidelines for method validation. Conclusions: The method was applied for the quantification of benzyl isothiocyanate in siwak extract, dental care powder, mouth wash, and toothpaste claimed to contain siwak. The developed method was found specific, simple, selective, and reliable for routine use in quality control analysis of different commercially available herbal care products.
Abbreviations used: RP18: Reversed phase C18, HPLC: High performance liquid chromatography, UV: Ultra violet, r2: regression coefficient, ICH: international conference on harmonization, TLC: Thin layer chromatography, CHCl3: Chloroform, v/v: volume/volume, RSD: Relative standard deviation, LOD: Limit of detection, LOQ: Limit of quantification.
Keywords: Benzyl isothiocyanate, dental care products, International Conference on Harmonization guidelines, reversed phase C18 high-performance liquid chromatography, Salvadora persica
|How to cite this article:|
Abdel-Kader MS, Kamal Y T, Alam P, Alqarni MH, Foudah AI. Quantitative analysis of benzyl isothiocyanate in Salvadora persica extract and dental care herbal formulations using reversed phase C18 high-performance liquid chromatography method. Phcog Mag 2017;13, Suppl S3:412-6
|How to cite this URL:|
Abdel-Kader MS, Kamal Y T, Alam P, Alqarni MH, Foudah AI. Quantitative analysis of benzyl isothiocyanate in Salvadora persica extract and dental care herbal formulations using reversed phase C18 high-performance liquid chromatography method. Phcog Mag [serial online] 2017 [cited 2021 Oct 24];13, Suppl S3:412-6. Available from: http://www.phcog.com/text.asp?2017/13/51/412/216342
- A simple, accurate and precise method was developed for the analysis of the antimicrobial agent benzyl isothiocyanate in Salvadora persica (Siwak) extract and selected dental care herbal formulations using RP18 HPLC
- Amount of benzyl isothiocyanate will indicate the efficacy of Siwak products
- The method subject to ICH validation guidelines.
| Introduction|| |
Family Salvadoraceae comprises three genera Azima, Dobera, and Salvadora.Salvadora persica (siwak) is one of the most popular medicinal plants throughout the Muslim world.,,S. persica with its fibrous branches have been promoted by the World Health Organization for oral hygiene. The spread of Islamic culture had a significant influence on the use of S. persica. Among 182 plant species suitable for preparing tooth brushing sticks, S. persica is the most extensively used. The extracts of S. persica roots have significant antimicrobial activity.,, The main antimicrobial component is the sulfur-containing volatile component benzyl isothiocyanate.
We report here on the development of a high-performance liquid chromatography (HPLC) method for the quantification of benzyl isothiocyanate in S. persica and dental care products labeled to contain siwak using reversed phase C18 column. This method provides a tool to evaluate the medical value of any siwak product based on its benzyl isothiocyanate contents.
| Subjects and Methods|| |
Samples, chemicals, solvents, and standards
Standard benzyl isothiocyanate [Figure 1] was purchased from Sigma-Aldrich, St. Louis, MO, USA. HPLC grade acetonitrile and methanol (E. Merck, Darmstadt, Germany) were used for the analysis. Deionized water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Orthophosphoric acid (H3 PO4) 88% was purchased from Fisher Scientific Company (UK).
Plant material and dental care herbal formulations
The roots of S. persica, family Salvadoraceae were purchased from the local market at Al-Kharj city in March 2016. The plant material was identified by Dr. Mohammad Atiqur Rahman, Taxonomist of the Medicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Voucher specimen (#9011) was deposited at the herbarium of this center. Dental care herbal products labeled to contain siwak extract were purchased from the local market at Al-Kharj city, Saudi Arabia. Others formulations were purchased from Hyderabad, India.
Extractions procedure for plant materials
The fresh roots of S. persica (20 g) were cut into small pieces and extracted with ethanol (3 mL × 100 mL) by maceration. Extracts were combined, evaporated under reduced pressure using rotary vacuum evaporator, transferred to 20 mL volumetric flask, volumes were completed using ethanol. Solutions were kept in refrigerator till the time of analyses.
Preparation of dental care herbal products for analysis of benzyl isothiocyanate
Accurately weighed 10 g from each product labeled to contain siwak extract was separately extracted with ethanol (3 mL × 70 mL) for 30 min. The ethanol extracts from each sample were combined and separately concentrated under reduced pressure using rotary vacuum evaporator. The concentrates of each sample were separately reconstituted in accurately measured 10 mL of ethanol and stored under refrigeration until the chromatographic analysis. A volume of 200 mL of mouth wash product labeled to contain siwak extract were extracted with CHCl3 three times, 100 mL each. The combined CHCl3 soluble fractions were evaporated under reduced pressure and transferred to 10 mL volumetric flask with ethanol.
High performance liquid chromatography chromatographic conditions
The HPLC analysis was performed on a Waters Alliance e2695 separating module (Waters Co., MA, USA) using ultra violet detector (Waters 2998) with autosampler and column oven. The instrument was controlled using “EMPOWER” software installed with equipment for data collection and acquisition. Compounds were separated on a C18 reverse phase column (250 mm × 4.6 mm, particle size 5 μm) maintained at room temperature. Acetonitrile and water in the ratio of 1:1 were used as the mobile phase. The flow rate was kept as 1 mL/min. The detection wavelength was 190 nm and the injection volume was 10 μL. All chromatographic operations were carried out at ambient temperature.
The guidelines of the International Conference on Harmonization (ICH) were applied for the validation of the proposed HPLC method.
A stock solution containing 1000 μg/mL of benzyl isothiocyanate was prepared in ethanol and different aliquot were prepared to get known concentrations starting from 0.1 to 500 μg/mL. The calibration graph was plotted using peak area versus drug concentration [Figure 2]. For assessing the linearity, the least square regression equation was used to calculate the correlation coefficient [Table 1].
Recovery of benzyl isothiocyanate was determined by the standard addition method where extra amounts of benzyl isothiocyanate standard (0, 50, 100, and 150%) were added to preanalyzed samples and the mixtures were reanalyzed using the proposed method [Table 2].
To evaluate precision, repeatability and intermediate precision were carried out. Repeatability of the method was evaluated by carrying out six independent assays of a test sample against benzyl isothiocyanate standard and calculating the percent relative standard deviation (RSD). Intra- and inter-day precisions were done by repeating the analysis on the same day (intra-day precision) and on three consecutive days (inter-day precision). Assay for each analysis was calculated and %RSD was determined [Table 3] and [Table 4].
The robustness of the method was carried out by purposefully altering the chromatographic conditions and observing the changes. Here, we made variations in the flow rate of the mobile phase and the detection wavelength and observed the variation in retention time and area of the peak [Table 5].
|Table 5: Robustness of the method by changing detecting wavelengths and temperature of column (n=6)|
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Limit of detection and limit of quantification
Limit of detection (LOD) (signal-to-noise ratio = 3.3) and limit of quantification (LOQ) (signal to noise ratio = 10) for the method has been determined based on signal-to-noise ratio for both compounds as per the ICH guidelines.
| Results and Discussion|| |
To find out a suitable mobile phase for the analysis of benzyl isothiocyanate in the ethanol extract of siwak and selected dental care herbal formulations, different trials had been carried out. Different compositions of mobile phase with methanol and water; acetonitrile and water have been applied in different ratios. Since the absorbance maxima suitable for quantitation of benzyl isothiocyanatein was found as 190 nm, buffers were avoided from the mobile phase, by considering the fact that wavelengths below 210 can suffer from interference due to impurity present in buffer, solvents which may obscure the peak of interest. Finally, chromatographic separation was carried out successfully in the isocratic mode using a mixture of acetonitrile: Water (50:50, v/v) as mobile phase. Retention time of benzyl isothiocyanatein was found as 9.322 ± 0.3 min [Figure 3]. The column temperature was ambient. The column was equilibrated with the mobile phase flowing at 1.0 mL/min for about 30 min before injection.
|Figure 3: High-performance liquid chromatography chromatogram of benzyl isothiocyanatein standard at 190 nm|
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Calibration graph was plotted on the basis of triplicate analysis of each calibration solutions using peak area against concentration and was found linear in the range of 0.5–500 μg/mL with a good linear relationship of 0.9977 ± 0.0011 [Figure 2]. The linear regression data for the calibration plot are indicative of a good linear relationship between peak area and concentration over a wide range [Table 1].
Recovery study for the proposed method was conducted by spiking previously analyzed test solution with benzyl isothiocyanate standard. The recovery of the method was found in the range of 98.9–101.3%. The values of % recovery, standard deviation and %RSD are listed in [Table 2].
The repeatability and intermediate precisions were calculated and reported in terms of %RSD in [Table 2] and [Table 3]. Intermediate precision include data of intra- and inter-day. The low values of %RSD indicate the reproducibility of the method, which can be adopted in any laboratory for the routine analysis of benzyl isothiocyanate in crude drug and different herbal formulations [Table 3] and [Table 4].
There was no significant change in the retention time and area of benzyl isothiocyanate was found when the flow rate of the mobile phase and detection wavelength were slightly changed. The low values of the RSD, shown in [Table 5], indicated the robustness of the method.
Limit of detection and limit of quantification
The LOD and LOQ were determined by signal to noise ratio method and found to be 0.15 μg/mL and 0.55 μg/mL, respectively.
Analysis of benzyl isothiocyanate in samples
The proposed validated method was applied for analysis of benzyl isothiocyanate in the ethanol extract of siwak [Figure 4] and selected dental care herbal formulations labeled to contain siwak extract. The peak areas of triplicate samples were analyzed by regression equation obtained from calibration plot to get the content of benzyl isothiocyanate in samples. The benzyl isothiocyanate content in the ethanol extract of siwak was found as 0.071% w/w. At the same time, in dental care formulations, the peak of benzyl isothiocyanate was not detected, and hence, it was reported as absent. It was also demonstrated that peaks of benzyl isothiocyanate in siwak extract was well resolved and did not merged with any impurity or any other constituent of drug.
|Figure 4: High-performance liquid chromatography chromatogram of benzyl isothiocyanatein in siwak sample at 190 nm|
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| Conclusion|| |
This HPLC method reported here is accurate, precise, reproducible, and specific with good sensitivity. The mobile phase used in the method was simple without using buffer and resulted in symmetric peaks at reasonable retention time. Extraction procedure was straight forward and no prior treatment was required. The method can be successfully used for routine analysis of benzyl isothiocyanate in crude drug and in herbal formulations without interference.
This project was supported by Sheikh Abdullah bin Zaid bin Ghonaim Research Chair of Prophetic Medicine, Developing and Manufacturing Natural Products, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Kingdom of Saudi Arabia.
Financial support and sponsorship
Sheikh Abdullah bin Zaid bin Ghonaim Research Chair of Prophetic Medicine, Developing and Manufacturing Natural Products.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mabberley DJ. Mabberley's Plantbook: A Portable Dictionary of Plants, their Classifications and Uses. 3rd
ed. Cambridge: University Press; 2008.
Sofrata A, Lingström P, Baljoon M, Gustafsson A. The effect of miswak extract on plaque pH. An in vivo
study. Caries Res 2007;41:451-4.
Ezmirly ST, Cheng JC, Wilson SR. Saudi Arabian medicinal plants: Salvadora persica
. Planta Med 1979;35:191-2.
Al-Otaibi M, Al-Harthy M, Gustafsson A, Johansson A, Claesson R, Angmar-Månsson B. Subgingival plaque microbiota in Saudi Arabians after use of miswak chewing stick and toothbrush. J Clin Periodontol 2004;31:1048-53.
World Health Organization. Prevention of Oral Diseases. Geneva: WHO; 1987.
Bos G. The miswãk, an aspect of dental care in Islam. Med Hist 1993;37:68-79.
Elvin-Lewis M. The therapeutic potential of plants used in dental folk medicine. Odontostomatol Trop 1982;5:107-17.
Al lafi T, Ababneh H. The effect of the extract of the miswak (chewing sticks) used in Jordan and the Middle East on oral bacteria. Int Dent J 1995;45:218-22.
Almas K. The antimicrobial effects of extracts of Azadirachta indica
(Neem) and Salvadora persica
(Arak) chewing sticks. Indian J Dent Res 1999;10:23-6.
Almas K, Al-Bagieh N, Akpata E. In vitro
antimicrobial effects of extracts of freshly cut and 1-month-old miswak (chewing stick). Biomed Lett 1997;56:145-9.
Sofrata A, Santangelo EM, Azeem M, Borg-Karlson AK, Gustafsson A, Pütsep K. Benzyl isothiocyanate, a major component from the roots of Salvadora persica
is highly active against gram-negative bacteria. PLoS One 2011;6:e23045.
International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use: Harmonised Triplicate Guideline on Validation of Analytical Procedures: Methodology, Recommended for Adoption at Step 4 of the ICH Process on November 1996 by the ICH Steering Committee, IFPMA, Switzerland; 1996.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]