Box–Behnken supported development and validation of robust HPTLC method: an application in estimation of punarnavine in leaf, stem, and their callus of Boerhavia diffusa Linn
Authors
Wasim Ahmad
Department of Pharmacy, Mohammad Al-Mana College for Medical Sciences, Al-Safa, Dammam, 34222 Kingdom of Saudi Arabia
Rasheeduz Zafar
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, 110062 India
Abuzer Ali
Department of Pharmacognosy, College of Pharmacy, Taif University, Taif, Saudi Arabia
Ameeduz Zafar
Department of Pharmaceutics, College of Pharmacy, Jouf University, Al-Jouf, Sakaka, Saudi Arabia
Mohammad Ansari
Department of Epidemic Disease Research, Institutes for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, 31441 Saudi Arabia
Md Sarafroz
Department of Pharmaceutical Chemistry College of Clinical Pharmacy Imam, Abdulrahman Bin Faisal University, Dammam, 31441 Saudi Arabia
Mohd Amir
Department of Natural Product & Alternative Medicines College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, 31441 Saudi Arabia
Niyaz Ahmad
Department of Pharmaceutics, College of Clinical Pharmacy Imam, Abdulrahman Bin Faisal University, Dammam, 31441 Saudi Arabia
Ibraheem Husain
Department of Pharmacology, Mesco Institute of Pharmacy, Uttar Pradesh, Amroha, India
Kamran Ashraf
Faculty of Pharmacy, University Teknologi MARA (UITM), Cawangan Selangor, 42300 Kampus Puncak Alam, Seleangor Darul Ehsan Malaysia
Boerhavia diffusa (BD) Linn. (Nyctaginaceae) is one of the most commonly used herbs in the Indian traditional system of medicine for the urinary disorders. The aim of the current investigation was to carry out initiation, development, and maintenance of BD callus cultures and quantitative estimation of punarnavine in plant and callus extracts. Leaves and stem of BD were used as explant for the tissue culture studies using Murashige and Skoog (MS) basal medium. MS Media comprising 2,4-Dichlorophenoxy acetic acid (2,4-D) (1 ppm) and 2,4-D (1 ppm) + Indole-3-acetic acid (IAA) (1.0 ppm) were found to yield friable callus from leaf explant; similarly, 2,4-D (0.3 ppm) + IAA (0.75 ppm) + Kinetin (0.3 ppm) and 2,4-D (0.5 ppm) + Naphthalene acetic acid (NAA) (1.5 ppm) + Kinetin (0.3 ppm) were found to yield friable callus from the stem explant. High-performance thin-layer chromatography method was been developed for the quantitative estimation of punarnavine (Rf = 0.73) using mobile phase containing toluene: ethyl acetate: formic acid in the ratio (7.0:2.5:0.7, v/v/v) at 262 nm. The validated method was found linear (r2 = 0.9971) in a wide range (100–1000 ng spot−1), precise, accurate, and robust. The values of limit of detection, LOD = 30.3 ng spot−1, and limit of quantification, LOQ = 100.0 ng spot−1. The robustness of the method was proved by applying the Box–Behnken design (BBD). The developed method found appropriate for the quality control of medicinal plants containing punarnavine as a constituent.
Keywords: Boerhavia diffusa, Punarnavine, HPTLC, Callus culture
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