Quantification of Berberine in different Berberis Species and their Commercial Samples from Herbal Drug Markets of India through HPTLC

A simple, precise, and convenient HPTLC method has been established for the analysis of Berberine, the major marker compound extracted from the root and stem of different Berberis species and their commercial samples in the name of Daruharidra. Chromatography was performed on silica gel 60F254 plates with n-propanol:water:formic acid (90:8.0:0.4) as mobile phase. Detection and quantification were performed densitometrically at λmax = 360 nm with berberine as external standard. The method is characterized by high sensitivity and linearity over wide range of concentrations. Berberine concentration in different species and their commercial counterpart were calculated. This will be utilized by pharmaceutical industries for the bioprospection of allied Berberis species for commercial exploitation and batch to batch consistency of raw materials.


1-Introduction
High-performance thin-layer chromatography (HPTLC) is commonly used for identification, assay, purity testing and for content uniformity of raw materials (herbal and animal extracts, fermentation mixtures, drugs and excipients) and formulated products (pharmaceuticals, cosmaceuticals, nutraceuticals). It has also emerged as an important analytical tool for detection and characterization of the active ingredients in crude drug formulations. In HPTLC, densitometry has been used to test purity [1], for assay of pharmaceutical dosage forms [2], and to obtain a chromatogram for herbal fingerprinting [3,4].
Berberis aristata DC. known, as 'Daruharidra' in Ayurveda is a versatile medicinal plant used singly or in combination with other medicinal plants for treating a variety of ailments like jaundice, enlargement of spleen, leprosy, rheumatism, fever, morning/evening sickness and snakebite [5][6][7][8][9][10]. In addition, the decoction of root or stem of Berberisknown as 'Rasaut' is specifically used in eye disease, skin disorders and indolent ulcers [11][12][13]. Although the roots of B. aristata are considered as the genuine drug, the study revealed that different species of Berberis viz. B. asiatica, B. chitria, and B. lycium are also being used as Daruharidra in different parts of the country. The study also shown that most of the market materials sold as Daruharidra consist of mostly the mixture of root & stem parts different Berberis species [14][15][16].
In view of its great importance as major ingredient in different herbal formulations worldwide, a detailed HPTLC study of four Berberis species viz. B. aristata DC., B. asiatica Roxb. ex DC., B. chitria Lindl. and B. lycium Royle collected from wild and ten commercial samples which are implicated as Daruharidra procured from various important crude drug markets of India viz. Aligarh, Amritsar, Bangalore -I, Bangalore -II, Delhi, Hyderabad, Jammu, Lucknow, Trichur, Varanasi has been carried out with the aim to authenticate and check the adulteration/substitution in different commercial crude drug samples. Berberine was used as external standard.

2-1 Plant Material
Roots and stems of the plant were collected from Dhanulti region of Uttarakhand, India and were authenticated by Dr A.K.S. Rawat, Scientist, NBRI, Lucknow, India and ten commercial samples procured from Aligarh, Amritsar, Bangalore-1, Bangalore-2, Delhi, Hyderabad, Jammu, Lucknow, Tirchur and Varanasi markets of India. The crude samples were submitted to drug depository of the Institute along with the voucher specimen numbers. (Table 1&2)

2-3-3 Chromatographic conditions
Chromatography was performed on 20 cm × 10 cm glass-backed HPTLC plates coated with 0.25-mm layers of silica gel Si 60F254 (E. Merck, Germany). Different volumes of standard solution and 5 μl of the extract solutions were applied to the plates as bands, 6 mm long and 8 mm apart, by using Camag Linomat V (Switzerland) sample applicator equipped with a 100μl micro syringe.

2-3-4 Detection, quantification and calibration of Berberine
Plates were developed to a distance of 80 mm, with n-propanol:water:formic acid (90:8.0:0.4), as mobile phase, in a 20 cm × 10 cm Camag glass twin-trough chamber previously saturated with mobile phase vapor; the temperature was 25°C and the relative humidity was 37%. After removal of the plates from the chamber completely dried in air at room temperature (25C) and peak areas for the samples and standard were recorded by densitometry in absorbance/reflectance mode at λ max = 360 nm, by means of a Camag TLC Scanner 3 equipped with WINCATS version 3.2.1 software. Typical chromatograms are shown in Figure 1-21 and the calibration plot obtained by diluting standards in different concentrations (µL).

2-3-5 Validation
The precision of the scanner was checked by scanning the same spots five times and the coefficient of the variance was calculated. The repeatability of the method was also established by applying 5g per spot of each standard solution five times and the coefficient of variance was calculated. The limit of detection and quantification were also determined. (Table  3&4) A p r i l 0 4 , 2014

3-Results and Discussion
Different compositions of the mobile phase were checked and the desired resolution of Berberine with reproducibility of peaks was achieved by using n-propanol:water:formic acid (90:8.0:0.4) (v/v). Under these conditions the Rf of Berberine was 0.32 and the compound was well resolved from other components of the extract.
The calibration curve of Berberine was linear and in the range of 1μg to 13μg. Linear regression and Rf of Berberine are given in Table 3. The amount of Berberine in the roots of different Berberis species and it market samples were also presented in Fig. 22

4-Conclusions
Berberine, one of the major alkaloids was quantified through HPTLC densitometric method and it was found more in roots as compared to stem i.e. 2.25 -5.20% and 1.02-2.01% respectively. Its concentration was also varied from species to species i.e. maximum in roots of B. chitria (5.20%) followed by B. lycium (3.99%), B. aristata (3.55%) and B. asiatica (2.25%).
An extensive market survey was done to check the quality of raw material (adulteration/substitution) available in different parts of the country. On morphological studies it was observed that most of these commercial samples are either mixture of two species or substituted with some other species than B. aristata. This was also confirmed with the analysis done through HPTLC (Table 2 & Fig. 22). Thus this study will be a useful tool for checking adulteration/substitution of Berberis in pharmaceutical preparations. It will also be useful for maintaining batch to batch consistency of crude samples/herbal formulations commercialized by pharmaceutical industries. A p r i l 0 4 , 2014