Bergenin determination in different extracts by high-performance thin-layer chromatographic densitometry.

AIM: Bergenin is an active secondary metabolite, found in Bergenia ligulata, family Saxifragaceae, which is an important medicinal plant used in the traditional system of medicine. It is distributed throughout the South and East Asia and some European countries, usually growing on high altitude in the Himalayan region and known as Pashanbhed (meaning "to break the stone"). The rhizome of B. ligulata has been used since long time in different traditional formulations of kidney and liver disorders. Due to its exhaustive use in the traditional system, it is commonly adulterated with the rhizome of other plants which do not contain its chemical marker bergenin. Hence, we developed high-performance thin-layer chromatographic (HPTLC) method for quantification of bergenin in B. ligulata which can be used for its quality control.
MATERIALS AND METHODS: A sensitive HPTLC method has been developed for the estimation of bergenin in different extracts of B. ligulata and its traditional formulations. Precoated HPTLC silica gel plates were used as stationary phase, and chloroform: methanol: acetic acid (8:1:1, v/v/v) was used as mobile phase.
RESULTS: The Rf value of bergenin was found to be 0.28 ± 0.03. Detection and quantification were performed by densitometry at 276 nm. The calibration plot was linear in the range of 200-5000 ng of bergenin with the correlation coefficient of (r (2)) 0.999, which confirms good linearity. The content of bergenin in methanolic and acetone extracts was found to be 5.51 ± 0.14 and 5.76 ± 0.16, respectively.
CONCLUSION: The method can be applied for quality control and standardization of B. ligulata and its traditional formulations as well as for checking the presence of adulterants.

Bergenin is an active secondary metabolite, found in Bergenia ligulata, family Saxifragaceae, which is an important medicinal plant used in the traditional system of medicine. It is distributed throughout the South and East Asia and some European countries, usually growing on high altitude in the Himalayan region and known as Pashanbhed (meaning “to break the stone”). The ethno-botanical and ethno-medicinal literature states that in Ayurveda and Unani medicines, the roots of B. ligulata possess astringent, tonic, antiscorbutic, and laxative properties[1] and is recommended for ulcers, dysuria, spleen enlargement, cough and fever,[2] and for kidney and liver disorders.[3] Alcoholic extract of the plant exhibited significant anti-inflammatory, analgesic, and diuretic activity.[45678] Due to its exhaustive use in the traditional system, it is commonly adulterated with the rhizome of other plants which do not contain its chemical marker bergenin. Because of its great importance as major ingredients in different formulations worldwide, we developed high-performance thin-layer chromatographic (HPTLC) method for quantification of bergenin in B. ligulata which can be used for its quality control.

Materials and Methods

Drug and chemicals

Bergenin standard was procured from Sigma-Aldrich having the purity of 99%. B. ligulata was procured from the local drug market and identified by Dr. S. Ahmad, Department of Pharmacognosy and Phytochemistry, Faculty of Medicine, Hamdard University, New Delhi. A sample specimen was deposited in the herbarium of the Bioactive Natural Product Laboratory, (Specimen no-/BL/BNPL/2012). All chemicals and reagents used were of analytical grade and purchased from Merck Chemicals, India.

Sample preparation

The samples were prepared by refluxing 1.0 g of dried powdered drug in methanol and acetone separately for 2 h. Solutions were filtered and cooled. The extracts were evaporated to dryness and reconstituted in 5.0 mL of methanol and acetone.

High-performance thin-layer chromatographic instrumentation and general conditions

The sample is spotted in the form of bands of width 5.0 mm using Camag Linomat Applicator (Hamilton, Switzerland) on a precoated silica gel aluminum plate thin-layer chromatography (TLC) F254 (20 cm × 10 cm). The mobile phase consisted of chloroform: methanol: acetic acid (8:1:1, v/v/v). Densitometric scanning was performed on Camag TLC scanner III in the wavelength of 276 nm operated by WINCATS Software 6 (CAMAG, Switzerland). The slit dimension was kept at 5 mm × 0.45 mm at 10 mm/s scanning speed.

Results and Discussion

In the preliminary TLC experiments, bergenin was found to be one of the major compounds in the rhizome of B. ligulata, considering the importance of bergenin.[910] In the present work, a simple, sensitive HPTLC method was developed for the estimation of bergenin as a marker compound. TLC procedure was optimized with a view to develop a method for determining bergenin in different extracts. Initially, chloroform: Methanol in various ratios was tried. When chloroform and methanol tried with formic acid in the ratio of 8:1:1, v/v/v, a good spot was observed but the Rf was nearby about 0.8. So, the mobile phase again modified and instead of formic acid, acetic acid is used. When chloroform, methanol, and acetic acid used in the ratio of 8:1.1, v/v/v, a compact, resolved and well-defined peaks of standard as well as sample [Figure 1] was observed with an Rf value of 0.28 ± 0.03.

Figure 1

(a) High-performance thin-layer chromatographic chromatogram of standard bergenin (4000 ng/spot), 276 nm wavelength showing an Rf value of 0.28; (b) high-performance thin-layer chromatographic chromatogram of methanolic extract of Bergenia ligulata; (c) high-performance thin-layer chromatographic chromatogram of acetone extract of Bergenia ligulata

Calibration curve of bergenin by high-performance thin-layer chromatographic

The linear regression data for the calibration curve (n = 3) as shown in Table 1 produced a good linear relationship over the concentration range 200–5000 ng/spot with respect to the peak area [Figure 2]. The concentration of bergenin in the sample was calculated by using this regression equation [Table 1].

Table 1

Calibration data for standard bergenin

Figure 2

Calibration curve for standard bergenin

Analysis of bergenin in different extracts

The quantitative estimation of bergenin content was carried out by HPTLC WinCats software using the calibration curve with respect to the peak area of standard spots which were found to be 5.51% ± 0.14% and 5.76% ± 0.16% w/w.

Conclusion

The simple HPTLC method developed for the estimation of the biomarker bergenin has the applicability in the quantification of bergenin and can be applied for quality control and standardization of B. ligulata and its traditional formulations as well as for checking the presence of adulterants.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.