Use of bergenin as an analytical marker for standardization of the polyherbal formulation containing Saxifraga ligulata
Hulikal Basavarajaiah Pushpalatha1, Kumar Pramod2, Rajendran Devanathan1, Ramachandran Sundaram1
1 Formulation and Development, Research and Development Center, The Himalaya Drug Company, Makali, Bengaluru, India
2 Department of Pharmaceutics, JSS College of Pharmacy, JSS University, Sri Shivarathreeswara Nagar, Mysore, Karnataka, India
|Date of Submission||07-Jul-2014|
|Date of Acceptance||14-Nov-2014|
|Date of Web Publication||27-May-2015|
Hulikal Basavarajaiah Pushpalatha
Research and Development Center, The Himalaya Drug Company, Makali, Bengaluru - 562 162, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Bergenin is the principle constituent of the well-known medicinal plant Saxifraga ligulata. Bergenin has anti-inflammatory, antipyretic, antiviral, immunostimulant, antihyperglycemic, and antioxidant properties. In this study, the presence of bergenin in Saxifraga ligulata and the formulations was identified using high performance thin layer chromatograpy fingerprinting technique. Objective: To develop a novel quantitative method for the estimation of bergenin using high performance liquid chromatography. Materials and Methods: The compound was separated, characterised and quantified using authentic reference standard. The method was validated per ICH guidelines for the parameters of accuracy, precision, linearity, limit of detection, limit of quantification and robustness. Results: The method was found to be accurate, linear (r2 = 0.998) and precise (%RSD <2%). The limits of detection (0.001%) and quantification (0.002%) were found to be suitable for detection and quantification of bergenin in commercial formulations. Conclusion: The developed methods are suitable for the quality control applications of Saxifraga ligulata containing formulations.
Keywords: Bergenin, dosage forms, high-performance liquid chromatography, high-performance thin layer chromatography, Saxifraga ligulata
|How to cite this article:|
Pushpalatha HB, Pramod K, Devanathan R, Sundaram R. Use of bergenin as an analytical marker for standardization of the polyherbal formulation containing Saxifraga ligulata. Phcog Mag 2015;11, Suppl S1:60-5
|How to cite this URL:|
Pushpalatha HB, Pramod K, Devanathan R, Sundaram R. Use of bergenin as an analytical marker for standardization of the polyherbal formulation containing Saxifraga ligulata. Phcog Mag [serial online] 2015 [cited 2022 Jan 22];11, Suppl S1:60-5. Available from: http://www.phcog.com/text.asp?2015/11/42/60/157690
| Introduction|| |
The plant Saxifraga ligulata, well-known as "Pashanbheda" in the indigenous system of medicine has a number of therapeutic benefits. S. ligulata has been widely recognized for its role in dissolving kidney stones.  It is also effective in the treatment of fever, eye ailments, dysentery and diarrhea, piles, inflammation, and chronic ulcers. Currently available data on Pashanbheda are limited and further research is needed to identify other key constituents with therapeutic properties. , Extensive survey in and around Kumaon region of Himalaya revealed that this plant is distributed in mixed vegetation on the rocky slopes in moist and shady habitats; predominantly on the northern and western slopes. ,, The root, rhizome, and whole plant of S. ligulata are used in the therapy for kidney and bladder stones and urinary problems. ,,,
Rhizome is the main part or source of drug. In this bitter, has a useful effect in cough and cold.  Flowers are consumed in the form of pickles. With honey, whole plant of S. ligulata is applied to gums during teething in infants to allay irritation.  S. ligulata has been reported to exhibit various pharmacological activities and thus has several traditional uses. It is used as an antidiabetic, antipyretic, , and as a tonic.  Ethnobotanical study conducted in the upper Siran Valley of Pakistan shows that S. ligulata (but pewa) is used as diuretic,  and hepatoprotective. Alcoholic extract of S. ligulata showed anticancer, antiprotozoal, diuretic, antiscorbutic, antilithiatic, ,, litholytic, and anti-inflammatory activities in a dose-dependent manner in rats.  S. ligulata is used in the formulation of herbal composition. Its composition is useful for caring the skin around the eyes. , The whole plant of S. ligulata showed activities like calcium oxalate monohydrate growth inhibition, decreased calcium phosphate nucleation, calcium oxalate inhibition, diuretic, hypermagneseuric, and antioxidant effect in in vitro and in vivo models. 
Bergenin is a trihydroxy benzoic acid glycoside with an IUPAC nomenclature as (2 R,3S,4S,4aR,10bS) -3, 4, 8, 10 -tetrahydroxy -2 -(hydroxymethyl) -9 -methoxy -3, 4, 4a, 10b -tetrahydro -2H -pyrano [3,2 -c] isochromen -6 -one. -tetrahydroxy -2 -(hydroxymethyl) -9 -methoxy -3, 4, 4a, 10b -tetrahydro -2H -pyrano [3,2 -c] isochromen -6 -one. Various methods have been published for quantitative estimation of bergenin in S. ligulata. Several protocols employing high -performance liquid chromatography (HPLC) have been reported for estimation of bergenin in S. ligulata. However, the methods described hitherto have several limitations like extensive sample preparation. , In this study, an accurate, simple, and reproducible HPLC method was developed and validated for the determination of bergenin in S. ligulata herb extract and dosage form.
| Materials and Methods|| |
Whole plant of S. ligulata was authenticated by Dr. Kannan, Botanist, The Himalaya Drug Company, (Bangalore, India). Extracts and formulations were prepared by the Department of Formulation Development, The Himalaya Drug Company.
Standard bergenin (total purity 98%) was procured from ChromaDex. Methanol and acetonitrile were of HPLC grade was procured from Rankem. Milli-Q water generated by the Waters System Model ZMQX5V0001 was used during the analysis. Methanol was used as a solvent for the preparation of standards and samples, acetonitrile (solvent-A) and purified water (solvent-B) (75:25, v/v) were used as mobile phase for HPLC analysis. All solutions used for HPLC analysis were filtered through 0.45 μm membrane filter using Millipore type filtration unit. A C18 ODS LUNA-phenomenex (250 mm × 4.60 mm) 5 μ column was used as the stationary phase.
Precoated silica gel 60 F 254 plates with a thickness of 0.2 mm were used (E-Merck, Mumbai) for thin layer chromatographic assay. Toluene: Ethyl acetate: Formic acid in a ratio of 6:6:1 was used as the mobile phase.
A CAMAG Linomat V instrument was used for a sample application; reprostar IV was used for photo-documentation.
A Shimadzu Prominence HPLC system, comprising quaternary pumps, photodiode array (PDA) auto-injector was used for HPLC analysis. Liquid chromatography (LC) solution software (Shimadzu corporation, JAPAN) was used for integration and calibration.
High-performance liquid chromatography
About 100 mg of finely powdered S. ligulata plant and dosage forms powder and 50 mg of S. ligulata extracts were weighed separately in a 100 ml beaker. About 30 ml of methanol was added and sonicated for about 2-3 min and allowed to settle. The extracts were decanted separately into 50 ml volumetric flasks. The residues were again extracted twice in a similar fashion with 10 ml of methanol each time. The extracts were combined with their respective volumetric flasks. The volume was made up with methanol and filtered through 0.45 μm syringe filter and used for further analysis.
High-performance thin layer chromatography fingerprinting
One-gram sample was dissolved with methanol and made up to 100 ml with the same solvent and used for high-performance thin layer chromatography (HPTLC) analysis. Standard bergenin at 1 mg/ml concentration in methanol was used.
High-performance thin layer chromatography fingerprinting
High-performance thin layer chromatography
10 μl of sample and standard were spotted on CAMAG Linomat V of 12 mm bandwidth on a precoated silica gel 60 F 254 plate of 0.2 mm thickness. The plate was developed in the solvent system of toluene: Ethyl acetate: Formic acid in a ratio of 6:6:1. Identification of bergenin was captured at 254 nm. The Rf value was identified at 0.13 with dark blackish brown spot [Figure 1]a and b.
High-performance liquid chromatography
The HPLC system (Shimadzu prominence quaternary pumps), PDA auto-injector and C18 ODS LUNA- phenomenex (250 mm × 4.60 mm) 5 μ column was used at 40°C temperature. Binary elution was carried out with acetonitrile: Purified water, 75:25, v/v at a flow rate of 0.8 ml/min, detection was at 275 nm using PDA. LC solution software was used for integration and calibration. Evaluation was via peak areas with linear regression.
Estimation of bergenin
To estimate the bergenin content in S. ligulata plant, tablets and extract samples, 20 μl aliquots of the sample were injected into HPLC. The HPLC analysis was continued for 30 min. The content of bergenin in dosage form was calculated by linear regression and mean percentages were calculated from triplicate experiments. The percentage of bergenin was calculated using 0.1 mg/ml of bergenin standard curve. The values were tabulated as shown in [Table 1]. The chromatograms are shown in [Figure 2]a-d.
|Figure 2: (a) Bergenin Standard Chromatogram (b) Sample Saxifraga ligulata plant Chromatogram (c) Sample Chromatogram (d) Sample Saxifraga ligulata Extract Chromatogram|
Click here to view
Calibration curve of standard and sample bergenin
Bergenin standard solution was prepared in methanol at a concentration of 1 mg/ml (stock solution). Working standard solutions were prepared by diluting standard stock solution with methanol in the concentration range 20-100 μg/ml. 20 μl from each working standard solution was injected in triplicates. Calibration curve was generated by linear regression based on peak areas.
0.2 mg/ml-4 mg/ml of bergenin tablet solution was prepared in methanol. 20 μl from each solution was injected in triplicates. Calibration curve was generated by linear regression based on concentration and peak areas.
The developed HPLC method was validated as per the principles of International Conference on Harmonization guidelines.
Accuracy was determined by spiking known concentrations of bergenin in the formulation sample at the rate of 10%, 20%, and 30% over the observed concentration. The experiment was done in triplicates. The results were expressed as mean recovery ± % relative standard deviation (RSD).
Interday and intraday precision of bergenin estimation were determined using two different instruments on two different days. The estimations were performed in triplicates. The results were expressed as mean assay ±%RSD.
In order to check if the method response follows a linear regression on a 5-point calibration curve was constructed for the sample and standard. Since the polyherbal formulations available in the market may show wide range of bergenin content due to different proportions of S. ligulata, we have constructed the linearity curve in wide range from 10% to 200% of the expected concentrations of the current formulation.
Limit of detection
Limit of detection was calculated using the standard error of the slope method and 3.3 times of such error was considered as a limit of detection.
Limit of quantification
Limit of quantification was calculated using the standard error of the slope and 10 times of such error was considered as a limit of quantification.
| Results and Discussion|| |
The chemical composition of herbal extracts are very complex, evaluating the quality of these extracts can only be assured by the use of validated analytical methods for identification and quantification of the active ingredients. The HPLC methods for the quantitative estimation of bergenin were validated with regard to their precision, accuracy and linearity.
Triplicates of three different concentrations of bergenin tablet on the same day (intraday) and different days (inter day) were carried out, integrated and expressed as % RSD. The results noted in [Table 1], reveal no significant intraday and interday variations. The % RSD for intraday and interday analysis was found to be in the range, bergenin 0.10-0.58, which is <2%. The accuracy of the method was determined from recovery studies. The preanalyzed sample was spiked with three different concentrations of bergenin, 10%, 20%, and 30%, and the mixtures were analyzed by the proposed method. The bergenin recoveries were in the range of 92.51-95.72 as reported in [Table 2]. The linearity of the sample curve was evaluated by injecting five concentrations ranging from 10% to 200% that is, 0.2, 1, 2, 3, 4 mg/ml solutions [Table 3]. Peak area and concentrations were subjected to least square linear regression analysis to calculate the calibration equation and correlation coefficient. Linearity was obtained over a concentration range of 10-200% of the sample. The linearity of standard and sample calibration graphs is shown in [Figure 3]a and b. The results are mentioned in [Table 4]. In the theoretical plates, asymmetry observed were within the limits of USP.  Reproducibility of bergenin content in three different formulations is provided [Table 5]. This optimized RP-HPLC method serves as the best quality control tool for qualitative and quantitative evaluation of bergenin in polyherbal formulations containing S. ligulata.
Application of high-performance liquid chromatography methods
The HPLC method developed serves as a very precise and robust tool for quantitative determination of bergenin. This method can be adopted to assess the quality of the drugs which can add more value to herbal research.
| Conclusion|| |
The developed HPLC and HPTLC methods can be utilized for the qualitative and quantitative estimation of bergenin in S. ligulata herb samples, extracts and dosage forms. The methods developed are simple, sensitive, and statistically validated for linearity, accuracy, and precision.
| Acknowledgment|| |
The authors are thankful to Dr. Prafulla S Chaudhari, R and D Center, and Dr. Jayashree Keshav, Ms. Shruthi V Kumar, Scientific Publications, The Himalaya Drug Company, Bangalore, India, for their support. Also, acknowledge the support of Dr. Shivakumar HG, Principal, JSS College of Pharmacy, JSS University, Mysore, India.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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