Simultaneous Equation Method for Estimation of Rutin Trihydrate and Berberine Chloride in Synthetic Mixture and Polyherbal Formulation

 

 Dr Palak Chaudhary*, Dr. Harsha U. Patel

Shri Satsangi Saketdham “Ram Ashram”, Group of Institutions, Faculty of Pharmacy,

Vadasma, Mehsana, Gujarat, India.

 

ABSTRACT:

The present study was focused on establishing and validating a simple, accurate, reliable and economical UV spectrophotometric method for the simultaneous determination of Rutin trihydrate (RUT) and Berberine Chloride (BER) in synthetic mixture and in antidiabetic polyherbal formulation. Rutin and Berberine from herbal plants are used as antidiabetic drug to prevent and manage Diabetes. The developed method gives best results in terms of linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The method was validated as per ICH guidelines. The linearity range for Rutin trihydrate and Berberine Chloride were found to be 10 to 50 μg/ ml and 10 to 50 μg/ml respectively. The values of LOD were 0.062 μg/ ml and 0.076 μg/ml and the values of LOQ were found to be 0.187 μg /ml and 0.229 μg/ ml for Rutin trihydrate and Berberine chloride respectively.

 

 

INTRODUCTION:

Diabetes mellitus (DM) is a group of metabolic disorder characterized by hyperglycemia resulting from the defects in insulin secretion, insulin action or both. A chronic hyperglycemic condition in diabetes is associated with long term damage, dysfunction, and failure of various organs, such as eyes, kidneys, nerves, heart and blood vessels. It is the most common serious metabolic disorder and is considered to be one of the five leading causes of death in the world.1-3 In last few years herbal medicines and related drugs are highly used and gaining popularity day by day all over the globe. The medicinal herbs have certain therapeutic agents which help to prevent and cure the disease. It is in tradition of Indian culture to use these herbs in health care system.4 

 

Rutin and Berberine from herbal plants are used as antidiabetic drug to prevent and manage DM. Fig. 1 and 2 shows the structure of RUT and BER respectively.

 

 

Rutin is a polyphenolic bioflavonoid and has wide range of pharmacological applications due to its significant antioxidant properties. Rutin has multispectrum pharmacological benefits for the treatment of various chronic diseases such as cancer, diabetes, hypertension and hypercholesterolemia.^5-7 Berberine is an isoquinqline alkaloid found in Tinospora cordifolia and many other plants. It lowers the elevated blood glucose level as effectively as metformin. Berberine is a bioactive marker used to treat many health problems like, diabetes, bacterial infection, intestinal problems, inflammation, hyperlipemia, congestive cardiac failure, etc.^8,9

 

From the literature it is revealed that various analytical methods for the determination of either RUT or BER have been reported. HPLC, UV Spectrophotometry, RP-UFLC and Capillary Electrophoresis methods have been reported for determination of RUT individually or in combination with other component.^10-15 For determination of BER many methods like HPLC, UV Spectrophotometry, HPTLC and LC-Tandem Mass spectrometry have been reported individually or in combination with other markers.^16-20

 

A comprehensive literature survey revealed that there is no suitable method reported for simultaneous estimation of Rutin trihydrate and Berberine Chloride. This paper describes the development and validation of UV spectrophotometric method for simultaneous determination of Rutin trihydrate and Berberine chloride.

 

 

Fig. 1: Structure of Rutin trihydrate (RUT)

     

 

Fig. 2: Structure of Berberine Chloride (BER)

 

METERIALS AND METHODS:

RUT and BER standard markers were procured from Sigma Aldrich and herbal formulation (Diabgul from Uniray Life Sciences) was procured from a local pharmacy. AR grade methanol (Ranchem) was used for development purpose.

 

Spectroscopic Analysis was carried out on a UV/VISIBLE 2450 (Shimadzu) double beam UV-Visible spectrophotometer with software of UV Probe version 2.34. The zero order absorption spectra were recorded over the wavelength range of 200-400 nm, against solvent blank, in quartz Cuvettes with 1 cm diameter. A Semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

 

Preparation of standard solutions:

Accurately weighed RUT (10 mg) and BER (10 mg) were individually transferred to a 100 mL volumetric flask and dissolved in 25 ml of methanol. Sonicate for 15 min and diluted to the mark with methanol to obtain a standard stock solution (100 µg/ml).

 

Simultaneous Equation Method (Vierordt’s Method)²¹:

Two wavelengths selected for the method are 258 nm and 349 nm that are absorption maximas of RUT and BER respectively in methanol. The stock solutions of both the drugs were further diluted separately with methanol to get a series of standard solutions of 10-50 μg/ml concentrations. The absorbances were measured at the selected wavelengths and absorptivities for both the drugs at both wavelengths were determined as mean of five independent determinations. The absorptivity value of the drug is the ratio of absorbance at selected wavelength with concentration of drug. Concentrations in the sample were obtained by using following equations----

C_x = A₂a_y1 – A₁a_y2 / a_x2a_y1 – a_x1ay₂   ---------------------- (1)

C_y = A₁a_x2 – A₂a_x1 / a_x2a_y1 – a_x1a_y2   ---------------------- (2)

 

Where, A₁ and A₂ are absorbances of mixture (test) at 258 nm and 349 nm respectively,

ax₁ and ax₂ are absorptivities of RUT at λ₁ and λ₂ respectively and

ay₁ and ay₂ are absorptivities of BER at λ₁ and λ₂ respectively.

C_x and C_y are concentrations of RUT and BER in test respectively.

 

Selection of wavelength:

Methanol is selected as a solvent based on solubility of drugs. The solutions were prepared. And scanning of UV spectrum was done to in the range of 200-400 nm to determine the wavelength of maximum absorption for both the drugs RUT and BER.

 

Application of proposed method for determination of RUT and BER in Synthetic Mixture:

The synthetic Mixture is prepared as:

 

_{Table 1: Composition of Synthetic Mixture}

 

Take Synthetic powder equivalent to 10mg of RUT and BER in 100ml volumetric flask. Dissolve it in 25ml methanol, sonicate for 15 min and make upto the mark with methanol. Shake vigorously and filter the solution. Finally the solution had the concentration 100μg/ml and 100μg/ml respectively for RUT and BER. After that from this solution 1ml was pipette out and diluted up to 10 ml with methanol. So the concentration was 10μg/ml and 10μg/ml for RUT and BER respectively. RUT and BER were measured and assay was calculated.

 

_{Table 2: Assay Recovered of Synthetic Mixture}

 

Application of proposed method for determination of RUT and BER in Poly Herbal Formulation

Take the Diabgul capsules and remove content from it. From that take 5 gm of powder formulation and dissolve in 50 ml of methanol. Macerate for 1 hour. Sonicate for 60 min at room temperature. Filter the extract and estimate the content of RUT and BER present in formulation. Measure the absorbance at λ₁ and λ_2. The concentration of RUT and BER was calculated using the simultaneous equation.

 

Table 3: Result of Formulation Analysis

 

METHOD VALIDATION:

The developed method is validated as per ICH guidelines.

 

Linearity:

The linearity response was determined by analyzing five independent levels of calibration curve in the range of 10-50μg/ml and 10-50μg/ml for RUT and BER respectively (n=6). Stock solution cconcentration of RUT was 100µg/ml and BER was 100µg/ml. From this solution, take 1, 2, 3, 4 ml and 5 ml diluted with 10 ml Methanol to make the concentration 10, 20, 30, 40, 50 µg/ml of RUT and BER respectively. This solution was used for mixture in which take 1 ml RUT solution from Stock and 1 ml BER solution from stock in 1 flask and dilute up to 10 ml it give 1:1 mixture. Calibration curves and Regression data are shown for the same.

 

Precision:

Intraday precision:

The mixed solution containing concentrations 10, 20, 30 μg/ml for RUT and 10, 20, 30μg/ml for BER was analysed with three replicate (n=3) each on same day and % RSD was calculated.

 

Interday Precision:

The mixed solution containing concentrations 10, 20, 30 μg/ml for RUT and 10, 20, 30 μg/ml for BER was analysed with three replicate (n=3) per day for consecutive 3 days and % RSD was calculated.

 

Accuracy:

Accuracy is the closeness of the test results obtained by the method to the true value. Recovery study was carried out by addition of standard drug to the sample at 3 different concentration levels (80%, 100%, 120%).  At each level three determinations were done. % Recovery was calculated as shown in Table 7.

 

Limit of Detection and Limit of Quantitation:

The Limit of detection and quantitation of the developed Method was assessed by analyzing five replicates of standard solutions containing concentrations 10 μg/ml for RUT and 10μg/ml BER. The LOD and LOQ were calculated as LOD = 3.3*σ/S, and LOQ = 10*σ/S, where σ is the standard deviation of the lowest standard concentration and S is the slope of the standard curve. % RSD was calculated.

 

RESULT AND DISCUSSION:

The overlapping spectra of RUT, BER and their standard mixture (1:1) are shown in Fig. 3. The spectral peak of both the drugs was well separated. For eatimation of RUT and BER, Vierordt’s method is used and 258 nm (λ1) and 349 nm (λ2) was selected as the analytical wavelength respectively. For calculation using the equation, wavelength is selected where one component gives maximum absorbance while the other one gives minimum absorbance. RUT and BER obeyed the Beer’s law between the range of 10-50 µg/ml and 10-50 µg/ml respectively. The calibration curves were plotted at selected wavelengths and the developed method was validated for different validation parameters as per ICH guidelines. The %RSD values were satisfactory and indicate that the method is precise and accurate.

 

Table 4: Linear Regression Data (n=6)

Parameters	Rutin Trihydrate	Berberine Chloride
Wavelength (nm)	258 nm	349 nm
Linearity	10-50 µg/ml	10-50 µg/ml
Linear regression equation a
Intercept (c)	-0.0432	0.0121
Slope (m)	0.0836	0.081
Correlation coefficient (R2)	0.9997	0.9997
LOD	0.062	0.076
LOQ	0.187	0.229

    ^a y=mx+c

 

_{Table 5:  Intraday precision data for estimation of RUT and BER*(n=3)]}

Sr. No.	Conc. (µg/ml)	Absorbance at 258nm	%RSD	Absorbance at 349nm	% RSD
1	10	0.810±0.006	0.78	0.816±0.004	0.06
2	20	1.634±0.008	0.51	1.640±0.013	0.77
3	30	2.465±0.017	0.70	2.472±0.011	0.44

 

 

 

Fig. 3: Overlain zero order spectra of RUT (10µg/ml), BER (10µg/ml) and their standard mixture (10µg/ml) using Methanol as solvent

 

Fig. 4: Calibration Curve for Standard Mixture at 258nm & 349nm

_{Table 6: Interday precision data for estimation of RUT and BER*(n=3)]}

Sr. No.	Conc. (µg/ml)	Absorbance at 258nm	%RSD	Absorbance at 349nm	% RSD
1	10	0.797±0.002	0.21	0.811±0.006	0.79
2	15	1.630±0.004	0.22	1.637±0.012	0.75
3	20	2.472±0.007	0.27	2.478±0.011	0.44

 

_{Table 7: Recovery Data for Accuracy (n=3)}

Sr. No.	Level of Spiking	Conc. Taken (µg/ml)	Amount of standard added (µg/ml)		Amount found (µg/ml)		% Recovery ± SD
			RUT	BER	RUT	BER	RUT	BER
1	Control	10	-	-	10.17	10.14	101.67±0.080	101.42±0.040
2	80%	10	8	8	18.32	18.09	101.76±0.037	100.52±0.020
3	100%	10	10	10	20.35	20.10	101.75±0.041	100.52±0.023
4	120%	10	12	12	22.39	22.11	101.77±0.045	100.52±0.025

 

CONCLUSION:

Accepting that HPLC method is expensive in hardware and chromatographic reagents, and more expensive compare to UV method. Simultaneous equation method has the advantages like it is simple, rapid, direct, economic and not requiring sophisticated techniques or instruments. The method developed is rapid, sensitive, specific, accurate and repeatable. It possesses significant linearity, accuracy, precision within acceptable range of RSD. As no method is available for combined estimation, this method can be used for the routine QC analysis of polyherbal formulation having RUT and BER. The method is simple, inexpensive, requires an easy sample preparation and gives reliable results for estimation.

 

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Received on 17.05.2019           Modified on 08.06.2019

Accepted on 29.06.2019         © RJPT All right reserved