DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF NIGELLA SATIVA SEED OIL AND GINGER EXTRACT IN THE SAME DOSAGE FORM

DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF NIGELLA SATIVA SEED OIL AND GINGER EXTRACT IN THE SAME DOSAGE FORM

B. Snehalatha*, M. Momin, A. V. Mishal and T. R. Kale

Quality Assurance Department, Oriental College of Pharmacy, Plot no.3,4,5; Sector-2, Near Sanpada Railway Station, Sanpada, Navi-Mumbai- 400705. India.

ABSTRACT:The combination of Nigella sativa seed oil and ginger extract is used as antiulcer agent and to treat abdominal pain, diarrhoea and flatulence. The paper describes validated simultaneous equation method for the simultaneous estimation of Nigella sativa seed oil and Ginger extract in a combined dosage form. Nigella sativa seed oil and Ginger extract were found to have absorption maxima at 256 and 282 nm respectively in 5% SLS solution. Both these drugs obeyed Beer's law in the concentration range of 100-500μg/mL. The high values of correlation coefficients (r²) indicated good linearity of calibration curve for both the drugs. The results of analysis have been validated statistically and by recovery study the value of standard deviation ranging from 97-99.21 % for Nigella sativa seed oil and 98-101.6 % for ginger extract were indicative of the accuracy and precision of the proposed method. The proposed method was successfully applied for the determination of nigella sativa seed oil and Ginger extract in the pharmaceutical formulation. This method was found to be simple, sensitive, accurate, precise and economical and applicable for the simultaneous determination of Nigella sativa seed oil and Ginger extract in combined dosage form.

Ion exchange column chromatography, Amino acids, Determination, Hydrolysis

INTRODUCTION: Nigella sativa is an annual flowering plant, native to south and southwest Asia. Nigella sativa oil contains an abundance of conjugated linoleic (18:2) acid, thymoquinone,  dithymoquinone, melanthin, nigilline, damascenine, and tannins. Melanthin is toxic in large doses and nigelline is paralytic, so this spice must be used in moderation. Thymoquinone, found in the seed oil extract of N. sativa, has been shown to have anti-neoplastic effects in rats and mice and in cultured human cells from several types of cancer, including pancreatic ductal adenocarcinoma.

It has protective antioxidant and anti-inflammatory ^1-2 effects, and promotes apoptosis (cell death) of the cancer cells.

Ginger - the "root," or actually the rhizome, of the plant Zingiber officinale has been a popular spice and herbal medicine for thousands of years. It has a long history of being used as medicine in Asian, Indian, and Arabic herbal traditions. In China, for example, ginger has been used to help digestion and treat stomach upset, diarrhea, and nausea for more than 2,000 years.

Ginger is native to Asia where it has been used as a cooking spice for at least 4,400 years.It has been used to help treat the common cold, flu-like symptoms, headaches, and painful menstrual periods ³. Ginger has been used to treat diseases related to gastrointestinal tract such as flatulence, indigestion, nausea and vomiting ^4-6.

The ginger extract composes of oleoresin containing volatile oils of gingerols, shogoals, α-zingiberene, β- bisabolene, β- sesquiphellandrene and ar-curcumene ⁷.

Literature survey revealed that various analytical methods such as UV spectroscopy, HPLC, pulse polarography can be used for determination of two drugs. The UV spectrophotometric analysis⁸ is often preferred in quality control testing and ordinary laboratories due to its broader availability, suitability and ease of use.

The present study involves developing and validating spectrophotometric method for simultaneous determination of floating beads containing Zingiber officinale extract and Nigella sativa seed oil as a drug candidate, which remain in stomach or upper part of GIT for prolonged period of time, therefore the maximum drug release is maintained at desired site.

MATERIALS AND METHODS:

Instrument & Apparatus

A double beam UV-visible Spectrophotometer (Shimadzu, UV-1800, Japan), attached to a computer software UV probe 3.2, with a spectral width of 2 nm, wavelength accuracy of 0.5 nm and pair of 1 cm matched quartz cells, Analytical balance (Sartorius Balance), volumetric flasks, pipettes of borosilicate glass.

Reagents and Materials

Nigella sativa seed oil was extracted with ether in laboratory, Ginger extract was received as a gift sample from Konark Herbals and Health Care, Nani Daman, Sodium Lauryl Sulphate, Distilled water, Whatman filter paper

Preparation of diluents

Sodium Lauryl Sulphate solution (5%). [Dissolve 5 gm of Sodiul Lauryl Sulphate (SLS) in 100 mL of water]

Preparation of Standard Stock Solutions

Accurately weighed Nigella sativa seed oil (100 mg) and Ginger extract (100 mg) was transferred to a separate 10 mL volumetric flask and dissolved and diluted to the mark with SLS solution to obtain a standard solutions having concentration Nigella sativa seed oil (10 mg/mL) and Ginger extract (10 mg/mL).

Method:

In simultaneous equation method, five working standard solutions having concentration 100, 200, 300, 400, 500 μg/mL for both Nigella sativa seed oil and Ginger extract (100 mg) were prepared in SLS solution and λmax of Nigella sativa seed oil and Ginger extract (100 mg) were calculated, absorptivity coefficients were calculated using calibration curve. The concentration of two drugs in the mixture can be calculated using following equations;

…………. (1)

………… (2)

Where, Cx and Cy= Concentration of Nigella sativa seed oil and Ginger extract respectively.

A1 = Absorbance of mixture at 256 nm

A2 = Absorbance of mixture at 282 nm

ax1 = Absorptivity of Nigella sativa seed oil at 256 nm

ax2 = Absorptivity of Nigella sativa seed oil at 282 nm

ay1 = Absorptivity of Ginger extract at 256 nm

ay2 = Absorptivity of Ginger extract at 282 nm

Method Validation:

Linearity

Calibration curves were plotted over a concentration range of 100 – 500 μg/mL for both Nigella sativa seed oil and Ginger extract. Accurately measured standard working solutions of Nigella sativa seed oil (0.1, 0.2, 0.3, 0.4 and 0.5 mL) and Ginger extract (0.1, 0.2, 0.3, 0.4 and 0.5 mL) were transferred to a series of 10 mL of volumetric flasks and diluted to the mark with SLS solution, and the absorbance was measured at 256 nm and at 282 nm for both drug. The calibration curves were constructed by plotting absorbance vs. concentration and the regression equations were calculated.

Precision:

Intraday Precision

Mixed solutions containing 100-500 μg/mL of Nigella sativa seed oil and 100-500 μg/mL of Ginger extract were analyzed 3 times on the same day and % RSD was calculated.

 Interday Precision

Mixed solutions containing 100-500 μg/mL Nigella sativa seed oil and 100-500 μg/mL of Ginger extract were analyzed on 3 different days and % RSD was calculated.

Accuracy

The accuracy of the method was determined by calculating recoveries of Nigella sativa seed soil and Ginger extract in mixture by the standard addition method. Known amounts of standard amount of Ginger extract was added at 50, 100 and 150 % levels to pre-quantified sample solutions of 1000 μg/mL Nigella sativa seed oil + 1000 μg/mL Ginger extract mixture. The absorbance of Nigella sativa seed oil and Ginger extract were recorded at λ₁ and λ_2. The percentage recovery was calculated by measuring the absorbance of both drug at their absorbance maxima and fitting these values into simultaneous equation. Each response was average of three determinations.

Limit of Detection and Limit of Quantitation

The limit of detection (LOD) and the limit of quantitation (LOQ) of the drug were derived by calculating the signal-to noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ = the standard deviation of the response,

S = Slope of calibration curve.

Analysis of Ginger extract and Nigella sativa seed oil in Combined Dosage Form

Beads equivalent to 0.3 gm of Nigella sativa seed oil and 0.1 gm of Ginger extract were weighed and transferred into 10 mL volumetric flask. Using this solution, dilution equivalent to 200μg/mL was prepared. Absorbance of the resulting solution was measured at 256 nm and 282 nm against SLS solution, relative concentration of two drugs in the sample was calculated using above equations (1) and (2).

RESULTS AND DISCUSSION:

In simultaneous equation method, the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all wavelengths, which was fulfilled in case of both these drugs. The two wavelengths used for the analysis of the drugs were 256 nm (λmax of Nigella sativa seed oil) and 282 nm (λmax of Ginger extract) at which the calibration curves were prepared for both the drugs. The overlain UV absorption spectra of Nigella sativa seed oil (256 nm) and Ginger extract (282 nm) in SLS solution is shown in (Figure 1).

FIGURE 1: OVERLAIN ABSORPTION SPECTRA OF NIGELLA SATIVA SEED OIL (256 NM) AND GINGER EXTRACT (282 NM) IN SLS SOLUTION

Validation of the Proposed Method

Calibration curve

Linear correlation was obtained between absorbance versus concentrations of Nigella sativa seed oil and Ginger extract in the ranges of 100 – 500 μg/mL. Regression parameters are mentioned in table 1 and the calibration curves of these two drugs at 256 nm and 282 nm were validated by the high value of correlation coefficients of regression (Table 1).

TABLE 1: REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION PARAMETER FOR THE PROPOSED METHOD

% RSD = Percent relative standard deviation

LOD     = Limit of detection

LOQ     = Limit of quantitation

SD        = Standard deviation

n           = number of replicates

The validation parameters were studied at all the wavelengths for the proposed method. Accuracy was determined by calculating the recovery and the mean was determined (Table 2). The method was

successfully used to determine the amounts of Nigella sativa seed oil and Ginger extract present in the beads (Table 3 and Table 4).

TABLE 2: RECOVERY DATA FOR THE PROPOSED METHOD

*SD is Standard deviation and n is number of replicates.

TABLE 3: FORMULATION COMPOSITION

 TABLE 4: ANALYSIS OF NIGELLA SATIVA SEED OIL AND GINGER EXTRACT BEADS

CONCLUSIONS: The developed simultaneous equation method is found to be simple, sensitive, accurate and precise and can be used for routine analysis of Nigella sativa seed oil and Ginger extract. The developed method was validated as per ICH guidelines. Statistical analysis proved that the method is repeatable and selective for the analysis of Nigella sativa seed oil and Ginger extract in their combined pharmaceutical formulations.

ACKNOWLEDGEMENTS: The authors are thankful to Konark Herbals and Health Care, Nani Daman, India for providing gift sample of Ginger extract for research. The authors are thankful to Oriental College of Pharmacy College, Sanpada, Navi Mumbai, India for providing all the facilities to carry out the work.

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   How to cite this article:

   Snehalatha B, Momin M, Mishal AV and Kale TR: Development and Validation of Spectrophotometric Method for Simultaneous Estimation of Nigella Sativa Seed Oil and Ginger Extract in the Same Dosage Form.Int J Pharm Sci Res2014; 5(12): 5235-39.doi: 10.13040/IJPSR.0975-8232.5 (12).5235-39.

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