INTRODUCTION:

There has been considerable public and scientific interest in the use of phyto-chemicals derived from dietary components to combat human diseases. Its usefulness is referred to in the ancient Hindu scriptures. It is used in food, turmeric can be mixed with other natural compounds such as slaked lime and used topically as a treatment for wounds, inflammation and tumours.¹

Cinchona is a genus of family Rubiaceae, native to tropical South America. They are large shrubs or small trees growing to 5-15 meters tall with evergreen foliage. The bark of cinchona produces several alkaloids, the most important alkaloid, quinine (Figure 1), has certain febrifuge properties. Quinine was used in the battle against malaria since the 1620's. The bark of wild species may yield a quinine content of as high as 7%, whereas cultivated crops yield contents up to 15%.²

Of many species of cinchona, only four species have economic value for the production of quinine: C. calisaya, C. legeriana, C. officianalis and C. succirubra. Quinine [130-95-O] is a weak base (pKi = 4.32 and pK2 = 8.4) with the following structure:

Figure 1. Structure of Quinine (C20H24N2O2; MW = 324)

Quinine is used, an antiseptic, and lotions. Quinine is chiefly used as a tonic in the treatment of falciparum malaria. Quinine is chosen where the ailment has become highly resistant to other anti-malarial drugs. Quinine shows various activities like antibacterial, antipyretic, cardiovascular stimulant, local anesthetic and analgesic properties. Quinine is used to prevent cardiac arrythmias and is used in tonic beverages, which are mixed with alcohols for bitter taste. Quinine is one of the most useful alkaloids for pharmaceutical purposes.³

A lot of work have been done for the determination of quinine in pharmaceuticals and biological fluids by HPLC / RP-HPLC.^4,5,6But a few reports are available for the determination of it in crude bark extracts as well as in marketed formulations. Mroczek and Glowniak performed separation of quinine on to TLC and HPTLC from extract of cinchona cortex and pharmaceutical preparations using quaternary mobile phase.^7,8 International pharmacopoeia reports a ternary mobile phase for quinine separation. Here, we developed a method for extraction as well as quantitative determination of quinine from bark of Cinchona species using binary mobile phase. The method is not only fast but also provides data for large scale processing of quinine from its raw material. This method will be helpful in quality control and quantitative studies for various industries as well as in the search for development of high yielding plant varieties either by plant breeding or biotechnological studies. Fluorescence under UV light is a characteristic of the majority of natural coumarins. These compounds are very easily detected, since they give characteristic fluorescent colors under UV light, which are intensified by further treatment with ammonia vapors. Scopoletin is a coumarin, which exhibits a blue violet fluorescence under UV light.^9,10,11,12

In the past 5 years, a number of publications have appeared on the determination of quinine, 60% of these dealing with body fluids and 25% with beverages and pharmaceutical preparations. Chromatographic techniques have mostly been used: gas chromatography¹³, gas chromatography-mass spectrometry^14,15, thin layer chromatography^16,17 and high-performance liquid chromatography.¹⁸

Habb-e-Bukhar is a polyherbal unani formulation, consisting of four ingredients of plant origin and it is widely used Daf-e-Humma, Moauriq and Humma-e-Hadda which is official in Unani pharmacopoeia. The major active component is quinine. From such polyherbal formulations separation, identification and estimation of chemical components is very difficult. Habb-e-Bukhar is used as antipyretic. It is given in fever due to elephantiasis and malaria.¹⁹

MATERIALS AND METHODS:

Plant Material and chemicals

Cinchona spp. bark was purchased from local market of Raipur, Chhattisgarh. Bark was pulverized to a fine powder in a mechanical blender. This fine powder was utilized for experimental purpose. All solvents and chemicals used in this study were of analytical grade. Reference standard of quinine was purchased from E. Merck, Germany.

Preparation of the unani formulation

Habb-e-Bukhar, three laboratory batches (coded HLS-I, HLS-II) were prepared in laboratory according to reported method of Unani formulary of India²⁹. The available commercially brand HMS-A, HMS-B of Habb-e-Bukhar was procured from local Pharmacy.

General experimental conditions

Instrument: Shimadzu RF-5301 PC

EM Wavelength: 372.0nm

EM Wavelength: 456.0nm

Slit Width: EX:5.0nm EM:5.0nm

Scan Speed: Super

Sensitivity: Low

Response Time: Auto

Shutter: Auto, Closed

Procedure for spectra measurements

Figure 2: Excitation and Emission spectra of quinine

The fluorescence emission spectra were obtained at room temperature. A preliminary analysis was carried out to determine the wavelength at which maximum intensity is exhibited by pure compound. For this purpose, a 100 µg/ml sample of pure quinine was prepared in methanol. This was scanned spectrofluorimetrically to obtain the excitation and emission wavelengths. The λ_max shown by quinine had an excitation at 372 nm and an emission at 456 nm. (Figure 2)

Standard sample preparation and calibration curve

A stock solution of quinine was prepared by dissolving 10 mg of quinine in a 100 mL volumetric flask containing methanol, sonicated for 10 minutes with ultrasonicator. A calibration curve was plotted between increasing amounts of quinine and their intensities. A straight line was obtained between 1.0 to 7.0 µg/ml. Correlation coefficients (r) were 0.9995. The linear regression equation was found to be y = 25.786x + 1.9679. (Figure 3)

Figure 3: Calibration curve of quinine

Soxhlet extraction and test sample preparation

Soxhlet extraction of 10 g (14 mesh) powdered bark was performed on water bath with 200 mL of corresponding solvent for 10 hrs. Extract thus obtained was concentrated in vacuo via rotavapor and re-dissolved in methanol and volume made up to 100 mL. This solution was taken as test sample for quantification purpose.

Method Validation

Precision

The intra-day precision of the method was validated with a standard solutions (concentrations 1.0, 3.0 and 6.0 mg/ml) of quinine under the selected optimal conditions three times a day. For inter-day precision, measurements one time a day on three consecutive days were conducted. All of the measurements were expressed as relative standard deviations (RSD).

Repeatability

Six independently prepared sample solutions (concentrations 1-7 mg/ml) of quinine with the same amount were analyzed and the variations within six measurements were calculated for evaluation of repeatability. The processes of the measurements were in accordance with the Standard sample preparation of quinine.

Recovery

Standards of quinine with the known amounts in solutions were spiked to the Habb-e-Bukhar solution of which the contents of quinine had been determined before the addition of the standard chemical. Then, quinine marker compound in Habb-e-Bukhar sample solutions were extracted, processed and quantified in accordance with the established procedures, and finally the recovery rates were calculated. Same procedures were applied for the three different marketed formulations.

Determination of quinine concentration in formulations

100 mg chinchona powder was weighed accurately and extracted with 10 ml methanol with vigorous shaking. It was then filtered and the volume made up to 100 ml with methanol. Then, this solution was further diluted to 100 ml and analyzed in the spectrofluorimeter and the intensity of the fluorescence was recorded. The concentration of quinine in the extract sample was determined from the standard curve.

RESULTS AND DISCUSSION:

Method Validation

Precision

The repeatability of sample application and measurement of peak area were expressed in terms of R.S.D. % for quinine. The results depicted in Table 1 showed that no significant intra- and inter-day variation was observed in the analysis of quinine at three different concentration levels 1, 3 and 6 µg/ml. The R.S.D. % for intra- and inter-day analysis was found to be less than 2 % in all the cases.

Linearity and range

For linearity, five different concentrations of quinine were used in a working range of 1.0-7.0 μg/ml. Linear regression equations and correlation coefficient (r) values for test quinine presented in Table 3. The method showed good linearity in the given range.

Limit of detection and quantitation (LOD and LOQ)

The LOD with signal/noise ratio of 3:1 was found to be 0.1768 μg/ml for quinine. The LOQ with signal/noise ratio of 10:1 was found to be 0.5411 μg/ml for quinine. (Table 3)

Recovery studies

The proposed method when used for extraction and subsequent estimation of quinine from unani formulations form after spiking with 50, 100 and 150 % of additional standard quinine afforded recovery between 97.34-99.40 % (Table 2). The data of validation parameters are listed in Table 3.

Table 1. Intra- and inter-day precision of method (n=6).

Amount (µg/ml)	Intra -day precision		Inter-day precision
Amount (µg/ml)	S.D. of intensity	R.S.D. %	S.D. of intensity	R.S.D. %
1.0	0.0251	0.001974	0.35102232	0.027590
3.0	0.034881	0.001291	0.104003	0.003846
6.0	0.025033	0.0004626	0.032042	0.000592

Table 2. Recovery Studies (n=6).

Excess drug added to the analyte (%)	Theoretical content (μg/ml)	Content found (μg/ml)	Recovery (%)	S.D.	R.S.D. (%)
50	1.0	1.45	97.34	0.6804	0.3421
100	3.0	5.87	97.84	1.0308	0.3461
150	6.0	14.91	99.40	4.9886	1.2615

Table 3. Summary of validation parameters.

Parameters	Data of quinine
Linearity Range Linear equation Slop (m) Intercept (C) Correlation coefficient	1.0-7.0 (μg/ml) Y= mX+C 25.786 1.9679 0.9995
Limit of detection	0.1768
Limit of quantitation	0.5411
Recovery (n=6)	98.19 %
Precision (R.S.D. %) Repeatability of application (n=6) Inter-day (n=6) Intra-day (n=6)	0.001923 0.010676 0.001242

CONCLUSION:

The improved method is useful for ‘in process’ analytical method for quinine determination as well as for the screening purposes and also provides useful information towards development of extraction technology for processing of quinine. This method represents an improved approach for quinine determination taking into consideration of fingerprint development and validation of quinine in herbal formulation. It offers the advantages of speed, simplicity and selectivity.

ACKNOWLEDGEMENT:

The authors are highly grateful to FIST scheme, {F. No. (SR/FST/LS1-013/2010)} Govt. of India, New Delhi for supporting infrastructure and UGC, [F. No: 34-131/2008 (SR)] Govt. of India, New Delhi for financial assistance under major research project.

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Received on 22.10.2011 Modified on 12.12.2011

Asian J. Research Chem. 5(1): January 2012; Page 14-17

Simple and Rapid Spectrofluorometric Fingerprint Development of Quinine in Unani Formulation

INTRODUCTION: