PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR ALOE POLYSACCHARIDES AS A MEASURE OF ALOE VERA QUALITY IN HERBAL FORMULATION Patel J*, Nayak B, Gaudani R, Modi H and Shah C Shree Swaminarayan Sanskar Pharmacy College, At & post Zundal, Ta. Dist. Gandhinagar-382 421. ABSTRACT Spectroscopic method development based on Aloe polysaccharide and Congo red dye interaction is presented in this work. The binding interaction between and Congo red was investigated by experiments and theoretical calculation. The results show that coaction of the electrostatic and hydrophobic interactions between Congo red and contributes to Congo red complex chromogenic reaction. Based on this interaction, method for spectrophotometric determination of with Congo red is proposed. The maximum absorbance of the complex is at 496-500 nm. Range is 100 to 2000 μg/ml (9.09 to181.82μg/ml in the test solution) with a correlation coefficient of 0.9910, the method applied for evaluation of marketed Aloe vera gel containing formulation. Keywords: Aloe vera, (Aloe polysaccharide), Congo red, Spectrophotometric method. INTRODUCTION The present work relates to spectrophotometric assay method for quantitative determination of a bioactive polysaccharide in a marketed Aloe vera (Aloe barbadensis Miller belonging to liliaceae family) product. Aloe vera leaf has 2 major components, yellow latex (exudates) from pericyclic cells containing mainly anthraquinones used as laxative and the clear inner leaf gel from parenchyma cells containing polysaccharide having physiological and pharmacological effects. When Aloe vera gel is poorly prepared, the polysaccharide compound is degraded. Degradation is indicated by decreased average molecular size, deacetylation of the polysaccharide resulting in loss of its solubility [1]. Spectrophotometric method is used for quantitative determination of aloe polysaccharides. The light absorption maximum wavelength of Congo red in 1 % (w/v) aqueous solution is approximately 488 nm. Aromatic group of dye could be attracted into the hydrophobic micro region of aloe polysaccharides through hydrophobic interaction. www.pharmasm.com IC Value 4.01 2654
Complex formed by electrostatic and hydrophobic interaction which can be shifted to higher wavelength with maximum absorbance [2]. MATERIALS AND METHODS Extraction of the gel from the plant is done without alkaline media (aqueous media). Loss on drying is done at 100 C up to consecutive weight. Comparison of extractive value of both alkaline and aqueous media is done. [3, 4] Method Development: Preparation of Aloe polysaccharide stock solution Freeze-dried aloe polysaccharide (40 mg) dissolved in 20 ml distilled water to a concentration of 2 mg/ml as main stock solution. For calibration standards, spiking solution prepared 10 to 2000 μg/ml by diluting appropriate aliquots of the stock solution with distilled water. To each tube will be added 5 ml Congo red reagent (0.01%w/v) & mild vortexing at room temperature for 20 min. Congo red solution λmax measurement Congo red 0.01% solution and 0.1% solution were prepared and its λmax measured it was around 488nm was observed. solution (plant) λmax measurement 100μg/ml stock solution of isolated from Aloe barbadensis plant was prepared and its λmax was measured it was around 210-230 nm was observed. and Congo red (0.01%w/v) reaction mixture λmax measurement- Aloe polysaccharides stock solution (100μg/ml) and congo red 0.01% reaction mixture prepared as per method described and its λmax measured. Calibration standard range selection- Trial 1: Range 10,20,50,80,100 μg/ml, Trial 2: Range 100, 200, 500, 800, 1000 μg/ml and Trial 3: Range 100, 200, 500, 800, 1000, 1200, 1500, 1800, 2000 µg/ml [3, 4] Method Validation: Preparation of Linearity Curve This concentration (2 mg/ml) is used as the main stock solution. Spiking solutions of 100, 200, 500, 800, and 1000, 1200, 1500, 1800μg/ml is prepared. www.pharmasm.com IC Value 4.01 2655
TABLE 1: SPIKING SOLUTIONS PREPARATION Conc. Volume taken Congo red Final volume Final conc. 100 0.5 5 5.5 9.09 200 0.5 5 5.5 18.18 500 0.5 5 5.5 45.45 800 0.5 5 5.5 72.73 1000 0.5 5 5.5 90.91 1200 0.5 5 5.5 109.09 1500 0.5 5 5.5 136.36 1800 0.5 5 5.5 163.64 2000 0.5 5 5.5 181.82 Specificity check The usual adulterants have been polysaccharides that are a fraction of the cost of Aloe vera, e.g., Guar gum and tragacanth etc. None of these reacts strongly to the assay. Guar gum 2000μg/ml and tragacanth2000 μg/ml stocks were prepared. Samples prepared as per preparation of stock. Precision [Intraday and Interday precision] Intraday precision: Intraday precision was carried out by taking three different concentration (100, 1000, 2000 μg/ml) repeated for 3 times in a day. Interday precision: Inter day precision was carried out by taking 3 different concentration (100, 1000, 2000 μg/ml) repeated on 3 different days. Accuracy [Recovery] Accuracy of the method was determined by recovery study by at three different levels (100, 1000, 2000 μg/ml). [3, 4] Method Evaluation: Method evaluation is done by measuring Aloe polysaccharide known quantity in marketed formulation. Formulation A and Formulation B are analyzed by UV visible spectrophotometry method as per developed method. RESULT AND DISCUSSION Loss on drying [5] www.pharmasm.com IC Value 4.01 2656
Sr. No Wt of petridish (gm) TABLE 2: LOSS ON DRYING Wt of petridish + polysaccharides (3ml) (gm) Wt of polysaccharides (gm) Constant wt of polysaccharides obtained after drying (gm) %LOD 1 12.8 15.8 3.0 2.59 13.67 2 13.5 16.5 3.0 2.71 11.67 3 13.2 16.2 3.0 2.69 11.33 Observation: Average % LOD Aloe vera gel was observed around 10-15%. Extractive value [5] TABLE 3: EXTRACTIVE VALUE Sr no. Parameter Average value in %w/w Reported value 1 alcohol extractive value 24% 25-30% 2 water extractive value 42% 50% Observation: Average % of water extractive value was observed higher than alcohol. Congo red solution λmax measurement Figure 1 Congo red 0.01% solution Figure 2 Congo red 0.1% solution Observation: Congo red 0.01% solution gives the sharp peak. solution and solution & Congo red (0.01%w/v) reaction mixture λmax measurement www.pharmasm.com IC Value 4.01 2657
Figure 3 solution (100μg/ml) Figure 4 and Congo red (0.01%w/v) reaction mixture Calibration standard range selection Trial-1: Range10 µg/ml to 100 µg/ml Observation: Less absorbance was measured at higher standard concentration. It was decided to go for higher calibration curve range to obtained good correlation and R 2 value. Trial-2: Range100 µg/ml to 1000 µg/ml Observation: Good result was observed calibration curve data were also found satisfactory, but still absorbance was found to be rising as per increasing the concentration. Trial-3: Range100 µg/ml to 2000 µg/ml TABLE 4: SELECTION CALIBRATION STANDARD RANGE stocks Volume Congo red Final Final conc. of Conc. μg/ml taken (0.01%w/v) ml volume μg/ml Absorbance 100 0.5 5 5.5 9.09 0.034 200 0.5 5 5.5 18.18 0.062 500 0.5 5 5.5 45.45 0.150 800 0.5 5 5.5 72.73 0.251 1000 0.5 5 5.5 90.91 0.315 1200 0.5 5 5.5 109.09 0.380 1500 0.5 5 5.5 136.36 0.498 1800 0.5 5 5.5 163.64 0.650 2000 0.5 5 5.5 181.82 0.734 www.pharmasm.com IC Value 4.01 2658
Observation: Good result was observed, Absorbance response as well calibration curve data was found be satisfactory. Method Development: Linearity Curve Figure 5 Graph of Linearity Curve Observation: Linearity range 9.09μg/ml-181.82 μg/ml of Aloe polysaccharide was found to be linear, correlation co-efficient 0.9890 for abs and concentration. Specificity check Specificity Sample TABLE 5: SPECIFICITY CHECK Congo red dye 0.01% (0.5 mg/ml) Guargum (0.5 mg/ml) Tragacanth (0.5 mg/ml) Abs sample 1 5ml 0.5ml 0.5ml NA 0.094 Sample 2 5ml 0.5ml NA 0.5ml 0.098 Sample sample 5ml 0.5ml NA NA 0.095 as such Observation: There was no effect of adulterant like Guargum and Tragacanth on absorbance. www.pharmasm.com IC Value 4.01 2659
Precision [Intraday and Interday precision]: TABLE 6: INTRADAY PRECISION Conc Absorbance Mean SD %RSD (n=3) 0.045 100 0.045 0.046 0.045 0.001 1.274 0.485 1000 0.485 0.490 0.487 0.003 0.593 0.710 2000 0.722 0.720 0.717 0.006 0.896 Observation: % RSD of different concentration of sample at deferent times in a day was shown within limit. TABLE 7: INTERDAY PRECISION Conc Absorbance Mean SD %RSD (n=3) 0.056 100 0.055 0.054 0.055 0.001 1.818 0.503 1000 0.506 0.495 0.501 0.006 1.134 0.723 2000 0.728 0.733 0.728 0.005 0.687 Observation: % RSD of different concentration of sample at different days was shown within limit. Accuracy [Recovery] Conc. Vol taken Congo red Final volume TABLE 8: ACCURACY Final conc. www.pharmasm.com IC Value 4.01 2660 Abs Specified conc. Cal conc. %Accuracy 100 0.5 5 5.5 9.09 0.039 9.09 8.56 94.16 1000 0.5 5 5.5 90.91 0.490 90.91 89.16 98.07 2000 0.5 5 5.5 181.82 0.733 181.82 184.08 101.24
Observation: % Accuracy of different concentration of samples at different levels was shown within limit. Method Evaluation: Quantitative Evaluation of Formulations TABLE 9: PREPARATION OF SPIKING STOCK SOLUTIONS Final Conc.(μg/ ml) Abso rbanc e Conc. Volume Taken Congo red Final Volume Cal Conc. 100 0.5 5 5.5 9.09 0.034 9.19 200 0.5 5 5.5 18.18 0.062 19.39 500 0.5 5 5.5 45.45 0.150 52.86 800 0.5 5 5.5 72.73 0.251 75.39 1000 0.5 5 5.5 90.91 0.315 104.37 1200 0.5 5 5.5 109.09 0.380 110.80 1500 0.5 5 5.5 136.36 0.498 156.14 1800 0.5 5 5.5 163.64 0.650 175.35 2000 0.5 5 5.5 181.82 0.734 215.03 Gel-A 0.5 5 5.5 unknown 0.203 68.00 Gel-B 0.5 5 5.5 unknown 0.485 152.22 Observation: in marketed formulations A & B was found to be 68.00 μg/ml and 152.22μg/ml respectively. Quantitative Evaluation of Formulation-A &Formulation-B [Pure Aloe vera Skin Gel by Health Secure (India) Pvt. Ltd., (labeled claim 90%w/w of 60g)] [Aloe vera Gel by Patanjali Ayurved (India) Ltd., (labeled claim 90 %w/w of 150ml)] Formulation Pure Aloe Vera Skin Gel-A Label claim(g) of Aloe vera extract TABLE 10: FORMULATION-A Amt of Should be Present(μg) Amt of estimated(μg) (n=3) 54 81 68 54 81 70 54 81 75 Assay results % recovery 83.95 % www.pharmasm.com IC Value 4.01 2661
Formulation Aloe Vera Gel-B Label claim(g) of Aloe Vera extract TABLE 11: FORMULATION-B Amt of Should be Present(μg) Amt of estimated(μg) (n=3) 135 202.5 152.2 135 202.5 148.8 135 202.5 158.4 Assay results % recovery ±S.D (n=3) 75.16 % Observation: The Marketed Formulations A-Pure Aloe vera Skin Gel and B-Aloe vera Gel % assay of aloe polysaccharide was found to be 83.95%& 75.16% respectively.3 DISCUSSION The current method is based on the molecular interaction between aloe polysaccharide and Congo red a coloring agent reference taken from Reaction of Congo Red with bioactive Aloe polysaccharides reported by Eberendu, A.N.R., & McAnalley, B.H. (1996) U.S. Patent with some modification including sample preparation without using alkaline medium and second with extended calibration curve range. Results of method showed that Congo red absorption maximum shifted to 496 nm wavelength in presence of aloe polysaccharides without using alkaline medium making it possible to quantify the bioactive polysaccharide present in the Aloe-vera containing product. Developed method has larger calibration curve range from 100 to 2000 μg/ml and satisfactory absorption intensity was found from lower level of calibration standard to higher calibration standard,the color complex remain stable for longer times. The advantage of this method is that samples can be prepared and qualitatively and quantitatively measured immediately can also be prepared and measured after some time without any significant change in color intensity. CONCLUSION The current method is based on the molecular interaction b/w aloe polysaccharide and Congo red with some modification including sample preparation without using alkaline medium & extended calibration curve range. Results of method showed that Congo red absorption maximum shifted to longer wavelength in presence of aloe polysaccharides www.pharmasm.com IC Value 4.01 2662
without using alkaline medium making it possible to quantify. Satisfactory absorption intensity was found from lower level of calibration standard to higher calibration standard. Manufacturer adulterates the degraded products with non-aloe polysaccharides which can be detected by this method. ACKNOWLEDGEMENT Authors are extremely gratefully to Management of Shree Swaminarayan Sanskar Pharmacy College, Zundal, for providing the necessary facilities and help in carrying out this work. REFERENCES 1. Joseph B & Justin S: Pharmacognostic and Phytochemical properties of Aloe vera. Journal of ISSN 2010; 4: 106-110. 2. Eberendu Alexis NR & McAnalley BH: Colorimetric assay for bioactive polysaccharide. U.S. patent 1996: Patent number: 5,512,488. 3. Eberendu AR et al: Quantitative colorimetric analysis of aloe polysaccharides as a measure of Aloe vera quality in commercial products. Journal of AOAC international 2005; 88: 1-3. 4. Bansal & Anthony: Method validation. Journal of A 2007; 9: 109-114. 5. Biren Shah & Sheth AK: Textbook of Pharmacognosy & Phytochemistry. Elsevier; 1 st edition, 2010, 110. For Correspondence: Jaimin Patel Email: jaiminrx@yahoo.in www.pharmasm.com IC Value 4.01 2663