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UV Spectrophotometric method for characterization of curcumin loaded nanostructured lipid nanocarriers in simulated conditions: Method development, in-vitro and ex-vivo applications in topical delivery
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117392
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UV Spectrophotometric method for characterization of curcumin loaded nanostructured lipid nanocarriers in simulated conditions: Method development, in-vitro and ex-vivo applications in topical delivery Vamshi Krishna Rapalli 1, Vedhant Kaul 1, Srividya Gorantla, Tejashree Waghule, Sunil Kumar Dubey, Murali Monohar Pandey, Gautam Singhvi* Department of Pharmacy, Birla Institute of Technology & Science, Pilani 333031, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 April 2019 Received in revised form 6 July 2019 Accepted 14 July 2019 Available online 15 July 2019
Curcumin the extract obtained from the dried rhizome of turmeric, Curcuma longa is a hydrophobic phenol that delivers numerous pharmacological actions like anti-inflammatory, anti-microbial and anti-oxidant, anti-psoriasis, antidiabetic, anticancer. But curcumin has low bioavailability issues that accompany low aqueous solubility, further, when administered orally, >90% of the drug degrades rapidly in the alkaline medium. Administering the drug topically can bypass the problem as well as first-pass metabolism and therefore delivering the drug at the targeted site of action. Encapsulating curcumin in nanostructured lipid nanocarriers (NLC) is an excellent novel strategy. Further, these NLC provides both the controlled release and helps in the enhanced permeation of the drug through the skin's physiological barrier, stratum corneum. For the NLC characterization, a reliable method must be developed that can accurately and precisely determine the drug content in the formulation and also for its in-vitro and ex-vivo characterization. This experiment describes the analytical validation parameters described as per International Conference of Harmonization guidelines to develop a method using the UVeVisible spectroscopy. The method was developed in two solvent systems i.e. methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. Methanol solvent was used for the determination of curcumin in the NLC formulation via determining the encapsulation efficiency and 6.4 pH phosphate buffer with 1.5% polysorbate 80 solvent was used for in-vitro and ex-vivo characterization of the developed NLC formulation (cream and gel). These methods were validated in response to linearity, the limit of detection, the limit of quantification, precision, accuracy, repeatability, and specificity. © 2019 Elsevier B.V. All rights reserved.
Keywords: Curcumin UV spectroscopy method Nanostructured lipid nanocarriers In-vitro Ex-vivo Skin permeation
1. Introduction Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) (Fig. 1) is a hydrophobic phenol obtained from the dried rhizome of turmeric, Curcuma longa, belonging to the Zingiberaceae family [1]. Curcumin is widely used as a natural yellow pigment in the food industry for its coloring, flavoring and preservative usefulness [2]. Curcumin possesses excellent biological and pharmaceutical actions such as anti-inflammatory, antioxidant, Abbreviations: SC, stratum corneum; NLC, nanostructured lipid nanocarriers; LOD, limit of detection; LOQ, limit of quantification; ICH, International Conference of Harmonization. * Corresponding author. E-mail addresses: [email protected] (V.K. Rapalli), f2015954@ pilani.bits-pilani.ac.in (V. Kaul), [email protected] (S. Gorantla), [email protected] (T. Waghule), [email protected]. ac.in (G. Singhvi). 1 Authors contributed equally. https://doi.org/10.1016/j.saa.2019.117392 1386-1425/© 2019 Elsevier B.V. All rights reserved.
anticancer, anti-diabetic, anti-arthritic, anti-angiogenic, anti-psoriasis and antimicrobial activities [3]. Curcumin is almost insoluble in acidic and neutral medium and comparatively soluble in alkaline medium. Although it shows appreciable solubility in the alkaline medium but >90% of the drug decomposes rapidly in basic pH conditions making it unstable at pH >7.4 [4]. Administering curcumin topically through the skin for its anti-inflammatory, anti-microbial and anti-oxidant role can be very efficacious for localized action. Skin serves as the major protective layer which is made up of three main layers i.e. the outermost epidermis, middle dermis and the deeper subcutaneous tissue (hypodermis). The epidermis is divided into five layers, stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. Drug penetration via the skin is limited due to the presence of the physiological barrier, stratum corneum (SC) which is relatively impermeable to foreign substances. Stratum corneum allows only certain molecules having lipophilic, smaller size and low molecular weight properties to pass through it (Fig. 2) [5e7].
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Fig. 1. Chemical structure of Curcumin.
Utilizing the novel nanocarriers like the nanostructured lipid nanocarriers (NLC) protects the drug from degradation and improves permeation by the occlusive effect. Due to hydrophobic nature and smaller size, NLC based formulations form an adhesive and occlusive thin layer over the outermost layer of the skin on topical application. This occlusive film reduces water evaporation from the skin and increases the gaps between corneocytes which help in the penetration of NLC into superficial layers of the skin and get embedded in the stratum corneum. Additionally, the hydration of skin favors the slow release of drug from the NLC deposited in the stratum corneum. This leads to the controlled release of drug in skin layers and provides local action of drug for prolong time as shown in Fig. 2 [8e12]. After the development of these nanocarriers system, there was a need for the characterization of nanocarriers which includes invitro release studies and the ex-vivo skin permeation studies are to be performed in biological pH. Different methods have been reported for the determination of curcumin in bulk and various pharmaceutical dosage forms. Ultraviolet-Visible spectroscopy [13e15], ultra-performance liquid chromatography [16], thin layer chromatography [17] and highperformance liquid chromatography [18e21] were reported for estimation of curcumin. The analytical techniques apart from UV spectrophotometry are expensive, required sophisticated instrument, complex processing, and time-consuming for estimation of the
curcumin in the biological matrix and quantify the drug release from nanoformulation at biological pH condition. For preformulation studies, routine formulation sample analysis, and characterization at industrial as well as academic research, UVevisible spectroscopy method is most preferred for the estimation of curcumin in formulation and bulk due to it's economic, rapid and easy to perform [15,22]. Reported UVevisible spectroscopic methods for curcumin were majorly developed in methanol solvent system for quantification in bulk drug and estimation of drug content in the formulation. With our best of knowledge, there is no such UV-method reported which can estimate the in-vitro and ex-vivo release of curcumin at biological pH medium from the nanocarriers based delivery systems. Therefore, there was a need for the development of such a method which can accurately and precisely estimate the drug release in order to mimic the human biological system or simulated conditions. To address the same, 6.4 pH phosphate buffer with 1.5% polysorbate 80 was chosen as it mimics skin condition and maintains sink condition. Since there are no such pre-developed methods of drug estimation in release samples using UVevisible spectroscopy. The aim of the study undertaken was to develop and validate a simple, fast, accurate and reliable curcumin UV-visible spectroscopic method in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 solvent system. The validation of the above-developed method was carried out as per the International Conference of Harmonization (ICH) guidelines Q2 (R1) [23,24]. Further, the developed method was applied for the quantification of curcumin in designed NLC. The method was extensively applied for the determination of entrapment efficiency, in-vitro release, and exvivo skin permeation study of the prepared NLC formulation. 2. Material and methods 2.1. Materials A Shimadzu UVevisible spectrophotometer (UV-1800) was used for all absorbance measurements. Curcumin was purchased from Sigma Aldrich. Methanol, Sodium dihydrogen phosphate monohydrate, and di-Sodium hydrogen phosphate were of HPLC grade and purchased from Merck Specialities Pvt. Ltd., Mumbai, India.
Fig. 2. The structure of skin with three major layers as outermost epidermis, middle dermis and the deeper subcutaneous tissue (hypodermis). Penetration of curcumin loaded NLC through the skin and release of curcumin in skin layers. The figure represents the permeation of curcumin loaded NLC into the skin due to the formation of thin film by NLC formulation. This film exhibit occlusive nature and further increase the permeation of NLC into Stratum Corneum.
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Polysorbate 80 was purchased from J.T bakers. Precirol ATO 5 and Labrafac M 1944 were obtained as gift samples from Gatteffose India, Pvt. Ltd. Carbopol® 974P NF was received as a gift sample from Lubrizol, India. 2.2. Method development 2.2.1. Determination of wavelength of maximum absorption A standard stock solution of curcumin 1 mg/mL solution was prepared in 50 mL of methanol. From the standard stock solution, 100 mg/mL secondary stock solutions were prepared in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. A 5 mg/mL solution was prepared in both media and UV spectroscopy scanning (800e200 nm) was performed using respective solvent systems as blank to determine the lmax of curcumin. Fig. 3 indicates the concentration vs absorbance in methanol and Fig. 4 indicates concentration vs absorbance in 6.4 pH phosphate buffer. 2.2.2. Analytical validation Analytical parameters including linearity, accuracy, specificity, robustness, precision limit of detection, and the limit of quantification were evaluated as per the ICH guidelines. Ultra-violet visible spectrometer (Shimadzu UV-1800) was utilized for method development operating with 1 nm spectral bandwidth. 2.2.3. Linearity and range The linearity of the analytical procedure was executed for six different concentrations (2, 4, 6, 8, 10 and 11 mg/mL) of curcumin in both methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. The experiment was performed for three days in duplicate (total n ¼ 6) in both solvent system. The obtained data were used to plot the linearity curve, regression equation and correlation coefficient equation was determined. The Limit of detection (LOD) and Limit of Quantification (LOQ) of the proposed UV method was determined. LOD and LOQ were calculated using the standard deviation of the response and the slope of the corresponding curve using the following equations: LOD: 3.3 (s/S); LOQ: 10 (s/S). Where; s represents the standard deviation of absorbance of sample and S represents the slope of the calibration curve [13]. 2.2.4. Precision The precision of the UV method was evaluated in terms of intraday and inter-day variations (intermediate precision). Precision levels were determined for the three different known concentrations of curcumin (4, 6 and 8 mg/mL), which were prepared from the two stock solutions. For determining intra-day precision, the
Fig. 3. UV-Spectroscopic spectrum of curcumin in methanol.
Fig. 4. UV-Spectroscopic spectrum of curcumin in 6.4 pH phosphate buffer.
absorbance of the three concentrations was measured three times a day in triplicate. And for inter-day precision, the absorbance was determined daily for 3 days in triplicate. The percent relative standard deviation (% RSD) was calculated for three different concentrations using the linearity curve [14]. 2.2.5. Repeatability The repeatability of the UV method was determined by determining the absorbance of the curcumin solution in the respective solvent system at the concentration of 4 mg/mL. The absorbance was determined six times. 2.2.6. Accuracy Accuracy is defined as the closeness of agreement between the true value and the analytical value. Using the standard addition method, a known amount of standard stock solution was added at different levels 80%, 100%, and 120% to the pre-analyzed solution of curcumin. The solutions were analyzed by the proposed method. Using the linearity curve, samples concentration was recalculated in triplicate [25]. 2.2.7. Specificity The specificity of the curcumin was evaluated by blank lipid nanoparticles. Blank lipid-based formulation (nanostructured lipid nanocarriers) was prepared and to the dispersion known amount of curcumin was added. The dispersion was mixed vigorously and 1 mL of the aliquot was withdrawn and subjected to lysis of nanoparticle and complete extraction of curcumin by methanol (up to 5 mL). Then it was subjected to filtration through a 0.22 mm filter and analyzed using the proposed method after required dilution [25]. 2.2.8. Application of the developed UV method for characterization of nanostructured lipid nanocarriers 2.2.8.1. Entrapment efficiency and drug loading. Nanostructured lipid nanocarriers of curcumin were prepared using emulsification technique followed by probe sonication method. Briefly, the batch quantity of solid lipid, liquid lipid and Curcumin were weighed in a clear glass vial and heated up to 80 C in a water bath to melt the lipid. The surfactant was dissolved in Milli-Q water and pre-heated up to 80 C in a separate vial and added to melted lipid and mixed thoroughly to form a hot primary emulsion. The formed primary emulsion was subjected to probe sonication and cooled down to room temperature for solidification of lipid nanoparticles [5]. The prepared formulation was analyzed for entrapment efficiency. To determine the entrapped and unentrapped drug the prepared formulation was subjected to centrifugation. The curcumin content in
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Fig. 5. Linearity plot of curcumin in methanol.
lipid nanocarriers and the unentrapped free drug was extracted using methanol and the curcumin content was estimated using the developed analytical method after filtering the sample through 0.22 mm filter. The trials were performed in triplicate. Drug concentration was calculated from the linearity curve equation obtained using methanol. Further, entrapment efficiency was calculated using the following equation [22]. %Entrapment efficiency ¼
ðAmount of drug taken for formulationÞ ðAmount of unentrapped drugÞ 100 ðTotal amount of drug in formulationÞ
2.2.8.2. Assay of curcumin loaded NLC embedded gel and cream formulations. The developed method was utilized for the estimation of curcumin content in nanostructured lipid nanocarriers embedded gel and cream based formulation. The curcumin loaded NLC based formulation was dissolved in methanol and water in a 9:1 ratio to extract the drug from the gel and cream based formulation. The gel and cream dissolved in extracting solvent composition and further diluted as per requirement (ensuring the theoretical value of drug content within the linearity range) and filtered through 0.22 mm membrane filter prior to analysis. The experiment was performed in triplicates for nanocarriers loaded gel and cream-based formulations.
2.2.8.3. In-vitro drug release estimation. The method developed in 6.4 pH phosphate buffer with 1.5% polysorbate 80 was evaluated to check its applicability for in-vitro drug release from curcumin loaded NLCs based gel and cream formulations. In-vitro drug release studies were performed through a dialysis membrane using Franz diffusion cell in 6.4 pH phosphate buffer with 1.5% polysorbate 80. Aliquots were collected in predetermined time points and the fresh buffer was replaced to maintain the sink condition. After 6 h of release study, the drug amount retained in the dialysis bag was estimated by extracting the drug from formulation to cross-check the accuracy of the developed method. Samples collected in different time points were filtered through a 0.22 mm filter and analyzed using developed method and drug concentration was determined by the developed method [26]. 2.2.8.4. Extraction of drug from the skin tissues. The developed method was investigated for its applicability in ex-vivo permeation studies. The known amount (200 mg/mL) of curcumin solution was spiked to homogenized skin tissue sample and centrifuged for separation of the tissue. The supernatant was withdrawn (1 mL), filtered through a 0.22 mm membrane filter, and diluted to get the concentration range within the linearity curve and analyzed for recovery in the skin tissue sample. The percentage recoveries of curcumin were calculated by comparing the extracted quantity of curcumin with the actual amount added to the tissue sample.
Fig. 6. Linearity plot of curcumin in 6.4 pH PBS buffer with 1.5% polysorbate 80.
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Table 1 Precision of Curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 (n ¼ 3). Con (mg/ ml)
Diluent
Methanol
Inter-day precision
Absorbance measured (mean ± SD)
RSD (%)
0.432 ± 0.0005 0.649 ± 0.0026 0.837 ± 0.0011 0.409 ± 0.0010 0.599 ± 0.0006 0.802 ± 0.0006
4 6 8 4 6 8
6.4 pH phosphate buffer with 1.5% polysorbate 80
Intra-day precision
Average potency Absorbance measured (%) (mean ± SD)
0.134 0.408 0.138 0.244 0.096 0.072
99.00 101.80 99.62 101.25 99.16 99.75
0.434 ± 0.0005 0.646 ± 0.0006 0.851 ± 0.0011 0.389 ± 0.0011 0.600 ± 0.0006 0.800 ± 0.0015
RSD (%)
Average potency (%)
0.133 0.089 0.136 0.243 0.096 0.191
99.50 101.10 101.25 101.75 99.33 99.50
Table 2 Accuracy of curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 (n ¼ 3). Diluent
Amount added (%)
Methanol
2.2.8.5. Distribution of curcumin in skin tissue. Ex-vivo skin permeation studies were performed on goat skin. The goat skin was collected from the slaughterhouse. After the removal of hair, fat and muscle debris of skin, washing was performed with phosphate buffer saline, the skin was placed on Franz diffusion cell. The NLC loaded topical formulation was applied on stratum corneum side of the skin facing towards the donor compartment. The assembly was placed on a magnetic stirrer at 300 rpm. The experiment was performed in triplicates. The samples were withdrawn at predetermined time points. The samples were analyzed using the developed UV method and the amount of drug permeated from formulations through goat skin was calculated. After ex-vivo drug release studies, the amount of drug retained in the skin was extracted by two methods. In the first method, tape stripping technique was utilized for the determination of drug content in stratum corneum, rest of the skin. In the second method complete drug retained in the skin was dissected into pieces and in both cases, the drug was extracted using methanol to determine the amount of drug retained in the skin tissue. The solution is filtered through a 0.22 mm filter and the amount of curcumin retained in skin tissue was estimated using the validated method [27]. 3. Results and discussion 3.1. Method development and validation
0.132 0.118 0.166 0.080 0.144 0.065
media was 1.0e11.0 mg/mL with a correlation coefficient (R2) >0.998. The LOD and LOQ of the proposed method for methanol were found to be 0.19 and 0.57 mg/mL respectively. Whereas LOD and LOQ for 6.4 pH phosphate buffer were found to be 0.11 and 0.31 mg/mL. 3.3. Intraday, interday precision, and repeatability Precision was determined in a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The intraday and interday precision results are shown in Table 1 and the results confirmed the reliability and stability of the developed method where all RSDs were <0.5%. Repeatability was determined by analyzing 4 mg/mL concentration of curcumin solution for six times and the percent amount found was calculated using the linearity curve. The % RSD was <0.5% in both methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. 3.4. Accuracy For accuracy determination, the results of recovery studies as shown in Table 2 were found to be within the range of 99.01e100.1% and the RSDs <0.5% which indicated the non-interference of excipients and the accuracy of the developed UV method.
The specificity of the curcumin was evaluated by blank lipid nanoparticles. The amount recovered from blank NLC formulation was 99.25 ± 0.28% this indicated the method was specific towards curcumin. There was no interference of excipients used in the lipidbased formulation. 3.6. Application of the developed UV method for characterization of nanostructured lipid nanocarriers
3.2. Linearity and range The calibration curve (Figs. 5 and 6) obtained was evaluated by its correlation coefficient. The linearity range of curcumin in both the Table 3 Entrapment efficiency study of curcumin in NLC (n ¼ 3).
57.04 ± 0.62 67.80 ± 1.16 70.70 ± 1.03
RSD (%)
3.5. Specificity
Curcumin was found to be water-insoluble and freely soluble in methanol, acetone, and acetonitrile. Based on solubility, stability, and application in formulation characterization, curcumin method was developed in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. The maximum absorption wavelength was determined in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 which was found to be 425 nm for both solvent systems.
% of Curcumin entrapped ± SD
100.00 ± 0.0010 100.60 ± 0.0010 99.72 ± 0.0015 100.00 ± 0.0006 99.75 ± 0.0012 100.00 ± 0.0006
80 100 120 80 100 120
6.4 pH phosphate buffer with 1.5% polysorbate 80
Recovery (%)
% of unentrapped Curcumin ± SD
% total amount of Curcumin recovered
43.76 ± 0.82 31.10 ± 0.93 28.36 ± 0.98
100.80 ± 0.72 98.90 ± 1.04 99.06 ± 1.01
% RSD 1.087 1.711 1.457
3.6.1. Entrapment efficiency and drug loading Curcumin entrapped in NLC were extracted and analyzed in triplicate for three different batches with different lipid composition. Table 4 Assay of curcumin in NLCs loaded gel and curcumin cream (n ¼ 3). Formulation
% assay ± SD
Nanocarriers loaded cream preparation (n ¼ 3) Nanocarriers loaded gel formulation (n ¼ 3)
100.07 ± 0.47 100.63 ± 0.62
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Table 5 Mass balance of the curcumin used in in-vitro release study. The total amount of drug used for release study The cumulative amount of drug released Amount of drug retained in a dialysis bag The total amount of drug recovered
1000 mg 614.46 ± 0.131 mg 356.91 ± 0.295 mg 971.37 ± 0.213 mg (97.14%)
The amount of curcumin entrapped and unentrapped was estimated with a validated analytical method. The results pertaining to entrapped and un-entrapped drug were mentioned in Table 3. The data demonstrated the high reproducibility (%RSD < 1.72) with percentage recovery of 98.90 ± 1.04 to 100.80 ± 0.72 indicated that the method was efficient and applicable for calculation of entrapment efficiency in nanocarriers based formulations of curcumin. 3.6.2. Assay of curcumin loaded NLC embedded gel and cream formulations Curcumin content in NLC based gel and cream based formulation preparations were analyzed for assay in triplicate. The assay results obtained from curcumin loaded NLC embedded gel and cream-based formulations are represented in Table 4. Results showed high reproducibility (% RSD < 0.01) and close to label claim
(100 ± 1%) of drugs. This indicated that the developed method can accurately and precisely determine the curcumin content without interference of the excipients or formulation matrix. Additionally, it also reflected that the method has the potential to determine the drug content in complex nanocarriers based formulation also. 3.6.3. In-vitro drug release estimation The in-vitro release study was performed in triplicate samples collected from the Franz diffusion cell were analyzed using the developed method. The amount of drug release was within the linearity range were analyzed directly and in case of high concentrations, the samples were diluted with release buffer before analysis. There was no interference of surfactants and buffers of release media in the quantification of curcumin. The cumulative amount of drug released was found to be 614.46 ± 0.131 mg in 6 h. The unreleased amount retained in the dialysis bag was extracted and estimated for mass balance (Table 5). About 97.14 ± 0.213% of the curcumin was recovered from the study. The results indicated that method was found to be appropriate to quantify the amount of drug release in presence of dissolution medium and can quantify the curcumin at early release points to maximum drug release during in-vitro drug release studies as shown in Fig. 7.
Fig. 7. In-vitro drug release study of curcumin loaded NLC in dissolution medium.
Fig. 8. Skin retention of curcumin in ex-vivo release study.
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3.6.4. Extraction of drug from the skin tissues and distribution of curcumin in skin tissue The known concentration of analyte solution was spiked into the homogenized skin tissue and analyzed after passing through a 0.22 mm membrane filter. The percentage recovery of curcumin was found to be 96.10 ± 0.41%. This indicated the developed method can be applied for skin permeation studies and tape stripping for evaluation of lipid nanocarriers based topical dosage form. The amount of drug retained in skin determined by tape stripping was found to be 39.228 ± 0.412 mg (n ¼ 3) as shown in Fig. 8. The amount of drug retained in the skin, determined by the direct method was found to be 40.698 ± 0.237 mg (n ¼ 3). Results of characterization studies indicated that the developed and validated UV spectroscopic method was simple, rapid, and accurate for estimation of the curcumin content in biological samples. 4. Conclusion The analytical method developed on UV spectrophotometer to determine the concentration of curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 was validated in terms of the linearity of the linear regression equation, accuracy, interday precision and intraday precision, repeatability of method, and sensitivity. Both the methods were reliable having RSDs <2%. Further, the method developed in methanol will be useful in estimating the encapsulation efficiency of the curcumin in the lipidbased formulations, which helps to determine the percent of the entrapped drug in the lipid structure. Whereas method developed in 6.4 pH phosphate buffer with 1.5% polysorbate 80 media can be utilized to determine the percent drug release from the lipid-based formulations. For both the applications, the developed methods were found to be accurate, simple and reliable. Declaration of Competing Interest The authors report no conflicts of interest. Acknowledgment Authors are thankful to Industrial Research Laboratory, BITS Pilani, Pilani campus for providing the facility to conduct the experiments. The presented work is not sponsored by any funding agency. References [1] A. Noorafshan, S. Ashkani-Esfahani, A review of therapeutic effects of curcumin, Curr. Pharm. Des. 19 (2013) 2032e2046. http://www.ncbi.nlm.nih.gov/ pubmed/23116311 (accessed April 19, 2019). [2] L. Hu, Y. Jia, F. Niu, Z. Jia, X. Yang, K. Jiao, Preparation and enhancement of oral bioavailability of curcumin using microemulsions vehicle, J. Agric. Food Chem. 60 (2012) 7137e7141, https://doi.org/10.1021/jf204078t. [3] S.J. Hewlings, D.S. Kalman, Curcumin: a review of its' effects on human health, Foods (Basel, Switzerland) 6 (2017), https://doi.org/10.3390/foods6100092. [4] Y.J. Wang, M.H. Pan, A.L. Cheng, L.I. Lin, Y.S. Ho, C.Y. Hsieh, J.K. Lin, Stability of curcumin in buffer solutions and characterization of its degradation products, J. Pharm. Biomed. Anal. 15 (1997) 1867e1876. [5] T. Waghule, V.K. Rapalli, G. Singhvi, P. Manchanda, N. Hans, S.K. Dubey, M.S. Hasnain, A.K. Nayak, Voriconazole loaded nanostructured lipid carriers based topical delivery system: QbD based designing, characterization, in-vitro and ex-vivo evaluation, J. Drug Deliv. Sci. Technol. 52 (2019) 303e315, https:// doi.org/10.1016/J.JDDST.2019.04.026. [6] G.M. Gelfuso, M.S. Cunha-Filho, T. Gratieri, Nanostructured lipid carriers for targeting drug delivery to the epidermal layer, Ther. Deliv. 7 (2016) 735e737, https://doi.org/10.4155/tde-2016-0059. [7] V. Girdhar, S. Patil, S. Banerjee, G. Singhvi, Nanocarriers for drug delivery: mini review, Curr. Nanomed. 8 (2018) 88e99, https://doi.org/10.2174/
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Anal. 20 (2009) 314e319, https://doi.org/10.1002/pca.1129. [22] R.A. da Silva-Buzanello, A.C. Ferro, E. Bona, L. Cardozo-Filho, P.H.H. de Araújo, F.V. Leimann, O.H. Gonçalves, Validation of an Ultravioletevisible (UVeVis) technique for the quantitative determination of curcumin in poly(l-lactic acid) nanoparticles, Food Chem. 172 (2015) 99e104, https://doi.org/10.1016/ J.FOODCHEM.2014.09.016. [23] International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Ich Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1), n.d. [24] G. Singhvi, P. Kalantare, D. Harish, R.N. Saha, Spectrophotometric determination of nor-epinephrine serotonin reuptake inhibitor (SNRI) drug milnacipran in pure and in dosage forms, Asian J. Chem. 25 (2013) 3682e3686, https://doi.org/10.14233/ajchem.2013.13716. [25] P.V. Kadam, C.L. Bhingare, R.Y. Nikam, S.A. 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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
UV Spectrophotometric method for characterization of curcumin loaded nanostructured lipid nanocarriers in simulated conditions: Method development, in-vitro and ex-vivo applications in topical delivery Vamshi Krishna Rapalli 1, Vedhant Kaul 1, Srividya Gorantla, Tejashree Waghule, Sunil Kumar Dubey, Murali Monohar Pandey, Gautam Singhvi* Department of Pharmacy, Birla Institute of Technology & Science, Pilani 333031, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 April 2019 Received in revised form 6 July 2019 Accepted 14 July 2019 Available online 15 July 2019
Curcumin the extract obtained from the dried rhizome of turmeric, Curcuma longa is a hydrophobic phenol that delivers numerous pharmacological actions like anti-inflammatory, anti-microbial and anti-oxidant, anti-psoriasis, antidiabetic, anticancer. But curcumin has low bioavailability issues that accompany low aqueous solubility, further, when administered orally, >90% of the drug degrades rapidly in the alkaline medium. Administering the drug topically can bypass the problem as well as first-pass metabolism and therefore delivering the drug at the targeted site of action. Encapsulating curcumin in nanostructured lipid nanocarriers (NLC) is an excellent novel strategy. Further, these NLC provides both the controlled release and helps in the enhanced permeation of the drug through the skin's physiological barrier, stratum corneum. For the NLC characterization, a reliable method must be developed that can accurately and precisely determine the drug content in the formulation and also for its in-vitro and ex-vivo characterization. This experiment describes the analytical validation parameters described as per International Conference of Harmonization guidelines to develop a method using the UVeVisible spectroscopy. The method was developed in two solvent systems i.e. methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. Methanol solvent was used for the determination of curcumin in the NLC formulation via determining the encapsulation efficiency and 6.4 pH phosphate buffer with 1.5% polysorbate 80 solvent was used for in-vitro and ex-vivo characterization of the developed NLC formulation (cream and gel). These methods were validated in response to linearity, the limit of detection, the limit of quantification, precision, accuracy, repeatability, and specificity. © 2019 Elsevier B.V. All rights reserved.
Keywords: Curcumin UV spectroscopy method Nanostructured lipid nanocarriers In-vitro Ex-vivo Skin permeation
1. Introduction Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) (Fig. 1) is a hydrophobic phenol obtained from the dried rhizome of turmeric, Curcuma longa, belonging to the Zingiberaceae family [1]. Curcumin is widely used as a natural yellow pigment in the food industry for its coloring, flavoring and preservative usefulness [2]. Curcumin possesses excellent biological and pharmaceutical actions such as anti-inflammatory, antioxidant, Abbreviations: SC, stratum corneum; NLC, nanostructured lipid nanocarriers; LOD, limit of detection; LOQ, limit of quantification; ICH, International Conference of Harmonization. * Corresponding author. E-mail addresses: [email protected] (V.K. Rapalli), f2015954@ pilani.bits-pilani.ac.in (V. Kaul), [email protected] (S. Gorantla), [email protected] (T. Waghule), [email protected]. ac.in (G. Singhvi). 1 Authors contributed equally. https://doi.org/10.1016/j.saa.2019.117392 1386-1425/© 2019 Elsevier B.V. All rights reserved.
anticancer, anti-diabetic, anti-arthritic, anti-angiogenic, anti-psoriasis and antimicrobial activities [3]. Curcumin is almost insoluble in acidic and neutral medium and comparatively soluble in alkaline medium. Although it shows appreciable solubility in the alkaline medium but >90% of the drug decomposes rapidly in basic pH conditions making it unstable at pH >7.4 [4]. Administering curcumin topically through the skin for its anti-inflammatory, anti-microbial and anti-oxidant role can be very efficacious for localized action. Skin serves as the major protective layer which is made up of three main layers i.e. the outermost epidermis, middle dermis and the deeper subcutaneous tissue (hypodermis). The epidermis is divided into five layers, stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. Drug penetration via the skin is limited due to the presence of the physiological barrier, stratum corneum (SC) which is relatively impermeable to foreign substances. Stratum corneum allows only certain molecules having lipophilic, smaller size and low molecular weight properties to pass through it (Fig. 2) [5e7].
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Fig. 1. Chemical structure of Curcumin.
Utilizing the novel nanocarriers like the nanostructured lipid nanocarriers (NLC) protects the drug from degradation and improves permeation by the occlusive effect. Due to hydrophobic nature and smaller size, NLC based formulations form an adhesive and occlusive thin layer over the outermost layer of the skin on topical application. This occlusive film reduces water evaporation from the skin and increases the gaps between corneocytes which help in the penetration of NLC into superficial layers of the skin and get embedded in the stratum corneum. Additionally, the hydration of skin favors the slow release of drug from the NLC deposited in the stratum corneum. This leads to the controlled release of drug in skin layers and provides local action of drug for prolong time as shown in Fig. 2 [8e12]. After the development of these nanocarriers system, there was a need for the characterization of nanocarriers which includes invitro release studies and the ex-vivo skin permeation studies are to be performed in biological pH. Different methods have been reported for the determination of curcumin in bulk and various pharmaceutical dosage forms. Ultraviolet-Visible spectroscopy [13e15], ultra-performance liquid chromatography [16], thin layer chromatography [17] and highperformance liquid chromatography [18e21] were reported for estimation of curcumin. The analytical techniques apart from UV spectrophotometry are expensive, required sophisticated instrument, complex processing, and time-consuming for estimation of the
curcumin in the biological matrix and quantify the drug release from nanoformulation at biological pH condition. For preformulation studies, routine formulation sample analysis, and characterization at industrial as well as academic research, UVevisible spectroscopy method is most preferred for the estimation of curcumin in formulation and bulk due to it's economic, rapid and easy to perform [15,22]. Reported UVevisible spectroscopic methods for curcumin were majorly developed in methanol solvent system for quantification in bulk drug and estimation of drug content in the formulation. With our best of knowledge, there is no such UV-method reported which can estimate the in-vitro and ex-vivo release of curcumin at biological pH medium from the nanocarriers based delivery systems. Therefore, there was a need for the development of such a method which can accurately and precisely estimate the drug release in order to mimic the human biological system or simulated conditions. To address the same, 6.4 pH phosphate buffer with 1.5% polysorbate 80 was chosen as it mimics skin condition and maintains sink condition. Since there are no such pre-developed methods of drug estimation in release samples using UVevisible spectroscopy. The aim of the study undertaken was to develop and validate a simple, fast, accurate and reliable curcumin UV-visible spectroscopic method in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 solvent system. The validation of the above-developed method was carried out as per the International Conference of Harmonization (ICH) guidelines Q2 (R1) [23,24]. Further, the developed method was applied for the quantification of curcumin in designed NLC. The method was extensively applied for the determination of entrapment efficiency, in-vitro release, and exvivo skin permeation study of the prepared NLC formulation. 2. Material and methods 2.1. Materials A Shimadzu UVevisible spectrophotometer (UV-1800) was used for all absorbance measurements. Curcumin was purchased from Sigma Aldrich. Methanol, Sodium dihydrogen phosphate monohydrate, and di-Sodium hydrogen phosphate were of HPLC grade and purchased from Merck Specialities Pvt. Ltd., Mumbai, India.
Fig. 2. The structure of skin with three major layers as outermost epidermis, middle dermis and the deeper subcutaneous tissue (hypodermis). Penetration of curcumin loaded NLC through the skin and release of curcumin in skin layers. The figure represents the permeation of curcumin loaded NLC into the skin due to the formation of thin film by NLC formulation. This film exhibit occlusive nature and further increase the permeation of NLC into Stratum Corneum.
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Polysorbate 80 was purchased from J.T bakers. Precirol ATO 5 and Labrafac M 1944 were obtained as gift samples from Gatteffose India, Pvt. Ltd. Carbopol® 974P NF was received as a gift sample from Lubrizol, India. 2.2. Method development 2.2.1. Determination of wavelength of maximum absorption A standard stock solution of curcumin 1 mg/mL solution was prepared in 50 mL of methanol. From the standard stock solution, 100 mg/mL secondary stock solutions were prepared in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. A 5 mg/mL solution was prepared in both media and UV spectroscopy scanning (800e200 nm) was performed using respective solvent systems as blank to determine the lmax of curcumin. Fig. 3 indicates the concentration vs absorbance in methanol and Fig. 4 indicates concentration vs absorbance in 6.4 pH phosphate buffer. 2.2.2. Analytical validation Analytical parameters including linearity, accuracy, specificity, robustness, precision limit of detection, and the limit of quantification were evaluated as per the ICH guidelines. Ultra-violet visible spectrometer (Shimadzu UV-1800) was utilized for method development operating with 1 nm spectral bandwidth. 2.2.3. Linearity and range The linearity of the analytical procedure was executed for six different concentrations (2, 4, 6, 8, 10 and 11 mg/mL) of curcumin in both methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. The experiment was performed for three days in duplicate (total n ¼ 6) in both solvent system. The obtained data were used to plot the linearity curve, regression equation and correlation coefficient equation was determined. The Limit of detection (LOD) and Limit of Quantification (LOQ) of the proposed UV method was determined. LOD and LOQ were calculated using the standard deviation of the response and the slope of the corresponding curve using the following equations: LOD: 3.3 (s/S); LOQ: 10 (s/S). Where; s represents the standard deviation of absorbance of sample and S represents the slope of the calibration curve [13]. 2.2.4. Precision The precision of the UV method was evaluated in terms of intraday and inter-day variations (intermediate precision). Precision levels were determined for the three different known concentrations of curcumin (4, 6 and 8 mg/mL), which were prepared from the two stock solutions. For determining intra-day precision, the
Fig. 3. UV-Spectroscopic spectrum of curcumin in methanol.
Fig. 4. UV-Spectroscopic spectrum of curcumin in 6.4 pH phosphate buffer.
absorbance of the three concentrations was measured three times a day in triplicate. And for inter-day precision, the absorbance was determined daily for 3 days in triplicate. The percent relative standard deviation (% RSD) was calculated for three different concentrations using the linearity curve [14]. 2.2.5. Repeatability The repeatability of the UV method was determined by determining the absorbance of the curcumin solution in the respective solvent system at the concentration of 4 mg/mL. The absorbance was determined six times. 2.2.6. Accuracy Accuracy is defined as the closeness of agreement between the true value and the analytical value. Using the standard addition method, a known amount of standard stock solution was added at different levels 80%, 100%, and 120% to the pre-analyzed solution of curcumin. The solutions were analyzed by the proposed method. Using the linearity curve, samples concentration was recalculated in triplicate [25]. 2.2.7. Specificity The specificity of the curcumin was evaluated by blank lipid nanoparticles. Blank lipid-based formulation (nanostructured lipid nanocarriers) was prepared and to the dispersion known amount of curcumin was added. The dispersion was mixed vigorously and 1 mL of the aliquot was withdrawn and subjected to lysis of nanoparticle and complete extraction of curcumin by methanol (up to 5 mL). Then it was subjected to filtration through a 0.22 mm filter and analyzed using the proposed method after required dilution [25]. 2.2.8. Application of the developed UV method for characterization of nanostructured lipid nanocarriers 2.2.8.1. Entrapment efficiency and drug loading. Nanostructured lipid nanocarriers of curcumin were prepared using emulsification technique followed by probe sonication method. Briefly, the batch quantity of solid lipid, liquid lipid and Curcumin were weighed in a clear glass vial and heated up to 80 C in a water bath to melt the lipid. The surfactant was dissolved in Milli-Q water and pre-heated up to 80 C in a separate vial and added to melted lipid and mixed thoroughly to form a hot primary emulsion. The formed primary emulsion was subjected to probe sonication and cooled down to room temperature for solidification of lipid nanoparticles [5]. The prepared formulation was analyzed for entrapment efficiency. To determine the entrapped and unentrapped drug the prepared formulation was subjected to centrifugation. The curcumin content in
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Fig. 5. Linearity plot of curcumin in methanol.
lipid nanocarriers and the unentrapped free drug was extracted using methanol and the curcumin content was estimated using the developed analytical method after filtering the sample through 0.22 mm filter. The trials were performed in triplicate. Drug concentration was calculated from the linearity curve equation obtained using methanol. Further, entrapment efficiency was calculated using the following equation [22]. %Entrapment efficiency ¼
ðAmount of drug taken for formulationÞ ðAmount of unentrapped drugÞ 100 ðTotal amount of drug in formulationÞ
2.2.8.2. Assay of curcumin loaded NLC embedded gel and cream formulations. The developed method was utilized for the estimation of curcumin content in nanostructured lipid nanocarriers embedded gel and cream based formulation. The curcumin loaded NLC based formulation was dissolved in methanol and water in a 9:1 ratio to extract the drug from the gel and cream based formulation. The gel and cream dissolved in extracting solvent composition and further diluted as per requirement (ensuring the theoretical value of drug content within the linearity range) and filtered through 0.22 mm membrane filter prior to analysis. The experiment was performed in triplicates for nanocarriers loaded gel and cream-based formulations.
2.2.8.3. In-vitro drug release estimation. The method developed in 6.4 pH phosphate buffer with 1.5% polysorbate 80 was evaluated to check its applicability for in-vitro drug release from curcumin loaded NLCs based gel and cream formulations. In-vitro drug release studies were performed through a dialysis membrane using Franz diffusion cell in 6.4 pH phosphate buffer with 1.5% polysorbate 80. Aliquots were collected in predetermined time points and the fresh buffer was replaced to maintain the sink condition. After 6 h of release study, the drug amount retained in the dialysis bag was estimated by extracting the drug from formulation to cross-check the accuracy of the developed method. Samples collected in different time points were filtered through a 0.22 mm filter and analyzed using developed method and drug concentration was determined by the developed method [26]. 2.2.8.4. Extraction of drug from the skin tissues. The developed method was investigated for its applicability in ex-vivo permeation studies. The known amount (200 mg/mL) of curcumin solution was spiked to homogenized skin tissue sample and centrifuged for separation of the tissue. The supernatant was withdrawn (1 mL), filtered through a 0.22 mm membrane filter, and diluted to get the concentration range within the linearity curve and analyzed for recovery in the skin tissue sample. The percentage recoveries of curcumin were calculated by comparing the extracted quantity of curcumin with the actual amount added to the tissue sample.
Fig. 6. Linearity plot of curcumin in 6.4 pH PBS buffer with 1.5% polysorbate 80.
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Table 1 Precision of Curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 (n ¼ 3). Con (mg/ ml)
Diluent
Methanol
Inter-day precision
Absorbance measured (mean ± SD)
RSD (%)
0.432 ± 0.0005 0.649 ± 0.0026 0.837 ± 0.0011 0.409 ± 0.0010 0.599 ± 0.0006 0.802 ± 0.0006
4 6 8 4 6 8
6.4 pH phosphate buffer with 1.5% polysorbate 80
Intra-day precision
Average potency Absorbance measured (%) (mean ± SD)
0.134 0.408 0.138 0.244 0.096 0.072
99.00 101.80 99.62 101.25 99.16 99.75
0.434 ± 0.0005 0.646 ± 0.0006 0.851 ± 0.0011 0.389 ± 0.0011 0.600 ± 0.0006 0.800 ± 0.0015
RSD (%)
Average potency (%)
0.133 0.089 0.136 0.243 0.096 0.191
99.50 101.10 101.25 101.75 99.33 99.50
Table 2 Accuracy of curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 (n ¼ 3). Diluent
Amount added (%)
Methanol
2.2.8.5. Distribution of curcumin in skin tissue. Ex-vivo skin permeation studies were performed on goat skin. The goat skin was collected from the slaughterhouse. After the removal of hair, fat and muscle debris of skin, washing was performed with phosphate buffer saline, the skin was placed on Franz diffusion cell. The NLC loaded topical formulation was applied on stratum corneum side of the skin facing towards the donor compartment. The assembly was placed on a magnetic stirrer at 300 rpm. The experiment was performed in triplicates. The samples were withdrawn at predetermined time points. The samples were analyzed using the developed UV method and the amount of drug permeated from formulations through goat skin was calculated. After ex-vivo drug release studies, the amount of drug retained in the skin was extracted by two methods. In the first method, tape stripping technique was utilized for the determination of drug content in stratum corneum, rest of the skin. In the second method complete drug retained in the skin was dissected into pieces and in both cases, the drug was extracted using methanol to determine the amount of drug retained in the skin tissue. The solution is filtered through a 0.22 mm filter and the amount of curcumin retained in skin tissue was estimated using the validated method [27]. 3. Results and discussion 3.1. Method development and validation
0.132 0.118 0.166 0.080 0.144 0.065
media was 1.0e11.0 mg/mL with a correlation coefficient (R2) >0.998. The LOD and LOQ of the proposed method for methanol were found to be 0.19 and 0.57 mg/mL respectively. Whereas LOD and LOQ for 6.4 pH phosphate buffer were found to be 0.11 and 0.31 mg/mL. 3.3. Intraday, interday precision, and repeatability Precision was determined in a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The intraday and interday precision results are shown in Table 1 and the results confirmed the reliability and stability of the developed method where all RSDs were <0.5%. Repeatability was determined by analyzing 4 mg/mL concentration of curcumin solution for six times and the percent amount found was calculated using the linearity curve. The % RSD was <0.5% in both methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. 3.4. Accuracy For accuracy determination, the results of recovery studies as shown in Table 2 were found to be within the range of 99.01e100.1% and the RSDs <0.5% which indicated the non-interference of excipients and the accuracy of the developed UV method.
The specificity of the curcumin was evaluated by blank lipid nanoparticles. The amount recovered from blank NLC formulation was 99.25 ± 0.28% this indicated the method was specific towards curcumin. There was no interference of excipients used in the lipidbased formulation. 3.6. Application of the developed UV method for characterization of nanostructured lipid nanocarriers
3.2. Linearity and range The calibration curve (Figs. 5 and 6) obtained was evaluated by its correlation coefficient. The linearity range of curcumin in both the Table 3 Entrapment efficiency study of curcumin in NLC (n ¼ 3).
57.04 ± 0.62 67.80 ± 1.16 70.70 ± 1.03
RSD (%)
3.5. Specificity
Curcumin was found to be water-insoluble and freely soluble in methanol, acetone, and acetonitrile. Based on solubility, stability, and application in formulation characterization, curcumin method was developed in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. The maximum absorption wavelength was determined in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 which was found to be 425 nm for both solvent systems.
% of Curcumin entrapped ± SD
100.00 ± 0.0010 100.60 ± 0.0010 99.72 ± 0.0015 100.00 ± 0.0006 99.75 ± 0.0012 100.00 ± 0.0006
80 100 120 80 100 120
6.4 pH phosphate buffer with 1.5% polysorbate 80
Recovery (%)
% of unentrapped Curcumin ± SD
% total amount of Curcumin recovered
43.76 ± 0.82 31.10 ± 0.93 28.36 ± 0.98
100.80 ± 0.72 98.90 ± 1.04 99.06 ± 1.01
% RSD 1.087 1.711 1.457
3.6.1. Entrapment efficiency and drug loading Curcumin entrapped in NLC were extracted and analyzed in triplicate for three different batches with different lipid composition. Table 4 Assay of curcumin in NLCs loaded gel and curcumin cream (n ¼ 3). Formulation
% assay ± SD
Nanocarriers loaded cream preparation (n ¼ 3) Nanocarriers loaded gel formulation (n ¼ 3)
100.07 ± 0.47 100.63 ± 0.62
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Table 5 Mass balance of the curcumin used in in-vitro release study. The total amount of drug used for release study The cumulative amount of drug released Amount of drug retained in a dialysis bag The total amount of drug recovered
1000 mg 614.46 ± 0.131 mg 356.91 ± 0.295 mg 971.37 ± 0.213 mg (97.14%)
The amount of curcumin entrapped and unentrapped was estimated with a validated analytical method. The results pertaining to entrapped and un-entrapped drug were mentioned in Table 3. The data demonstrated the high reproducibility (%RSD < 1.72) with percentage recovery of 98.90 ± 1.04 to 100.80 ± 0.72 indicated that the method was efficient and applicable for calculation of entrapment efficiency in nanocarriers based formulations of curcumin. 3.6.2. Assay of curcumin loaded NLC embedded gel and cream formulations Curcumin content in NLC based gel and cream based formulation preparations were analyzed for assay in triplicate. The assay results obtained from curcumin loaded NLC embedded gel and cream-based formulations are represented in Table 4. Results showed high reproducibility (% RSD < 0.01) and close to label claim
(100 ± 1%) of drugs. This indicated that the developed method can accurately and precisely determine the curcumin content without interference of the excipients or formulation matrix. Additionally, it also reflected that the method has the potential to determine the drug content in complex nanocarriers based formulation also. 3.6.3. In-vitro drug release estimation The in-vitro release study was performed in triplicate samples collected from the Franz diffusion cell were analyzed using the developed method. The amount of drug release was within the linearity range were analyzed directly and in case of high concentrations, the samples were diluted with release buffer before analysis. There was no interference of surfactants and buffers of release media in the quantification of curcumin. The cumulative amount of drug released was found to be 614.46 ± 0.131 mg in 6 h. The unreleased amount retained in the dialysis bag was extracted and estimated for mass balance (Table 5). About 97.14 ± 0.213% of the curcumin was recovered from the study. The results indicated that method was found to be appropriate to quantify the amount of drug release in presence of dissolution medium and can quantify the curcumin at early release points to maximum drug release during in-vitro drug release studies as shown in Fig. 7.
Fig. 7. In-vitro drug release study of curcumin loaded NLC in dissolution medium.
Fig. 8. Skin retention of curcumin in ex-vivo release study.
V.K. Rapalli et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117392
3.6.4. Extraction of drug from the skin tissues and distribution of curcumin in skin tissue The known concentration of analyte solution was spiked into the homogenized skin tissue and analyzed after passing through a 0.22 mm membrane filter. The percentage recovery of curcumin was found to be 96.10 ± 0.41%. This indicated the developed method can be applied for skin permeation studies and tape stripping for evaluation of lipid nanocarriers based topical dosage form. The amount of drug retained in skin determined by tape stripping was found to be 39.228 ± 0.412 mg (n ¼ 3) as shown in Fig. 8. The amount of drug retained in the skin, determined by the direct method was found to be 40.698 ± 0.237 mg (n ¼ 3). Results of characterization studies indicated that the developed and validated UV spectroscopic method was simple, rapid, and accurate for estimation of the curcumin content in biological samples. 4. Conclusion The analytical method developed on UV spectrophotometer to determine the concentration of curcumin in methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80 was validated in terms of the linearity of the linear regression equation, accuracy, interday precision and intraday precision, repeatability of method, and sensitivity. Both the methods were reliable having RSDs <2%. Further, the method developed in methanol will be useful in estimating the encapsulation efficiency of the curcumin in the lipidbased formulations, which helps to determine the percent of the entrapped drug in the lipid structure. Whereas method developed in 6.4 pH phosphate buffer with 1.5% polysorbate 80 media can be utilized to determine the percent drug release from the lipid-based formulations. For both the applications, the developed methods were found to be accurate, simple and reliable. Declaration of Competing Interest The authors report no conflicts of interest. Acknowledgment Authors are thankful to Industrial Research Laboratory, BITS Pilani, Pilani campus for providing the facility to conduct the experiments. The presented work is not sponsored by any funding agency. References [1] A. Noorafshan, S. Ashkani-Esfahani, A review of therapeutic effects of curcumin, Curr. Pharm. Des. 19 (2013) 2032e2046. http://www.ncbi.nlm.nih.gov/ pubmed/23116311 (accessed April 19, 2019). [2] L. Hu, Y. Jia, F. Niu, Z. Jia, X. Yang, K. Jiao, Preparation and enhancement of oral bioavailability of curcumin using microemulsions vehicle, J. Agric. Food Chem. 60 (2012) 7137e7141, https://doi.org/10.1021/jf204078t. [3] S.J. Hewlings, D.S. Kalman, Curcumin: a review of its' effects on human health, Foods (Basel, Switzerland) 6 (2017), https://doi.org/10.3390/foods6100092. [4] Y.J. Wang, M.H. Pan, A.L. Cheng, L.I. Lin, Y.S. Ho, C.Y. Hsieh, J.K. Lin, Stability of curcumin in buffer solutions and characterization of its degradation products, J. Pharm. Biomed. Anal. 15 (1997) 1867e1876. [5] T. Waghule, V.K. Rapalli, G. Singhvi, P. Manchanda, N. Hans, S.K. Dubey, M.S. Hasnain, A.K. Nayak, Voriconazole loaded nanostructured lipid carriers based topical delivery system: QbD based designing, characterization, in-vitro and ex-vivo evaluation, J. Drug Deliv. Sci. Technol. 52 (2019) 303e315, https:// doi.org/10.1016/J.JDDST.2019.04.026. [6] G.M. Gelfuso, M.S. Cunha-Filho, T. Gratieri, Nanostructured lipid carriers for targeting drug delivery to the epidermal layer, Ther. Deliv. 7 (2016) 735e737, https://doi.org/10.4155/tde-2016-0059. [7] V. Girdhar, S. Patil, S. Banerjee, G. Singhvi, Nanocarriers for drug delivery: mini review, Curr. Nanomed. 8 (2018) 88e99, https://doi.org/10.2174/
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