Stability of Curcumin Improved in Hydrophobic Based Deep Eutectic Solvents

 

T. Raja Sekharan^1,2, R. Margret Chandira¹*, S.C. Rajesh³, Shunmugaperumal Tamilvanan⁴, C.T. Vijayakumar⁵,

B.S. Venkateswarlu¹

¹Department of Pharmaceutics, Vinayaka Mission’s College of Pharmacy,

Vinayaka Mission's Research Foundation (Deemed to be University), Salem-636308, Tamil Nadu, India.

²Department of Pharmaceutics, Sankaralingam Bhuvaneswari College of Pharmacy,

Anaikuttam-626130, Sivakasi, Tamil Nadu, India.

³Department of Pharmaceutical Analysis, Sankaralingam Bhuvaneswari College of Pharmacy,

Anaikuttam-626130, Sivakasi, Tamil Nadu, India.

⁴National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati,

Sila Katamur (Halugurisuk), Changsari, Kamrup, Assam, 781101, India.

⁵Department of Polymer Technology, Kamaraj College of Engineering and Technology,

K. Vellakulam, 625701, Tamil Nadu, India.

 

ABSTRACT:

Turmeric is a commonly known natural spice that contains many phytoconstituents. Among which Curcumin is a polyphenol present in turmeric responsible for many pharmacological actions. Curcumin is still used as a traditional medicine in fields such as Ayurvedic, Siddha, and Unani. Though Curcumin has a large number of activities, it has disadvantages, such as small shelf life due to poor chemical stability, poor absorption results in less bioavailability, less water solubility, rapid metabolism results in quick elimination from the systemic circulation. A Deep eutectic solvent (DES) is a new class of solvents. Hydrophobic DES can be used for dissolving water-insoluble compounds. DES can be prepared when two solid components mixed in a particular proportion are converted into liquid. DES can be used as a solvent for dissolving water-insoluble compounds and to increase the stability. In this work initially, curcumin linearity studies were conducted in different buffers. A buffer showing maximum absorbance was selected from the linearity studies. Then, DES was prepared by combining Camphor:Menthol (1:1) (CM-DES), Camphor:Thymol (1:1) (CT-DES) and, Menthol:Thymol (1:1) (MT-DES). The stability of curcumin in different DES was determined from the stock and working solutions in benchtop condition (room temperature) and, refrigerator condition (5±3°C). Only working solution stability was determined in the in vitro media temperature (37±2°C). From this study, it was concluded that 50 mM Sodium dihydrogen orthophosphate with 0.5% SLS at pH 5.5 showed maximum absorbance value compared with other buffers, so it was selected for further studies. From stability studies, it was found that curcumin in CM-DES was found to be stable in both stock and working solutions compared to the other two CT-DES and MT-DES.

 

 

INTRODUCTION:

Toxic effects of synthetic medications have drawn awareness to turn toward natural medicines¹. The demand for herbal medicines is increasing rapidly due to their lack of side effects. Further as healthcare costs continue to escalate, the attraction for low-cost remedies has stimulated consumers to reevaluate the potential of alternatives². Turmeric (Curcuma longa) belongs to the Zingiberaceae family, is broadly utilized as a zest, food, additive and traditional medication and as a family unit solution for different illnesses in Asian nations, including China and southeast Asia^3-5. The name turmeric is derived from the Latin word terra merita or turmeryte⁶. Curcumin, the main constituent present in turmeric⁷ along with other chemical constituents known as curcuminoids. The major curcuminoids present in turmeric are 77% Curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin (curcumin III), and the recently identified cyclocurcumin⁸. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), also called diferuloylmethane, is the main natural polyphenol present in turmeric⁹.

 

Many of the research work recommended curcumin has been used in the avoidance and curing of various many illnesses^10-12. Curcumin can be used to the following illness like anti-proliferative, anti-invasive, and anti-angiogenic agent, chemopreventive agent, as a mediator of chemoresistance and radioresistance. As a curative effect in wound healing, cardiovascular diseases, pulmonary diseases, parkinson’s disease, alzheimer’s disease, and in various types of arthritis, atherosclerosis, chronic anterior uveitis, colon cancer, familial adenomatous polyposis, hypercholesteremia, inflammatory bowel disease, pancreatic cancer, pancreatitis, psoriasis, ulcerative colitis¹², potent anti-oxidant, anti-inflammatory, anti-cancer, antidiabetic, hepatoprotective, anti-spasmodic¹³, cholesterol lowering¹⁴, anti-coagulant¹⁵ etc.

 

Even though Curcumin has a large number of action, its usage is limited in the foodstuff industry and pharma companies because of its poor solubility and low stability in different environment¹⁶. Its bioavailability is also poor and it remains a major challenge due to the presence of olefinic groups present in its structure this β-diketone of poor aqueous solubility rendering it of relatively low bioavailability¹⁷. Curcumin is sensitive to light and temperature, which limits its applications¹⁶. The degradation of curcumin is pH-dependent, at neutral and under alkaline conditions it degrades more speedily, at acidic conditions, it degrades slowly, with less than 20% the following 30 min to one hr^18-21. Protecting the stability of curcumin is important for effective pharmacological action²².

 

Eutectic solvents are useful in numerous areas in the pharmaceutical field like to increase the drug solubility, permeation, and absorption²³. DES is a new class of neoteric solvents that are said to be “green solvent” made from low-cost ingredients, which entered the year 2003^24,25. DES is defined as a combination of two or more ingredients normally solid at room temperature, but when combined at a particular molar ratio, converted into liquid at room temperature²⁶. Among the two ingredients one is hydrogen bond donor (HBD) and another one hydrogen bond acceptor (HBA)²⁷. Tomasz Jeliński et al²², the previously improved curcumin stability with the help of hydrophilic based eight natural DES. In this study, an attempt was made to determine the stability of curcumin in three hydrophobic-based DES prepared with Camphor:Menthol, Camphor:Thymol and Menthol:Thymol in equal proportion (1:1) with the help of suitable buffer.

 

MATERIALS AND METHODS:

Curcumin was obtained from Sami laboratories limited. Camphor and Thymol were purchased from Sisco Research Laboratories Pvt. Ltd, Maharastra, Menthol was procured from Reachem Laboratory chemicals private limited, Chennai. Methanol was purchased from Himedia Laboratory Pvt. Ltd. Mumbai. Distilled water was purchased from Rajco Pvt. Ltd., Sivakasi. All other chemicals used in this study for preparing different buffers were of analytical grade.

 

Selection of buffer:

Curcumin has poor solubility and stability in aqueous media. So initially, the suitable buffer on the basis of good solubility and stability was determined. Different trials have been made with different buffers pH according to different works of literature. The standard curve for Curcumin was performed in methanol, acetate phosphate buffer pH 4.5, phosphate buffer pH 7.4, phosphate buffer pH 6.8, phosphate buffer pH 5.5, phosphate buffer pH 5.5 with 0.5% SLS, 50 mM Sodium dihydrogen orthophosphate with 0.5% SLS with a pH of 5.5. The linearity of concentrations ranges from 1–10 µg/mL was made with different above said solutions. The solution with maximum absorbance was taken for further studies as they have the maximum solubility of Curcumin. For analyzing the drug content, various analytical methods like UV spectroscopy, HPLC method, HPTLC method, etc., are in use. According to ICH guidelines, drug analysis can be determined by simple, sensitive, quick, and accurate. UV spectroscopy fulfills the above condition and it is acceptable for regular testing of drugs^28,29,30. Therefore, in this study curcumin solubility studies were determined with the help of double beam UV-Visible spectrophotometer (Shimadzu- 1800, Japan).

 

Curcumin in methanol and in different pH:

Curcumin is freely soluble in methanol. According to different works of literature, methanol satisfied the conditions like relative to peak quality and noninterference at the specified wavelength^28,31. 10-mg curcumin was initially dissolved in 10-mL methanol (Curcumin stock solution of 1 mg/mL).  From the curcumin stock solution 10 mL was pipetted out and make up to 100 mL with the same methanol (Curcumin intermediate solution of 100 µg/mL). From the curcumin intermediate solution, 1 to 10 mL was pipetted out in a separate 100 mL volumetric flask and make up the volume with respective buffer solutions to give a concentration of 1 to 10 µg/mL. The buffers used for this study are phosphate buffer pH 5.5^32a, phosphate buffer pH 6.8^32b, phosphate buffer pH 7.4, in acetate buffer pH 4.5, phosphate buffer pH 5.5 with 0.5% SLS and 50 mM sodium dihydrogen orthophosphate with 0.5% SLS.

 

Preparation of DES:

Three types of DES were prepared by combining Camphor:Menthol (1:1) (CM-DES); Camphor:Thymol (1:1) (CT-DES) and Menthol:Thymol (1:1) (MT-DES). About equal quantities of the eutectic forming ingredients are accurately weighed and transferred to a small beaker, and it was mixed well with the help of magnetic stirrer at 500 rpm until a clear liquid was formed.

 

Curcumin in DES:

10-mg Curcumin was initially dissolved in 10 mL of CM-DES (Curcumin stock solution of 1 mg/mL) and dissolved with the help of magnetic stirrer with slight heat.  Applying slight heat increases the solubility of curcumin rapidly when compared to without heat. Similarly, a curcumin stock solution in other types of DES (CT-DES and MT-DES) was prepared. 10 mL of curcumin stock solution (prepared with CM-DES, CT-DES, and MT-DES) was further made up to 100-mL methanol (Curcumin intermediate solution of 100 µg/mL). From the curcumin intermediate solution, 1 to 10 mL was pipetted out in a separate 100 mL volumetric flask and make up the volume with the best buffer to give a concentration of 1 to 10 µg/mL. The stability of curcumin in best buffer was performed with the help of bioanalytical method templates and validation^34,35.

 

Benchtop stability:

Bench top stability for Curcumin in three types of DESs (CM-DES, CT-DES, and MT-DES) in stock and working solution was performed by keeping the solution at the bench top at room temperature for about 25±3°C at a time interval of 1, 2, 3, 4, 5, 6, 12, and 24 h in triplicate. The stock solution stability was determined by making a dilution according to preparation of Curcumin intermediate (50 µg/mL) solution in methanol and preparation of Curcumin working solution (5 µg/mL) in 50 mM sodium dihydrogen ortho-phosphate with 0.5%w/v SLS solution. 50 mM sodium dihydrogen ortho-phosphate with 0.5%w/v SLS solution was selected for the last dilution as this solution has better absorbance than other buffers solution at 430 nm. The sample readings were compared with freshly prepared stock solution and analyzed with the help of UV double beam spectrophotometer.

 

Refrigerator stability (5±3°C):

Curcumin (in three different DES) in stock and working solution was prepared according to the above said procedure and kept in refrigerator maintaining a temperature of 5±3°C for about 48 hrs. The reading was taken at 2, 4, 6 and 8 days in triplicates. The samples readings were compared with freshly prepared stock solution and analyzed with the help of UV double beam spectrophotometer.

 

Stability in in-vitro media temperature (37°C±2°C):

Curcumin (in three DES) in stock and working solutions was prepared according to the above said procedure and kept at 37±2°C (dissolution temperature) for about 14 h. The reading was taken at 1, 2, 4, 6, 8, 10, 12, and 14 h to check the stability of Curcumin.

 

RESULTS AND DISCUSSION:

Curcumin in Methanol:

Curcumin is freely soluble in methanol. Curcumin showed more amount of absorbance at a concentration of 1–10 µg/mL at 425 and 430 nm, respectively. The linearity for curcumin concentrations ranges from 1–10 µg/mL was made with methanol and different buffers. The absorbance of another buffer pH was compared with methanol absorbance.  Acetate buffer pH 4.5, phosphate buffer pH 5.5 (with and without SLS), 6.8, 7.4, and 50 mM Sodium dihydrogen orthophosphate with 0.5 % SLS (pH 5.5) was performed and the reading was shown as in Fig. 1. The comparative readings of Curcumin in different pH are given in the table 1.

 

Phosphate buffer pH 5.5 with 0.5% SLS was initially prepared with potassium dihydrogen phosphate, disodium hydrogen phosphate, and SLS in sufficient distilled water. However, we are unable to get a clear solution, a precipitate formed this is due to the interaction between sodium present in SLS and potassium present in potassium dihydrogen phosphate. So we replaced potassium dihydrogen phosphate with sodium dihydrogen phosphate. When potassium dihydrogen phosphate was replaced with sodium dihydrogen phosphate, we got a clear liquid. There was no change in the pH. Buffer having maximum absorbance was selected for further stability studies.  Among the different buffer pH, 50 mM Sodium dihydrogen orthophosphate with 0.5% SLS was selected for further studies due to maximum absorbance value compared to another pH.

 

Table 1: Comparative readings of Curcumin in different buffer pH solutions

*Linearity was determined for three times

 

Fig. 1: Linearity of Curcumin in methanol, phosphate buffer pH 6.8, in phosphate buffer pH 7.4, in acetate buffer pH 4.5, in phosphate buffer pH 5.5, in phosphate buffer pH 5.5 with 0.5 % SLS and in 50 mM Sodium dihydrogen orthophosphate with 0.5 % SLS

 

Benchtop stability studies:

Benchtop stability for curcumin in-stock solution prepared from different DES (CM-DES, CT-DES, and MT-DES) and working solution in 50 mM Sodium dihydrogen orthophosphate with 0.5% SLS buffer was performed to determine curcumin stability while working in the laboratory temperature and compared with a freshly prepared stock solution. It helps in determining the curcumin stability in stock, the working solution while working at least for a while of 24 h at a period of 1, 2, 3, 4, 5, 6, 12, and 24 h.

 

Curcumin benchtop stability studies in CM-DES in the stock solution were found to be between 100.824±0.799 and 98.220±0.262% and for working solution it was found to be between 100.824±0.799 and 98.220±0.262. These values are within the acceptance criteria of 98%-102%. The percentage relative standard deviation (% RSD) was found to be 0.28 for a stock solution and 0.27 for the working solution. Based on the acceptance criteria results Curcumin in CM-DES was found to be stable for 24 h in both stock and working solutions.

 

Curcumin benchtop stability studies in CT-DES in the stock solution were found to be between 101.192±0.455 and 95.664±0.207% and for working solution it was found to be between 101.092±0.958 and 94.969±0.500%. These values are within the acceptance criteria of 98%-102%. The percentage relative standard deviation (% RSD) was found to be 0.47 for a stock solution and 0.92 for the working solution. Based on the acceptance criteria results Curcumin in CT-DES was found to be stable for 12 h in both stock and working solutions.

 

Curcumin benchtop stability studies in MT-DES in the stock solution were found to be between 101.035±1.028 and 83.367±1.056% and for working solution it was found to be between 100.401±0.601 and 75.351±1.102%. Based on the acceptance criteria results, curcumin in MT-DES was stable for 4 h in a stock solution and only 2 h in working solution.

 

The results of the benchtop stability of curcumin in different DES from stock and the working solution are shown in  table 2.

 

Table 2: Benchtop stability of Curcumin in different DES from stock and working solution

*Performed three times

 

Refrigerator temperature stability studies:

Curcumin stability in different DESs at 5±3°C was determined by placing the stock and working solution in a refrigerator for 8 days and samples were analyzed for every 2 days interval with a freshly prepared stock solution. Curcumin in CM-DES in the stock solution was found to be between 100.561±0.374% and 98.384±0.318% and for working solution, it was found to be between 100.495±0.799% and 98.154±0.228%. Based on the acceptance criteria range Curcumin in CM-DES was found to be stable for 8 days in both stock and working solutions.

 

Curcumin in CT-DES in the stock solution was found to be between 100.563±0.599% and 96.690±0.901% and for working solution it was found to be between 100.132±0.303% and 95.829±0.358%. Based on the acceptance criteria range Curcumin in CT-DES was found to be stable for 6 days in a stock solution and 4 days in the working solution.

 

Curcumin in MT-DES in the stock solution was found to be between 100.802±0.810% and 92.084±0.417% and for working solution, it was found to be between 100.301±0.459% and 91.850±0.810%. Based on the acceptance criteria range Curcumin in MT-DES was found to be stable for 4 days in a stock solution and 2 days in the working solution. The stability data of curcumin in different DES from stock and the working solution placed in a refrigerator at 5±3°C are shown in table 3.

 

Table 3: Stability data of Curcumin in different DES from stock and the working solution placed in refrigerator at 5±3°C

*Performed three times

 

Stability studies in in-vitro media temperature:

Stability for curcumin in working solution in in vitro media temperature was performed to check the curcumin stability in the media at the time of in vitro release studies for a period of 24 hrs. It was checked in a different time interval of 1, 2, 4, 8, 12, 16, 20, and 24 h by comparing it with a freshly prepared working solution.  The working solution prepared from CM-DES in 50 mM Sodium dihydrogen orthophosphate containing 0.5% SLS was found to be between 100.462±0.318% and 98.121±0.099%. Based on the acceptance criteria data the results clarify that Curcumin was stable for a period of 24 hrs at 37±2°C. The working solution was prepared from CT-DES and the solution was further diluted with 50 mM Sodium dihydrogen orthophosphate containing 0.5% SLS was found to be 100.397±0.455% and 86.759±1.098%. Based on the acceptance criteria data the results clarify that curcumin in CT-DES was stable for a period of 8 h at 37±2°C. The working solution was prepared from MT-DES and the solution was further diluted with 50 mM Sodium dihydrogen orthophosphate containing 0.5% SLS was found to be between 100.334±0.252% and 77.288±1.004%. Based on the acceptance criteria data the results clarify that curcumin in MT-DES was stable for a period of 2 h only at 37±2°C. The results of the stability of curcumin in the working solution in in vitro media temperature are shown in table 4.

 

Table 4: In vitro media temperature stability of curcumin in different DES from working solution

*Performed three times

 

CONCLUSION:

In this work, Curcumin standard curve was performed at different pH, based on the results it was confirmed that 50mM sodium dihydrogen ortho-phosphate with 0.5% SLS showed maximum absorbance compared to other pH. An attempt was made to determine the stability of curcumin in three hydrophobic based DES (CM-DES, CT-DES, and MT-DES) in a stock solution and in working solution prepared with 50mM sodium dihydrogen ortho-phosphate with 0.5% SLS. Curcumin in CM-DES was found to be stable in all stability parameters [benchtop (25±3°C), refrigerator (5±3°C), and in vitro media (37±2°C) temperature] both in-stock and working solution. Curcumin in CT-DES at benchtop condition was stable in stock solution and in working solution it was stable for 12 hrs. In refrigerator condition, curcumin in CT-DES was found to be stable for 6 days in a stock solution and 4 days in the working solution. In the in vitro media condition, it was stable for eight hrs only. Curcumin in MT-DES at benchtop condition was found to be stable for 4 h in a stock solution and only 2 h in the working solution. In refrigerator condition, curcumin was found to be stable for 4 days in a stock solution and 2 days in the working solution. In in vitro conditions, it was found to be stable for 2 h only. From this study, it was concluded that 50 mM sodium dihydrogen ortho-phosphate with 0.5% SLS is a best solution for making standard curve. In addition, also from the stability studies Curcumin was found to be stable in CM-DES in both stock and working solutions compared to the other two CT-DES and MT-DES.

 

ACKNOWLEDGEMENT:

The access to scientific literature and lab facility to perform the stability studies are provided jointly by Vinayaka Mission’s College of Pharmacy, Salem and Sankaralingam Bhuvaneswari College of Pharmacy, Sivakasi.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Accepted on 25.03.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6430-6436.