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Forced Degradation Studies of Aloe and Emodin by HPTLC

SINDHU NARAYANAN, ARUNA P. JADHAV* AND V. J. KADAM Department of Quality Assurance, Bharati Vidyapeeth’s College of Pharmacy, CBD Belapur, Navi Mumbai-400 614, India

Narayanan, et al.: Degradation Studies of and Emodin

Anthraquinones are natural phenolic compounds, which are reported to act as anti-aging, anti-inflammatory, antioxidant, anti-cancer, laxative and antitumor agents. They are abudant in plants like candle bush, aloes, cascara bark and rhubarb. The present work was to observe the effect of different forced degradation conditions by high-performance thin layer chromatography on potential markers i.e. aloe emodin and emodin. Both aloe emodin and emodin were subjected to various forced degradation studies such as oxidation, acid and alkaline hydrolysis, photolysis, hydrolytic and thermal degradation. Aloe emodin, was more susceptible to acid hydrolysis and degradation was found to a lesser extent under thermal degradation whereas significant degradation was observed under acid hydrolysis, lesser extent was observed under alkali hydrolysis for emodin. Forced degradation studies on aloe emodin and emodin gives information about its storage and intrinsic stability conditions considering the advanced pharmaceutical aspects of formulation.

Key words: , aloe emodin, emodin, forced degradation, high-performance thin layer chromatography

Aloe emodin [1,8-Dihydroxy-3-(hydroxymethyl)- This is an open access article distributed under the terms of the Creative 9,10-anthracenedione] and emodin [6-methyl-1,3,8- Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the trihydroxyanthraquinone] are anthraquinones naturally author is credited and the new creations are licensed under the identical terms. present in rhubarb (Rheum emodi), candle bush For reprints contact: [email protected] (Cassia alata), aloes (Aloe barbadensis dried leaf juice) and cascara (Cascara sagrada)[1]. They act as Accepted 29 November 2015 Revised 19 April 2015 *Address for correspondence Received 10 October 2014 E-mail: [email protected] Indian J Pharm Sci 2015;77(6):795-798

November - December 2015 Indian Journal of Pharmaceutical Sciences 795 www.ijpsonline.com antiinflammatory[2], laxative[3], antiaging, anticancer[4], Accurate, precise, robust HPTLC method was antioxidant[5] and antitumor agents. Aloe emodin developed using toluene: ethyl acetate: Formic acid, and emodin show cell cycle arrest, cytotoxic to 10:2:1 (v/v/v) as mobile phase. Good resolution cells and leads to apoptosis in human tongue and for aloe emodin and emodin was observed with breast cancer cells thus acting as antiproliferative Rf values at 0.37±0.03 and 0.55±0.03, respectively agents. There are many herbal marketed formulations, (fig. 1). Linear relationship was found to be in which have aloe emodin and emodin as major the concentration range of 300-800 ng/spot constituents such as Amlycure D.S. capsules, N-Zime (y=9.4274x+906.14 with r²=0.9993) for aloe emodin syrup, Divya Udarkalp churna, and LIV-SR syrup. and 150-400 ng/spot (y=13.031x-115.96 with Considerable interest on herbal drugs has been r²=0.9996) for emodin. aroused because of their beneficial effects on humans. The aim of the present work was to carry out forced On treating the solutions with 0.1 N HCl for 2 h, degradation studies and to provide the information the peak areas corresponding to aloe emodin and on stability inherent conditions of the potential emodin decreased and additional peaks was observed markers i.e. aloe emodin and emodin and to ascertain at Rf 0.52, 0.71 for aloe emodin and 0.01, 0.36, the degradation pathways. The force degradation 0.58 for emodin. This shows that aloe emodin and studies of drug substances should be carried out in emodin undergoes degradation under acidic conditions various conditions such as effect of light, humidity, (fig. 2a and b). On treating the solutions with 0.1 N elevated temperature, oxidizing agents, acid and base NaOH for 2 h, aloe emodin and emodin was found degradation (across a range of pH values) according less susceptible to base degradation. Additional peaks to stability guidelines by ICH Q1AR (International were observed at Rf 0.01, 0.64 for aloe emodin and Conference on Harmonisation). The effect of various 0.01, 0.39, 0.69 for emodin. Oxidative degradation forced degradation conditions was observed using was observed in presence of 6% v/v of hydrogen high-performance thin layer chromatography (HPTLC) peroxide when exposed for 3 h and the HPTLC [6] analysis . There is no method reported for analysis chromatogram shows additional peaks at Rf 0.56, 0.68 of forced degradation studies of aloe emodin and for aloe emodin and 0.63 for emodin. Aloe emodin emodin. and emodin shows moderate degradation when exposed to dry heat at 105º for 8 h. Additional peaks

Analytical grade reagents: ethyl acetate, formic were observed at Rf 0.02, 0.65 for aloe emodin and acid, toulene and methanol were obtained from 0.01, 0.08, 0.65 for emodin. Aloe emodin undergoes S.D. Fine-Chem Limited, Mumbai, India. Aloe hydrolytic degradation to a large extent at 80º for 8 h emodin and emodin standards were procured from and shows additional peak at 0.62 Rf whereas emodin Total Herb Solutions, Mumbai. Aloe emodin and shows moderate degradation and additional peak was emodin stock solutions (1000 µg/ml) were prepared observed at 0.31. Aloe emodin and emodin shows less by dissolving accurately weighed 10 mg of each standard in 10 ml methanol. TLC plates pre-coated with silica gel 60 F254 was used as stationary phase. Standard and sample solutions were applied as a band to the 10×10 cm plate (6.0 mm wide) by using Camag Linomat V sample applicator (100 µl Hamilton syringe). Development of plate was done ascending to a distance of 80 mm with mobile phase at room temperature (24±2°) in Camag glass twin-trough chamber. The chamber was saturated for 30 min previously and the plates were scanned at 263 nm after development with Camag TLC Scanner- 3 using the deuterium lamp.

In situ HPTLC spectral overlaying of aloe emodin and emodin was taken and isoabsorptive point Fig. 1: HPTLC chromatogram of standard aloe emodin and emodin. Chromatogram of aloe emodin and emodin at Rf 0.37 and 0.55, i.e 263 nm was selected as scanning wavelength. respectively by TLC.

796 Indian Journal of Pharmaceutical Sciences November - December 2015 www.ijpsonline.com degradation when exposed to sunlight or UV-254 nm intrinsic stability of aloe emodin and emodin. Care for 8 h. Degradant peaks were observed at Rf 0.45, should be taken when stored under acidic conditions 0.65 for aloe emodin and 0.70 for emodin. The as both drugs undergo rapid degradation. These concentration of unaffected aloe emodin and emodin studies and observations regarding stability of aloe was calculated under several forced degradation emodin and emodin may help during storage and conditions based on the area under curve (Table 1). in its modern and traditional formulation aspects in pharmacy. The forced degradation studies on aloe Forced degradation studies on aloe emodin and emodin and emodin was performed and it can be emodin were carried out and it was found that aloe concluded that these findings provide an insight and emodin was more susceptible to acid (29.22%) and information about the storage and intrinsic stability water degradation (36.23%), moderate to oxidation conditions of aloe emodin and emodin with respect induced degradation (61.87%) and to lesser extent to the advanced formulation aspects. to day light (85.74%) and dry heat (89.23%) induced degradation. Emodin was found to be more Acknowledgements: susceptible to acid (23.88%) degradation. Moderate Authors are thankful to the Anchrom Laboratory, degradation was observed in water (70.22%), Mumbai for giving training on HPTLC instrument. oxidation (76.68%) and dry heat (82.05%) induced degradation and to lesser extent to day light (86.54%) Financial support and sponsorship: and base (95.332%) induced degradation. The HPTLC Nil. technique developed for both drugs estimation resolves the degradation products under all forced Conflicts of interest: degradation conditions thus providing information on There are no conflicts of interest.

a b Fig. 2: (a) HPTLC chromatogram of standard aloe emodin subjected to acid degradation. Subjected to acid degradation and

Rf of degradants=0.52, 0.71. (b) HPTLC chromatogram of standard emodin subjected to acid degradation. Subjected to acid degradation and

Rf of degradants=0.01 0.36 0.58.

TABLE 1: FORCED DEGRADATION DATA OF ALOE EMODIN AND EMODIN Exposure conditions Time (h) Aloe emodin Emodin

Recovery (%) Rf of degradation Recovery (%) Rf of degradation aloe emodin products emodin products Acid (0.1 N HCl) reflux at 80° 2 29.22 0.52, 0.71 23.88 0.01, 0.36, 0.58 Base (0.1 N NaOH) reflux at 80° 2 81.93 0.01, 0.64 95.33 0.01, 0.39, 0.69

H2O2, (6% v/v) 3 61.87 0.56, 0.68 76.68 0.63 Dry heat (105°) 8 89.23 0.02, 0.65 82.05 0.01, 0.08, 0.65 Water, reflux at 80° 8 36.23 0.62 70.22 0.31 Photostability‑day light 8 85.74 0.46, 0.65 86.54 0.70 Each result is an average of three measurements. The markers aloe emodin and emodin was exposed to different forced degradation conditions as per ICH guidelines. ICH: International Conference on Harmonisation

November - December 2015 Indian Journal of Pharmaceutical Sciences 797 www.ijpsonline.com REFERENCES 4. Pecere T, Gazzola MV, Mucignat C, Parolin C, Vecchia FD, Cavaggioni A, et al. Aloe-emodin is a new type of anticancer agent with selective activity 1. Evans WC. Trease and Evans Pharmacognosy. 15th ed. London (UK): against neuroectodermal tumors. Cancer Res 2000;60:2800-4. Elsevier Publishers; 2004. 5. Vargas F, Díaz Y, Carbonel K. Antioxidant and scavenging activity of 2. Park MY, Kwon HJ, Sung MK. Evaluation of aloin and aloe-emodin as emodin, aloe-emodin, and on free-radical and reactive oxygen anti-inflammatory agents in aloe by using murine macrophages. Biosci species. Pharm Biol 2004;42:342-8. Biotechnol Biochem 2009;73:828-32. 6. Reich E, Schibli A. High-Performance Thin-Layer Chromatography for 3. Zhang HQ, Zhou CH, Wu YQ. Effect of emodin on small intestinal peristalsis the Analysis of Medicinal Plants. 1st ed. Switzerland: Thieme Medical of mice and relevant mechanism. World J Gastroenterol 2005;11:3147-50. Publishers; 2006.

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