Journal of Applied Pharmaceutical Science Vol. 7 (01), pp. 129-134, January, 2017 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2017.70117

ISSN 2231-3354

Preparative HPLC fractionation of cassia Water Extract and their in-vitro Antimalarial Activities

Mutaz Akkawi1, Saleh Abu-Lafi2*, Hanadi Attieh1, Qassem Abu-Remeleh1, Sadam Makhamra3, Mutaz Qutob4

1Life Sciences Department, College of Science and Technology, Al-Quds University, Abu-Dies, Palestine. 2Faculty of Pharmacy, Al-Quds University, P. O. Box 20002, Abu-Dies, Palestine. 3Chemistry Department, Faculty of Science and Technology, Al-Quds University, Abu-Dies, Palestine. 4Department of Earth and Environmental Sciences, College of Science and Technology, Al-Quds University, Abu-Dies, Palestine.

ABSTRACT ARTICLE INFO

Article history: Due to the development of antimalarial resistance strains of Plasmodium falciparum, the search for new Received on: 16/08/2016 antimalarial drugs is immediately needed. Such drugs might work by obstructing the heme detoxification Revised on: 03/09/2016 pathway in the malaria parasite, a vital requirement for parasite existence in host erythrocytes. The present Accepted on: 02/10/2016 study is aimed to investigate the antimalarial activity of four preparative HPLC fractions from Cinnamomum Available online: 31/01/2017 cassia water extract using semi-quantitative in vitro micro-assay that is based on the inhibition of

ferriprotoporphyrin IX (FP) biomineralization. A reversed phase C18 inch preparative column and water- Key words: acetonitrile binary solvent mixture mobile phase were used to collect the fractions at 275 nm. UHPLC-MS Preparative HPLC, revealed that the major compounds in water crude Cinnamomum cassia extract are and cinnamic acid. Cinnamomum cassia, Containing coumarin as a major compound, fraction 2 showed superior efficacy compared to chloroquine and 2- Malaria, β-Hematin, mercaptopyrimidine positive controls. Fraction 3 which contains cinnamic acid showed moderate activity, while Hemozoin, Chloroquine. fraction 4 was the least potent.

INTRODUCTION The situation has been further deteriorated particularly in Asia,

Malaria is still considered to be a serious public health Africa and South America due to the emergence of resistant Plasmodium strains to almost all available drugs, specifically concern in tropical and subtropical areas (WHO, 2015). It is chloroquine, amodiaquine, sulphadoxine-pyrimethamine, caused by the transmission of the plasmodium parasite through the bite of infected anopheles mosquitos. Humans can be infected artemisinin and its derivatives. Consequently, searching for new by five species of plasmodium genera, namely, P. falciparum, P. alternative, effective leads and eventually affordable novel new vivax, P. ovale, P. malariae and P. knowlesi. However, drugs from medicinal origin is highly demanded. infections caused by p. falciparum species are the most fatal is one of the well-known and widely used which has gained

(Rathore, 2006). In 2015, 95 countries and territories had recent attention due to its important medicinal traits. Among the numerous increased health benefits reported on cinnamon are ongoing malaria transmission (WHO Fact sheet, 2016). Despite the progress toward minimizing malaria cases and deaths, it antimicrobial (Matan et al., 2006), antifungal (Wang et al., 2005), remains a cause of severe morbidity and mortality (WHO, 2015). antitermitic (Tung et al., 2010), nematicidal (park et al., 2005), insecticidal (Cheng et al., 2009), anti-inflammatory (Tung et al.,

2008), antioxidant (Mathew and Abraham, 2006), antiulcerogenic (Shiraga et al., 1988), anti-diabetic (Kim et al., 2006) and * Corresponding Author anticancer activities (Lu et al., 2010). Moreover, it has been Prof. Saleh Abu-Lafi, Faculty of Pharmacy, Al-Quds University, reported to have an influence on neurological disorders such as P.O. Box 20002, Abu-Dies, Palestine. Tel. and Fax: + 970-2-2790413 Email: sabulafi @ science.alquds.edu Parkinson’s and Alzheimer’s diseases (Petersona et al., 2009).

© 2017 Mutaz Akkawi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 130 Akkawi et al. / Journal of Applied Pharmaceutical Science 7 (01); 2017: 129-134

Further studies showed that it reduces the risk of colon Chromatographic analysis cancer (Wondrak et al., 2010), increases blood circulation in the Analytical HPLC, Preparative HPLC and UHPLC-MS uterus and improve tissue regeneration (Minich and Msom, 2008), Instrumentations treats toothaches and removes foul breath (Gupta et al., 2011; Analytical samples were analyzed using HPLC of Waters Jakhetia et al., 2010). In our previous work on Cinnamomum Alliance (e2695 separations module), which is equipped with 2998 cassia, it was found that powdered bark water infusion exhibit Photo diode Array (PDA). Data acquisition and control were effective inhibitory effects on heme bio-crystallization, obstructing carried out using Empower 3 chromatography data software hemozoin (malaria pigment) formation (Attieh et al., 2015). The (Waters, Germany). The Preparative High Pressure Liquid current work however is aimed to collect preparative HPLC Chromatography (Prep-HPLC) system consisted of 3535 fractions using reversed phase inch column and to explore their quaternary gradient module equipped with 996 PDA detector. The effect on hemozoin formation. UHPLC chromatography was performed under reverse phase conditions using a TSQ Quantum Access MAX (Thermo MATERIALS AND METHODS Scientific, San Jose, CA, USA) which includes a Dionex Pump with a degasser module, an Accela PDA detector and an Accela Materials Autosampler. The chromatographic separations were performed on Dimethyl sulfoxide (DMSO), chloroquine diphosphate a Kinetex™ column (C8, 2.6 µm particle size, 100 Å pore size, , sodium acetate, coumarin, cinnamic acid, glacial acetic acid 100 x 2.1 mm) from Phenomenex, USA. A UHPLC and hemin chloride were procured from Sigma-Aldrich, Israel. SecurityGuard™ cartridge (C8, for 2.1 mm ID column) was from Cinnamon (Cinnamomum cassia) used was from a local Phenomenex, USA. The injection volume was 10 μL, the oven Palestinian market. All the research has been conducted at the temperature was maintained at 35°C (Attieh et al., 2015; Kang et Faculty of Science & Technology and the Faculty of Pharmacy in al., 2014). Al-Quds University, 2016.

Preparation of extract Chromatographic conditions Two grams of Cinnamomum cassia fine-powdered bark Crude Cinnamomum cassia from pure water was run on was soaked in 150 ml of distilled hot water (at 90°C), left for 30 reversed phase ODS column of Waters (XBridge, 4.6 ID x 150 minutes at room temperature and then filtered using MN 615.Ø110 mm, 5 μm) with a guard column of XBridge ODS (20 mm x mm filter paper. The resultant extract was evaporated using (IKA 4.6mm ID, 5 μm). The mobile phase consisted of a binary solvent WEREK RV06-ML) rotary evaporator at 70°C under reduced mixture of 0.5% acetic acid solution (A) and acetonitrile (B) in pressure. Then, it was lyophilized using Labconco freeze drier linear gradient mode. A 100% of solvent A was initially used and (USA), until constant weight was achieved. The final dried extract then descended to 70% A in 40 minutes, then to 40% A in 20 was stored in an amber bottle and kept in the fridge until minutes, finally to 10% A in 2 minutes and remained there for 6 fractionated by preparative HPLC (Akkawi et al. 2014). minutes and then back to the initial conditions in 2 minutes. The HPLC system was equilibrated for 5 minutes with the initial acidic In-vitro semi-quantitative test for screening of anti-malarial water mobile phase (100 % A) before injecting the next sample. activity All the samples were filtered with a 0.45 m PTFE filter. The As per the protocol described by Deharo (Deharo et al., PDA wavelengths range was from 210-500 nm. The flow rate was 2002), a mixture containing 50 μl of 0.5 mg/ml haemin chloride 1 ml/min. Injection volume was 10 l and the column temperature (freshly dissolved in DMSO), 100 μl of 0.5 M sodium acetate was at room temperature. The preparative HPLC experiments buffer (pH 4.4) and 50 μl of the tested anti-malarial drug solution were run on ODS column (Agilent PrepHT C18, 22.2 x 250 mm, or control, was incubated in a normal non-sterile 96-well flat 10 μm). bottom plate at 37ºC for 18-24 hours. It is imperative that the The gradient program started from 95% water, 5% solutions be added to the plate in this order. The plate was then acetonitrile to 100 % acetonitrile in 17 minutes and back to the centrifuged for 10 minutes at 4000 rpm. The supernatant was starting mobile phase composition in 4 minutes. The flow rate was removed and the pH of reaction was measured. The final pH of the 12 ml/minutes, the injection volume was 1000 micro l, the column mixture should be between 5.0 and 5.2. The wells were washed temperature was set at room temperature and the PDA with 200 μl DMSO per well to remove free haemin chloride. The wavelengths ranged from 200-450 nm (Attieh et al., 2015; Kang et plate was centrifuged again, discharging the supernatant al., 2014). afterwards. The β-haematin remaining was then dissolved in 200 The UHPLC-MS chromatographic separation was μl of 0.1 M NaOH to form an FP that could be measured achieved using a linear gradient program at a constant flow rate of spectrophotometrically. Finally, the absorbance was 0.4 mL/min over a total run time of 70 min. The mobile phase determined at 405 nm using an ELISA reader (Stat Fax-2100). gradient program was the same as the HPLC except for the use of Ultrapure water was used as negative control, whereas chloroquine 0.1% formic acid instead of acetic acid. Samples were detected by dissolved in ultrapure water was used as positive control. a TSQ Quantum Access Max mass spectrometer in positive and Akkawi et al. / Journal of Applied Pharmaceutical Science 7 (01); 2017: 129-134 131 negative ion mode using Electron Spray ionization (ESI) and full Typical chromatogram of the crude extract at 275 nm is scan acquisition. Air was produced (SF 2 FF compressor, Atlas shown in Figure 1. Extended gradient conditions were Copco, Belgium). Purified nitrogen was used as source and intentionally used in order to separate all the compounds present in exhaust gases. Cinnamomum cassia extract at a satisfactory resolution. Two major slightly soluble compounds were eluted at Cinnamon preparative sample preparation 27.5 and 36.2 minutes while five other minor compounds were When 500 mg of crude Cinnamomum cassia was eluted earlier (Figure 1). Standard cinnamic acid which is known dissolved in 5 ml water, the solution turned turbid but when 5 ml to present in Cinnamomum cassia was injected and eluted at 36.2 of ethanol was added a clear solution was obtained which was minutes. Its UV-Vis spectrum exactly matched the peak eluted at directly filtered through PTFE 0.45 m membrane filter before 36.2 minutes from cinnamon (Figure 2). The other peaks injection. One ml of the clear solution was directly injected to the present in Cinnamomum cassia water extract and their preparative HPLC and four fractions were collected. Fraction 1 corresponding overlaid UV-Vis profile from 210-500 nm is was from 0-6.21 minutes, fraction 2 was from 6.21-14.20 minutes, depicted in Figure 1. fraction 3 was from 14.20-14.80 minutes and finally fraction 4 was The crude water extract of Cinnamomum cassia was also from 14.8-22 minutes, the end of the run. analyzed by ultra-performance liquid chromatography coupled to photodiode array and mass spectrometry (UHPLC-PDA-MS). A RESULTS AND DISCUSSION full scanned MS in the positive ESi mode revealed the identity of the peak eluted at 27.5 minutes to be coumarin (eluted at 17 Cinnamomum cassia consists of a broad range of minutes using the UHPLC-MS mobile phase conditions). The compounds such as , cinnamic acid, cinnamyl protonated m/z [M+H]+ ion shows peak at 147.31 Da, indicating alcohol, cinnamyl acetate, eugenol and to a large extent of coumarin presence (Figure 3). In general, are known to coumarin (Wang et al. 2013). Some of these compounds, have anti-malarial properties (Cubukcu et al., 1990 and Noster et particularly the volatile ones, can be separated and determined al., 1990). using GC-MS. High Performance Liquid Chromatography (HPLC) Preparative HPLC-PDA was utilized in the reversed however is better technique to separate the more polar compounds phase mode using an inch column in order to collect four fractions present in Cinnamomum cassia which dissolves in water. (Figure 4). Ten successive injections of a total of 500 mg Herbal therapies of Cinnamomum cassia are usually concentration were completed. At 275 nm, fraction 1 mainly prepared for diabetic patients by infusion of the powdered bark showed a major peak at about 2.1 minutes while fraction 2 part of the plant. The dissolved active and inactive ingredients contained an overlapped mixture of peaks including coumarin, alike are then released into the water solvent. The crude apparently because of the concentration overloading conditions. lyophilized Cinnamomum cassia extract was injected into an Fraction 3 however includes almost pure cinnamic acid and analytical HPLC chromatograph in the reversed phase mode. fraction 4 only showed a small peak at 16 minutes (Figure 4).

220.2 276.9 276.9 287.6 0.14

307.9

2 7 .5 7 6 3 6 .2 0 3 276.9 338.9 0.12

278.1 273.4 0.10 310.3 323.4 395.1407.1407.1 426.5428.9 454.3455.5 468.9 488.3498.1

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 nm

0.08 15.434 Peak 1 17.405 Peak 2 18.690 Peak 3

19.682 Peak 4 AU 21.037 Peak 5 21.274 Peak 6 27.575 Peak 7 0.06 30.079 Peak 8 36.204 Peak 9 1 7 .4 0 6 0.04 1 8 .6 9 0

3 0 .0 8 0

1 5 .4 3 6

2 1 .0 3 7 1 9 .6 8 4

0.02 2 1 .2 7 3

0.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Fig. 1: HPLC-PDA chromatogram of all the peaks present in crude water extract of Cinnamon at 275 nm & their overlaid UV-Vis spectra between 210-500 nm.

132 Akkawi et al. / Journal of Applied Pharmaceutical Science 7 (01); 2017: 129-134

0.30 O

0.25 OH

0.20

trans-cinnamic acid - 36.320 AU 0.15

0.10 O O

0.05

0.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Fig. 2: Overlaid HPLC-PDA chromatogram of crude water extract of Cinnamon (blue) and cinnamic acid standard (black) dissolved in 50% ethanol at 275 nm. The overlaid spectra of standard cinnamic acid with the peak from Cinnamon is depicted at the right corner.

D:\service\...\data\2017 04 2016\Ci_2 04/07/2016 18:09:12 UV at 272 nm RT: 0.00 - 69.98 SM: 7B A 17.63 NL: 6.69E5 650000 Channel A 600000 UV Ci_2 550000 26.96 500000

450000 400000 350000

uAU 300000 17.04 250000 200000 150000 15.99 55.42 100000 55.83 20.15 56.11 50000 2.47 15.22 20.53 25.42 0.76 9.88 13.19 27.60 30.58 34.28 34.79 36.61 37.58 56.51 62.58 5.40 8.41 9.12 40.27 42.17 43.33 45.44 46.78 49.05 50.57 52.36 57.57 63.53 67.35 0 Ci_2 #4285-4423 RT: 17.56-18.12 AV: 70 SB: 287 18.64-21.00 NL: 2.33E5 0 2 4 6 8 10 12 14 16 18 F:20+ c ESI22 Q1MS24 [50.000-300.000]26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 91.37 Time (min) 100 + RT: 0.00 - 70.00 SM: 7BB [M+H] 17.70 NL: 100 80 4.37E5 147.31 m/z= 90 146.50-147.50 60 F: + c ESI 92.34 80 Q1MS [50.000- 300.000] MS 40 103.33 70 Ci_2

Positive RelativeAbundance 60 20 118.38 199.48 157.42 222.56 287.23 56.14 50 65.41 121.41 270.36 mode 0 40 50 100 150 200 250 300 RelativeAbundance m/z 30

55.87 20 62.25

10 18.08 1.38 5.68 12.14 13.22 16.74 19.15 22.10 25.70 27.08 30.65 31.46 33.06 34.87 37.27 40.98 43.86 49.43 55.70 62.64 63.90 67.19 69.38 4.91 8.11 9.43 40.74 44.39 47.23 53.85 56.82 60.69 61.90 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 Time (min)

Fig. 3: UHPLC-PDA of the Cinnamon water extract at 272 nm (A). The extracted ion chromatogram in the positive ESi mode of the 17.7 minutes peak (B), + shows protonated coumarin at m/z of [M+H] of 147.31 Da

Akkawi et al. / Journal of Applied Pharmaceutical Science 7 (01); 2017: 129-134 133

2.20 2.20 210.8 273.5 2.00 2.00 1.80 1.60 1.40

1.20 309.3 AU 1.80 14.400 1.00 0.80 0.60 1.60 0.40 0.20 0.00 1.40 250.00 300.00 350.00 400.00 nm 1.20

AU 1.00

0.80 O 0.60 OH 0.40 0.20

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Minutes Fig. 4: Typical preparative RP-HPLC-PDA chromatogram of water extract of Cinnamon. 1 ml was injected at flow rate of 12 ml/min at 275 nm. Fraction 1, from 1-6 minutes, fraction 2 from 6-14 minutes, fraction 3 from 14-15 minutes and fraction 4 was from 15 to the end of the elution 22 minutes.

Fig. 5: Efficiency of the four fractions obtained from preparative HPLC separation of Cinnamon water extract, dissolved in water at concentration of 1mg/ml, compared to negative control (water) and positive controls (CQ-chloroquine 0.1mg/ml & MP 1 mg/ml), showing the absorption values of dissolved β-hematin (alkaline hematin) at 405 nm using ELISA reader as per Deharo et al. semi-quantitative method. Each result represents an average of 16 individual experiments.

Using the ferriprotporphyrin bio-mineralisation inhibition Fraction 2 consists of a mixture of compounds that were clearly test, this study compared the inhibitory effect of four preparative seen in the analytical HPLC profile (Figure 1). In the preparative HPLC fractions of water extract on beta-hematin formation in a chromatogram however, these compounds were eluted together cell-free system. The mechanism of inhibition is probably through preceding fraction 3 which is almost pure cinnamic acid formation of a complex between active ingredients in these compound. Three preparative HPLC fractions out of four from the extracts and ferriheme; this complex prevents the formation of water extracts of Cinnamomum cassia bark were shown to possess beta-hematin. Several heme-binding anti-malarial compounds, in-vitro antimalarial activities (Figure 5). Results obtained were such as chloroquine, efficiently inhibit this process, and this is compared to positive (chloroquine (CQ) of 0.1 mg/ml and 2- believed to be the predominant mechanism by which these drugs mercaptopyrimidine (2-MP) of 1 mg/ml) and negative (ultrapure work as antimalarials (Akkawi et al. 2014; Attieh et al., 2015). All H2O) controls. According to the semi-quantitative method the fractions were subjected to in-vitro assays. Fraction 2 performed, the absorption is inversely proportional to drug which contains coumarin as a major compound appeared to efficiency and to hemozoin content; the lower the absorption, the be the most effective in inhibiting the formation of beta-hematin. more efficient the drug and the stronger the inhibitory effect 134 Akkawi et al. / Journal of Applied Pharmaceutical Science 7 (01); 2017: 129-134 towards hemozoin formation. Figure 5 showed that the most active Kim SH, Hyun SH, Choung SY. Anti-diabetic effect of cinnamon fraction is number 2 that contains mainly coumarin and the least is extract on blood glucose in db/db mice. J Ethnopharmacol, 2006; 104(1-2): 119-123. fraction 4. Fraction 3 which contains cinnamic acid is fairly Kwon HK, Jeon WK, Hwang JS, Lee CG, So JS, Park JA, Ko BS, active. Previous studies have shown that cinnamic acid derivatives Im SH. Cinnamon extract suppresses tumor progression by modulating angiogenesis and the effector function of CD8+ T cells. Cancer Lett, 2009; have an effect on the in vitro growth of Plasmodium falciparum 278(2): 174-182. and on the permeability of the membrane of malaria-infected Lu J, Zhang K, Nam S, Anderson RA, Jove R, Wen W. Novel erythrocytes (Kanaani and Ginsburg, 1992). Coumarins also have angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling. Carcinogenesis, 2010; 31(3):481-488 been found to exhibit anti-malarial activity (Cubukcu et al., 1990 Matan N, Rimkeeree H, Mawson J, Chompreeda P, and Noster et al., 1990). The in-vitro activity guided preparative Haruthaithanasan V, Parker M. Antimicrobial activity of cinnamon and HPLC fractions of Cinnamomum cassia results support these oils under modified atmosphere conditions. Int J Food Microbiol, 2006; 107(2): 180-185. findings. The water extracts of Cinnamomum cassia revealed Mathew S, Abraham TE. In vitro antioxidant activity and potential activity even at low concentration of infusions and scavenging effects of extract assayed by different decoction (Attieh et al., 2015). However, fraction 2 of the current methodologies. Food Chem Toxicol, 2006; 44(2): 198-206. Minich St and Msom L. Chinese Herbal Medicine in Women’s study even showed superior activity in comparison to the crude Health, Women’s Health, 2008. extract as manifested by a high capability to inhibit β-hematin Noster S, Kraus L: In vitro antimalarial activity of Coutarea latiflora and Exostema caribaeum extracts on Plasmodium falciparum. Planta Med; formation in vitro. 1990, 56: 63-65. Park IK, Park JY, Kim KH, Choi KS, Choi IH, Kim CS, Shin SC. CONCLUSION Nematicidal activity of plant essential oils and components from (Allium sativum) and cinnamon (Cinnamomum verum) oils against the pine wood Cinnamomum cassia consists of numerous active water nematode (Bursaphelenchus xylophilus) Nematology, 2005; 7: 767-774. soluble compounds which has significant influence in inhibition of Petersona DW, Georgea RC, Scaramozzinoa F, LaPointea NE, Andersonb RA, Gravesa DJ, Lewa J. Cinnamon extract inhibits tau aggregation hemozoin formation. Water crude extract contains two major associated with Alzheimer’s disease in vitro. J Alzheimers Dis, 2009; 17: 585– compounds, coumarin and cinnamic acid. Coumarin as a major 597. compound in water Cinnamomum cassia extract showed the best Rathore D. Strategies for Malaria Control. VBI Scientific Annual Report, 2006; 49-53. antimalarial effect. Cinnamic acid which is also abundant in Shiraga Y, Okano K, Akira T, Fukaya C, Yokoyama K, Tanaka S, Cinnamomum cassia presented a moderate antimalarial effect. Fukui H, Tabata M. Structures of potent antiulcerogenic compounds from Cinnamomum cassia. Tetrahedron, 1988; 44: 4703-4711. ACKNOWLEDGEMENT Tung YT, Chua MT, Wang SY, Chang ST. Anti-inflammation activities of and its constituents from indigenous cinnamon We are grateful to Dr. Pierre Lutgen (IFBV-BELHERB, (Cinnamomum osmophloeum) twigs. Bioresour Technol, 2008; 99(9): 3908– Luxembourg) for his helpful discussions and insightful comments. 3913. Tung YT, Yen PL, Lin CY, Chang ST. 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