New Gingerol Derivative against Human Colorectal Carcinoma Caco-2 Cells

ABSTRACT:

In this article we report the isolation, characterization and evaluation of in vitro anti-colon cancer activity of a new gingerol derivative, namely (5'r£)-5-hydroxy-1-(4-hydroxy-3- methoxyphenyl)tetradec-8-en-3-one, (N6), and a known sesquiterpene (1,2- Dihydroxybisabola-3,10-diene,N7), isolated from Zingiber officinale rhizomes for the first time, together with other five known compounds; 6-gingerol (N1), 8-gingerol (N2), 10- gingerol (N3), 4-gingerol (N4) and 4'-0-methyl-6-gingerol (N5). The isolated compounds were identified using different spectroscopic techniques (1D and 2D-NMR, MS and IR). A modified chromatographic method was established and optimized to isolate the major gingerols (N1, N2, and N3) in grams. Thereafter the cytotoxic activity of the isolated compounds was evaluated against colorectal carcinoma cell line, Caco-2. It was observed that anti-colon cancer activity of N2 was superior to that of 5-FU, the classic reference cytotoxic agent. Additionally, the length of the aliphatic chain in 8-gingerol (N2) is optimum for the anticancer activity and any decrease, as in N1 and N4, or increase, as in N3, in the side chain length leads to gradient decrease in the cytotoxic activity. Following this further, methylation of phenolic OH group (N5), leads to a dramatic decrease in the anticancer activity. Furthermore, loss of aromaticity (N7), results in complete loss of the cytotoxic activity. On the other hand, introducing a n-bond in the aliphatic side chain enhances the anti-cancer activity as depicted in N6. Taken together, the aromaticity, the side chain length as well as the presence of free phenolic group contribute significantly to the anti-colon cancer activity of gingerol and its derivatives. These results open up a new window in the rational design of gingerol-based semisynthetic drugs with improved anticolon cancer activity.

1. Introduction

Ginger (the rhizomes of Zingiber officinale Roscoe), a member of the tropical and sub-tropical zingiberaceae, is globally one of the most commonly used spice. In addition to its use as a spice and condiment, ginger is also of use as a medicinal agent in the various traditional systems of medicine [1, 2]. Ginger possesses a wide array of medicinal uses and is observed to be effective against unrelated ailments. Laboratory studies have shown that ginger possesses free radical­scavenging, antioxidative, anti-inflammatory, antimicrobial, antiviral, gastroprotective, antidiabetic, antihypertensive, cardioprotective, anticancer, chemopreventive, and immunomodulatory effects [1, 2]. Currently, there is a renewed interest in ginger, and several scientific investigations aimed at isolation and identification of the active constituents of ginger and scientific verification of its pharmacological actions [1]. Ginger’s numerous biological activities have been attributed mainly to gingerols and shogaols, the major pungent principles found in ginger [3, 4].

In view of the aforementioned significant bioactivities, large quantities of the pure compounds are urgently needed for further pharmacological studies. However, obtaining the pure compounds by conventional column chromatography separation methods is a challenge because of their structure similarity and unstable chemical properties [5-7]. This study develops a modified economic, rapid, and efficient method of isolation and purification to afford a high yield of gingerols with high purity to fulfill the requirement of obtaining gingerol-derived compounds. Using this optimized method, we were able to report here in the isolation of a new gingerol derivative (N6) from the dried rhizomes of Zingiber officinale, together with five known gingerols (N1-N5), in addition to a sesquiterpene (N7) which is reported for the first time from this natural source.

Extensive research over the past decade has dominated by multiple synthetic chemotherapeutic drugs that are non-invasive but display low selectivity and hence deadly adverse effects. The current cornerstone of adjuvant and palliative chemotherapy for colorectal cancer is 5-FU [9]. However, such compound shows several side effects ranging from, myelotoxicity, gastrointestinal disturbances, cardiotoxicity, and hepatotoxicity [10]. These limitations direct towards the finding of a more effective and safe drug which may raise the therapeutic benefits for patients.

Human colorectal-carcinoma is the third most common cancer diagnosed in both men and women worldwide [8]. Treatment options are limited with poor efficacy and marked patient-to- patient variation in therapeutic outcomes. In most cases, surgical resection and organ transplantation remain the only curative treatment options that may involve very expensive and invasive procedures with considerable limitations. Therefore, developing an effective therapy is of prime importance.

Chemoprevention by plant-derived compounds or dietary phytochemicals has emerged as an accessible and promising approach to cancer control and management [11-15]. Of the many phytochemicals displaying a wide array of biochemical and pharmacological activities, 6- gingerol, is the major pharmacologically active component in ginger. Recently, several lines of evidence suggest that 6-gingerol is effective in the suppression of transformation, hyperproliferation, and inflammatory processes that initiate and promote carcinogenesis, as well as the later steps of carcinogenesis, namely, angiogenesis and metastasis, and it also affect several molecular targets, and it cause modulation of several cellular pathways in the tumor cells [16-18]. It was reported that 6-gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase [18], but there is no thoroughly study to date has profiled the anti-colon cancer activity of other gingerol derivatives. The proposed protective role of ginger and its bioactive constituents in tumor development may prevail in the intestinal tract as it has a long tradition of being very effective in alleviating symptoms of gastrointestinal problems. Preclinical studies have shown that ginger possesses carminative, gastroprotective, antiulcerative, and antiemetic properties, and it prevents epigastric discomfort, dyspepsia, stomachache, abdominal spasm, and cancer of the gastrointestinal system. It also has been recommended to combat nausea, and improve the gastrointestinal side effects associated with cancer chemotherapy [1, 19-20]. Therefore, the current study aims to evaluate the potential anti-colon cancer activity of isolated set of gingerols in comparison with 5-fluorouracil. Furthermore, this study pursues to delineate the relationships between structure elements of gingerol derivatives and their anti-colon cancer activity, in an attempt to help in a rational design of semisynthetic compounds with improved anti-colon cancer activity.

2. Results and discussion 2.1 Chemistry

The methanolic extract of the rhizomes of Zingiber officinale Roscoe was suspended in water and partitioned successively with petroleum ether, methylene chloride, ethyl acetate and n-butanol. The methylene chloride soluble fraction was subjected to a series of chromatographic techniques, leading to the isolation of a new gingerol related compound (N6) together with six known compounds (N1-N5), and N7 which is the first time to be isolated from ginger. The known compounds were identified as 6-gingerol (N1), 8-gingerol (N2), 10-gingerol (N3), 4-gingerol (N4), 4'-0-methyl-6-gingerol (N5), and 1,2- Dihydroxybisabola-3,10-diene (N7), Figure1, by comparison of their spectroscopic data with that reported in literature [21-25]. Studies of the isolation and purification of gingerols are scarce in the literature [26]. The major gingerols (6-, 8-, and 10-gingerol) were reported to be isolated in the range of milligrams with high purity using complicated chromatographic techniques as HSCCC [5], or semi-preparative HPLC [26]. Fortunately, it is firstly reported in our study to isolate the major gingerols in the range of grams using medium pressure liquid chromatography MPLC and reversed phase Ci8 silica gel. This method can be considered as a modification for [26] new methodology for purification of gingerols using HPLC system, Luna-Ci8, and methanol- water (75:25, v/v) as the mobile phase. In our study the best mobile phase was methanol- water (70:30, v/v) for isolation of the major gingerols, but if it is needed to isolate 4-gingerol, the best mobile phase will be methanol- water (60:40, v/v) as if methanol- water (70:30, v/v) system was used; 4-gingerol and 6-gingerol will be eluted as a mixture.

Compound N6 was obtained as dark yellow oil. Its molecular formula is C21H32O4 as deduced from EI-MS, [M]+ peak at m/z 348.4 and [M-H2O]+^ ion peak at m/z 330.3. The IR (neat) spectrum displayed absorption bands indicating hydroxy (3437cm-1), carbonyl (1707 cm-1), and aromatic (1604 cm-1) functionalities. The NMR spectra of compound N6 (table 1) is close to that of gingerols specially 10-gingerol (N3), as the 1H-NMR spectrum of N6 exhibited signals due to a methoxy at ô 3.80 (3H, s, H-7') and three aromatic protons at ô 6.74 (1H, d, J= 8.0 Hz, H-5'), 6.58 (2H, overlapped, H-2',and 6') which suggests the presence of a 4-hydroxy-3-methoxyphenyl group. This was confirmed also by the presence of a methoxy (5 55.9, C-7') and 6 aromatic carbons (5 146.4, 144.0, 132.6, 120.7, 114.4, 111.0) in the 13C- NMR spectrum (table 1). Furthermore, the presence of a carbonyl carbon (S 211.3, C-3) and a hydroxymethine carbon (5 67.2, C-5) indicated that it is a gingerol derivative. 13C- NMR spectrum of compound N6 differs than that of N3 by the presence of only 10 aliphatic carbon signals instead of 12 carbons for the side chain and presence of two olefinic carbon signals (5 128.7 and 130.9) indicating an unsaturation position. The presence of an extra double bond was also revealed from the ^-NMR spectrum that showed two olefinic protons at 5 5.23 (1H, overlapped dq, J= 16.0, 6.4 Hz, H-8), and 5.29 (1H, overlapped dq, J= 16.0, 6.4 Hz, H-9). The position of the double bond at C-8 and the assignments of the two olefinic protons were confirmed based on HSQC and HMBC spectra, which revealed that the olefinic protons, H-8 and H-9 showed HMBC correlations with aliphatic carbon signals at 5 23.3 (C-7) and 27.2 (C-10). Additional HMBC correlations (Table 1) between the protons at 5 1.30 (H-6a) and 1.41 (H-6b) with the carbons C-7, C-4, C-5, and C-8, and between the protons at 5 2.01 (H-7) with the carbons C-6, C-5, C-8, and C-9, and between the protons at ô 1.93 (H-10) with the carbons C-11, C-12, C-8, and C-9 were used to assign the position of the double bond. Thus the structure of N6 was established as (5',£)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)tetradec-8-en-3-one, which is a new gingerol derivative.

Compound N7 was obtained as colorless needles [from methanol- water (6:4)] with melting point 110-115 °C. Its molecular formula is C15H26O2 as deduced from EI-MS, [M]+ peak at m/z 238 and [M-H2O]+^ ion peak at m/z 220. The IR (neat) spectrum displayed absorption bands indicating hydroxy functionality (3262 cm-1). The 1H-NMR spectrum (table 2) shows the presence of two olefinic protons at ô 5.04 (1H, t, J= 7.2 Hz, H-10) and 5.47 (1H, brs, H-4) and four methyl groups; three vinylic CH3 groups at ô 1.54, 1.61, 1.74 representing protons H-13, H-12, H-15 respectively and one CH3 doublet at ô 0.74 (J= 6.8 Hz) assigned to H-14. The two proton signals at ô 3.88 and 3.90 indicated the presence of two oxygenated methine carbons as revealed from the two carbon signals at ô 68.0 and 69.2 in the 13C-NMR spectrum confirming the presence of a glycol moiety. HMBC spectrum correlations between the proton signal at ô 1.61 (H-12 ) with carbon signals at ô 124.6 (C- 10), 131.4 (C-11), and 25.7 (C-13), the proton signal at ô 0.74 (H-14) with the carbon signals at Ô 40.6 (C-6), 30.5 (C-7), and 35.2 (C-8), the proton signal at ô 1.74 (H-15) with carbon signals at Ô 68.0 (C-2), 136.8 (C-3), and 130.0 (C-4), between the proton signal at ô 3.88 (H- 1/2) with the carbon signals at ô 20.5 (C-15), C-4, and C-3 were used to assign the positions of the CH3 groups, the two double bonds, and the glycol moiety. The chemical shift values of carbons of this compound were the same as those of the known compound 1,2- Dihydroxybisabola-3,10-diene mentioned by Gachetei al. 2011 [25] which is the only reference for the spectroscopic data of this compound. Although this compound was reported by Wang et al. 2008 [27], they did not mention its spectroscopic data. It is worth noted that there was a difference in the assignment of the proton signal at ô 1.45 (1H, dt, J = 3.6, 13.8 Hz) reported by Gachetet al. 2011 [25]. As they attributed this signal to H-6, while in our 1H- NMR spectrum, the corresponding signal appeared at ô 1.34 (1H, dt, J= 3.2, 13.6 Hz) was assigned to H-5a and that was confirmed by the HMBC correlation from H-5a (S 1.34 ) to C- 7, C-6, and C-1. Neither Gachetet al. nor we were able to determine the relative or the absolute configuration of this compound as the crucial signals, that is, H-1/H-2 were overlapping and suitable reference data could not be found. On the basis of the spectroscopic data and comparison with literature data [25], the structure of compound N7 was determined to be 1,2-Dihydroxybisabola-3,10-diene, which is isolated for the first time from ginger.

2.2 Evaluation of cytotoxic activity

The cytotoxic effects of the isolated compounds against colorectal carcinoma were evaluated in a cell-based assay using Caco-2 cells and compared to that of 5-FU, a drug extensively used in adjuvant and palliative chemotherapy for colorectal cancer. Using this approach, dose response and time course cytotoxicity of the standard agent, 5-FU, were initially carried out by MTT assay. The dose-response effect of 5-FU was more evident after 72 hours of incubation with an IC50 value of 60 цМ than at 48 hours with IC50 value of 158.5 цМ, whereas at 24 hours an IC50 was not reached with 5-FU at any concentration tested (25-250 цМ). Therefore, 72 hours has been chosen as the incubation period for the dose-viability response of all tested compounds.

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