Article pubs.acs.org/JAFC
An Anthocyanin-Rich Extract from Hibiscus sabdariffa Linnaeus Inhibits N‑Nitrosomethylurea-Induced Leukemia in Rats Tsung-Chang Tsai,† Hui-Pei Huang,‡,⊥,# Yun-Ching Chang,⊥,#,∥ and Chau-Jong Wang*,⊥,#,∥ †
Superintendent Office, Antai Medical Care Cooperation, Antai Tian-Sheng Memorial Hospital, Pingtung, Taiwan Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan ⊥ Institute of Biochemistry and Biotechnology, Medical College, Chung Shan Medical University, Taichung, Taiwan # Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan ‡
ABSTRACT: A previous study reported that anthocyanins from roselle (Hibiscus sabdariffa L.) showed significant anticancer activity in human promyelocytic leukemia cells. To explore the antitumor effect of anthocyanin, a roselle bioactive polyphenol in a rat model of chemical-induced leukemia was assayed. Anthocyanin extract of roselle (Hibiscus anthocyanins, HAs) was supplemented in the diet (0.1 and 0.2%). This study was carried out to evaluate the protective effect of HAs on Nnitrosomethylurea (NMU)-induced leukemia of rats. The study employed male Sprague−Dawley rats (n = 48), and leukemia was induced by intravenous injection of 35 mg kg−1 body weight of NMU dissolved in physiologic saline solution. The rats were divided into four groups (n = 12): control, NMU only, and HAs groups that received different doses of HAs (0.1 and 0.2%) daily, orally, after NMU injection. After 220 days, the animals were killed, and the following parameters were assessed: morphological observation, hematology examination, histopathological assessment, and biochemical assay. When compared with the NMU-only group, HAs significantly prevented loss of organ weight and ameliorated the impairment of morphology, hematology, and histopathology. Treatment with HAs caused reduction in the levels of AST, ALT, uric acid, and MPO. Also, the results showed that oral administration of HAs (0.2%) remarkably inhibited progression of NMU-induced leukemia by approximately 33.3% in rats. This is the first report to demonstrate that the sequential administration of HAs followed by NMU resulted in an antileukemic activity in vivo. KEYWORDS: Hibiscus sabdariffa Linnaeus, anthocyanins, chemoprevention, leukemia, animal model, N-nitrosomethylurea
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INTRODUCTION Malignant neoplasm is characterized by unregulated cell division that presents more than 100 diverse clinical pathologies.1 Even though substantial advances and intensive efforts have focused on improving treatments, the efficacy of cancer therapies remains poor overall, and cancer is still a leading cause of death worldwide. In Taiwan, for instance, cancer was recently ranked first among the 10 leading causes of death (Department of Health, Executive Yuan, Taiwan, 2012). Leukemia is one of the most threatening diseases today. Most leukemia patients are typically treated with regimens that are based on (or at least include) chemotherapy or radiotherapy.2 Epidemiological evidence has shown an association between diets rich in fruits and vegetables and a lower incidence of cancer and cardiovascular disease.3,4 Because the plants are purported to be nontoxic, low-cost, available in agricultural areas, and culturally acceptable, their effectiveness in the treatment of cancer has been widely studied. Therefore, the development of a safe and more effective therapeutic strategy is very necessary. Hibiscus sabdariffa L. is a member of the Malvaceae family, known commonly as “roselle”. This species belongs to the genus Hibiscus represented by 250 species.5 It is cultivated in tropical and subtropical countries (including Taiwan) and is considered an important medicinal plant in some parts of the world. As a folk medicine, it is claimed to be effective against kidney stones and urinary bladder stones. It is also used for its © XXXX American Chemical Society
hypocholesterolemic, antifungal, antimicrobial, antihypertensive, and antispasmodic actions.6−8 Therefore, roselle could be considered as a functional food or a source of nutraceutical constituents, as daily consumption of its extract can be beneficial to human health. Roselle extracts have been demonstrated to have a broad range of therapeutic effects9 such as anticancer.10 These effects have been attributed to the various constituents of roselle including anthocyanins, flavonoids, polysaccharides, organic acids, and some minerals.11 The importance of the roselle calyx resides mainly in its anthocyanin content (2.52 g/100 g).12 Hibiscus anthocyanins (HAs) were identified as having delphinidin-3-sambubioside (70% of the anthocyanins) and cyanidin-3-sambubioside as the major pigments, with delphinidin-3-glucoside and cyanidin-3glucoside as the minor ones.9 Many biological activities of anthocyanins, such as antihypertensive,13 antioxidant,14 and hypocholesterolemic effects and hepatoprotective activities,15 of roselle have been investigated. We and our colleagues’ previous studies showed that roselle possessed multiple effects, including (i) antioxidation: roselle extract was shown to inhibit the development of atherosclerosis by protecting against oxidative stress in rats. Protocatechuic Received: November 22, 2013 Revised: January 27, 2014 Accepted: January 28, 2014
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Figure 1. Effect of HAs on gross appearance and organ weight of NMU-treated rats. (A) Protocol of the animal model (experimental design). Rats were injected with NMU every 2 weeks for 6 times (arrow). The day of the first injection is designated day 0. HAs were given orally at day 0 every day until sacrifice. NMU at 35 mg/kg iv and animals after iv injection were monitored for blood data every 30−40 days from the caudal vein (triangle). The details were as described under Materials and Methods. (B) All of the animals were sacrificed, and the livers and spleens were immediately fixed in 6% formaldehyde for specimen preparation. The arrows indicate cancer cell invasion in liver tissue shown with a gray-white hyalinized look. (C) Data represented as net weight of organ (liver or spleen)/body weight (OW/BW). The indicated days are counted after iv NMU and recorded once about 30−40 days. These values were expressed as the mean ± SD, n = 12. (#) p < 0.01 and (##) p < 0.001, compared with control group. (∗) p < 0.05 and (∗∗) p < 0.005, compared with NMU-treated group.
invasion, and metastasis.21,22 (v) Antidiabetes effects roselle extracts are worthy of further investigation toward development as an adjunct therapy for diabetes or metabolic syndromes.23,24 (vi) Other activities of H. sabdarif fa polyphenols (HPE) give them potential as anti-inflammatory agents.25 Recently, Pen et al. and Lee et al. illustrated the anti-insulin resistance properties of HPE and its effect on hypoglycemia, hypolipidemia, and antioxidation.26,27 The demonstration of the beneficial effects of dietary polyphenols on health is becoming an important issue. Although most cellular studies have proven their biological effects at the in vitro level, the correlation between the in vivo observed effects and the presence of the responsible metabolites is still unclear. Therefore, the aim of this study
acid (a phenolic acid extract from roselle) was reported to inhibit LDL oxidation induced by copper or a NO donor.16 (ii) Antihyperlipidemia and antiatherosclerosis effects of roselle extract reduced foam cell formation, reduced smooth muscle cell migration, and lowered calcification in the blood vessels of treated rabbits.17 HAs markedly inhibited oxidation of oxLDL by CuSO4, perhaps by CD36 down-regulation in vitro.18,19 (iii) Hepatoprotection effect of roselle extract was shown to exert the hypolipidemic effect via inhibiting lipogenesis and promoting hepatic lipid clearance.20 (iv) Anticancer inhibitory effect of roselle extracts on carcinogenesis demonstrated that it possesses potential as a cancer chemopreventive agent against apoptosis evasion, insensitivity to antigrowth signals, tissue B
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analyzer (model XE-2100, Sysmex). Blood smears were stained with Liu and Papanicolaou stain for cytological examination. Biochemical Data Measurement. Analyses of all biochemical parameters in serum were examined with an autoanalyzer (Olympus AU2700, Olympus) using commercial reagent kits. The aspartate aminotransferase (AST/GOT), alanine aminotransferase (ALT/ GPT), and uric acid were measured by enzymatic methods (AST, ALT, kinetic UV test; and UA, enzymatic color test). Pathological Examination. Biopsies of liver and spleen were immediately obtained after the animals were sacrificed (day 220). A portion of them was fixed in 10% formalin solution, processed using routine histology procedures, embedded in paraffin, cut in 5 μm sections, and placed on a glass microscope slide for analysis. The samples were stained with hematoxylin and eosin (H&E) for morphological evaluation and then examined microscopically. Immunohistochemistry. Immunohistochemical investigations were carried out on paraffin-embedded sections. Sections were dried in an oven at 50 °C overnight, deparaffinized in xylene, rehydrated in a graded alcohol series, immersed in citrate buffer (pH 6.0), and incubated in an autoclave for 25 min. Sections were then removed, allowed to cool at room temperature for 20 min, and rinsed with running water and distilled water for a total of 10 min. They were then blocked for the endogenous peroxidase measurement by incubating in a solution of 3% hydrogen peroxide (H2O2) for 10 min. Tissue sections were washed with PBS and then immunostained with primary antibodies for 30 min. The antigen−antibody complex was visualized by an avidin−biotin−peroxidase complex method followed by diaminobenzidine tetrahydrochloride (DAB) as a chromogen. After washing, sections were counterstained with Gill’s hematoxylin, washed with tap water, mounted with Permount (Merck, Darmstadt, Germany), and examined by light microscopy. For confirmation that the immunocystochemical procedure was performing correctly, antibodies against MPO were tested. Statistical Analysis. All data are presented as means ± SD. The statistically significant differences compared with the untreated control group or NMU group were calculated by Student’s t test. Differences with probability value 180 mg/day in humans.40 Anthocyanins have received considerable attention because of their wide range of potential health-promoting properties,41 including cancerprevention effects.42 Indeed, intact anthocyanin forms (glycosides) have been detected in rat and human plasma as well as in urine after oral administration.43 However, low bioavailability of anthocyanins, much less than 0.1% of intake, was suggested by the low concentration measured in plasma and urine after anthocyanin ingestion in some short-term studies.44 Therefore, we wanted to ensure a sufficiently high intake and, thus, selected a daily dose of HAs of 100−200 mg/kg (approximately equal to 0.1−0.2%). On the other hand, our purpose was to assess the influence of HAs in rats fed an anthocyanin-rich diet for a relatively long period. This study was to evaluate the potential chemoinhibitory effects of HAs on leukemia. In this 220 day study we observed that dietary supplementation of 0.1 and 0.2% HAs decreased the WBC counts and uric acid levels by 30−70% in a dose-dependent manner. Our data demonstrate that an anthocyanin-rich diet (HAs) in fact is sufficient to enhance the antileukemia response, which is consistent with other studies. For instance, anthocyanin-rich berry extract significantly inhibits leukemia cell proliferation at 10−100 μg/mL, which corresponds to anthocyanin concentrations between 10−6 and 10−5 M.45 There is some controversy about its bioavailabilty, but recent evidence indicates that despite low bioavailability, plasma concentrations of anthocyanins appear sufficient to induce changes in gene expression and signal transduction in vivo46 in a manner suggesting a positive role in physiological functions and health outcomes. Several in vivo studies have provided evidence that plant foods have a notable impact on the absorption and metabolism of anthocyanins. In view of the above lower bioavailability of anthocyanins in patients, this was ascribed to the plasma or urinary recovery. For example, Harada et al. and Oki et al. found that 0.01−0.03% of purple sweet potato anthocyanins given by oral administration can be absorbed into plasma of both rat and human.47,48 Ohnishi et al. pointed out that the recovery of administered cranberry juice anthocyanins in the urine of humans was estimated as 5%,49 whereas other researchers recovered between 1.8 and 2% of strawberry anthocyanins.50 Moreover, a previous study of 15 patients with coronary artery disease tested for total urinary recovery of administered cranberry juice anthocyanins. The recovery was in the range of 0.078−3.2% among the volunteers.46 Although anthocyanin bioavailability appears low, it could have been underestimated, because the metabolites and breakdown products of anthocyanins have not yet been identified.44 In addition, some research is needed to determine if anthocyanins alone or from their synergistic interactions with other phenolic compounds play a role in the inhibition of cancer. We believe such questions will be answered through continuing investigations in future decades. The present results demonstrated that the HAs induced significant anticancer activity in a NMU-induced rat leukemia model. Although a cancer cell killing mechanism for HAs in human leukemia (HL-60) cells has been suggested,9,28,51,52 clinical trials evaluating the use of roselle as a chemopreventive or therapeutic agent are lacking. Separation of the phenolic compounds of the roselle extract and in vivo experiments to determine whether HAs could be an effective anticancer agent for leukemia are important. Hence, this is the first report of the
Table 1. Inhibitory Effects of HAs on NMU-Induced Rats Tumorigenesis treatmenta
leukemia tumor incidenceb (%)
control NMU only NMU + HAs 0.1% NMU + HAs 0.2%
0/12 (0%) 11/12 (91.7%) 7/12 (58.3%) 4/12 (33.3%)
ratio of death 0/12 6/12 3/12 1/12
(0%) (50%) (25%) (8.3%)
a
First time iv NMU and treated various concentrations of HAs (0.1 and 0.2%) on the same day. bTumor incidence is equal to the number of dead rats that had leukemia combined with the number of tumor rats.
of progression. Leukemia can be divided into four common types: chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML). N-Methyl-N-nitrosourea (NMU), a DNA alkylating agent, acts as a potent carcinogen and mutagen to various tissues. O6-Methylguanine adducts have been proposed to be the critical lesion in a variety of NMUinduced tumors. In particular, Huggins et al. found that NMU specifically elicited a high incidence of leukemia in Sprague− Dawley rats.36 Therefore, NMU is a broad-spectrum carcinogen capable of inducing tumors in various target organs, including leukemia. According to a previous paper, blood samples were collected and used to evaluate cytological examination (through Liu and Papanicolaou stains) and morphological changes of rat livers and spleens by histological evaluation (through H&E staining) and to classify the leukemia of poorly differentiated blasts (through an immunohistochemical analysis).37 The results showed that the NMU-induced group was positive for MPO, but negative for CD3, CD15, CD20, and CD34. NMUinduced leukemogenesis in a S-D rat suggests a more definite way to classify leukemia M3 (APL, acute promyelocytic leukemia). NMU characteristically induces an early and high incidence of leukemia and malignant lymphomas in young rats when provided at relatively high doses, resulting in a high mortality rate of the animals and limiting the conduct of longterm experimental studies.38 In this study, we used the animal model of NMU-induced leukemia to detect the influence of HAs. Mortality rates varied from 50% in the NMU-only group to 8.3% with the HAs 0.2% group through the whole course (Table 1). NMU-related macroscopic observations included pale liver, heart, kidney, and bone marrow and enlargement of the spleen, thymus, and mandibular and mesenteric lymph nodes, which correlated with the diagnosis of lesions of the hematopoietic system.39 Figure 1B shows that the livers and spleens from the NMU-exposed rats were abnormally enlarged, but with treatment of HAs both organs were significantly restored to nearly normal. Subsequently, we detected the count of ALT, AST, WBC, RBC, PLT, and Hb levels in the blood. These results display that HAs can significantly diminish elevated serum ALT and AST as well as complete blood count levels in rats exposed to NMU (Figure 2). Finally, we investigated the presence of leukemic cells in the peripheral smear using Liu and Papanicolaou stains; in addition, the severity of tissue injury resulting from leukemic cell infiltration was also assessed by histological detection (H&E and MPO assay). We found HAs can effectively abrogate NMU-induced leukemic cell infiltration and subsequent tissue damage (Figures 3 and 4). Thus, evaluation of the protective effect of anthocyanin extract of roselle on leukemia might shed light on drug discovery or alternative therapy. G
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antileukemic activity of HAs in vivo. Although further studies are needed to examine the mechanisms in detail, this study supports the contention that dietary intake of bioactive food compounds such as HAs may prevent cancer and reduce cancer risk.
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AUTHOR INFORMATION
Corresponding Author
*(C.-J.W.) Mail: Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, South District, Taichung 402, Taiwan. Phone: +886 4 24730022, ext. 11670. Fax: +886 4 23248167. E-mail: wcj@ csmu.edu.tw. Author Contributions ∥
Y.-C.C. and C.-J.W. contributed equally to the main findings of the manuscript.
Funding
This work was supported by grants from the National Science Council (NSC 98-2313-B-040-005-MY2) and Chung Shan Medical University, Antai Tian-Sheng Memorial Hospital (CSMU-TSMH-101-01), Taiwan. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank all of our colleagues who participated in the present project.
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ABBREVIATIONS USED HAs, Hibiscus anthocyanin; NMU, N-nitrosomethylurea; AST, aspartate aminotransferase; ALT, alanine aminotransferase; H&E stain, hematoxylin and eosin stain; IHC stain, immunohistochemical stain; MPO stain, myeloperoxidase stain; CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; PLT, platelet; Hb, hemoglobin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; O6meG, O6-methylguanine
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REFERENCES
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dx.doi.org/10.1021/jf405235j | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
