squamosa seeds extract containing annonaceous acetogenin
5 CHEN Yong, XU Shasha, CHEN Jianwei, WANG Yu, XU Huiqing, FAN Naibing,
(College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210046) Abstract: Seeds
of Annona squamosa L. have been used in the south of China as a folk remedy to treat “malignant
sores” (cancer). To investigate the chemical constituents and the anti-tumor activity of the
10 standardized A. squamosa seeds extract in vitro and in vivo. Annonaceous acetogenin profiles of the standardized extract were determined by using Fourier transform infrared (FT-IR) and high performance liquid chromatography (HPLC) techniques. The anti-tumor activity of the extract was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity in vitro and H22 hepatoma cells transplantation tumor model in vivo. The FT-IR spectroscopy showed the
15 presence of annonaceous acetogenin compounds in the extract. Two major annonaceous acetogenins: 12, 15-cis-squamostatin-A and bullatacin were identified and quantified by HPLC. The seed extract showed significant anti-tumor activity against four human tumor cell lines, especially for MCF-7 (IC50. 0.25 μg/ml) and Hep G2 (IC50. 0.36 μg/ml) cells in vitro. The extract inhibited the growth of H22 tumor cells in mice with a maximum inhibitory rate of 69.55% by oral administration. A. squamosa
20 seed extract showed significant anti-tumor activities against human hepatoma cells in vitro and in vivo, indicating a potential for developing the extract as a novel anti-liver cancer drug. Key words: chemistry of Chinese Medicine; Annona squamosa; Anti-tumor; HPLC; Annonaceous acetogenin
25 0 Introduction
Cancer is a major problem which still remains unresolved. Therapies developed along with the principles of modern medicine are often limited in their efficacy, carry the risk of adverse effects and are often very costly, especially for the developing world . Botanical extracts have
been used as a source of medicinal agents for thousands of years. The seeds extract of Annona  30 squamosa was used in the south of China as a folk remedy to treat “malignant sores” (cancer) .
Recently, phytochemical and pharmacological studies on A. squamosa seeds have shown that the
major bioactive compounds are annonaceous acetogenins, which have a strong antitumor activity [3-7]. Our previous work showed that the total content of annonaceous acetogenins was higher in the seeds of A. squamosa than in four other species (A. glabra, A. muricata, A. reticulate and A.  35 bullata) . However, few standardized seeds extracts of A. squamosa have been available so far
as compared with those of other Annona species. For example, an encapsulated extract of A. triloba has been effectively used as a botanical supplement to treat certain cancers .
In the present work, seeds extract of A. squamosa was prepared with a simple method.
Annonaceous acetogenin profiles and anti-tumor activity of the extract were also investigated in
40 vitro and in vivo.
Foundations: Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Material Medica (No. P09018); the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. ysxk-2010); Doctoral Fund of Ministry of Education of China (No. 20113237110009) and 2011 Program sponsored for scientific innovation research of college graduate in Jiangsu province (No. 790). Brief author introduction:CHEN Yong (1987-), doctoral candidate, Chinese medicine chemical analysis Correspondance author: LI Xiang (1953-), tutor of a Ph.D. student, Chemistry of Chinese Medicine. E-mail: firstname.lastname@example.org
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1 Materials and methods
  45 12,15-cis-squamostatin-A and bullatacin (Fig. 1) were isolated from the seeds of
Annona squamosa L. in our laboratory. Both molecular structures were characterized based on spectroscopic analysis. Each compound was determined by HPLC analysis and confirmed by LC-MS, NMR spectroscopy. The purity of each compound reached above 98%. 5-Fluorouracil (5-Fu) and cyclophosphamide (CTX) were used as anti-tumor agents in vitro and in vivo,
50 respectively, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma (Shanghai, China). HPLC grade methanol was purchased from Hanbang Science and Technology Company (Nanjing, China) and the deionized water was obtained from the Milli-Q Plus 185 system (Millipore, Bedford, MA, USA). Other chemicals were all of analytical grade.
Fig. 1. Representative HPLC of annonaceous acetogenins in A. squamosa seeds extract. Peaks: (1)
12,15-cis-squamostatin-A and (2) bullatacin. The mobile phase was: 83% solvent A (methanol) and 17%
solvent B (deionized water).
1.2 Preparation of seeds extract
60 Seeds of A. squamosa were collected from Guangdong Province in October 2009 and identified by Prof. Jian-wei Chen (Nanjing University of Chinese Medicine, Jiangsu, China). The sample was authenticated and deposited at the herbarium of the Pharmaceutical College of Nanjing University of Chinese Medicine, Jiangsu (No. 083). Dried seeds (50 g, 40-mesh) were soaked in ethanol (1000 ml) for 3 days and filtered through filter paper. The residue was further
65 extracted twice. The combined filtrates were concentrated using a rotary evaporator at 45?C. The ethanol extract was dissolved in water and partitioned with equal volume of ethyl acetate twice. The ethyl acetate portion was concentrated using a rotary evaporator at 45?C, giving a yield of 10-12%. The extract was diluted to a series of desired concentrations for the experiment.
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1.3 FT-IR and HPLC analysis of annonaceous acetogenin compounds
70 FT-IR spectra were recorded from the dried seeds extract (2 mg) in KBr pellets on a
-1NEXUS-470 FT-IR spectrophotometer in the range of 4000-500 cm. The dried seeds extract was dissolved in HPLC grade methanol (3 mg/ml), filtered through a 0.45 μm membrane filter, and
subjected to qualitative and quantitative analysis by using Agilent 1200 liquid chromatography system (Agilent technologies, CA, USA) consisting of double pump (G1312A), auto-sampler
(ALS G1329A), diode-array detector (G1315A) and Agiletn LC 3D ChemStation Software 75  (B.03.01). Chromatographic analysis was carried out under the conditions described previously with minor modifications. The column configuration consisted of an Agilent Zorbax Extend
RP-Ccolumn (250 mm × 4.6 mm, 5 μm). Detection wavelength was set at 220 nm. The mobile 18 phase consisted of methanol (83%) and deionized water (17%). The flow rate was 1.0 ml/min. The
column temperature was maintained at 30?C. The standard solutions of bullatacin and 80
squamostatin-A were prepared with HPLC grade methanol. Working standard solutions 12,15-cis- were prepared by diluting standard solution with methanol to give six different concentrations in
the range of 0.224 – 2.24 mg/ml (bullatacin) and 0.0524-0.524 mg/ml (12,15-cis-squamostatin-A) for calibration curves. The standard solutions were filtered through a 0.45 μm membrane prior to
injection. Chromatographic peaks were confirmed by comparing their retention times with the 85
reference standards and by DAD spectra (200-600 nm). Quantification was performed by peak integration using the external standard method. The calibration curve for bullatacin was: y = 4.66 33 × 10c + 56.97 (= 0.9995) and 12,15-cis-squamostatin-A: y = 5.891 × 10c - 24.71 (= R?R? 0.9993). All chromatography was performed at room temperature and in triplicates. The limit of
detection (LOD) and limit of quantification (LOQ) were calculated based on the standard 90
deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve. 1.4 Cell lines and animals
A-549 (human lung carcinoma), Hela (human cervix carcinoma), MCF-7 (human breast 95
carcinoma) and HepG2 (human hepatoma carcinoma) cell lines were provided by the Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica and cultured with DMEM medium (HyClone Laboratories Inc., Logan, UT) containing 10% fetal calf serum (Sijiqing, Hangzhou, China) and penicillin and streptomycin 100 U/ ml, at 37?C with 5% CO. 2
Male Kunming mice [SCXK (Shanghai) 2007-0005] weighting 18-22 g were purchased from Slac 100
laboratory animal Co. Ltd. (Shanghai, China). All the procedures were approved by Animal Ethical Council of Nanjing University of Chinese Medicine. The mice were maintained under laboratory conditions at 25?C under a normal 12h/12h light/dark cycle with humidity of 55% and fed with food and water ad libitum. The mice were allowed, at least, 5 days to adapt to the
laboratory environment before experiments. 105
1.5 In vitro anti-tumor activity [12, 13] Five-day in vitro MTT cytotoxicity tests against human tumor cell lines were carried
out at the Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, using A-549, Hela, MCF-7 and HepG2 cell lines, with 5-Fu as a positive control. Briefly, 4cells were incubated in 96-well plates (1 × 10cells/well) containing 100 μl of the culture medium. 110
After overnight growth, cells were treated with various concentrations of the seeds extract for 5
days. The cells were harvested and washed with PBS and incubated with 20 μl MTT (5mg/ml) for
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4 h, and finally, 150 μl of DMSO were added to each well. The maximum absorbance was detected at 570 nm by an ELISA plate reader (Model680, BIO-RAD, USA). The inhibition rate 115 values were calculated by Probit analysis . was calculated and IC50
1.6 In vivo anti-tumor activity At least 10 mice were used for each sample and each dose. Mean values and S.D. were
determined by standard methods . The significant difference was estimated by the standard
Anti-tumor effect on Hwas observed in normal male Kunming mice. The test was 120 22  performed by observing the effect on the growth of the tumor as described previously . A dose 7of 0.2 ml of aseptic Hcells (about 1.0 × 10/ml) was implanted subcutaneously at the right groin 22 of mice. 24 h after the tumor implantation, the test sample dissolved in olive oil was provided once a day by oral administration for 10 days with CTX as a positive control and olive oil as a negative control. After treatments, all rats were sacrificed, and the tumors and spleen were dissected and 125
weighed. The tumor growth inhibition rate was calculated as: inhibition rate (%) = 100 × (C - T)/C, where C is the average tumor weight of the control group and T is the tumor weight of the treated
sample group. Spleen index = (spleen weight/final bodyweight) × 100. 2 Results130
2.1 FT-IR spectra and HPLC analysis of annonaceous acetogenin compounds FT-IR spectroscopy has been shown to be a powerful tool for the study of the physicochemical properties of annonaceous acetogenins and extract from A. cornifolia . The IR -1 absorption bands at 3422, 1742 and 1465 cmshowed the presence of hydroxyl, carbonyl and
double bond that are the characteristic spectral data of annonaceous acetogenin compounds (Fig. 135
Fig. 2. FT-IR spectrum of A. squamosa seeds extract.
Amongst annonaceous acetogenins, 12,15-cis-squamostatin-A (47.98 ? 2.42 mg/g) and
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140 bullatacin (256.18 ? 13.61 mg/g) were identified as major compounds (Tab. 1). From the
annonaceous acetogenins profiling by HPLC analysis, several peaks representing annonaceous acetogenin compounds were observed. The presence of 12,15-cis-squamostatin-A (1) and bullatacin (2) was indentified through their retention times i.e. about 12.48 and 30.57 min, respectively (Fig. 1).
Tab. 1. Content of annonaceous acetogenins in A. squamosa seeds extract. 145 abcomposition LOD LOQ A. squamosa compounds (mg/g)(μg/ml)(μg/ml)
12,15-cis-squamostatin-A 47.98 ? 2.42 0.042 0.127 Bullatacin 256.18 ? 13.61 0.011 0.033 a b Note: limit of detection. limit of quantification.
2.2 In vitro anti-tumor activity The anti-tumor activity of the extract against the growth of four human tumor cell lines was assayed and the ICdata were shown in Tab. 2. The extract exhibited significantly higher 50
anti-tumor activity against the MCF-7 (IC. 0.25 μg/ml) and Hep G2 (IC. 0.36 μg/ml) cells than 150 5050
against other cells. The extract’s dose-dependent inhibition against MCF-7 and HepG2 cells was
shown in Fig. 3.
Tab. 2. ICvalues of A. squamosa seeds extract and fluorouracil against four human tumor cell lines. 50
IC(μg/ml)50 Sample A549 Hela MCF-7 HepG2 -1-13.2 13.0 2.5×10 3.6×10 A. squamosa seed extract -1-1-1-3 3.2×10 1.4×10 2.2×10 8.2×10 5-Fu
155 Fig. 3. Comparison of the cell growth inhibition potential of A. squamosa seeds extract versus the standard control fluorouracil against MCF-7 and HepG2 cells. The values were inhibition rates (%) with a mean of triplicates at each point. The bars represent standard deviation. The concentration values were Log [dose] with units of μg/ml.
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2.3 In vivo anti-tumor activity The results in Tab. 3 showed that the extract exhibited an anti-tumor effect against Hwhen 22
administered by oral. The inhibition rate of the extract reached 69.55% at a dose of 18 mg/kg/day, higher than that of CTX (63.75%) administered by intraperitoneal injection at a higher dose of 30
Tab. 3. Effect of A. squamosa seeds extract on Hsolid tumor growth. 22 Dose Tumor weight Inhibition Sample Spleen index (mg/kg)(g)(%)
Control 0.53 ? 0.18 0.82 ? 0.06 a aCTX 30 0.19 ? 0.0763.75 0.30 ? 0.16 The extract 4.5 0.31 ? 0.10 40.42 0.66 ? 0.21 9 0.31 ? 0.10 40.82 0.63 ? 0.10 a 18 0.16 ? 0.0669.55 0.62 ? 0.30 a Note: p < 0.001 (Student’s t-test)
170 3 Discussion Nowadays, exploration of novel anti-tumor drugs is a key issue for mankind. Treating cancer
with botanical extracts, such as A. squamosa, has been shown to be an easy and effective approach. Phytochemical and pharmacological studies on the A. squamosa seeds have shown that the major bioactive compounds in the extract are annonaceous acetogenins, which are well known for
inhibition of tumor cells that are resistant to multiple drugs. An encapsulated extract of A. triloba 175 has been effectively used as a botanical supplement to treat certain cancers . However, few standardized extracts of A. squamosa seeds are available. Previous studies reported its pharmacological activities, such as antioxidant, anti-psoriatic, larvicidal and anthelmintic activities [17-20]. In the present work, two main compounds of annonaceous acetogenins:
12,15-cis-squamostatin-A and bullatacin were identified and quantified by HPLC analysis from 180 the extract of A. squamosa seeds. Bullatacin, a bistetrahydrofuran annonaceous acetogenin known as the most potent inhibitor of the mitochondrial respiratory chain complex I, exhibited 300 times more effective than taxol as tested in vivo, and was used to control the quality of standardized [9, 17]extract . 12, 15-cis-squamostatin-A and bullatacin also showed significant anti-tumor activity [10, 11]against various tumor cell lines . 185 The extract showed significantly higher anti-tumor activity against the MCF-7 and HepG2 cells. Furthermore, the anti-tumor effect of the extract was also investigated by in vivo mice bearing Hhepatoma cells transplantation tumor model, which is one of the most common tumor 22 models employed in the anti-tumor research . CTX, which has been extensively used in clinical
application, has strong side effects such as significant decrease in body and spleen weight in mice. 190
Compared with CTX, the extract inhibited the growth of Htumor cells in mice with a maximum 22 inhibitory rate of 69.55% and no side effects were observed, suggesting that the extract of A.
squamosa seeds may be a potential candidate for a novel anti-liver cancer drug.
In this paper, the chemical constituents and the anti-tumor activity of the standardized A. 195
squamosa seeds extract were investigated in vitro and in vivo. Two major annonaceous
acetogenins: 12, 15-cis-squamostatin-A and bullatacin were identified and quantified by HPLC. A.
squamosa seed extract showed significant anti-tumor activities, indicating a potential for
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developing the extract as a novel anti-liver cancer drug.
200 Acknowledgements (Optional) This work was supported by grants from Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Material Medica (P09018), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010), Doctoral Fund of Ministry of Education of China (20113237110009) and 2011 Program sponsored for
scientific innovation research of college graduate in Jiangsu province (790). We are thankful to Dr. 205
Houman Fei (Saint Mary’s University, Canada) for the revision of the English content of the
manuscript. References  Sabir S M, Ahmad S D, Hamid A, Khan M Q, Athayde M I, Santos D B, Boligon A A, Rocha J B T. 210 Antioxidant and hepatoprotective activity of ethanolic extract of leaves of Solidago microglossa containing
polyphenolic compounds[J]. Food Chemistry, 2012, 131, 741-747.  Guangdong Food and Drug Administration. Guangdong Chinese Materia Medica standards[S], vol. I. Guangdong science and technology Press, Guangdong, 2004, pp.194.  Bermejo A, Figadere B, Zafra-Polo M C, Barrachina I, Estornell E, Cortes D. Acetogenins from Annonaceae: 215 recent progress in isolation, synthesis and mechanisms of action[J]. Natural Products Reports, 2005, 22, 269-303.
 Liaw C C, Wu T Y, Chang F R, Wu Y C. Historic perspectives on Annonaceous acetogenins from the chemical bench to preclinical trials[J]. Planta Medica, 2010, 76, 1390-1404.  CHEN J W, CHEN Y, LI X. Beneficial aspects of Custard Apple (Annona squamosa L.) seeds, in Preedy, V.R., Watson, R.R., Patel V.B. (Eds.), Nuts & Seeds in Health and Disease Prevention[M]. Elsevier Academic Press, 220 London, Burlington, San Diego, 2011, pp. 439-445.
 LI Y F, FU L W. Antitumor effects of annonaceous acetogenins[J]. Chinese Pharmacology Bulletin, 2004, 20, 245-247.  LIU H X, HUANG G R, ZHANG H M. Annonaceous acetogenin mimics bearing aterminal lactam and their cytotoxicity against cancer cells[J]. Bioorganic & Medicinal Chemistry Letters, 2007, 17, 3426-3430. 225  YANG H J, LI X, TANG Y P, ZHANG N, CHEN J W, CAI B C. Supercritical fluid CO2 extraction and
simultaneous determination of eight annonaceous acetogenins in Annona genus plant seeds by HPLC-DAD method[J]. Journal of Pharmaceutical and Biomedical Analysis, 2009, 49, 140-144.  McLaughlin J L. Paw paw and cancer: Annonaceous acetogenins from discovery to commercial products[J]. Journal of Natural Products, 2008, 71, 1311-1321.
230  YANG H J, ZHANG N, Li X, CHEN J W, CAI B C. Structure-activity relationships of diverse annonaceous
acetogenins against human tumor cells[J]. - Bioorganic & Medicinal Chemistry Letters, 2009, 19, 2199-2202.  CHEN Y, CHEN J W, LI X. Cytotoxic bistetrahydrofuran annonaceous acetogenins from the seeds of Annona squamosa[J]. Journal of Natural Products, 2011, 74, 2477-2481.  Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and 235 cytotoxicity assays[J]. Journal of Immunological Methods, 1983, 65, 55-63.
 Scudiero D A, Shoemaker R H, Paull K D, Monks A, Currents M R, Seniff D, Boyd M R. Evaluation of a
soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines[J]. Cancer Research, 1988, 48, 4827-4833.  Finney D J. Probit analysis[M], third ed. Cambridge University Press, Cambridge. 1980.
 Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S, Suzuki M. Antitumor effect of 240 hexa-N-acetylchitohexaose and chitohexaose[J]. Carbohydrate Research, 1986, 151, 403-408.  QIN C Q, ZHOU B, ZENG L T, ZHANG Z H, LIU Y, DU Y M, XIAO L. The physicochemical properties and antitumor activity of cellulase-treated chitosan[J]. Food Chemistry, 2004, 84, 107-115.  Lima L A R, Pimenta L P S, Boaventura M A D. Acetogenins from Annona cornifolia and their antioxidant
capacity[J]. Food Chemistry, 2010, 122, 1129-1138. 245
 Saelee C, Thongrakard V, Tencomnao T. Effects of Thai medicinal herb extracts with anti-psoriatic activity
on the expression on NF-κB signaling biomarkers in HaCaT keratinocytes[J]. Molecules, 2011, 16, 3908-3932.  Kamaraj C, Bagavan A, Elango G, Zahir A A, Rajakumar G, Marimuthu S, Santhoshkumar T, Rahuman A A. Larvicidal activity of medicinal plant extracts against Anopheles subpictus & Culex tritaeniorhynchus[J]. Indian
Journal of Medical Research, 2011, 134, 101-106. 250
 Kamaraj C, Rahuman A A, Elango G, Bagavan A, Zahir A A. Anthelmintic activity of botanical extracts against sheep gastrointestinal nematodes, Haemonchus contortus[J]. Parasitology Research, 2011, 109, 37-45.
 WU D D, GAO Y F, CHEN L X, QI Y M, KANG Q Z, WANG H L, ZHU L Y, YE Y, ZHAI M X. Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice[J]. Peptides, 2010,
31, 850-864. 255
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260 陈勇;徐莎莎;陈建伟;王玉;许惠琴;范乃兵;李祥 ;南京中医药大学;药学院;南京;210046； 摘要;番荔枝种子在中国南方民间被用于治 疗恶疮肿痛。本论文为了研究番荔枝种子提取物 的化学成分及抗肿瘤作用。运用 FT-IR 和 HPLC 技术来确定其主要化学成分？在体外；用 MTT 法研究了其抑制人肿瘤细胞活性？在 体内；研究了其对 H22 肿瘤移植小鼠肿瘤生长抑 制作用。结果 FT-IR 图谱显示番荔枝种子
提取物含有番荔枝内酯类化合物；运用 HPLC 技术 确定了其中主要成分为 12, 15-cis-
265 squamostatin-A 和 bullatacin；并对这两个主要成分进行了 含量测定。在体外抗肿瘤活性测 试中；番荔枝种子提取物对四种人肿瘤细胞显示出了一定的 抑制活性；尤其对 MCF-7 (IC50. 0.25 μg/mL)和 Hep G2 (IC50. 0.36 μg/mL)细胞？在体内抗肿 瘤活性测试中；高剂量 的番荔枝种子提取物对 H22 肿瘤移植小鼠肿瘤生长抑制率达到 69.55%。番荔枝内酯提取物显示出强大的体内外抗肝肿瘤细胞活性；表明其可开发成为一 种新的抗肝癌药物。 270 关键词;中药化学？番荔枝？抗肿瘤？高效液相？番荔枝内酯
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