Nghiên cứu tác dụng gây apoptosis của một số cao chiết dược liệu trên dòng tế bào ung thư

BỘ Y TẾ VIỆN DƯỢC LIỆU BÁO CÁO TỔNG KẾT ĐỀ TÀI CƠ SỞ 2019 NGHIÊN CỨU TÁC DỤNG GÂY APOPTOSIS CỦA MỘT SỐ CAO CHIẾT DƯỢC LIỆU TRÊN DÒNG TẾ BÀO UNG THƯ Chủ nhiệm đề tài PGS TS Nguyễn Thị Thu Hương Đơn vị Trung tâm Sâm Dược liệu Tp Hồ Chí Minh Thời gian thực 12/2018 đến 11/2019 Tp Hồ Chí Minh – 11/2019 BỘ Y TẾ VIỆN DƯỢC LIỆU BÁO CÁO TỔNG KẾT ĐỀ TÀI CƠ SỞ 2019 NGHIÊN CỨU TÁC DỤNG GÂY APOPTOSIS CỦA MỘT SỐ CAO CHIẾT DƯỢC LIỆU TRÊN DÒNG TẾ BÀO UNG THƯ CHỦ NHIỆM ĐỀ TÀI TRƯỞNG ĐƠN VỊ VIỆN DƯỢC LIỆU Tp Hồ Chí Minh, 11/2019 MỤC LỤC DANH MỤC CÁC CỤM TỪ - KÝ HIỆU VIẾT TẮT i DANH MỤC BẢNG iii DANH MỤC CÁC HÌNH .iv TÓM TẮT v SUMMARY vi PHẦN A: CÁC THÔNG TIN CHUNG vii I TĨM TẮT NỘI DUNG, CƠNG VIỆC CHỦ YẾU ĐÃ ĐẠT ĐƯỢC vii II CÁC SẢN PHẨM CỦA ĐỀ TÀI viii 2.1 Kết nghiên cứu viii 2.2 Hình thức, cấp độ cơng bố kết viii III TÌNH HÌNH SỬ DỤNG KINH PHÍ ix IV ĐỘI NGŨ CÁN BỘ THỰC HIỆN ĐỀ TÀI………………………………….…ix PHẦN B: NỘI DUNG BÁO CÁO TỔNG KẾT x MỞ ĐẦU x CHƯƠNG I TỔNG QUAN 1.1 Tổng quan dược liệu có tiềm kháng ung thư 1.1.1 Tổng quan Trâm bầu (Combretum quadrangulare Kuz.) 1.1.2 Tổng quan Trâm mốc (Syzygium cumini (L.) Skeels) 1.1.3 Tổng quan Gừng gió (Zingiber zerumbet (L.) Sm.) 1.1.4 Tổng quan Mảnh cộng (Clinacanthus nutans) 1.1.5 Tổng quan Ké đầu ngựa (Xanthium strumarium L.) 1.2 Tổng quan ung thư 12 1.2.1 Giới thiệu chung ung thư 12 1.2.2 Khái quát apoptosis 14 1.2.3 Những thay đổi tế bào apoptosis 18 1.2.4 Liệu pháp thảo dược hỗ trợ điều trị ung thư 20 CHƯƠNG II ĐỐI TƯỢNG, NỘI DUNG VÀ PHƯƠNG PHÁP NGHIÊN CỨU 25 2.1 Nguyên vật liệu 25 2.1.1 Nguyên liệu 25 2.1.2 Thiết bị, dụng cụ hóa chất 26 2.2 Phương pháp nghiên cứu 27 2.2.2 Phương pháp chiết ngấm kiệt Error! Bookmark not defined 2.2.3 Phương pháp nuôi cấy tế bào 28 2.2.4 Phương pháp thử độc tính tế bào thực nghiệm SRB 33 2.2.5 Phương pháp nhuộm huỳnh quang kép acridine orange – ethidium bromide (AO – EB) 34 2.2.6 Phương pháp phân mảnh DNA 35 2.2.7 Phương pháp xử lý số liệu thực nghiệm đánh giá kết 36 CHƯƠNG III KẾT QUẢ VÀ THẢO LUẬN 37 3.1 Hiệu suất chiết xuất dược liệu 37 3.2 Khả gây độc tế bào ung thư HepG2 in vitro cao chiết dược liệu 37 3.3 Khả cảm ứng apoptosis tế bào ung thư HepG2 cao chiết Ké đầu ngựa 39 CHƯƠNG IV KẾT LUẬN VÀ ĐỀ NGHỊ 46 4.1 Kết luận 46 4.2 Kiến nghị 46 TÀI LIỆU THAM KHẢO 47 DANH MỤC CÁC CỤM TỪ - KÝ HIỆU VIẾT TẮT Viết tắt AAPH ABTS AGS AO Apaf-1 APO-1 AqE ATP ATPase BA BaP Bid CAD CARD CD95 ChE CHO CPT CrE CT26WT CTL dATP DFF D-GalN DISC DMEM DMSO DNA DNAse DPPH DR DRs Tiếng Anh 2,2'-azobis(2-amidinopropane) dihydrochloride 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) Adenocarcinoma gastric cell line Acridine orange Adapter protein apoptotic proteaseactivating factor-1 Apoptosis antigen Aqueous extract Adenosine triphosphate Adenosine triphosphatase Betulinic acid Benzo(a)Pyrene Pro-apoptosis protein Caspase Activated Dnase N-terminal Caspase Recruitment Domain Cluster of differentiation 95 Chloroform extract Chinese hamster ovary cells Tiếng Việt 2,2'-azobis(2-amidinopropan) dihydrochlorid 2,2'-azino-bis (acid 3-ethylbenzothiazolin-6sulphonic) Dòng tế bào ung thư dày Acridin cam Dịch chiết nước Adenosin triphosphate Enzym xúc tác chuyển hóa ATP Axit betulinic Benzo(a)Pyren Caspase hoạt hóa Dnase Dịch chiết chloroform Tế bào buồng trứng chuột hamster Trung Quốc Camptothecin Camptothecin Crude extract Dịch chiết thô Murine colorectal carcinoma cell Tế bào ung thư biểu mô đại tràng line chuột Cytotoxic T-lymphocytes Tế bào lympho T gây độc Deoxy-ATP nucleotide Nuleotid deoxy-ATP DNA fragmentation factor Nhân tố phân mảnh DNA D-galactosamine D-galactosamin Death-inducing signaling complex Phức hợp tín hiệu truyền cảm ứng gây chết Dulbecco’s modified eagle medium Dimethyl sulfoxide Dimethyl sulfoxid Deoxyribonucleic acid Acid deoxyribonucleic Deoxyribo nuclease Enzym xúc tác phân cắt DNA 1,1–diphenyl–2–picrylhydrazyl 1,1–diphenyl–2–picrylhydrazyl Death receptor Thụ thể chết Death receptors Thụ thể chết i EAE EB EC50 ED50 EDTA EMEM FADD FBS GPT HDAC Hela HEPES HepG2 HIV HO-1 HSV HT50 IARC ICAD IkBa iNOS LPS MTT NF-κB NQO1 Nrf2 PARP SRB TNFR1 TNF-α TRADD Ethyl acetate extract Ethidium bromide Half maximal effective concentration Effective dose 50 Ethylenediaminetetraacetic acid Eagle’s minimal essential medium FAS-associated death domain Fetal bovine serum Serum glutamic pyruvic transaminase Histon deacetylase Dịch chiết ethyl acetat Ethidium bromid Nồng độ hiệu 50% Liều hữu hiệu trung bình Acid ethylenediaminetetraacetic Huyết thai bị Nhóm thuốc ức chế histon deacetylase Hela cells Tế bào ung thư cổ tử cung 4-(2-hydroxyethyl)-1Acid 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid piperazineethanesulfonic Liver hepatocellular carcinoma cell Dòng tế bào ung thư gan line Human immunodeficiency virus Virus gây suy giảm miễn dịch Heme oxygenase-1 Herpes simplex virus Virus herpes simplex Half-hemolysis time Thời gian tán huyết trung bình International Agency for Research Cơ quan nghiên cứu ung thư quốc on Cancer tế Inhibitor of caspase activated DNAse IkappaBalpha Inducible nitric oxide synthase Yếu tố cảm ứng tổng hợp nitric Lippopolysaccharide Lippopolysaccharid dye compound 3-(4,5- Chất nhuộm Dimethylthiazol-2Dimethylthiazol-2-yl)-2,5yl)-2,5-diphenyltetrazolium diphenyltetrazolium bromidefor bromidefor nuclear factor kappa-light-chain- Yếu tố nhân kappa B enhancer of activated B cells NAD(P)H quinone oxidoreductase Nuclear factor erythroid 2-related Yếu tố liên quan đến NF-E2 factor Poly (ADP-ribose) polymerase Protein chuyển hóa sửa chữa DNA Sulforhodamine B TNF receptor-1 Thụ thể TNF-1 Tumor necrosis factor alpha Yếu tố hoại tử khối u alpha TNF receptor-1-associated death domain ii DANH MỤC BẢNG Bảng 1.1 Một số hợp chất có nguồn gốc thực vật nghiên cứu thử nghiệm lâm sàng điều trị ung thư 21 Bảng 2.1 Mã số lưu mẫu dược liệu nghiên cứu 25 Bảng 3.1 Hiệu suất chiết cao chiết dược liệu 37 Bảng 3.2 Phần trăm gây độc tế bào HepG2 cao chiết camptothecin……… 38 iii DANH MỤC CÁC HÌNH Hình 1.1 Cây Trâm bầu (Combretum quadrangulare)………………………………… Hình 1.2 Cây Trâm mốc (Syzygium cumini)…………………………………………… Hình 1.3 Cây Gừng gió (Zingiber zerumbet)…………………………………………….5 Hình 1.4 Cây Mảnh cộng (Clinacanthus nutans)…………… …………………………7 Hình 1.5 Cây Ké đầu ngựa (Xanthium strumarium)……………………… ………… Hình 1.6 Các đường apoptosis…………………………………………………… 15 Hình 1.7 Sự thay đổi hình thái tế bào apoptosis…………………………… 19 Hình 2.1 Bột nguyên liệu khơ từ dược liệu nghiên cứu………………………… 26 Hình 2.2 Dịng tế bào HepG2 (HB-8065, ATCC®)………………………………… 29 Hình 3.1 Hình thái tế bào bào HepG2 xử lý với cao chiết nồng độ 100 µg/ml CPT 0,07 µg/ml sau 48 ……………………………………………………… 38 Hình 3.2 Hình thái tế bào ung thư HepG2 xử lý với cao chiết phần mặt đất Ké đầu ngựa nồng độ khác sau 60 giờ… ……………………….………….40 Hình 3.3 Đặc điểm apoptosis tế bào HepG2 xử lý với cao chiết ethanol 96% từ phần mặt đất Ké đầu ngựa nồng độ sau 60 giờ…………………….…….41 Hình 3.4 Kết điện di phân mảnh DNA tế bào HepG2 sau 60 xử lý với cao chiết từ phần mặt đất Ké đầu ngựa nồng độ khác nhau………… …… 42 iv TÓM TẮT Ung thư nguyên nhân gây tử vong đứng thứ hai toàn giới Dược liệu nguồn tiềm phát triển thuốc kháng ung thư thông qua chế cảm ứng apoptosis gây kháng phân bào Nghiên cứu tiến hành khảo sát khả gây độc tế bào phương pháp SRB dòng tế bào ung thư gan HepG2 cao chiết ethanol 96% từ dược liệu: Trâm bầu, Trâm mốc, củ Gừng gió, phần mặt đất Ké đầu ngựa phần mặt đất Mảnh cộng Từ chọn dược liệu tiềm để tiếp tục khảo sát khả cảm ứng apoptosis tế bào ung thư HepG2 Kết cho thấy, nồng độ 100 μg/ml, có cao chiết ethanol từ phần mặt đất Ké đầu ngựa (KĐN) Trâm bầu thể hoạt tính gây độc tế bào HepG2 với phần trăm gây độc 50% Cao chiết cồn 96% từ KĐN có khả gây độc tế bào ung thư mạnh số dược liệu khảo sát với IC50 81,69 μg/ml Kết khảo sát khả cảm ứng apoptosis cho thấy cao chiết cồn 96% từ KĐN gây apoptosis tế bào HepG2 thông qua thay đổi hình thái tế bào, đặc nhân xuất thể apoptotic sau 60 xử lý Như vậy, số dược liệu khảo sát, phần mặt đất Ké đầu ngựa có khả kháng dòng tế bào ung thư HepG2 tiềm Các kết đề tài cung cấp liệu khoa học tin cậy, làm tiền đề cho việc định hướng nghiên cứu tác dụng gây apoptosis số cao chiết dược liệu dòng tế bào ung thư v SUMMARY Cancer has become the second leading cause of human death globally Herbal medicines have been used as the potential source of medical treatment through apoptosis inducing mechanism or inhibition of mitosis This research was carried out to survey the cytotoxic effect of 96% ethanol extract from five potential herbs on liver cancer HepG2 cell lines using SRB method: Combretum quadrangulare Kuz leaves, Syzygium cumini (L.) Skeels leaves, Zingiber zerumbet (L.) Sm roots, aerial parts of Clinacanthus nutans, Xanthium strumarium L Therefore, the most promising herb would be selected to continuously undergo apoptosis inducing evaluation Results showed that, at a concentration of 100 μg/ml, only the ethanol extract from the aerial parts of Xanthium strumarium L (Xs) and Combretum quadrangulare Kuz leaves exhibited HepG2 cytotoxic activity with toxicity percentage over 50% The Xs’s extract had the strongest ability to cause cancer cell toxicity among surveyed herbs with IC50 of 81.69 μg/ml The results of the second apoptosis inducing evaluation indicated that Xs induced apoptosis on HepG2 cells via changes in cell morphology, characteristics and genomic DNA fragmentation after 60 hours treatment In present study, out of five potential herbs surveyed, the Xs’s extract reveals apoptosis inducing ability The results suggest that this herb may be potential for treatment of cancer as supplements The present findings provide reliable scientific, which may contribute to the effort of finding potential anticancer herbal drugs based on apoptosis inducing mechanism vi Tuyên, Lâm Bích Thảo, Nguyễn Hồng Dũng, Phùng Thị Thu Hường, Nguyễn Thái Biềng, Lê Văn Minh (2019), Khảo sát thành phần hóa học hoạt tính sinh học cao chiết hạt trâm mốc, Tạp chí Y học TP HCM, 23(4), 268-276 Nopsiri W., Chansakaow S., Putiyanan S., Natakankitkul S S., Santiarworn D (2014), Antioxidant and anticancer activities from leaf extracts of four Combretum species from Northern Thailand, Chiang Mai University Journal of Natural Sciences, 13(2):195-205 Thi-Phuong Nguyen, ThucHuy Duong, Huu-Hung Nguyen (2018), Cytotoxic activity of Combretum quadrangulare leaf extracts on HepG2 cancer cell line, Journal of Science and Technology-NTTU, 31-33 Văn Ngọc Hướng, Vương Văn Trường (2012), Các hợp chất có tác dụng sinh học Gừng gió (Zingiber zerumbet Sm.), Tạp chí Dược học, 10/2012, 438(52), 10-13 Sharifah Sakinah S., Tri Handayani S., Azimahtol Hawariah L (2007), Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio, Cancer Cell International, 7, 10 Ng P Y., Chye S M., Ng C H., Koh R Y., Tiong Y L., Pui L P., Tan Y H., Lim C S., Ng, K Y (2017), Clinacanthus nutans hexane extracts induce apoptosis through a caspase-dependent pathway in human cancer cell lines, Asian Pacific Journal of Cancer Prevention: APJCP, 18(4), 917-926 11 Ciulei I (1982), Methodology for analysis of vegetables drugs, Ministry of Chemical Industry, Bucarest, Roumania, 67 12 Chumark P., Panya K., Yupin S., Srichan P., Noppawan M (2008), The in vitro and ex vivo antioxidant properties, hypolipidaemic and antitherosclerotic activities of water extract of Moringa oleifera Lam Leave, Journal of Ethnopharmacology, 119, 439-436 13 Oyaizu M (1986), Studies on product of browning reaction prepared from glucose amine, Japan Journal of Nutrition, 44, 307-315 14 Nguyen M N., Ho Huynh T D (2016), Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects, BMC Complementary and Alternative Medicine, 16, 220 15 Moon J.-K., Shibamoto T (2009), Antioxidant assays for plant and food components, Journal of Agricultural and Food Chemistry, 57(5), 1655-1666 16 Houghton P., Fang R., Techatanawat I., Steventon G., Hylands P J., Lee C C (2007), The sulforhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity, Methods, 42(4), 377-387 17 Lee M T., Lin W C., Yu B., Lee T T (2017), Antioxidant capacity of phytochemicals and their potential effects on oxidative status in animals - A review, Asian-Australasian Journal of Animal Sciences, 30(3), 299-308 18 Panche A N., Diwan A D., Chandra S R (2016), Flavonoids: an overview, Journal of Nutritional Science, 5, e47 19 Yuan C S., Wang C Z., Wicks S M., Qi L W (2010), Chemical and pharmacological studies of saponins with a focus on American ginseng, Journal of Ginseng Research, 34(3), 160-167 Original Article Phytochemical analysis and anticancer activity of the above ground portion extract of Xanthium strumarium L on HepG2 cancer cell line Ly Hai Trieua, Nguyen Thi Thu Huonga, Truong Trieu Minha, Yuxia Zhaob, Le Van Minha* Affiliation a Research Center of Ginseng and Medicinal Materials, National Institute of Medicinal Materials, Ho Chi Minh City, Vietnam b Collaborative Scientific Research Centre, Shanghai University of Medicine and Health Sciences, Shanghai, 201318, China *Corresponding author: Dr Le Van Minh Research Center of Ginseng and Medicinal Materials National Institute of Medicinal Materials Ho Chi Minh City, Vietnam E-mail: lvminh05@gmail.com Phone: 0937326123 Abstract Cancer is the second most common cause of human deaths worldwide Recent studies have disclosed that plant extracts exhibit anticancer activity through various mechanisms This study is to investigate the secondary metabolites, antioxidant and anticancer activities of the above ground portion extract of X strumarium (AGP-XS) The phytochemical characteristics of AGP-XS were deteminated by qualitative chemical tests Total polyphenol and flavonoid contents of dried powdered material and crude extract were determined by colorimetric methods The AGP-XS was evaluated for in vitro antioxidant activity by DPPH assay The IC50 of AGP-XS on HepG2 cancer cell line was determined by sulforhodamine B (SRB) assay Apoptosis induction activity of AGP-XS was determined by acridine orange– ethidium bromide (AO–EB) dual staining Results illustrated that AGP-XS mainly contains flavonoids, alkaloids, tannins, triterpenes, and saponins Ethanol extracts had highest content of polyphenol (84.86 mg gallic acid equivalent/g dry weight), and exhibited the great total antioxidant activity (IC50 = 184.13 μg/mL) and antiproliferative activity on HepG2 cancer cell lines (IC50 = 81.69 μg/mL) Furthermore, the characteristics of apoptosis including shrinkage of the cell and apoptotic bodies were found following 60 hours of AGPXS treatment The data suggest that AGP-XS had antioxidant potential and antiproliferative effect This antiproliferative activity was associated with an increase of apoptosis Theses findings warrant further research on X strumarium as a potential chemotherapeutic agent for cancer Keywords: Xanthium strumarium L., phytochemical analysis, antioxidant, anticancer, apoptosis, HepG2 Abbreviations: AGP-XS, the above ground portion extract of X strumari; DPPH, 1,1diphenyl-2-picrylhydrazyl; SRB, sulforhodamine B; AO–EB, acridine orange–ethidium bromide; IC50, Half maximal inhibitory concentration Introduction An imbalance of systems oxidation and anti-oxidation caused by reactive oxygen species (ROS) such as superoxide, hydroxyl, and peroxyl, reactive nitrogen species (RNS) such as nitric oxide and peroxynitrite, can damage the DNA and lead to the oxidation and peroxidation of lipid and proteins in cells These can cause some chronic and degenerative diseases such as cancer, diabetes mellitus, alzheimer’s, parkinson’s… (Xu et al., 2017) Hepatocellular carcinoma or liver cancer is the most popular widespread cancer, accounting for approximately 90% (Kumar et al, 2011) It causes high annual mortality rates, especially in Southeast Asia, where viral hepatitis is endemic (Newell et al., 2008) Some current treatment therapies include chemotherapy, radiotherapy and chemically derived drugs However, treatment like using chemotherapy can cause many side effects and adversely affects the health of patients (Greenwell et al., 2015) Simultaneously, the major problem of chemotherapy therapies to treat hepatocellular carcinoma is the cancer resistance mechanism, due to the increase in multidrug-resistant protein and apoptotic protein reduction (Chen et al., 2007) Therefore, more effective methods for cancer control and apoptosis induction are needed, contributing to cancer treatment According to World Health Organization, over 60% of the global population and around 80% in developing countries use traditional and medicine plants for their treatment purposes (Shrestha et al., 2003) Many previous studies have shown that herbs have a very important role in cancer treatment, helping to control cancer and induce apoptosis in cancer cell (Greenwell et al., 2015, Ochwang’I et al., 2014) Xanthium strumarium L (Asteraceae) has been used as a traditional herbal on the treatment of various ailments Pharmacological studies have demonstrated that the compounds and extracts from X strumarium have extensive activities, such as anticancer, antibacterial, antifungal, antioxidant, antitumor, anti-inflammatory, antinociceptive, antilipidemic, hypoglycaemic, and other activities X strumarium has been reported to contain sesquiterpenoids, polyphenols, flavonoids, steroids, glycosides, triterpenoids, alkaloids, anthroquinones, and coumarins (Fan et al., 2019) Previous studies have also shown that the compounds and extracts from X strumarium presented anticancer activitiy (Fan et al., 2019, Tao et al., 2017, Al-Mekhlafi et al., 2017) However, there have been no reports regarding the evaluation of anti-proliferation activity and activating apoptosis of above ground portion extract of X strumarium extract on HepG2 cancer cell line Hence, the aim of this study was to consider in vitro antioxidant and anticancer activities on HepG2 cancer cell line of the above ground portion extract of X strumarium extract (AGP-XS) At the same time, this study determines the phytochemical characteristics of AGP-XS by qualitative chemical tests and total polyphenol and flavonoid contents Materials and methods 2.1 Plant material and extraction The above ground portion of X strumarium (stems and leaves) were collected on May 2018 from Ho Chi Minh City Dried powdered material (Loss on drying is 10.14%) was extracted (The ratio is 1: 10) with ethanol at room temperature for 24 hours in percolator apparatus The extract was collected at a rate of mL/min and concentrated using a rotary evaporator at 60 °C under reduced pressure to obtaine crude ethanol extract, which corresponds to a yield of 11.73% 2.2 Chemicals, reagents, apparatus and equipment Ethanol 96% (OPC Pharmaceutical Company), Methanol (Merck), Folin-Ciocalteu’s phenol reagent, aluminum chloride, quercetin (HPLC ≥ 98%), gallic acid (HPLC ≥ 98%), DPPH reagent (1,1-diphenyl-2-picrylhydrazyl), and ascorbic acid were purchased from SigmaAldrich® Co Ltd (USA) 2.3 Phytochemical analysis Preliminary qualitative phytochemical analysis was carried out to identify the secondary metabolites present of AGP-XS The screening was performed according to the method of Cuilei (Cuilei, 1982) with minor modifications for alkaloids, flavonoids, tannins, triterpenoids, saponins, coumarins, anthraquinones, anthocyanosides, proanthocyanidins, lipids, volatile oils, carotenoids, reducing agents, and organic acids The qualitative results are expressed as (+) for the presence and (−) for the absence of phytochemicals 2.4 Estimation of total phenolic content The total phenolic content was estimated by Folin Ciocalteu’s method as previously described using gallic acid as a standard with slight modifications (Chumark et al., 2008) Briefly, 200 μL test sample was mixed with 6 mL of double distilled water and 500 μL of Folin-Ciocalteu’s reagent After 5 min, 1.5 mL of sodium carbonate solution (20% w/v) was added to the mixture and the volume was made up to 10 mL with distilled water The reaction was kept in the dark for hours at room temperater The absorbance was measured at 758 nm and all determinations were carried out in triplicate The total polyphenol content was calculated from the calibration plot (Y = 0.0097x – 0.0278, R2 = 0.997) and expressed as mg of gallic acid equivalent (GAE)/g of dry mass 2.5 Estimation of total flavonoids content The flavonoids content was determined based on the aluminum chloride colorimetric method using quercetin as reference compound (Nguyen et al., 2015) Stock quercetin solution was prepared by dissolving 1.0 mg quercetin in 10 mL methanol, then the standard quercetin solutions were prepared by serial dilutions using methanol Briefly, an amount of mL diluted extract or standard quercetin solutions was separately mixed with 1 mL of 2% aluminum chloride and the volume was made up to 10 mL with methanol Then, the solution was mixed and incubated for 15 minutes at room temperature The absorbance of the reaction mixtures was measured at 454 nm with an UV–Vis spectrometer The measurements were performed in triplicate The total flavonoids content was calculated from the calibration plot (Y = 0.0219x – 0.0554, R2 = 0.998) and expressed as mg of quercetin equivalent (QE)/g of dry mass 2.6 In vitro antioxidant activity assay The antioxidant activity was determined by DPPH free radical scavenging assay based on a previously described method (Chumark et al., 2008) Briefly, mL reaction mixture in methanol containing 0.5 mL of varying concentrations of extract and 0.5 mL of 0,6 mM DPPH reagent was kept in the dark at room temperature for 30 minutes Then, the absorbance was measured at 516 nm and all tests were performed in triplicate Ascorbic acid (Sigma Co Ltd, USA) was used as a positive control The results were expressed as IC50 values for each sample Percentage of the radical scavenging activity was estimated using the expression: Where Ac is the absorbance of the blank (without test extract) and At the absorbance of the sample (with test extract) 2.7 Cell lines and cell culture HepG2 cells (HB-8065) were purchased from the American Type Culture Collection (Manassas, Rockville) Cells were cultured in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% (v/v) FBS, mM L-glutamine, 20 mM HEPES, 0.025 μg/mL amphotericin B, 100 IU/mL penicillin G, and 100 μg/mL streptomycin at 37 °C and 5% CO2 These chemicals were purchased from Sigma-Aldrich® Co Ltd (USA) Cells used in this study were between passages and 20 2.8 SRB assay The assay was performed as previously described (Nguyen et al., 2016) Cells were loaded at a density of 104 cells/well in 96-well plates and cultured for 24 h before being incubated with AGP-XS at different concentrations for 48 hours Treated cells were fixed with cold 50% (w/v) trichloroacetic acid (Merck) solution for 1–3 hours, washed and stained with 0.2% (w/v) SRB (Sigma) for 20 minutes After five washes with % acetic acid (Merck), protein-bound dye was solubilized in 10 mM Tris base solution (Promega) After that, optical density values were recorded at 492 nm and 620 nm using a plate reader (Synergy HT, Biotek Instruments) The percentage of the growth inhibition (I%) was estimated using the expression: In which ODt and ODc are the optical density value of the test sample and the control sample, respectively Camptothecin (Calbiochem) was used as a positive control 2.9 AO/EB double staining Cells seeded in 6-well plates (2 × 105 cells/well) were grown at 37 °C, 5% CO2 for 24 hours Cells were then treated with AGP-XS at concentration of 60, 80 and 100 μg/mL After 60 hours treatments, cells were washed with PBS and stained with a solution of AO-EB (100– 100 μg/mL) Cell morphology was observed by fluorescence microscopy 2.10 Data analysis The results were expressed in terms of mean ± SEM (Standard error of the mean) using MS Excel 2016 software Data were analyzed by Graphpad Prism software (Inc., La Jolla, CA, USA) and Systat statistical software (Inc., SigmaStat) using t-test and One-way ANOVA following by Student-Newman-Keuls test Results 3.1 Phytochemical analysis The extract was subjected to qualitative chemical tests for the identification of various secondary metabolites present in AGP-XS The results showed that the AGP-XS the possible presence of flavonoids, alkaloids, tannins, triterpenes, saponins, volatile oils, and organoic acids (Table 1) 3.2 Total polyphenol and flavonoid contents Total polyphenol and flavonoid contents of dry powder and AGP-XS of X strumarium were expressed as mg Quercetin equivalents/g and mg Gallic acid equivalents/g of dry mass, sequentially Table represents the analytical data for polyphenol and flavonoid contents of the dry powder and AGP-XS 3.3 In vitro antioxidant activity There is conglomerating evidence that reactive free radicals cause cell damage, which is one of the causes of aging and leading to a lot of diseases such as Alzheimer’s, Parkinson’s and cancer Thus, antioxidants have get significant consideration due to the potential of reducing adverse effects of ROS In this study, DPPH assay was used to confirm the free radical scavenging proficiency of AGP-XS, which are commonly applied to evaluate the antioxidant activity of plant extracts Result illustrated that the scavenging abilities of AGPXS was concentration-dependent and at concentration of 187.5 μg/mL, the AGP-XS presented over 50% inhibition against DPPH free radical by 50.3% The IC50 values for DPPH radicals with AGP-XS (184.13 μg/mL) was higher than ascorbic acid (4.37 μg/mL) (Fig 1) 3.4 Cytotoxicity of AGP-XS on HepG2 cancer cell line To investigate the anticancer potential of AGP-XS against human cancer cell line, HepG2 cancer cell line was treated with divergent concentrations of AGP-XS (40, 60, 80, 100 μg/mL) for 48 hours The result of SRB assay revealed that the AGP-XS decreased the percent viability of HepG2 cells in a concentration-dependent manner (Table 3) AGP-XS exhibited IC50 value of 81.69 μg/mL, whereas the IC50 of Camptothecin was 0.079 μg/mL These results divulged morphological changes and contraction of cells leading to cell death induced by AGP-XS in the HepG2 cancer cell line compared to control cells (Supplement) 3.5 AGP-XS induced apoptosis on HepG2 cells The ability to induce apoptosis of AGP-XS was detected using acridine orange/ethidium bromide (AO/EB) dual staining on HepG2 treated with extract Results indicated that HepG2 cells showed some characteristics of apoptosis after 60 hours treatment with AGPXS AGP-XS or CPT treated cells exhibited nuclear condensation and apoptotic bodies (Fig 3) Simultaneously, there was a decrease in the number of cells and changes in cell morphology at concentrations of AGP-XS and CPT compared to control cells after 60 hours treatment (Fig and 3) Discussion In recent years, the use of medicinal materials in cancer control and treatment has been increasingly concerned due to the diverse presence of secondary metabolites with multiple biological properties (Greenwell et al., 2015, Ochwang’I et al., 2014) Preliminary phytochemical analysis illustrated the presence of secondary metabolites in AGP-XS such as flavonoids, alkaloids, tannins, triterpenes, saponins, volatile oils, and organoic acids This result is similar to previous study, both leaves and stems of X strumarium contain flavonoids, alkaloids, tannins, triterpenes, saponins, and cardiac glycosides (Kamboj et al., 2014) These secondary metabolites are announced to have a lot of biological and therapeutic activities (Lee et al., 2017) Among the various phytochemicals, polyphenols compounds have attracted the attention of scientists as well as its application in different fields such as pharmaceutical, nutraceutical, health, and cosmetic industries These compounds are common in the plants that can be considered as the part of the daily diet and are attracted as natural antioxidants (Panche et al., 2016) In this study, AGP-XS was determined total polyphenol and flavonoid contents using colorimetric methods based on the gallic acid and quercetin standard, respectively Result indicated that AGP-XS exhibited the total polyphenol and flavonoid contents with 84.86 mg gallic acid equivalent/g dry weight and 3.66 mg quercetin equivalent/g dry weight, sequentially Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are thought to play an important role in human diseases Overloading free radicals causes many negative impacts on biological systems It can combine and oxidize biological molecules such as proteins, lipids, and carbohydrates This causes damage to cells, tissues and organs, leading to cancer progression (Xu et al., 2017) In the present study, antioxidant activity of AGP-XS was evaluated using DPPH free radical scavenging assay and compared with ascorbic acid as positive control Result demonstrated that the AGP-XS presented DPPH free radical scavenging with IC50 value of 184.13 μg/mL Previous studies have shown that polyphenols, flavonoids, and tannin are considered as sources of antioxidants and scavenging activity Polyphenols or flavonoids has been shown to have antioxidant activity and inhibit the initiation or spread of oxidative reaction chains via donating a hydrogen molecule of hydroxyl groups, resulting in a more stable, less-reactive radical (Panche et al., 2016) The anticancer activity of AGP-XS was also investigated through sulforhodamine B (SRB) assay and acridine orange–ethidium bromide (AO–EB) dual staining on HepG2 cancer cell line The SRB assay is used in order in screening to evaluate the toxicity of the crude extracts from AGP-XS The SRB assay is a simple and sensitive colorimetric method for measuring the cytotoxicity of toxic substances and plant extracts against cancer cell lines The anionic dyes SRB will bind electrostatically with the positively charged part of the protein The amount of binding dye will reflect the total protein of the cells In the assay, the cells were fixed, washed and stained with SRB After that, the SRB will bind to the cell’s protein, which is dissolved to form a pink solution The optical density of the solution correlates with the total protein or number of cells The change in number of cells compared to the control (Camptothecin) reflects the cytotoxicity of the test samples (Houghton et al., 2007) The result revealed that AGP-XS showed cytotoxic activity on HepG2 with IC50 value of 81.69 μg/mL In addition, results also indicated that AGP-XS induced apoptosis on HepG2 cells through changes in cell morphology and apoptotic bodies formation after 60 hours treatment HepG2 cells showed some characteristics of apoptosis after 60 hours treatment with AGP-XS such as a decrease in the number of cells and changes in cell morphology, nuclear and chromatin condensation and late apoptotic cells with condensed or fragmented chromatids The presence of alkaloids, polyphenols, sesquiterpenoids and terpenoids in AGP-XS may be related to anticancer activity The alkaloids from many medicinal herbs have been shown anticancer activitiy with different mechanisms of action (Lu et al., 2012) Anthraquinones, coumarin and flavonoid belonging to the polyphenols group have been shown to have anticancer, antioxidant, and anti-inflammatory activities (Morsy et al., 2017, Niedzwiecki et al., 2016, Malik et al., 2016, Panche et al., 2016) Terpenoids are known as antitumor and anti-inflammatory compounds properties (Salminen et al., 2008) Sesquiterpenoids have many important biological activities, which are plentiful in X strumarium, exhibiting strong activities with antitumor, antibacterial, antiviral, and anti-inflammation (Vasas et al., 2011) Xanthatin and xanthinosin, sesquiterpenoids isolated from X strumarium was found to confer anticancer activity (Ramírez-Erosa et al., 2007, Liu et al., 2018) Therefore, the anticancer activity of the AGP-XS may be attributed to the contribution of these individual chemical components The anticancer ability can be considered the main pharmacological effect of X strumarium and has been extensively studied on types of cancer such as lung cancer, liver cancer, breast cancer, and cervical cancer Similar to present study, many previous studies also demonstrated that leaves, stems and roots extracts as well as isolated compounds such as xanthatin, xanthinosin, 8-epixanthatin-1α,5α-epoxide, and 1β-hydroxyl-5α-chloro-8-epi-xanthatin from X strumarium exhibited anti-proliferation and induced apoptosis activity on various cancer cell lines such as MDA-MB-231, MCF-7, HepG2, A549, HeLa, and HTC-15 via different signal pathways (Fan et al., 2019) This is the first study to demonstrate the anticancer activity of the above ground portion extract of X strumarium on HepG2 cancer cell line Results indicated that AGP-XS contains a wide variety of secondary metabolites that expressed antioxidant and anticancer activities based on the experiments performed However, more scientific evidence is needed to comprehensively evaluate the biological effects of AGP-XS Conclusion The present study demonstrated that the antioxidant and anticancer activities of the above ground portion extract of X strumarium These results collectively indicated that the above ground portion extract of X strumarium induced apoptosis on HepG2 cancer cell line via evaluating in cell morphological characteristics This results study also showed that the benefits of using medicinal plant in the topical treatment and administration of cancer Acknowledgments The authors express the gratitude to the National Institute of Medicinal Materials, Vietnam for the financial support under grant number 09/2019/HĐ-ĐTCS-TTS Conflict of interest statement All the authors of this manuscript declare that there is no conflict of interest References Al-Mekhlafi, F A., Abutaha, N., Mashaly, A M A., Nasr, F A., Ibrahim, K E., Wadaan, M A (2017) Biological activity of Xanthium strumarium seed extracts on different cancer cell lines and Aedes caspius, Culex pipiens (Diptera: Culicidae) Saudi J Biol Sci., 24, 817-821 Chen, Y B., Yan, M L., Gong, J P., Xia, R P., Liu, L X., Lu, S C., Zhang, J G., Zeng, D P., Xie, J G., Yang, J Y., Yan, L N (2007) Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1 Chinese Med J (Engl), 120, 703-707 Chumark, P., Panya, K., Yupin, S., Srichan, P., Noppawan, M (2008) The in vitro and ex vivo antioxidant properties, hypolipidaemic and antitherosclerotic activities of water extract of Moringa oleifera Lam Leave Journal of Ethnopharmacology, 119, 439-436 Ciulei I., 1982 Methodology for Analysis of Vegetables Drugs Ministry of Chemical Industry; Bucarest, Roumania, 67 Fan, W., Fan, L., Peng, C., Zhang, Q., Wang, L., Li, L., Wang, J., Zhang, D., Peng, W., Wu, C (2019) Traditional Uses, Botany, Phytochemistry, Pharmacology, Pharmacokinetics and Toxicology of Xanthium strumarium L.: A Review Molecules (Basel, Switzerland), 24(2), 359 Greenwell, M., Rahman, P K (2015) Medicinal Plants: Their Use in Anticancer Treatment International journal of pharmaceutical sciences and research, 6(10), 4103-4112 Houghton, P., Fang, R., Techatanawat, I., Steventon, G., Hylands, P J., Lee, C C (2007) The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity Methods, 42(4), 377-387 Kamboj, A., Atri, P., Saluja, A K (2014) Phytochemical screening, in-vitro evaluation of antioxidant and free radical scavenging activity of leaves, stems and roots of Xanthium strumarium L.,(Compositae) British Journal of Pharmaceutical Research, 4(1), 1-22 Kumar, M., Zhao, X., Wang, X W (2011) Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: One step closer to personalized medicine? Cell Biosci., 1, Lee, M T., Lin, W C., Yu, B., Lee, T T (2017) Antioxidant capacity of phytochemicals and their potential effects on oxidative status in animals - A review Asian-Australasian journal of animal sciences, 30(3), 299-308 Liu, R., Shi, D., Zhang, J., Li, X., Han, X., Yao, X., Fang, J (2018) Xanthatin Promotes Apoptosis via Inhibiting Thioredoxin Reductase and Eliciting Oxidative Stress Mol Pharm., 15, 32853296 Lu, J J., Bao, J L., Chen, X P., Huang, M., Wang, Y T (2012) Alkaloids isolated from natural herbs as the anticancer agents Evidence-based complementary and alternative medicine: eCAM, 2012, 485042 Malik, E M., Müller, C E (2016) Anthraquinones As Pharmacological Tools and Drugs Medicinal Research Reviews, 36(4), 705748 Morsy, S A., Farahat, A A., Nasr, M., Tantawy, A S (2017) Synthesis, molecular modeling and anticancer activity of new coumarin containing compounds Saudi pharmaceutical journal: SPJ: the official publication of the Saudi Pharmaceutical Society, 25(6), 873-883 Newell, P., Villanueva, A., Friedman, S L., Koike, K., Llovet, J M (2008) Experimental models of hepatocellular carcinoma J Hepatol., 48, 858-879 Nguyen, M N., Ho-Huynh, T D (2016) Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects BMC complementary and alternative medicine, 16, 220 Nguyen, T T., Parat, M O., Hodson, M P., Pan, J., Shaw, P N., Hewavitharana, A K (2015) Chemical Characterization and in Vitro Cytotoxicity on Squamous Cell Carcinoma Cells of Carica papaya Leaf Extracts Toxins (Basel), 8(1), Niedzwiecki, A., Roomi, M W., Kalinovsky, T., Rath, M (2016) Anticancer Efficacy of Polyphenols and Their Combinations Nutrients, 8(9), 552 Ochwang’I, D O., Kimwele, C N., Oduma, J A., Gathumbi, P K., Mbaria, J M., Kiama, S G (2014) Medicinal plants used in treatment and management of cancer in Kakamega County Kenya Journal of Ethnopharmacology, 151, 1040-1055 Panche, A N., Diwan, A D., Chandra, S R (2016) Flavonoids: an overview Journal of nutritional science, 5, e47 Ramírez-Erosa, I., Huang, Y., Hickie, R A., Sutherland, R G., Barl, B (2007) Xanthatin and xanthinosin from the burs of Xanthium strumarium L as potential anticancer agents Can J Physiol Pharmacol., 85, 1160-1172 Salminen, A., Lehtonen, M., Suuronen, T., Kaarniranta, K., Huuskonen, J (2008) Terpenoids: natural inhibitors of NF-kB signaling with anti-inflammatory and anticancer potential Cell Mol Life Sci., 65, 2979-2999 Shrestha, P M., Dhillion, S S (2003) Medicinal plant diversity and use in the highlands of Dolakha district, Nepal Journal of Ethnopharmacology, 86(1), 81-96 Tao, L., Cao, Y., Wei, Z., Jia, Q., Yu, S., Zhong, J., Wang, A., Woodgett, J.R., Lu, Y (2017) Xanthatin triggers Chk1-mediated DNA damage response and destabilizes Cdc25C via lysosomal degradation in lung cancer cells Toxicol Appl Pharmacol., 337, 85-94 Vasas, A., Hohmann, J (2011) Xanthane sesquiterpenoids: Structure, synthesis and biological activity Nat Prod Rep., 28, 824-842 Xu, D P., Li, Y., Meng, X., Zhou, T., Zhou, Y., Zheng, J., Zhang, J J., Li, H B (2017) Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and Resources International journal of molecular sciences, 18(1), 96 Table Preliminary phytochemical analysis results of AGP-XS via chemical reactions Metabolites AGP-XS Alkaloids + Flavonoids + Tannins + Triterpenoids + Saponins + Coumarins Anthraquinones (+): the presence; (−): the absence Metabolites Proanthocyanidins Anthocyanosids Lipids Volatile oils Carotenoids Organic acids Reducing agents AGP-XS + + - Table Total polyphenol and flavonoid contents in AGP-XS Sample Polyphenols (mg GAE/g d.w.) Flavonoids (mg QE/g d.w.) 4.68 ± 0.19 1.23 ± 0.02 84.86 ± 5.13a 3.66 ± 0.08a Dry powder Extract a p < 0.05 significantly different, mg GAE/g d w.: mg of gallic acid equivalents/1 g of dry weight, mg QE/g d w.: mg of quercetin equivalents/1 g of dry weight Table Cytotoxicity of AGP-XS on HepG2 cancer cell line Conc (μg/mL) AGP-XS % cytotoxicity 40 60 80 100 12.44 ± 2.22 30.22 ± 1.14 45.57 ± 3.72 73.27 ± 3.35 IC50 (μg/mL) 81.69 ± 1.55 Fig DPPH scavenging activity of AGP-XS and ascorbic acid All values are reported as means ± SEM (n = 3) Fig Morphological transforms were observed in HepG2 cancer cells treated without (Control) or with different concentrations of AGP-XS (60, 80 and 100 μg/mL) and Camptothecin (CPT) for 60 hrs Fig Apoptotic characteristics of Hep-G2 cells treated with AGP-XS at different concentrations (60, 80 and 100 μg/mL) and Camptothecin (CPT) for 60 hrs by AO-EB dual staining Red arrows indicate condensed or fragmented chromatins; yellow arrows indicate late apoptotic cells with condensed or fragmented chromatins