Evaluation of anti-proliferative activity of medicinal plants used in Asian Traditional Medicine to treat cancer

Evaluation of anti-proliferative activity of medicinal plants used in Asian Traditional Medicine to treat cancer

Journal of Ethnopharmacology 235 (2019) 75–87 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier...

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Journal of Ethnopharmacology 235 (2019) 75–87

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm

Evaluation of anti-proliferative activity of medicinal plants used in Asian Traditional Medicine to treat cancer

T

Yin-Yin Siewa, Hui-Chuing Yewa, Soek-Ying Neoa, , See-Voon Seowb,c, Si-Min Lewa, Shun-Wei Lima, Claire Sophie En-Shen Lima, Yi-Cheng Nga, Wei-Guang Seetoha, Azhar Alid, ⁎ Chay-Hoon Tane, Hwee-Ling Koha, ⁎

a

Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore c Affiliated National University Cancer Institute, National University Health System, Singapore 119074, Singapore d Cancer Science Institute of Singapore, 14 Medical Drive, Singapore 117599, Singapore e Department of Pharmacology, Yong Loo Lin School of Medicine, 16 Medical Drive, Block MD3, #04-01S, Singapore 117600, Singapore b

ARTICLE INFO

ABSTRACT

Keywords: Cancer Cytotoxicity Traditional medicine Asia and Oceania

Ethnopharmacological relevance: The extensive biodiversity of plants in Southeast Asia and inadequate research hitherto warrant a continued investigation into medicinal plants. On the basis of a careful review of fresh medicinal plant usage to treat cancer from previous ethnobotanical interviews in Singapore and from the traditional uses of the indigenous plants, fresh leaves of seven locally grown medicinal plant species were evaluated for anti-proliferative activity. Aim of the study: To evaluate the anti-proliferative activity of local medicinal plant species Clausena lansium Skeels, Clinacanthus nutans (Burm. f.) Lindau, Leea indica (Burm. f.) Merr., Pereskia bleo (Kunth) DC., Strobilanthes crispus (L.) Blume, Vernonia amygdalina Delile and Vitex trifolia L. Materials and method: Fresh, healthy and mature leaves of the seven medicinal plants were harvested from various locations in Singapore and Malaysia for Soxhlet, ultrasonication and maceration extractions in three different solvents (water, ethanol and methanol). Cell proliferation assay using water soluble tetrazolium salt (WST-1) assay was performed on twelve human cancer cell lines derived from breast (MDA-MB-231, T47D), cervical (C33A), colon (HCT116), leukemia (U937), liver (HepG2, SNU-182, SNU-449), ovarian (OVCAR-5, PA1, SK-OV-3) and uterine (MES-SA/DX5) cancer. Results: A total of 37 fresh leaf extracts from seven medicinal plants were evaluated for their anti-tumour activities in twelve human cancer cell lines. Of these, the extracts of C. lansium, L. indica, P. bleo, S. crispus, V. amygdalina and V. trifolia exhibited promising anti-proliferative activity against multiple cancer cell lines. Further investigation of selected promising leaf extracts indicated that maceration methanolic extract of L. indica was most effective overall against majority of the cancer cell lines, with best IC50 values of 31.5 ± 11.4 µg/mL, 37.5 ± 0.7 µg/mL and 43.0 ± 6.2 µg/mL in cervical C33A, liver SNU-449, and ovarian PA-1 cancer cell lines, respectively. Conclusion: The results of this study provide new scientific evidence for the traditional use of local medicinal plant species C. lansium, L . indica, P. bleo, S. crispus, V. amygdalina and V. trifolia in cancer treatment. These results highlight the importance of the upkeep of these indigenous plants in modern society and their relevance as resources for drug discovery.

Abbreviations: CL, Clausena lansium; CN, Clinacanthus nutans; DMSO, dimethyl sulfoxide; IC50, half maximal inhibitory concentration; LI, Leea indica; Mac E, maceration 70% (v/v) ethanol; Mac M, maceration 70% (v/v) methanol; NCI, National Cancer Institute; PB, Pereskia bleo; SC, Strobilanthes crispus; Sox E, Soxhlet 70% (v/v) ethanol; Sox M, Soxhlet 70% (v/v) methanol; Sox W, Soxhlet water; Ult E, ultrasonication 70% (v/v) ethanol; Ult M, ultrasonication 70% (v/v) methanol; Ult W, ultrasonication water; VA, Vernonia amygdalina; VT, Vitex trifolia; WST-1, Water soluble tetrazolium salt ⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-Y. Siew), [email protected] (H.-C. Yew), [email protected] (S.-Y. Neo), [email protected] (S.-V. Seow), [email protected] (S.-M. Lew), [email protected] (S.-W. Lim), [email protected] (C.S.E.-S. Lim), [email protected] (Y.-C. Ng), [email protected] (W.-G. Seetoh), [email protected] (A. Ali), [email protected] (C.-H. Tan), [email protected] (H.-L. Koh). https://doi.org/10.1016/j.jep.2018.12.040 Received 1 December 2018; Received in revised form 24 December 2018; Accepted 24 December 2018 Available online 30 December 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.

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1. Introduction

2.4. General cell culture methods

Locally grown medicinal plants are commonly used in the treatment of cancer in Southeast Asian countries such as Singapore and Malaysia, although allopathic medicine is the primary form of healthcare. Due to Singapore's rapid urbanization, scarcity of land and scant local effort to document herbal knowledge particularly fresh medicinal plants, there is pressing need to show the importance of upkeep of fresh medicinal plants and conduct scientific research on these indigenous plants to contribute to improved healthcare, as well as to the discovery of potential new drugs from existing remedies. In a previous ethnobotanical study by our group, we documented the usage of some of these locally grown medicinal plants and noted that some of these herbs are specifically used by this part of the world (Siew et al., 2014). In this study, seven local medicinal plant species, namely Clausena lansium Skeels, Clinacanthus nutans (Burm. f.) Lindau, Leea indica (Burm. f.) Merr., Pereskia bleo (Kunth) DC., Strobilanthes crispus (L.) Blume, Vernonia amygdalina Delile and Vitex trifolia L., were selected for anti-proliferative effects screening after consideration of their traditional uses, reported usage and published literature. This study aimed to evaluate the anti-proliferative activity of various leaf extracts of seven local medicinal plant species against a panel of human cancer cell lines derived from breast, cervical, colon, leukemia, liver, ovarian, and uterine cancer.

Twelve human cancer cell lines derived from breast (MDA-MB-231, T47D), cervix (C33A), colon (HCT116), leukemia (U937), liver (HepG2, SNU-182, SNU-449), ovary (OVCAR-5, PA-1, SK-OV-3) and uterus (MES-SA/DX5) were used. OVCAR-5 cells were obtained from the National Cancer Institute (NCI, Frederick, USA), while the rest were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (ThermoScientific, USA) supplemented with 10% heat-inactivated fetal calf serum (ThermoScientific, USA) in 5% CO2 incubator at 37 °C in a humidified atmosphere. 2.5. Cell proliferation WST-1 assay Exponentially growing cells were plated in 96-well plates at optimized cell densities: SNU-182, SNU-449, SK-OV-3, MES-SA/Dx5 (7 × 103 cells/100 µL); MDA-MB-231 (1 × 104cells/100 µL); HepG2, T47D (1.1 × 104 cells/100 µL); HCT116 (2 × 104 cells/100 µL); PA-1 (2.5 × 104 cells/100 µL); OVCAR-5, C33A, U937 (3 × 104 cells/ 100 µL). Adherent cells were incubated overnight at 37 °C and 5% CO2 to allow attachment, while pre-incubation was not carried out for nonadherent U937 cells. After 48 h of treatment with the appropriate agent (extract/drug/vehicle control), media was aspirated and replaced with 10% v/v cell proliferation reagent WST-1 (Roche, Switzerland) in complete media. For U937 cells which are non-adherent, cells were plated in round-bottom 96-well plates and centrifuged 5 min × 125g prior to aspiration of media. Cells were incubated for 30–60 min at 37 °C and 5% CO2. The formazan dye produced was quantitated at 440 nm against a reference wavelength of 650 nm using a microplate reader (Tecan infinite M200 PRO, Switzerland). Cell viability was expressed as percentage of the vehicle control. The half maximal inhibitory concentration (IC50) values were determined using GraphPad Prism 6 (GraphPad Software, Inc., USA). The results were generated from three independent experiments and each experiment was performed in triplicate.

2. Material and methods 2.1. Plant sources Fresh, healthy and mature leaves were harvested for extraction. The leaves of C. lansium, P. bleo, V. trifolia, S. crispus, C. nutans and L. indica were obtained from Singapore while the leaves of V. amygdalina were obtained from Malaysia. Voucher specimens of the plants were deposited at the Department of Pharmacy Herbarium in National University of Singapore. The details of medicinal plants along with their voucher specimen codes are listed in Table 1.

3. Results and discussion 2.2. Identification of plants

Plants are promising sources of anticancer chemotherapeutic agents. The anti-proliferative activity of a total of 37 leaf extracts of 7 plant species was investigated. A summary of their scientific names, local common names, plant characteristics, traditional uses and pharmacological properties are presented in Table 1. Traditionally, all these plants have been used to treat cancer or tumour.

All the plant names have been checked with The Plant List (2013) at http://www.theplantlist.org. In addition, C. lansium and L. indica were identified with reference to the book “Encyclopedia of Medicinal Plants” (Zhao and Xiao, 2009) and the journal article “Leea L. (Vitaceae) Of Singapore” (Lok et al., 2011), respectively. S. crispus was identified by Dr. John Wood from Forest Herbarium, Oxford. V. trifolia and V. amygdalina was identified with reference to the “World Checklist of Selected Plant Families” (WCSP, 2015). P. bleo was identified with reference to “The IUCN Red List of Threatened Species” (Griffith et al., 2013). C. nutans was identified with reference to eFloras (eFloras, 2008).

3.1. Anti-proliferative activities of leaf extracts from seven plants Leaf extracts of seven plants obtained from various extraction methods were initially screened against a panel of human cancer cell lines comprising of breast, cervical, colon, leukemia, liver, ovarian, and uterine cancer. The plant extract concentrations tested were at 2 mg/ mL and the treatment duration was 48 h. The 48 h treatment duration is used by the National Cancer Institute's Developmental Therapeutics Program (DTP) for the preliminary in vitro screening of crude plant extracts in human tumour cell lines. The viability of control cells not treated with any extract was designated as 100%, and the extracttreated cells were expressed as percentage compared to the control. The percentages of cell survival presented in Table 2 are classified into four categories and colour-coded for clearer visual comparison: ≤ 20.0% cell viability denotes “strong anti-proliferative activity” (red); 20.1–50.0% cell viability denotes “moderately strong anti-proliferative activity” (orange); 50.1–75.0% cell viability denotes “weak anti-proliferative activity” (yellow); ≥ 75.1% cell viability denotes “no antiproliferative activity” (green).

2.3. Preparation of plant extracts Leaves were washed, blotted dry and blended using a dry grinder and extracted using Soxhlet, ultrasonication and maceration with water, 70% ethanol and 70% methanol. The crude extracts were filtered, concentrated under reduced pressure by rotary evaporator and lyophilized for 72 h to yield dried powder. All the dried extracts were stored under vacuum in desiccators at 25 °C. The dried extracts were dissolved in dimethyl sulfoxide (DMSO) and diluted to the desired concentration before addition to cells. The concentration of DMSO in these dilutions was restricted to no more than 0.4% to minimize potential effects of the solvent on cell growth. 76

Clinacanthus nutans (Burm.f.) Lindau Sabah Snake Grass Belalai Gajah 憂遁草 CN-0054

Clausena lansium (Lour.) Skeels Fool's Curry Leaf Wampee 黄皮 CL-001

1

2

Plant scientific names Common names Voucher specimen codes

No.

C. nutans is a tall, erect and sometimes rambling shrub. The stems are cylindric, leaf blade lanceolate to ovatelanceolate (Pieroni and Vandebroek, 2007).

C. lansium is a species of strongly scented evergreen tree 3 to 8 m tall. The leaves are smooth and dark green (Kuvatanasuchati et al., 2011).

Plant characteristics

The fresh leaves are used traditionally to treat insect and snake bites (Sakdarat et al., 2009). The ethanolic leaf extract have also been used clinically in the form of topical cream to treat Herpes genitalis and Herpes zoster lesions (Kongkaew and Chaiyakunapruk, 2011). In Singapore and Malaysia, numerous anecdoctal reports indicate the leaves are used to treat various cancers (Yong et al., 2013; Siew et al., 2014)

The leaves are used to treat asthma, hepatitis, cold, rheumatic arthralgia, gastrointestinal inflammation, malaria and cancer (Van Sam, 2008; Yang et al., 1988).

Traditional uses

Table 1 Characteristics, traditional uses and pharmacological properties of the seven medicinal plants studied.

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Anti-allergenic (Kow et al., 2017) Antibacterial (Chithra et al., 2016) Anticancer activity of whole plant extract against human erythroleukemia (K562) and human Burkitt's lymphoma (Raji) cell lines (Yong et al., 2013). The hexane extract demonstrated strong antiproliferative activity against non-small cell lung cancer (A549), nasopharygeal cancer (CNE1), liver cancer (HepG2) cell lines (Ng et al., 2017). The aqueous extract exerted a significant cytotoxic effect on HeLa cells (Yusmazura et al., 2017). The methanol extract demonstrated strong cytotoxic effect against liver Hep-G2 cancer cells and breast cancer (MDA-MB-231) cells (Quah et al., 2017). The methanol and ethyl acetate root extracts were found to inhibit the proliferation of MCF-7 breast cancer cells (Teoh et al., 2017). Crude methanol leaf extract exhibited cytotoxicity to the D24 melanoma cells (24 h EC50: 0.95 mg/mL and 72 h EC50: 0.77 mg/mL) (Fong et al., 2016). The water extract demonstrated an IC50 of 138.82 ± 0.60 µg/mL against A549 human lung carcinoma cells (Fazil et al., 2016) Anti-dengue virus activity (Sakdarat et al., 2017) Anti-inflammatory effect (Azam et al., 2017; Mai et al., 2016; Tan et al., 2016; Wanikiat et al., 2008) Antimicrobial activity (Yu et al., 2017) Anti-obesity activity (Abdulwahid et al., 2017) Antinociceptive activity (Rahim et al., 2016) Anti-oxidant (Alam et al., 2017; Kong et al., 2016; Sarega et al., 2016a, 2016b; Yong et al., 2013; Yu et al., 2017) Antiproliferative (Yong et al., 2013)

α-glucosidase inhibitory activity (Alam et al., 2017)

Anticancer activity of fruit peel extract against human gastric carcinoma (SGC7901), human hepatocellular liver carcinoma (HepG-2) and human lung adenocarcinoma (A-549) cancer cell lines (Prasad et al., 2009). The coumarins isolated demonstrated cytotoxicity against oral cavity KB, breast MCF7 and small cell lung NCI-187 cancer cells (Maneerat et al., 2010). 3-formylcarbazole isolated from the stem showed potent cytotoxic activity against leukemia K562, non-small lung H1299 and liver SMMC-7721 cancer cells (Jiang et al., 2013). Mafaicheenamine E isolated from the roots exhibited cytotoxicity against breast MCF-7 cancer cell line (Maneerat et al., 2012). The three amides isolated from the seeds were cytotoxic against oral cavity KB and small cell lung NCI-H187 cancer cells (Maneerat et al., 2011). Antidiabetic (Adebajo et al., 2009; Kong et al., 2018) Anti-inflammatory effect (Adebajo et al., 2009; Du et al., 2015; Shen et al., 2014; Shen et al., 2017; Xu et al., 2014) Antimicrobial (Adebajo et al., 2009; Xu et al., 2014) Anti-obesity (Huang et al., 2017) Anti-oxidant (Adebajo et al., 2009; Prasad et al., 2009) Hepatoprotective (Adebajo et al., 2009; Du et al., 2015) Insecticidal (Guo et al., 2016) Lipid-lowering (Kong et al., 2018) Neuroprotection (Liu et al., 2014; Liu et al., 2017) Tyrosinase inhibitor (Chai et al., 2017)

Pharmacological properties

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Plant scientific names Common names Voucher specimen codes

Leea indica (Burm. f.) Merr. Bandicoot Berry Memali 岩陀 LI-0109

Pereskia bleo (Kunth) DC. Rose Cactus Seven Star Needle 七星针 PB-0003

Strobilanthes crispus (L.) Blume Black Face General Pokok Pecah Beling 黑面将军 SC-0090

No.

3

4

5

Table 1 (continued)

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S. crispus is a woody spreading shrub that can easily reach more than 1 m in height in cultivation. The leaves are elliptical in shape and are rough to the touch (Bich and Tap, 2003).

P. bleo is a leafy cactus shrub about 2 m tall with large, orange flowers. The long thorns on the branches are up to 5–10 mm long. (Butterworth and Wallace, 2005).

L. indica is a perennial shrub with ribbed branches and swollen nodes. It can grow up to 16 m tall. The pinnately compound leaves are large and toothed (Lok et al., 2011).

Plant characteristics

The boiled leaves are used to treat cancer, diabetes and used as a laxative (Goh, 2004).

The leaves are used to treat cancer, diabetes, hypertension, body revitalization, gastric discomfort, ulcers and inflammatory conditions. Users consume either the raw leaves, or a concoction made from fresh leaves to treat a range of conditions (Malek et al., 2009). A review of botanical characteristics, traditional usage, chemical components, pharmacological activities, and safety of P. bleo has been published (Zareisedehizadeh et al., 2014).

The plant is used to treat leucorrhea, intestinal cancer, and uterus cancer (Saralamp, 1997). The dried leaves are consumed as tea beverage and are believed to be effective against cancer (Bourdy and Walter, 1992). The leaves are also used in diabetes and the ointment prepared from roasted leaves is used to relieve vertigo (Srinivasan et al., 2008; Dhar et al., 1968).

Traditional uses

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Anticancer activity of the S. crispus leaf extract against hormone dependent breast cancer cell line MCF-7 but not the non-hormone dependent breast cancer cell line MDA-MB-231 (Bakar et al., 2006; Chong et al., 2012). The essential oil from S. crispus was not cytotoxic against liver cancer (HepG2), colon cancer (Caco-2), MDA-MB-231 (Rahmat et al., 2006b). The methanol extract had the strongest cytotoxicity against Caco-2, followed by MDA-MB-231 and HepG2 (Rahmat et al., 2006a). The stem ethyl acetate and leaf water extract demonstrated IC50 value of 38 µg/ml and 23 µg/ml respectively against MCF-7 breast cancer cells (Gordani et al., 2017). The stem hexane extract exhibited IC50 of 38.80 ± 8.50 µg/mL and 42.50 ± 39.67 µg/mL against HepG-2 and MDA-MB-231 cells (Koh et al., 2017). The active sub-fraction of leaves demonstrated growth inhibitory effects similar to that of tamoxifen (Yaacob et al., 2015). γ-sitosterol isolated was cytotoxic against Caco-2, HepG2, and MCF-7 with IC50 values of 8.3, 21.8, and 28.8 μg/mL, respectively (Endrini et al., 2014). Antidiabetic (Fadzelly et al., 2006) Anti-inflammatory (Ling et al., 2016) Anti-obesity (Zawawi et al., 2016) Anti-oxidant (Bakar et al., 2006; Rahmat et al., 2006b) Enzyme inhibition (Pan et al., 2016)

Anticancer activity of alpha-tocopherol isolated from P. bleo against human nasopharyngeal epidermoid carcinoma cell line (KB) (Malek et al., 2009), anticancer activity of methanol leaf and stem extract against human breast carcinoma cell line (T-47D) (Tan et al., 2005). The extracts have no significant anti-proliferative effect against the mouse mammary cancer cells (4T1) (Er et al., 2007), human breast adenocarcinoma (MCF-7) and human colorectal adenocarcinoma (HT-29) cell lines (Wahab et al., 2009) Antimicrobial (Malek et al., 2009; Wahab et al., 2009) Antinociceptive (Abdul-Wahab et al., 2012) Cell-penetrating properties (Loo et al., 2017) Snake venom neutralising property (Otero et al., 2000)

Anticancer activity of the leaf extracts against Ehrlich Ascites Carcinoma (EAC) cells in Swiss albino mice, cervical epidermoid carcinoma (Ca Ski) (Raihan et al., 2012; Wong et al., 2011), however no anticancer activity was demonstrated against colon cancer cell lines (HT-29, HCT-15 and HCT-116) (Reddy et al., 2012). The methanol and ethanol leaf extracts were found to be selectively cytotoxic in vitro to DU-145 and PC-3 prostate cancer cell lines (Ghagane et al., 2017) Antimicrobial (Harun et al., 2016) Anti-oxidant (Harun et al., 2016; Saha et al., 2004) Antiviral (Ali et al., 1996) Phosphodiesterase inhibition (Temkitthawon et al., 2008)

Antiviral (Haetrakul et al., 2016; Haetrakul et al., 2018; Kongkaew and Chaiyakunapruk, 2011; Pongmuangmul et al., 2016) Immunomodulatory activity (Sriwanthana et al., 1996) Immunosuppressive activity (Le et al., 2017) Insulin resistance prevention (Sarega et al., 2016a, 2016b) Neuronal protection (Tsai et al., 2016; Wu et al., 2018)

Pharmacological properties

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Plant scientific names Common names Voucher specimen codes

Vernonia amygdalina Delile Angiotensin-Iconverting enzyme inhibition (Oboh et al., 2016) 南非叶 VA-0059

No.

6

Table 1 (continued)

V. amygdalina is a perennial shrub of 2–5 m in height. It has a rough bark with dense black straits, and elliptic leaves that are up to 20 cm long. The leaves are green and have a characteristic odour and bitter taste (Ijeh and Ejike, 2011).

Plant characteristics

The leaves are used to treat fever, hiccups, kidney problems, and stomach discomfort (Burkill, 1995). Professor Ernest Izevbigie from Jackson State University has patented a phytochemotherapeutic compositions produced from the aqueous extracts (and fractions thereof) isolated from V. amygdalina leaves. This formula was shown to inhibit the growth of neoplastic cells, including human breast cancer cells (U.S. Patent 6713098 and Izevbigie, 2004).

Traditional uses

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Antacid and carminative (Mbatchou et al., 2017) Antibacterial (Chikwendu et al., 2016; Enyi-Idoh et al., 2012; Evbuomwan et al., 2018; Ngumah et al., 2016; Oshim et al., 2016) Anti-benign prostatic hyperplasia (Ajayi et al., 2017) Anticancer activity of the leaf extract against breast cancer cell lines (MCF-7), BT-549, (Gresham et al., 2008; Izevbigie, 2003; Opata and Izevbigie, 2006; Oyugi et al., 2009; Wong et al., 2013; Yedjou et al., 2008; Yedjou et al., 2013). The root extract was cytotoxic against primary cells harvested from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients (Khalafalla et al., 2009). V. amygdalina significantly reduced the viability of MCF-7 cells in a dose-dependent response (Yedjou and Tchounwou, 2017). The leaf extract was found to enhance the chemotherapeutic vulnerability of breast cancer cells (Howard et al., 2016). The IC50 of the leaf extract against PC-3 prostate cancer cells was 196.6 µg/mL (Johnson et al., 2017). The IC50 of ethyl acetate extract against U-87 glioblastoma multiforme was 48.0 ± 26.72 μg/mL (Rohin et al., 2017). Anti-Chikungunya (Chan et al., 2016) Anti-cholera (Shittu et al., 2016) Antidiabetic effect (Adeoye et al., 2017b; Akah et al., 2004; Asante et al., 2016; Egedigwe et al., 2016a; Egedigwe et al., 2016b; Mgbeje et al., 2016b; Okon and Olatunbosun, 2017; Ojieh et al., 2016; Okoduwa et al., 2017; Otitolaiye et al., 2017) Anti-hyperlipidemia (Oluwafemi et al., 2016; Rani et al., 2016) Anti-inflammatory (Adedapo et al., 2014; Quasie et al., 2016) Antimalarial (Cissy et al., 2016; Iwalokun, 2008; Okpe et al., 2016; Omoregie and Pal, 2016; Razak et al., 2014) Anti-oxidant (Adedapo et al., 2014; Adeoye et al., 2017a; Adeoye et al., 2017b; Alara et al., 2017; Atangwho et al., 2013; Ho et al., 2015; Okon and Umoren, 2017; Olufunsho and Ayodele, 2017; Olugbuyiro et al., 2017; Oluwafemi et al., 2016) Antiparasitic (Tadesse et al., 1993) Antipyretic (Tijjani et al., 2017) Anxiolytic (Onasanwo et al., 2016) Chemoprotective effect (Adesanoye et al., 2016) Gastro-protective effect (Adefisayo et al., 2017) Haemoglobin level decrease (Kadir et al., 2018) Hepatoprotection (Adesanoye et al., 2016; Ho et al., 2015; Iwo et al., 2017; Kadiri, 2017; Mgbeje et al., 2016a) Hypoglygcaemic (Akah and Okafor, 1992; Atangwho et al., 2014) Neuroprotective effect (Ademosun et al., 2017; Ebuehi and Ajagun-Ogunleye, 2017) Vasorelaxant property (Ch’ng et al., 2017)

Bitter Leaf South African Leaf

Analgesic (Tijjani et al., 2017)

Immunomodulatory effects (Yankuzo et al., 2018)

Pharmacological properties

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Journal of Ethnopharmacology 235 (2019) 75–87

3.1.1. Clausena lansium The C. lansium Soxhlet leaf extracts showed strong or moderately strong anti-proliferative activity, that is less than 50% cell viability, in liver (SNU-182, SNU-449, HepG2), ovarian (PA-1), cervical (C33A), breast (MDA-MB-231, T47D) and colon (HCT116) cells (Table 2). Other cancer cell lines generally showed more than 75% cell viability in the C. lansium leaf extracts. In general, the organic solvent (less polar) extracts were more active, with Soxhlet extraction giving rise to more active extracts compared to ultrasonication. Our observations of the potency of Soxhlet organic solvent leaf extracts in liver cancer cell viability are comparable to that of fruit peel extracts from the same plant reported by Prasad et al. (2009). Ethyl acetate fraction of 50% ethanol fruit peel extract at a concentration of 50 µg/mL was reported to show 72.4 ± 0.65% inhibitory activity in HepG2 cells (Prasad et al., 2009). C. lansium has been traditionally used in the treatment of cancer (Yang et al., 1988; Van Sam, 2008). Interestingly out of 230 species of medicinal plants used by the local people in Ben En National Park, Vietnam, C. lansium is the only species reported for the treatment of cancer (Van Sam et al., 2008). The leaves and fruits are boiled in water and the decoction consumed. Given the promising anti-proliferative results shown here together with previous reports (see references in Table 1) and its traditional usage against cancer, further investigation of the leaves and fruit peels of C. lansium in particular against liver, ovarian, cervical, breast and colon cancer is necessary.

Woung healing (Ruslim et al., 2017; Nafiu et al., 2016) Anticancer activity of the fruit extract of V. trifolia against tsFT210 mouse mammary carcinoma cells (Li et al. 2005). The leaf extract was cytotoxic against HepG2 liver carcinoma and cervical carcinoma (HeLa) cells (Vasanthi et al., 2014). Vitetrifolin H, vitetrifolin I and monoterpenoid vitexoid isolated from the fruits inhibited HeLa cell proliferation (Wu et al., 2009). 3β-hydroxy30-al-urs-12-en-28-oic acid isolated from the fruits of V. trifolia showed significant cytotoxic activity against HL-60, SGC-7901, PANC-1, Eca-109 (Huang et al., 2016). Antifungal (Hernandez et al., 1999) Antioxidant (Sreedhar et al., 2010) Anti-bacterial (Hossain et al., 2001) Anti-inflammatory (Matsui et al., 2009; Bao et al., 2018) Free radical scavenging (Sreedhar et al., 2010) Hepatoprotective activity (Anandan et al., 2009) Insulin sensitizing effect (Nishina et al., 2017) Melanin synthesis inhibition (Lee et al., 2016) Mosquito larvicidal activity (Kannathasan et al., 2007) Topoisomerase I inhibitor (Luo et al., 2017) Tracheospasmolytic activity (Alam et al., 2002) Wound healing activitiy (Manjunatha et al., 2007) The leaves are used to treat rheumatism, dysentery, headache, fever, oral inflammation, infection and cancer (Samy et al., 2005; Matsui et al., 2009; Saklani et al. 2017). In Traditional Chinese Medicine, the fruits (Fructus Viticis) are specifically used to treat fever and headache, acute conjunctivitis, blurred vision and dizziness (The State Pharmacopoeia Commission of People's Republic of China, 2010; Li et al., 2005). V. trifolia is a large shrub, up to 8 m in height with the stems covered by soft hairs. The leaves composed of 3 linear leaflets ranging from 1 to 12 cm in length (Nadkarni, 1976). Vitex trifolia L. Simpleleaf Chastetree Legundi Lemuni 三叶蔓荆 VT-0101 7

Traditional uses Plant characteristics Plant scientific names Common names Voucher specimen codes No.

Table 1 (continued)

Pharmacological properties

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3.1.2. Clinacanthus nutans The C. nutans leaf extracts demonstrated either no or weak antiproliferative activity in the cell lines tested, that is 50–75% or more than 75% cell viability (Table 2), except for the Soxhlet methanolic extract with strong anti-proliferative activity against liver SNU-449 cancer cells (Table 2). It appeared to be the least active plant amongst the seven plants studied. A few studies on the anticancer effects of C. nutans extracts suggested that non-polar extracts generally have stronger anti-proliferative activity than polar extracts. For example, Yong et al. (2013) evaluated the anti-proliferative activity of aqueous, methanolic and chloroform leaf extracts against eight different cancer cell lines. The non-polar chloroform extract significantly inhibited the cell proliferation in the seven cancer cell lines (including HepG2), the aqueous extract inhibited five cell lines, and the methanol extract inhibited four cell lines. Ng et al. (2017) investigated C. nutans extracts from leaves and stems in five solvents (water, methanol, ethyl acetate, chloroform and hexane), and reported hexane and chloroform extracts had significant anti-proliferative activity in the three cancer cell lines tested (i.e. A549, CNE1 and HepG2 cells). Based on our findings and those of other groups (see references in Table 1), future anticancer studies could be focused on the more non-polar extracts. C. nutans is used in different parts of Asia to treat various ailments (Yong et al., 2013; Alam et al., 2016). An ethnobotanical study in Singapore found many who reportedly used the plant for cancer treatment, with the leaves decocted in water and then the liquid consumed (Siew et al., 2014). Layman testimonies and newspaper reports in Malaysia claimed the plant saved them from various cancers (Yong et al., 2013; Alam et al., 2016). More research is warranted to investigate its therapeutic claims. 3.1.3. Leea indica The L. indica leaf extracts prepared by Soxhlet, maceration and ultrasonication methods, remarkably demonstrated strong or moderately strong anti-proliferative activity against liver (SNU-182, SNU-449, HepG2), ovarian (PA-1, OVCAR-5, SK-OV-3), cervical (C33A), uterine (MES-SA/Dx5), breast (MDA-MB-231, T47D), colon (HCT116) cancer cells (Table 2). Interestingly, not all leaf extracts were active against leukemic U937 cells - all three Soxhlet extracts, maceration methanolic and ultrasonication methanolic extracts had strong or moderately strong anti-proliferative activity; whereas ultrasonication water showed no appreciable effect on cell viability. Regardless of extraction method 80

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Table 2 Effects of various leaf extracts (treated at 2 mg/mL) from seven plants on the percentage cell viability of human cancer cell lines. Data was derived from the average of 3 independent experiments, each with triplicate wells.

(continued on next page)

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Table 2 (continued)

and solvent of extraction, L. indica extracts were active against almost all cell lines tested, except for U937 cells. Traditionally, the dried leaves of L. indica are consumed as tea beverage and believed to be effective against cancer (Bourdy and Walter, 1992). The leaves have also been boiled or eaten raw for the treatment of benign growths (Siew et al., 2014). Our results here together with others (see references in Table 1) provide scientific data to its folk usage. To further evaluate the antiproliferative activity of L. indica, IC50 values were determined in these same panel of cell lines for Soxhlet (water, ethanolic, methanolic), and maceration methanolic extracts (see Section 3.2.2).

ovarian PA-1, and cervical C33A cancer cells. The ultrasonication methanolic leaf extract had strong anti-proliferative activity against C33A cancer cells. S. crispus is popularly consumed in Singapore as a medicinal herb, especially in the prevention and treatment of cancer (Siew et al., 2014). The leaves of this plant are typically boiled in water and then the decoction is drunk (Goh, 2004). Our work here together with other group findings (see references in Table 1) suggest further work is needed to investigate the folkloric usage of S. crispus in cancer. 3.1.6. Vernonia amygdalina The V. amygdalina methanolic leaf extracts showed generally stronger anti-proliferative activity compared to the aqueous leaf extracts (Table 2). Remarkably, methanolic leaf extracts exhibited strong or moderately strong anti-proliferative activity against all the cell lines tested (Table 2). The water leaf extracts were generally less active than organic leaf extracts. Traditionally, the leaves of V. amygdalina are used for treating cancer in Africa and Southeast Asia. In Ogun State, Nigeria, the leaves are used by traditional healers for the management of cancer (Soladoye et al., 2010). According to some users surveyed, the fresh leaves of V. amygdalina are chewed to treat cancer and diabetes (Siew et al., 2014). Based on the promising results presented in Table 2 and those of others (see references in Table 1), more studies are needed to understand its potential in cancer treatment. To further evaluate the anti-proliferative activity of V. amygalina, the Soxhlet and ultrasonication methanolic extracts were selected for the determination of IC50 values in the same panel of cancer cell lines (see Section 3.2.1).

3.1.4. Pereskia bleo The P. bleo leaf extracts generally showed strong or moderately strong anti-proliferative activity against breast (T47D), cervical (C33A), colon (HCT116), liver (SNU-182, SNU-449, HepG2), ovarian (PA-1) and uterine (MES-SA/Dx5) cancer cells (Table 2). The water extracts were generally less active compared to the organic solvent extracts, except against some cell lines (e.g. C33A, SNU449, PA-1 and MES-SA/Dx5). The IC50 values of P. bleo extracts on some of these cell lines have been previously reported by other groups: methanol extract was active against T47D with IC50 of 2 µg/mL (Tan et al., 2005) and HCT116 with IC50 of 41.6 µg/mL (Malek et al., 2009). An ethnobotanical survey carried out found many users who consumed the leaves of this plant for the treatment of cancer (Siew et al., 2014). Given its importance as a medicinal plant used to treat cancer, our results here along with others findings (see references in Table 1) suggest future research is required and more focus may be placed on selected cancer cell lines.

3.1.7. Vitex trifolia The V. trifolia leaf extracts generally demonstrated either strong or moderately strong anti-proliferative activity against the three liver cancer cell lines (SNU-182, SNU-449, HepG2), except for ultrasonication water extract which was weak to no appreciable effect (Table 2). The V. trifolia leaf extracts generally displayed strong anti-proliferative activity against ovarian PA-1 cells (with the exception of Soxhlet water extract). The methanolic and ethanolic leaf extracts showed more potent anti-proliferative effects than water extracts in the other ovarian

3.1.5. Strobilanthes crispus The S. crispus leaf extracts were generally not affecting the cell viability appreciably (mostly more than 75% viability), except for a few organic solvent extracts that displayed strong anti-proliferative activity against specific cancer cell lines (Table 2). For instance, the Soxhlet ethanolic extract had strong anti-proliferative activity against cervical C33A and breast MDA-MB-231 cells; while the Soxhlet methanolic leaf extract had strong anti-proliferative activity against liver SNU-449, 82

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cells. Hernandez et al. (1999) demonstrated that the dichloromethane leaf extract from sequential maceration was very cytotoxic against OVCAR-5 (ED50 = 2.9 µg/mL), however in the case of methanolic extract, the ED50 value was greater than 20 µg/mL. Hence, possible future work could involve extraction of the leaves with other solvents, e.g. dichloromethane, to investigate if the anti-proliferative activity could be further enhanced. The V. trifolia leaf extracts were generally potent against anti-proliferative activity in cervical C33A cells, breast MDAMB-231 and colon HCT116 cells (Table 2). The organic solvent extracts of V. trifolia demonstrated strong anti-proliferative activity against uterine MES-SA/Dx5 cells and leukemia U937 cells. The extracts of V. trifolia were less effective in inhibiting growth of T47D breast cancer cells compared with MDA-MB-231. In general, the water extracts were less active than the organic solvent extracts. In Indian traditional medicine, V. trifolia leaves are used to treat cancer, although there is no mention of which type of cancer (Umashanker and Shruti, 2011; Dhanamani et al., 2011). In addition to the leaves, the fruit of V. trifolia, is used as a folk medicine in parts of China to treat cancers (Saklani et al., 2017; Li et al., 2005). Our study here along with others (see references in Table 1) provide scientific evidence that supports the traditional use of V. trifolia in cancer treatment.

Table 3 IC50 values (µg/mL) of V. amygdalina Soxhlet methanolic (VA Sox M) and ultrasonication methanolic leaf extracts (VA Ult M). Cancer type

Breast Cervix Colon Leukemia Liver Ovary Uterus

Cancer cell lines

MDA-MB-231 T47D C33A HCT116 U937 HepG2 SNU-182 SNU-449 OVCAR-5 PA-1 SK-OV-3 MES-SA/Dx5

IC50 of V. amygdalina (µg/mL) VA Sox M

VA Ult M

653.4 ± 927.9 ± 472.4 ± > 1000 > 1000 786.9 ± 659.1 ± 340.3 ± > 1000 489.3 ± 695.8 ± 269.2 ±

646.3 ± 690.8 ± 347.5 ± 511.9 ± 618.2 ± 778.9 ± > 1000 556.0 ± > 1000 457.6 ± 820.4 ± 400.6 ±

26.1 54.8 27.9 98.3 51.1 37.9 22.1 30.6 37.7

53.7 59.7 60.7 19.4 69.3 94.1 89.7 73.4 102.3 82.0

Data are presented as mean ± SD from 3 independent experiments, triplicate for each.

had IC50 values close to the NCI criteria for crude extracts to select promising plants for further drug discovery (Suffness and Pezzuto, 1990). In colon HCT116 cancer cells, the IC50 values of L. indica extracts range between 71.7 and 100.8 µg/mL (Table 4). Our observations are similar to Reddy et al. (2012), who reported that the IC50 values of ethanol, hexane, ethyl acetate and water fractions against human colon HT-29, HCT-15 and HCT116 cancer cell lines were all greater than 100 µg/mL. This suggests L. indica extracts are not toxic against HCT116 cells. In breast cancer cells, we observed L. indica extracts had stronger anti-proliferative activity against MDA-MB-231 (IC50 of 79.8–97.0 µg/mL) than to T47D (IC50 of 146.4–241.2 µg/mL) (Table 4). These extracts, however, were not considered to be active according to the NCI criteria (Suffness and Pezzuto, 1990). Other studies also reported non-cytotoxicity of the plant extracts against breast cancer cell lines. Nurhanan et al. (2008) demonstrated that methanol extracts of L. indica did not exert any cytotoxicity in Sulforhodamine B assay against breast MCF-7 and T47D cancer cell lines (IC50 > 100 µg/mL in both cell lines). A study by Wong and Abdul Kadir (2011) using MTT cytotoxicity assay reported non-cytotoxicity of the ethyl acetate fraction in MCF-7 breast cancer cells (IC50 = 138.05 ± 19.16 μg/mL). We found that maceration methanolic extract of L. indica had good cytotoxicity against cervical C33A cancer cells (IC50 = 31.5 ± 11.4 µg/mL) (Table 4). Wong and Abdul Kadir (2011) demonstrated that the ethyl acetate fraction showed an IC50 value of 85.83 ± 6.01 μg/mL against the Ca Ski cervical cancer cells. They reported that mollic acid arabinoside isolated from the leaves of L. indica could induce mitochondria-mediated apoptosis in Ca Ski cells (Wong et al., 2012). The different IC50 values observed in these cervical cells may be due to the different cell lines tested. Amongst the three liver cancer cell lines tested, SNU-449 (mesenchymal) was most sensitive to the effects of the methanolic extract of L. indica obtained by maceration (IC50 = 37.5 ± 0.7 µg/mL), whereas HepG2 (epithelial) was most resistant (IC50 = 180.8 ± 41.2 µg/mL) to the extract (Table 4). Wong and Abdul Kadir (2011) demonstrated the poor cytotoxicity of the extracts towards HepG2 as significant decrease of cell viability was observed only at an extract concentration of 100 μg/mL and above. The IC50 values of the extracts for OVCAR-5 and SK-OV-3 were generally higher than that of PA-1 (Table 4). For maceration methanolic extract, the IC50 value in OVCAR-5 (IC50 = 122.5 ± 13.8 µg/mL) was higher than that of SK-OV-3 (IC50 = 82.1 ± 9.4 µg/mL). To the best of our knowledge, this is the first report of the evaluation of the anti-proliferative activity of L. indica on cancer cell lines of the ovary, uterus and leukemia.

3.2. Evaluation of IC50 values for V. amygdalina and L. indica leaf extracts Based on the promising preliminary screening results in Table 2, two plant species V. amygdalina and L. indica, were further investigated against the panel of 12 cancer cell lines for the half-maximal inhibitory concentration (IC50) values of selected extracts. 3.2.1. IC50 values of V. amygdalina leaf extracts The IC50 values of the methanolic Soxhlet and ultrasonication leaf extracts of V. amygdalina were evaluated in the 12 cancer cell lines and are presented in Table 3. The IC50 values of V. amygdalina extracts ranged from 269.2 µg/mL to greater than 1000 µg/mL (Table 3). Hence according to the National Cancer Institute (NCI) guideline (Suffness and Pezzuto, 1990), these extracts were considered as not cytotoxic. Our results show that the IC50 values for V. amygdalina Soxhlet and ultrasonication extracts in breast MDA-MB-231 cancer cells were 653.4 ± 26.1 µg/mL and 646.3 ± 53.7 µg/mL, respectively (Table 3). In contrast studies by Wong et al. (2013) reported that IC50 values for MDA-MB-231 were 83, 53, 46 µg/mL at 24, 48 and 72 h respectively. On the other hand, Izevbigie (2003) reported IC50 value of 5.68 ± 0.2 μg/mL for V. amygdalina extract in MCF-7 cells. One plausible explanation for the different IC50 values may be due to different extraction methods used. In the study by Wong et al. (2013), fresh leaves soaked in 80% ethanol were homogenized, left for 1 h and the extracted mixture was then filtered, and finally lyophilized. Izevbigie (2003) soaked leaves in cold water (1:1) overnight at 4 °C, crushed leaves by a gentle means to a mixture, filtered to remove particulate matter and filtration through a 0.45-μm filtration unit for sterilization. However using the same extraction method by Izevbigie (2003), Opata and Izevbigie (2006) reported an IC50 value of 218 μg/mL in the MCF-7 cells. Further investigation is needed to confirm the activity of V. amygdalina extracts in cancer cells. 3.2.2. IC50 values of L. indica leaf extracts The IC50 values of Soxhlet water, Soxhlet ethanolic, Soxhlet methanolic and maceration methanolic leaf extracts of L. indica were determined in the 12 cancer cell lines, and presented in Table 4. Organic solvent extracts of L. indica generally had stronger anti-proliferative activity than the aqueous extracts. In particular, the maceration methanolic extract showed the lowest IC50 values in a majority of the cell lines. Two cell lines, namely liver SNU-449 (IC50 = 37.5 ± 0.7 µg/mL) and cervical C33A (IC50 = 31.5 ± 11.4 µg/mL) cancer cells (Table 4),

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Table 4 IC50 values (μg/mL) of L. indica maceration methanolic (LI Mac M), Soxhlet ethanolic (LI Sox E), Soxhlet methanolic (LI Sox M), and Soxhlet water leaf extracts (LI Sox W). Cancer type

Cancer cell lines

IC50 of different L. indica extracts (µg/mL) LI Mac M

Breast Cervix Colon Leukemia Liver Ovary Uterus

MDA-MB-231 T47D C33A HCT116 U937 HepG2 SNU-182 SNU-449 OVCAR-5 PA-1 SK-OV-3 MES-SA/Dx5

97.0 241.2 31.5 89.1 129.5 180.8 83.4 37.5 122.5 43.0 82.1 52.4

± ± ± ± ± ± ± ± ± ± ± ±

LI Sox E 27.5 30.3 11.4 38.5 39.4 41.2 12.8 0.7 13.8 6.2 9.4 13.3

79.8 183.6 42.6 100.8 274.2 198.8 332.4 45.2 177.3 61.6 84.1 59.9

± ± ± ± ± ± ± ± ± ± ± ±

LI Sox M 11.7 47.2 14.5 15.1 18.3 52.7 59.6 9.3 13.7 2.4 7.7 16.0

85.8 146.4 76.6 71.7 154.7 175.2 166.4 107.9 105.2 133.9 114.0 55.4

± ± ± ± ± ± ± ± ± ± ± ±

LI Sox W 20.6 36.3 26.0 42.0 47.2 68.8 40.5 14.7 5.7 25.0 2.0 1.4

96.6 217.9 41.0 92.7 230.2 194.8 487.0 45.3 324.1 53.9 135.3 58.3

± ± ± ± ± ± ± ± ± ± ± ±

20.4 33.9 19.5 16.6 87.0 10.7 61.9 6.4 60.9 3.4 13.7 31.5

Data are presented as mean ± SD from 3 independent experiments, triplicate for each.

4. Conclusion

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In conclusion, the anti-proliferative activities of 37 leaf extracts from seven local medicinal plant species were investigated against 12 different human cancer cell lines derived from various tissues. Results indicated that some of the extracts of C. lansium, L . indica, P. bleo, S. crispus, V. amygdalina and V. trifolia exhibited promising anti-proliferative activity against multiple cancer cell lines, and evaluation of some of their IC50 values suggest the potential of these plant extracts for future investigation. These results provide new scientific evidence for the traditional use of local medicinal plants in the treatment of cancer, and highlight the importance of upkeep of these indigenous plants in contemporary society and their relevance as resources for drug discovery.

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CRediT authorship contribution statement Yin-Yin Siew: Investigation, Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing. Hui-Chuing Yew: Investigation, Formal analysis. Soek-Ying Neo: Investigation, Data curation, Formal analysis, Methodology, Supervision, Writing - review & editing. See-Voon Seow: Investigation, Formal analysis, Writing - review & editing. SiMin Lew: Investigation, Formal analysis. Shun-Wei Lim: Investigation, Formal analysis. Claire Sophie En-Shen Lim: Investigation, Formal analysis. Yi-Cheng Ng: Investigation, Formal analysis. Wei-Guang Seetoh: Investigation, Formal analysis. Azhar Ali: Writing - review & editing. Chay-Hoon Tan: Conceptualization, Writing - review & editing. Hwee-Ling Koh: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing - review & editing. Acknowledgements This study is supported by the National University of Singapore – Leeward Pacific Pte Ltd research collaboration grants 148-000-172-592 and R-148-000-140-592, and the National University of Singapore Provost Industrial PhD Programme Research Scholarship (to SYY). Classification Malignant disease and immunosuppression. 84

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