Cytotoxic Effects of Compounds Isolated from Ricinodendron Heudelotii

molecules

Article Cytotoxic Effects of Compounds Isolated from Ricinodendron heudelotii

Omolara F. Yakubu 1,2, Abiodun H. Adebayo 1,*, Titilope M. Dokunmu 1 , Ying-Jun Zhang 2,* and Emeka E.J. Iweala 1

1 Department of Biochemistry, College of Science and Technology, Covenant University, Canaan Land, Ota PMB 1023, Ogun State, Nigeria; [email protected] (O.F.Y.); [email protected] (T.M.D.); [email protected] (E.E.J.I.) 2 State Key Laboratory of Phytochemistry and Plant Resources of West China, Kunming Institute of Botany, Chinese Academy of Sciences. #132, Lanhei Road, Heilongtan, Kunming 650201, China * Correspondence: [email protected] (A.H.A.); [email protected] (Y.-J.Z.); Tel.: +234-80-3635-7197 (A.H.A.); +86-871-6522-3235 (Y.-J.Z.) Received: 10 August 2018; Accepted: 22 November 2018; Published: 2 January 2019

Abstract: This study was designed to explore the in vitro anticancer effects of the bioactive compounds isolated from Ricinodendron heudelotii on selected cancer cell lines. The leaves of the plant were extracted with ethanol and partitioned in sequence with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction was phytochemically studied using thin layer chromatography (TLC) and column chromatography (CC). Structural elucidation of pure compounds obtained from the ethyl acetate fraction was done using mass spectra, 1H-NMR, and 13C-NMR analysis. The isolated compounds were subsequently screened using five different cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7, SW-480, and normal lung epithelial cell line, BEAS-2B, to assess their cytotoxic effects. Nine compounds were isolated and structurally elucidated as gallic acid, gallic acid ethyl ester, corilagin, quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside, 1,4,6-tri-O-galloyl glucose, 3,4,6-tri-O-galloyl glucose, 1,2,6-tri-O-galloyl glucose, and 4,6-di-O-galloyl glucose. Corilagin exhibited the most cytotoxic activity with an IC50 value of 33.18 µg/mL against MCF-7 cells, which were comparable to cisplatin with an IC50 value of 27.43 µg/mL. The result suggests that corilagin isolated from R. heudelotii has the potential to be developed as an effective therapeutic agent against the growth of breast cancer cells.

Keywords: anticancer; Ricinodendron heudelotii; chemoprevention

1. Introduction Cancer is a major cause of death across the globe, affecting all age groups [1]. The World Health Organization has predicted that cancer cases will increase from 14 million in 2012 to 22 million in the next four decades [2]. It has caused a great sense of burden both to individual lives and the society at large [3]. The absolute cure of cancer remains a big challenge despite available anticancer agents, awareness campaigns, and prevention strategies. Breast and prostate cancer have the highest incidence in women and men, respectively [4]. It is therefore pertinent to keep exploring for novel chemotherapeutic agents that can evade drug-resistant cancer cells. Natural or natural based anti-cancer drugs, such as vincristine, paclitaxel, bleomycin, and others, have been clinically used over the years [5]. Ricinodendron heudelotii (family: Euphorbiceae) plant parts have been used in traditional medicine to treat cough, stomach ailments, and pain related to child birth. Decoctions and infusions are mostly prepared from the leaves and stem bark. Traditional practitioners prescribe it for women suffering from miscarriages [6]. Phytochemical screening of the leaf extract revealed the

Molecules 2019, 24, 145; doi:10.3390/molecules24010145 www.mdpi.com/journal/molecules Molecules 2019, 24, 145 2 of 11 presence of tannins, terpenoids, glycosides, and alkaloids, and it exhibited antimicrobial effects [7]. Two dinorditerpenoids, 1,2-dihydroheudelotinol and heudelotinone, and three other compounds, lupeol, E-ferullic acid octacosylate, and 3-methylmethylorsellinate, have been isolated from the stem bark and roots of R. heudelotii [8]. Seven new tetracyclic triterpenoids ricinodols with potential as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors has also been reported to be isolated from R. heudelotii [9]. Yakubu et al. [10] also reported the leaf extract to have regulatory effects on artemisinin toxicity when co-administered with artemisinin. This study aims to identify the chemical structure of isolated bioactive compounds in R. heudelotii as well as evaluate the compoundsfor in vitro cytotoxic activity against human leukemia (HL-60), human hepatocellular carcinoma (SMMC-7721), human lung adenocarcinoma (A549), human breast cancer cell (MCF-7), and colon adenocarcinoma (SW480) cell lines.

2. Results Nine compounds were isolated from the ethyl acetate fraction of R. heudelotii by employing column chromatography, including Sephadex LH-20 and MCI CHP20P gel.

2.1. Structural Elucidation of Isolated Compounds Structure elucidation of the isolated compounds was carried out using spectroscopic techniques: 1H- (600 MHz) and 13C-NMR (150 MHz) and DEPT along with 2D-NMR for some compounds (Figure1). Compound 1 was obtained as an off-white amorphous powder, showing a molecular − ion peak at m/z 170 [M] in the negative ESI-MS. The molecular formula, C7H6O5, was deduced from the combination of 1H-, 13C-NMR and DEPT spectroscopic data. Compound 1 was identified as gallic acid when the data was compared with those in the literature [11,12]. Compound 2, an off-white powder, showed a quasi-molecular ion peak at m/z 198 [M]− in the negative ESI-MS. 1 13 Combining with the H-, C-NMR and DEPT spectroscopic data, a molecular formula, C9H10O5, was deduced. Comparing the data with those in the literature, compound 2 was identified as gallic acid ethyl ester [13]. Compound 3 was obtained as a yellow amorphous powder, showing a molecular ion peak at m/z 633 [M]− in the negative ESI-MS. From 1H-, 13C-NMR, and DEPT spectroscopic data, the molecular formula of C27H22O18 was deduced. Comparing the data with those in the literature, compound 3 was identified as corilagin [14]. Compound 4 was obtained as a yellow amorphous powder. It showed a molecular ion peak at m/z 447 [M − H]− in the negative 1 13 ESI-MS while the molecular formula of C21H20O11, was deduced from the H-, C-NMR, and DEPT spectroscopic data. Likewise, by comparing the data with those in the literature, compound 4 was quercetin-3-O-rhamnoside [15,16]. Compound 5 was obtained as a pale yellow amorphous powder showing a quasi-molecular ion peak at m/z 463 [M − H]− in the negative ESI-MS, and the molecular 1 13 formula of C21H20O12, was deduced from H-, C-NMR, and DEPT spectroscopic data. By comparing the data with those in the literature, compound 5 was identified as myricetin-3-O-rhamnoside [12,17]. Compound 6 was obtained as an off white powder, showing a quasi-molecular ion peak at m/z 635 [M − H]− in the negative ESI-MS. Combining with the 1H-, 13C-NMR, and DEPT spectroscopic data, a molecular formula of C27H24O18 was deduced. By comparing the data with those in literature, compound 6 was identified as 1,4,6-tri-O-galloyl-β-D-glucose [18]. Compound 7 was obtained as a white amorphous powder. It showed a molecular ion peak at m/z 636 [M − H]− in the negative ESI-MS. Combining the 1H-, 13C-NMR, and DEPT spectroscopic data, a molecular formula of C27H24O18 was deduced. The data obtained correlates with those in the literature and compound 7 was identified as 3,4,6-tri-O-galloyl-D-glucose [19]. Compound 8, an off-white amorphous powder with a molecular ion peak at m/z 635 [M − H]− in the negative ESI-MS. A molecular formula of 1 13 C27H24O18, was deduced from the H-, C-NMR, and DEPT spectroscopic data. Comparing the data with those in the literature, compound 8 was identified as 1,2,6-tri-O-galloyl-β-D-glucose [20,21]. Compound 9 was obtained as a white amorphous powder showing a molecular ion peat at m/z 483 [M − H]− in the negative ESI-MS. A molecular formula was deduced from the 1H-, 13C-NMR, Molecules 2019, 24, 145 3 of 11 and DEPT spectroscopic data. By comparing the data with those in the literature, compound 9 was identified as 4,6-di-O-galloyl-D-glucose [22,23].

2.2. Spectroscopic DataMolecules of Compounds 2018, 23, x FOR1–9 PEER REVIEW 3 of 11

− 1 Gallic acid (1): Off-white2.2.Spectroscopic amorphous Data of Compounds powder, C 17–H9 6O5, negative ESI-MS m/z: 170 [M] , H-NMR 13 (600 MHz, acetone-d6): δ 7.10Gallic (2H, acid s,(1 H-2,6),): Off-whiteC-NMR amorphous (150 powder, MHz, acetone-C7H6O5, negatived6): 121.0 ESI (C-1),-MS m/z 109.0: 170 (C-2,6),[M]−, 1H-NMR 145.9 (C-3,5), 138.3 (C-4),(600MHz, 168.0 acetone (C-7).-d6): δ 7.10 (2H, s, H-2,6), 13C-NMR (150MHz, acetone-d6): 121.0 (C-1), 109.0 (C-2,6), 145.9 (C-3,5), 138.3 (C-4), 168.0 (C-7). Gallic acid ethyl ester (2): Off-white amorphous powder, C9H10O5, negative ESI-MS m/z: − 198 [M]−, 1H-NMR (600 MHz,Gallic acetone-acid ethyld ester): δ (2):0.84Off-white (3H, t,amorphousJ = 7.1 Hz, powder, H-20), C 3.829H10O (2H,5, negative q, J = 7.1ESI- Hz,MS m/z H-1: 1980) [M] , 1 6 H 13 H-NMR (600MHz, acetone-d6): δH 0.84 (3H, t, J = 7.1 Hz, H-2′), 3.82 (2H, q, J = 7.1 Hz, H-1′) 7.05 (2H, 7.05 (2H, s, H-3,5); C-NMR (150 MHz, acetone-d6): δC 120.7 (C-1); 109.4 (C-2,6), 138.0 (C-4), 144.9 s, H-3,5); 13C-NMR (150 MHz, acetone-d6): δC 120.7(C-1); 109.4 (C-2,6), 138.0 (C-4), 144.9 (C-3, 5), 166.8 0 0 (C-3,5), 166.8 (C-7), 60.6(C-7), (C-1 60.6), (C 14.1-1′), (C-214.1 (C).-2′). − Corilagin (3): Off-whiteCorilagin amorphous (3): Off-white powder, amorphous C27H powder,22O18,negativeC27H22O18,negative ESI-MS ESIm/-zMS633 m/z [M 633− [MH] − H], −, 1H- 1 0 0 H-NMR (600, CD3OD):NMR δ(600,H 7.05 CD3OD): (2H, δ s,H 7.05 H-2 (2H,,6 ),6.69,s, H-2′,6 6.65′),6.69 (each, 6.65 (each 1H, 1H, s, HHDP-3”,3s, HHDP-3′′,3′′′), 6.36 6.36 (1H, (1H, d, J d,= 2.4 Hz, J = 2.4 Hz, H-1), 4.95H (1H,-1), 4.95 ddd, (1H,J = ddd 10.9,, J = 8.0, 10.9, 1.2 8.0, Hz, 1.2 H-6a),Hz, H-6a 4.80), 4.80 (1H, (1H, brs, brs, H-3), H-3), 4.51 (1H, (1H, t, t,J =J 9.6= 9.6 Hz, Hz, H-5), 4.46 - 13 H-5), 4.46 (1H, dd, J =(1H, 1.7, dd, 1.2 J = Hz, 1.7, H-4),1.2 Hz, 4.16 H-4), (1H, 4.16dd, (1H,J d=d 8.0,, J = 8 10.9.0, 10.9 Hz, Hz, H-6b), H-6b), 3.98 3.98 (1H,(1H, br brd,d, J J= =1.2 1.2 Hz Hz,, H 2); C- NMR (150MHz, CD3OD): HHDP 훿C 167.1 and 167.6 (C=O), 145.8, 145.1 (C-3′′, 3′′′), 144.9, 144.3 (C-5′′, H-2); 13C-NMR (150 MHz, CD OD): HHDP δ 167.1 and 167.6 (C=O), 145.8, 145.1 (C-3”,3 ), 144.9, 5′′′), 136.3, 136.23 (C-4′′, 4′′′), 116.2C , 116.1 (C-1′′, 1′′′), 124.1, 123.8 (C-2′′, 2’’’), 107.3, 106.7 (C-6′′, 6′′′); 144.3 (C-5”,5 ), 136.3,galloyl 136.2 (C-4”,4훿C 165.2 (C=O),), 116.2, 146.2 116.1 (C-3 (C-1”,1′, C-5′), 139.7), 124.1, (C-4′), 123.8 119.4 (C-2”,2 (C-1′), 109.7), 107.3, (C-2′, 106.7C-6’); glucose (C-6”,6 훿C);92.9 (C- 0 0 0 0 0 0 galloyl δC 165.2 (C=O),1), 78.3 146.2 (C- (C-33), 76.6,C-5 (C-5),), 72.4 139.7 (C (C-4-2), 64.2), 119.4(C-6), 62.8 (C-1 (C),-4). 109.7 (C-2 ,C-6 ); glucose δC 92.9 (C-1), 78.3 (C-3), 76.6 (C-5),Quercetin 72.4 (C-2),-3-O-rhamnoside 64.2 (C-6), 62.8(4):Yellow (C-4). amorphous powder, C21H20O11; negative ESI-MS m/z 447 [M − H]−, 1H-NMR (600 MHz, CD3OD): δ 7.33 (1H, d, J = 2.0 Hz, H-2′), 7.31 (1H, dd, J = 2.0, 8.3 Hz, H- Quercetin-3-O-rhamnoside (4): Yellow amorphous powder, C21H20O11; negative ESI-MS m/z 6′), 6.91 (1H, d, J = 8.3 Hz, H-5′), 6.19 (1H, s, H-6), 6.36 (1H, s, H-8), 5.34 (1H, s, H-1′′), 4.22 (1H, brs, H- 447 [M − H]−, 1H-NMR (600 MHz, CD OD): δ 7.33 (1H, d, J = 2.0 Hz, H-20), 7.31 (1H, dd, J = 2.0, 2′′), 3.76 (1H, dd, J = 3.2,3 9.4 Hz, H-3′′), 3.34 (1H, dd, J = 7.5, 9.4 Hz, H-4′′), 3.41 (1H, m, H-5′′), 0.94 (3H, 8.3 Hz, H-60), 6.91 (1H, d, J = 8.3 Hz, H-50), 6.19 (1H, s, H-6), 6.36 (1H, s, H-8), 5.34 (1H, s, H-1”), d, J = 6.1 Hz, H-6′′);13C-NMR (150 MHz, CD3OD): δC 18.7 (C-6′′), 71.1 (C-5′′), 70.6 (C3′′), 71.1 (C-2′′), 4.22 (1H, brs, H-2”), 3.7671.2 (C (1H,-4′′), dd,94.3 J(C=-8), 3.2, 98.7 9.4 (C Hz,-6), 102.1 H-3”), (C- 3.341′′), 104.1 (1H, (C dd,-10),J 115.7= 7.5, (C 9.4-2′),Hz, 115.8 H-4”), (C-5′), 120.8 3.41 (1H,(C-1′), 122.2 13 m, H-5”), 0.94 (3H, d,(CJ-6=′), 6.1134.3 Hz, (C-3), H-6”); 145.4 (CC-NMR-3′), 148.8 (150(C-4′), MHz, 156.7 (C CD-2),3 OD):157.3 (CδC-9),18.7 161.4 (C-6”), (C-5), 165.6 71.1 (C (C-5”),-7), 177.8 (C- 70.6 (C3”), 71.1 (C-2”),4). 71.2 (C-4”), 94.3 (C-8), 98.7 (C-6), 102.1 (C-1”), 104.1 (C-10), 115.7 (C-20), 115.8 (C-50), 120.8 (C-10), 122.2 (C-6Myricetin0), 134.3 3-O (C-3),-rhamnoside 145.4 (C-3(5):Y0ellow), 148.8 amorphous (C-40), 156.7 powder (C-2),, C21 157.3H20O12, (C-9), negative 161.4 ESI (C-5),-MS m/z: 463 [M − H]−, 1H-NMR (600 MHz, CD3OD): δ 6.95 (2H, s, H-2′,6′), 6.36 (1H, d, J = 1.6 Hz, H-6), 6.20 (1H, d, 165.6 (C-7), 177.8 (C-4). J = 1.6 Hz, H-8),5.31 (1H, s, H-1′′),3.80 (1H, dd, J = 3.2, 9.5 Hz, H-2′′), 3.55 (1H, m, H-3′′),, 3.17 (1H, m, O 5 13 m z Myricetin 3- -rhamnosideH-4′′), 3.34 (1H, ( ): m, Yellow H-5′′), amorphous 0.84 (3H, d, J powder, = 6.1Hz, H C-216′′H). 20CO-NMR12, negative (150 MHz, ESI-MS CD3OD):/ δ:c177.6 463 (C-4), − 1 0 0 [M − H] , H-NMR (600163.8 MHz,(C-7), 161.4 CD3 (COD):-5), 157.1δ 6.95 (C (2H,-9), 156.6 s, H-2 (C-,62), ),145.9 6.36 (C (1H,-5′, 3 d,′), 135.4J = 1.6 (C Hz,-4′), 135.2 H-6), (C 6.20-3), 119.7 (1H, (C-1′), d, J = 1.6 Hz, H-8), 5.31107.9 (1H, (C- s,2′), H-1”), 107.9 (C 3.80-6′), (1H,104.3 dd,(C-10)J =, 102.4 3.2, 9.5(C-1 Hz,′′), 98.8 H-2”), (C-8) 3.55, 93.7 (1H,(C-6), m, 71.9 H-3”), (C-4′′) 3.17, 71.3 (1H,(C-5′′), 70.8 (C-3′′), 70.3 (C-2′′), 17.8 (C-6′′). 13 m, H-4”), 3.34 (1H, m, H-5”), 0.84 (3H, d, J = 6.1 Hz, H-6”). C-NMR (150 MHz, CD3OD): δC 177.6 (C-4), 163.8 (C-7), 161.4 (C-5),1,4,6-T 157.1ri-O-galloyl (C-9),-β- 156.6D-glucose (C-2), (6): 145.9Off-white (C-5 amorphous0, 30), 135.4 powder (C-40),, 135.2C27H24O (C-3),18,negative 119.7 ESI-MS − 1 0 0 m/z 635 [M0 − H] ; H-NMR (600 MHz, CD3OD): δH 7.15, 7.10, 7.06 (each 2H, s, galloylH-2′,6′), 5.78 (1H, (C-1 ), 107.9 (C-2 ), 107.9d, J = (C-6 8.2 Hz,), 104.3 H-1), (C-10),5.22 (1H, 102.4 t, J = (C-1”),9.6 Hz, H 98.8-4), (C-8),4,45 (1H, 93.7 dd, (C-6), J = 12. 71.94, 2.0 (C-4”), Hz, H-6a 71.3), 4.22 (C-5”), (1H, dd, J = 70.8 (C-3”), 70.3 (C-2”),12.4 17.8, 4.9 (C-6”).Hz, H-6b), 4.06 (1H, ddd, J = 2.0, 4.9, 9.6 Hz, H-5), 3.84 (1H, dd, J = 9.2, 9.6 Hz, H-3), 3.65 (1H, 1,4,6-Tri-O-galloyl-dd, βJ -=D 8.2,-glucose 9.2 Hz, (H6-):2). Off-white amorphous powder, C27H24O18, negative ESI-MS − 1 0 0 m/z 635 [M − H] ; H-NMR3,4,6-Tri (600-O-galloyl MHz,-D- CDglucose3OD): (7):δOHff-7.15,white 7.10,amorphous 7.06 (eachpowder, 2H, C27H s,24 galloylO18, negative H-2 ESI,6 ),-MS m/z 5.78 (1H, d, J = 8.2 Hz,63 H-1),5 [M − 5.22H]−, 1 (1H,H-NMR t, J (600= 9.6 MHz, Hz, CD H-4),3OD): 4.45 δH (1H,7.07, 6.99,6.94 dd, J = 12.4,(each 2.02H, Hz,s,galloylH H-6a),-2 4.22′,6′), 4.42 (1H, (1H, m, H-6a), 4.26 (1H, m, H-6b), -glucose form: δH 4.76 (1/3H, d, J = 7.8 Hz, H-1), 3.56 (1/3H, dd, J = 9.5, 7.8 dd, J = 12.4, 4.9 Hz, H-6b), 4.06 (1H, ddd, J = 2.0, 4.9, 9.6 Hz, H-5), 3.84 (1H, dd, J = 9.2, 9.6 Hz, H-3), Hz, H-2), 5.45 (1/3H, t, J = 9.5 Hz, H-3), 5.35 (1/3H, dd, J = 9.5, 9.8 Hz, H-4), 4.07 (1/3H, m, H-5), - 3.65 (1H, dd, J = 8.2, 9.2 Hz, H-2). glucose form: δH 5.25 (2/3H, d, J = 3.6 Hz, H-1), 3.81 (2/3H, dd, J = 9.8, 3.6 Hz, H-2), 5.68 (2/3H, t, J = 9.8 3,4,6-Tri-O-galloyl-Hz,D H-glucose-3), 5.36 ( (2/37):H, Off-white t, J = 9.8 Hz amorphous,H-4), 4.47 (2/3H, powder, m, H- C5)27. H24O18, negative ESI-MS m/z − 1 0 0 635 [M − H] , H-NMR (6001,2,6-T MHz,ri-O-galloyl CD3OD):-β-D-glucoseδH 7.07, (8): 6.99, Off-white 6.94 (eachamorphous 2H, s, powder, galloyl C H-227H24,6O18),, negative 4.42 (1H, ESI-MS: m/z 635 [M − H]−, 1H-NMR (600 MHz, CD3OD): δH 7.10, 7.04, 7.00 (each 2H, s, galloyl H-2′,6′), 5.92 (1H, m, H-6a), 4.26 (1H, m, H-6b), β-glucose form: δH 4.76 (1/3H, d, J = 7.8 Hz, H-1), 3.56 (1/3H, dd, J = 9.5, 7.8 Hz, H-2), 5.45 (1/3H,d, J = t,8.5J Hz,= 9.5 H- Hz,1), 5.23 H-3), (1H, 5.35 dd, J(1/3H, = 8.5, 9.6 dd, Hz, JH=-2), 9.5, 4. 56 9.8 (1H, Hz, dd, H-4), J = 2.0, 4.07 12.2 (1/3H, Hz, H-6a), m, 4.47 H-5), (1H, dd, J = 4.8, 12.2 Hz, H-6b), 3.83 (1H, ddd, J = 2.0, 4.8, 9.6 Hz, H-5), 3.81 (1H, t, J = 9.6 Hz, H-3), 3.66 (1H, t, α-glucose form: δH 5.25 (2/3H, d, J = 3.6 Hz, H-1), 3.81 (2/3H, dd, J = 9.8, 3.6 Hz, H-2), 5.68 (2/3H, t, J = 9.6 Hz, H-4); 13C-NMR (150MHz, CD3OD): δC 63.1 (C-6), 69.6 (C-4), 72.7 (C-5), 74.4 (C-3), 74.2 (C- J = 9.8 Hz, H-3), 5.362), (2/3H, 93.2 (C t,-J1),= 117.0, 9.8 Hz, 119.1, H-4), 119.2 4.47 (galloyl (2/3H, C-1 m,′), 109.2, H-5). 108.8, 108.8 (galloyl C-2′,6′), 145.3, 145.4, 145.8 1,2,6-Tri-O-galloyl-(galloylβ-D -glucoseC-3′,5′), 138.6, (8): 138.7, Off-white 139.1 ( amorphousgalloyl C-4′), 164.1, powder, 164.9, C 165.727H24 (C=O).O18, negative ESI-MS: − 1 0 0 m/z 635 [M − H] , H-NMR4,6-Di-O (600-galloyl MHz,-β-D- CDglucose3OD): (9):OδHff-white7.10, amorphous 7.04, 7.00 powder, (each 2H, C20H s,20O galloyl14, negative H-2 ESI,6 -),MS: m/z 5.92 (1H, d, J = 8.5 Hz,483 H-1), [M − H] 5.23−, 1H (1H,-NMR dd, (600J MHz,= 8.5, CD 9.63OD): Hz, δ H-2),H 7.09, 4.567.07, 7.08, (1H, 7.06 dd, (eachJ = 2.0,1H, 12.2s, 2× galloyl Hz, H-6a), H-2′,6′), 5.13

4.47 (1H, dd, J = 4.8, 12.2 Hz, H-6b), 3.83 (1H, ddd, J = 2.0, 4.8, 9.6 Hz, H-5), 3.81 (1H, t, J = 9.6 Hz, 13 H-3), 3.66 (1H, t, J = 9.6 Hz, H-4); C-NMR (150 MHz, CD3OD): δC 63.1 (C-6), 69.6 (C-4), 72.7 (C-5), Molecules 2019, 24, 145 4 of 11

74.4 (C-3), 74.2 (C-2), 93.2 (C-1), 117.0, 119.1, 119.2 (galloyl C-10), 109.2, 108.8, 108.8 (galloyl C-20,60), 145.3, 145.4, 145.8 (galloyl C-30,50), 138.6, 138.7, 139.1 (galloyl C-40), 164.1, 164.9, 165.7 (C=O). 4,6-Di-O-galloyl-β-D-glucose (9): Off-white amorphous powder, C H O , negative ESI-MS: Molecules 2018, 23, x FOR PEER REVIEW 20 20 14 4 of 11 − 1 m/z 483 [M − H] , H-NMR (600 MHz, CD3OD): δH 7.09, 7.07, 7.08, 7.06 (each 1H, s, 2× galloyl 0 0 H-2(1H,,6 ),t, 5.13J = 9.4 (1H, Hz, t, H-4),J = 9.4 4.39 Hz, (1H, H-4), m, 4.39 H-6a), (1H, 4.18 m, H-6a),(1H, m, 4.18 H-6b), (1H,  m,-glucose H-6b), form:α-glucose 5.17 (1/2H, form: 5.17d, J = (1/2H, 3.7 d,Hz,J = 3.7H-1), Hz, 3.98 H-1), (1/2H, 3.98 t, (1/2H, J = 9.4 t,Hz,J = H-3), 9.4 Hz, 4.53 H-3), (1/2H, 4.53 dd, (1/2H, J = 3.7, dd, 9.6J =Hz, 3.7, H-2), 9.6 Hz,-glucose H-2), β form:-glucose δH 4.60 form: δH(1/2H,4.60 (1/2H,d, J = 7.8 d, Hz,J = H-1), 7.8 Hz, 3.89 H-1), (1/2H, 3.89 m, (1/2H, H-5), 3.70 m, H-5),(1H, t, 3.70 J = 9.3 (1H, Hz, t, JH-3),= 9.3 3.31 Hz, (1/2H, H-3), dd, 3.31 J (1/2H,= 9.3, 7.8 dd, J =Hz, 9.3, H-2). 7.8 Hz, H-2).

FigureFigure 1. 1.Structures Structuresof of compoundscompounds ((1–9) isolated from from Ricinodendronheudelotii.Ricinodendron heudelotii. 2.3. Bioassay 2.2. Bioassay InIn an anin in vitro vitrocytotoxicity cytotoxicity assay,assay, compoundscompounds 1–5 were applied applied to to HL-60, HL-60, SMMC-7721, SMMC-7721, A-549, A-549, MCF-7,MCF-7, and and SW-480 SW-480 cancer cancer cells cells toto assessassess theirtheir cytotoxiccytotoxic effects. Compound Compound 33 (Corilagin)(Corilagin) exhibited exhibited µ thethe most most cytotoxic cytotoxic activity activity against against MCF-7 MCF-7 cellscells withwith ICIC5050 valuesvalues of of 33.18 33.18 and and 25.81 25.81 μg/mLg/mL for for HL-60 HL-60 cells.cells. The The IC IC5050 valuevalue of compound 33 inin MCF-7 cells cells is is comparable comparable with with the the positive positive control control drug drug (cisplatin),(cisplatin), having having an an ICIC5050 valuevalue of of 27.43 27.43 (Table (Table 1).1). Corilagin Corilagin had had 29.19, 29.19, 4.91, 4.91, and and 49.82% 49.82% inhibition inhibition for forA-549, A-549, SMMC-7721, SMMC-7721, and and SW-480 SW-480 respectively. respectively. The The inhibition inhibition rate rate of ofother other compounds compounds (gallic (gallic acid, acid, gallicgallic acid acid ethyl ethyl ester, ester, quercetin-3- quercetin-3-O-rhamnoside,O-rhamnoside, myricetin-3- myricetin-3-OO-rhamnoside)-rhamnoside) wasless wasless than than 50% 50% at at the concentrationthe concentration of 40 µofM 40 (Table μM (Table2), suggesting 2), suggesting that they that showed they showed no signiﬁcant no significant inhibitory inhibitory activity activity against theagainst cancer the cell cancer lines; cell therefore, lines; therefore, their IC50 theirvalues IC50 werevalues not were measured. not measured. FiguresFigures2– 42–4 shows shows the the photomicrograph photomicrograph of theof the cytotoxic cytotoxic activities activities of compound of compound3 on 3 breast,on breast, colon, andcolon, leukemia and leukemia cancer cell cancer lines withcell lines relatively with similar relatively activity similar when activity compared when with compared cisplatin with (control cisplatin group). (control group).

Table 1. IC50 values (µg/mL) of Corilagin (3) isolated from R. heudelotii against some cancer cell lines.

Sample HL-60 MCF-7 SW480 BEAS-2B Corilagin (3) 25.81 ± 0.67 * 33.18 ± 0.76 * 37.04 ± 1.06 * 16.32 ± 1.43 Cisplatin 2.32 ± 0.07 27.43 ± 1.26 10.19 ± 1.03 — * Signiﬁcant (p < 0.05) when compared to the control (Cisplatin).

Molecules 2018, 23, x FOR PEER REVIEW 5 of 11 Molecules 2018, 23, x FOR PEER REVIEW 5 of 11

Table 1. IC50 values (μg/mL) of Corilagin (3) isolated from R. heudelotii against some cancer cell Table 1. IC50 values (μg/mL) of Corilagin (3) isolatedlines. from R. heudelotii against some cancer cell lines. Sample HL-60 MCF-7 SW480 BEAS-2B Corilagin(3)Sample 25.81±0.67* HL-60 33. MCF-718±0.76* 37.04±1.06* SW480 BEAS-2B 16.32±1.43 Corilagin(3)Cisplatin 25.81±0.67* 2.32±0.07 33. 27.43±1.2618±0.76* 37.04±1.06* 10.19±1.03 16.32±1.43 --- Molecules 2019, 24, 145 Cisplatin*Significant 2.32±0.07 (p<0.05) when 27.43±1.26 compared to 10.19±1.03the control (Cisplatin). --- 5 of 11 *Significant (p<0.05) when compared to the control (Cisplatin). Table 2. Percentage of inhibition (I%) of compounds 1–5 (40μM) isolated from R. heudelotii on different Table 2. Percentage of inhibition (I%) of compounds 1–5 (40 µM) isolated from R. heudelotii on different Tablecell lines. 2. Percentage of inhibition (I%) of compounds 1–5 (40μM) isolated from R. heudelotii on different cell lines. cell lines. Compounds HL-60 A-549 SMMC-7721 MCF-7 SW480 CompoundsCompounds1 HL-60 14.80±5.00 HL-60 A-54913.67±2.91 A-549 SMMC-7721 SMMC-7721 5.85±1.16 18.41±4.00MCF-7 MCF-7 10.04±3.51SW480 SW480 1 1 2 14.80 ± 14.80±5.00 12.26±2.295.00 13.67 13.67±2.91 11.82±0.14± 2.91 5.85 5.195.85±1.16±0.83± 1.16 18.41±4.00 24.76±3.30 18.41 ± 4.00 10.04±3.51 1.15±0.28 10.04 ± 3.51 2 2 3 12.26 ± 12.26±2.29 76.23±0.512.29 11.82 11.82±0.14 29.19±1.42± 0.14 5.19 4.915.19±0.83±1.43± 0.83 24.76±3.30 57.93±3.19 24.76 ± 3.30 1.15±0.28 49.82±3.80 1.15 ± 0.28 3 3 4 76.23 ± 76.23±0.510.51 4.56±2.61 29.19 29.19±1.42 5.49±2.01± 1.42 4.91 1.12±1.054.91±1.43± 1.43 57.93±3.19 14.95±0.82 57.93 ± 3.19 49.82±3.80 2.44±2.49 49.82 ± 3.80 4 4 5 4.56 ± 4.56±2.612.61 0.65±2.87 5.49 5.49±2.01 8.32±3.46± 2.01 1.12±1.05 1.121.62±1.05± 1.05 14.95±0.82 10.67±4.05 14.95 ± 0.82 2.44±2.49 12.45±3.66 2.44 ± 2.49 5 5 0.65 ± 0.65±2.872.87 8.32 8.32±3.46± 3.46 1.62±1.05 1.62 ± 1.05 10.67±4.05 10.67 ± 4.05 12.45±3.66 12.45 ± 3.66

Figure 2. Photomicrograph of breast cancer cell line (a) untreated, (b) treated with cisplatin, and (c) Figure 2. Photomicrograph of breast cancer cell line (a) untreated, (b) treated with cisplatin, and (c) treated Figuretreated 2. withPhotomicrograph compound3 (H&E of breast x100). cancer cell line (a) untreated, (b) treated with cisplatin, and (c) × treatedwith compound with compound3 (H&E3 (H&E100). x100).

Figure 3. Photomicrograph of colon cancer cell line (a) untreated, (b) treated with cisplatin, and (c) treated MoleculesFigure 2018, 233., Photomicrographx FOR PEER REVIEW of colon cancer cell line (a) untreated, (b) treated with cisplatin, and (6c )of 11 Figurewithtreated compound 3. withPhotomicrograph compound3 (H&E × 3100).(H&E of colon x100). cancer cell line (a) untreated, (b) treated with cisplatin, and (c) treated with compound 3(H&E x100).

FigureFigure 4. 4. PhotomicrographPhotomicrograph of ofleukemia leukemia cancer cancer cell cellline line(a) untreated, (a) untreated, (b) treated (b) treated with withcisplatin, cisplatin, and (andc) treated (c) treated with with compound compound 3 (H&E3 (H&E x200).× 200).

Molecules 2019, 24, 145 6 of 11

3. Discussion After many years of research on cancer, cancer still remains a destructive disease responsible for over one quarter of the deaths recorded globally. It is one of the major health obstacles, representing the second largest cause of mortality [24]. This is mainly because cancer chemotherapy is often mitigated by a high degree of toxicity to normal cells or failure of treatment due to the development of resistance to anticancer drugs. However, medicinal plants have been increasingly shown to possess anticancer properties, and this has recently become the subject of some scientific studies [25] because they are considered to be less toxic and can be effective for treatment of different diseases. To date, the therapeutic pre-clinical screening of medicinal plants against multiple cancer cells and characterization of the bioactive components in these plants are limited. Our study reports a bioactive compound with cytotoxicity effects in the leaf extracts of R. heudelotii and its potential to be developed for use for cancer treatment. Corilagin (compound 3) was isolated and structurally identified from the ethyl acetate fraction of the ethanolic extract of R. heudelotii leaves in our study. Only compound 3 showed significant and comparable cytotoxic property relative to cisplatin. Corilagin has been previously identified in several other plants, especially those of the Euphorbiceae family. It was first isolated from Caesalpinia coriaria in 1951, hence the name of the molecule [26]. It was later identified as a weak carbonic anhydrase inhibitor [27]. Studies have reported its antibacterial, hepatoprotective, anti-oxidant [28], anti-inflammatory [29], anti-hypertensive [30], and anti-tumor activities [31]. Yang et al. [32] reported that corilagin can block glioblastoma cells and stem-like cells’ proliferation. In this study, corilagin was found to exhibit an inhibitory effect on HL-60, MCF-7, and SW480 cell growth by 76.23, 57.93, and 49.82%, respectively (Table2). This could be linked to its ability to activate the Jun N-terminal kinase (JNK) signaling pathway [33]. When these pathways are turned on, it can lead to the activation of the proapoptotic β-cell lymphoma 2 (Bcl-2) protein through phosphorylation and/or upregulation of gene expression and hence mitochondrial cell death [34]. Breast cancer cells treated with corilagin showed reduced cell diameters when compared to the untreated cells, implying that the compound caused cell death of most of the cancerous cells (Plate 1). Corilagin is an ellagitannin, which have been reported to induce pro-apoptotic effects in in both MDA-MB-231 and MCF-7 breast cancer cells [35]. Kuo et al. [36] observed that ellagitannins from Terminalia arjuna bark extract induced apoptosis in A549 cells and in human breast adenocarcinoma MCF-7 cells via blockage of cell-cycle progression in the G0/G1 phase. The cytotoxic properties of tannins are characterised by the release of reactive oxygen species (ROS), which is followed by a reduction in the thioredoxin, superoxide dismutase, and in the level of redox active proteins [37]; this might explain the cytotoxic activity of compound 3. The chemopreventive activity of tannins, especially the ellagitannins and hydrolysable tannins, has been linked to activation of apoptosis [38]. According to the findings of Jia et al. [31], corilagin inhibited SKOv3ip by inducing cell cycle arrest and enhancing apoptosis in the cancer cell lines. Down regulation of cyclin B1 and phosphor-cdc 2 after corilagin treatment on cancer cells was discovered [31]. Corilagin has also been reported to cause the release of TNF-α inhibitor in inflammation [39], decrease the secretion of the transforming growth factor beta 1 (TGF-β1) in a dose dependent manner [31], and inhibit hepatitis C viral replication in vitro [40]. Corilagin enhanced apoptosis by inducing cell cycle arrest at the G2/M stage in ovarian cancer cells. Anti-cancer strategies have focused on possible ways of deactivating the G2/M checkpoint, thereby causing cancer cells to undergo mitosis, and leading to increased DNA damage and then cell death [41,42]. Ming et al. [43] also reported the inhibitory action of corilagin on hepatocellular carcinoma cells (HCC) at the G2/M phase by upregulating p53 and p21, and downregulating cyclin B1, cdc 2, and pAKT. Selectivity index (SI) signifies the differential activity of compounds; the greater the SI value, the more selective the compound [44]. The SI data, as shown in Table3, indicate that corilagin was non-selective to HL-60, MCF-7, and SW480 cancer cell lines. This suggests its general toxicity to the cell. Adverse effects caused by treatment and increased resistance to tumor cells, leading to treatment failure, has led to a Molecules 2019, 24, 145 7 of 11 search for therapeutic alternatives. Therefore, it is important to find selective bioactive compounds that could arrest tumor cell proliferation.

Table 3. Selectivity index of compound 3 (corilagin).

Sample HL-60 MCF-7 SW480 Corilagin (3) 0.63 0.49 0.44

4. Materials and Methods

4.1. General Experimental Procedures

1 2 D- and D-NMR spectra were recorded in CD3OD and acetone-d6 with Bruker Avance III600 spectrometer (Bruker, Fällande, Switzerland) operating at 600 MHz for 1H-NMR, and 150 MHz for 13C-NMR, respectively. Coupling constants are expressed in hertz (Hz), and chemical shifts are given on a δ (parts per million, ppm) scale with tetramethylsilane (TMS) as an internal standard. ESI mass spectra were recorded on a VG Auto Spec-300 spectrometer (Waters, Milford, MA, USA). Column chromatography (CC) was performed on Sephadex LH-20 (25–100 µm, GE Healthcare Bio-Science AB, Uppsala, Sweden), MCI-gel CHP20P (75–150 µm, Mitsubishi Chemical Co, Ltd., Tokyo, Japan), and Toyopearl HW-40F (Tosoh Co. Ltd., Tokyo, Japan). Thin-layer chromatography (TLC) was performed on silica gel H-precoated plates, 0.2–0.25 mm thick (Qingdao Haiyang Chemical Co., Qingdao, China), with benzene–ethylformate–formic acid (3:6:1 or 2:7:1, v/v), and compounds were detected by spraying with 2% ethanolic FeCl3 and 10% sulfuric ethanol solution followed by heating.

4.2. Plant Sample Collection Ricinodendron heudelotii leaves were collected within Covenant University premises Ota, Ogun, Nigeria. The plant was identiﬁed by Dr. J.O. Popoola (Botanist, Biological Science Department, Covenant University, Ota, Ogun State, Nigeria) and a voucher specimen was prepared and submitted in the Forest research Institute of Nigeria, Ibadan with voucher number, FHI 110573. Leaves were dried at room temperature (25 ◦C) and blended using an electric blender into a coarse powder.

4.3. Extraction and Solvent Partitioning ofthe Crude Extract The powdered leaf samples (10 kg) were extracted using 95% ethanol by macerationat room temperature, the ﬁltrate was further condensed under reduced temperature and pressure, and the yield of the extract obtained was 11.75%. The concentrated crude extract (1 kg) was suspended in 1 L distilled water and partitioned in sequence with petroleum ether (Pet; 8 L), ethyl acetate (EtOAc; 8 L), and n-butanol (8 L). The solvent fractions were concentrated to dryness to afford four (4) fractions as: Pet, EtOAc, n-butanol, and aqueous fractions. The EtOAc fraction was selected for subsequent isolation procedures.

4.4. Isolation of Compounds fromthe Partitioned Fraction The ethylacetate fraction (246 g) was loaded into a column containing Sephadex LH-20 (800 g). The eluent was a mixture of water and methanol in different ratios starting with 100:0 to 0:100. Fractions with similar TLC patterns were combined to afford 13 fractions. Fraction 11 (36 g) was chromatographed on a Sephadex LH-20 column and MCI-gel CHP20P eluting with water:methanol (4:1) to afford compounds 1 (8.53 mg), 2 (2.1 mg), and 3 (12 mg). Fraction 12 (3.8 g) was subjected to repeated column chromatography also eluting with H2O and methanol (60:1) to afford compound 4 (98 mg) and 5 (13.2 mg). Compounds 6 (9.9 mg), 7 (2.6 mg), 8 (2.7 mg), and 9 (4.4 mg) were obtained from fraction 13 (14.5 g) after repeated puriﬁcation by chromatography on a Sephadex LH-20, MCI-gel CHP20P and Toyopearl HW-40F columns eluting with H2O/methanol. Molecules 2019, 24, 145 8 of 11

4.5. Bioassay

4.5.1. Cell Culture Cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW-480, were obtained from the American Type Culture Collection Center (ATCC) (Manassas, VA, USA). They were cultured in RMPI-1640 or DMEM medium supplemented with 10% bovine serum albumin (Gibco, Grand Island, NY, USA). Prior to exposure to drugs, the cancer cell lines were cultured in a CO2 incubator for 48 h and the cell density was adjusted to 5 × 104 cells/well.

4.5.2. Sample Preparation

Compounds (1 mg/mL) were dissolved in either DMSO or H2O depending on their solubility. Samples (2 µL) were suspended in centrifuge tubes containing 48 µL of complete medium to make a ﬁnal volume of 100 µL.

4.5.3. Assay Procedures The cytotoxicity assay was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method by measuring the absorbance of formazan precipitate formed in the cells based on MTS reduction [45]. In this assay, cells were seeded into each of the wells of 96-well cell culture plates, incubated for 12h at 37 ◦C, and then the test compounds (40 µM) were added and allowed to incubate for 48 h. Each cell line was exposed to different concentrations (0.064, 0.32, 1.6, 8, and 40 µM) of the test compounds in triplicates using cisplatin-an anticancer drug as the positive control. The absorbance of the lysate was measured using a 96-well micro plate reader (Bio-Rad 680, Hercules, CA, USA) at 490 nm. Compounds with a growth inhibition rate of 50% were further evaluated at concentrations of 0.064, 0.32, 1.6, 8, and 40 µM in triplicates with cisplatin as positive control. The concentration that inhibited 50% of the cell growth were expressed as IC50, which was calculated using Reed and Muench’s method [46,47]. The percentage inhibition (I%) was calculated as follows:

Inhibition (%) = (Cc − Cs)/Cc × 100 (1) where Cc = viable cell count of negative control, Cs = viable cell count of sample, IC50 was calculated by the following equation:

Log10 (IC50) = (Log10 (CL) (IH − 50) + log10 (CH) (50 − IL))/(IH − IL) (2)

Log (IC ) IC50 = 10 10 50 ;IH: I% above 50%; IL: I% below 50%; CH: High drug concentration; and CL: Low drug concentration.

4.6. Selectivity Index

The selectivity index (SI) for the cytotoxicity of corilagin was calculated using the ratio of the IC50 value of the compound on a normal cell line to the IC50 value of the compound on cancer cell lines [48].

4.7. Statistical Analysis Samples are expressed as mean ± standard deviation or standard error of mean.

5. Conclusions In conclusion, we report signiﬁcant cytotoxic activities of corilagin isolated from leaf extract of R. heudelotii on a breast cancer cell line, and its potential for development as an anticancer agent to reduce the burden of cancer diseases globally. Molecules 2019, 24, 145 9 of 11

Author Contributions: O.F.Y. performed the isolation of the compounds, activity assay and the preparation of the manuscript. A.H.A. and E.E.J.I. designed the experiment and the principal investigators of the experiment. Y.-J.Z. participated in structural elucidation of compounds and direction of the experiment. T.M.D. made valuable revision of the manuscript. All authors approved the final version manuscript. Funding: This research was funded by the National Natural Science Foundation of China (No. 31470429) and the OWSD Postgraduate Fellowship. Acknowledgments: The authors are grateful to the staffs of the analytical and bioactivity screening groups at the State Key Laboratory of Phytochemistry & Plant Resources in West China, KIB, CAS, for measuring the spectroscopic data and cytotoxic activity. Conflicts of Interest: The authors declare no conflict of interest

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