Doctoral Dissertation
Anti-Blood Cancer Activities of Bioactive Compounds from Rice (Oryza
sativa var. Koshihikari) and an Invasive Weed (Andropogon virginicus)

LA HOANG ANH

Graduate School for International Development and Cooperation
Hiroshima University

September 2022

Anti-Blood Cancer Activities of Bioactive Compounds from Rice (Oryza
sativa var. Koshihikari) and an Invasive Weed (Andropogon virginicus)

D190581LA HOANG ANH

A Dissertation Submitted to
the Graduate School for International Development and Cooperationof Hiroshima University in Partial Fulfillmentof the Requirements for the Degree ofDoctor of Agriculture

September 2022

ACKNOWLEDGMENTSFirst and foremost, I would like to convey my heartfelt appreciation and gratitude to
my main academic supervisor, Associate Professor TRAN DANG XUAN for his
outstanding support and guidance. Throughout my doctoral course, his zeal and diligencehave always inspired and motivated me. He has taught me a wide range of abilities in bothacademics and sciences. He enlightened me from the most fundamental concept toaccomplish major results. He assisted me in overcoming obstacles to attain my objectives.
Furthermore, he has always stimulated and supported me in both study and life. It is myhonor to be his doctoral student, and I am forever grateful for an excellent pattern of a
successful researcher like him.
I would like to send my special gratitude to my sub-supervisors Professor LEE HAN
SOO and Associate Professor HOSAKA TETSURO for their valuable recommendations andsuggestions during my program. These valuable comments help me complete my study moresignificantly and comprehensively. I would like to extend my sincere appreciations toProfessor ICHIHASHI MASARU for the constructive comments and questions which
improve my doctoral dissertation.
I would like to compliment my deep thanks and appreciation to Assistant Professor
NGUYEN VAN QUAN for his instructions and encouragements in my experiments andacademic writing since the beginning of my doctoral course. I am immensely thankful for
his unfailing aid, which not only strengthened my academic performance but also assisted
me in balancing with other aspects of my life.
I would like to acknowledge Professor AKIYOSHI TAKAMI, Division Chief ofDepartment of Internal Medicine, Division of Hematology, Aichi Medical University for
permitting me to research at the Division of Hematology in term of collaboration project
between the Laboratory of Plant Physiology and Biochemistry, Hiroshima University andithe Laboratory of Hematology, Aichi Medical University. My distinctive gratefulness also
goes to him for his kind instructions and supports during my study.
I would like to specially thank Mr. VU QUANG LAM, a doctoral student at Aichi
Medical University for his enthusiastic supports through my experiments in the Laboratoryof Hematology, Aichi Medical University. I would like to express gratitude to Ms.
TOMOKO AMIMOTO, a technician of Natural Science Center for Basic Research andDevelopment (N-BARD), Hiroshima University for her significant expert advice, analyticalinsights, and decent training programs. I am grateful for the support and collaboration of all
members of the Xuan lab who are not only kind partners but also great friends. I gratefully
acknowledge the Japanese Government–Monbukagakusho (MEXT) Scholarship for funding
my studies at Hiroshima University. I would like to send great thanks to all staffs at Graduate
School for International Development and Cooperation (IDEC) for their unwavering
supports and readiness to help me whenever I need. Finally, I am incredibly thankful,especially my parents and my brother, who have always been a pillar and an everlastingsource of fuel for me to achieve my current goals.

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SUMMARY
1. Background
Blood cancer (1.2 million cases per year) has been a serious human disease during along history with a high mortality rate (58%). Therefore, this study was conducted toinvestigate promising candidates from plants for inhibiting blood cancer. Among plantsources, Adropogon virginicus is an invasive weed that distributes globally, threatening
agriculture and economics in many countries. Thus, research on pharmaceutical properties
may lead to further strategies to take advantage of this serious invasive weed that was once
thought to be of no use. On the other hand, rice (Oryza sativa) is an important food crop in
the world, which has been recently being received increasing attention for the research on
medicinal purposes. In rice, momilactones are high valuable diterpene lactones exhibiting
multiple biological benefits. Based on these reasons, we aimed (1) to explore potential
phytochemicals and pharmaceutical properties of the invasive weed A. virginicus with focus
on antioxidant, anti-skin aging, anti-diabetic, and anti-blood cancer properties and (2) todetermine the potentials of momilactones isolated from rice (O. sativa var. Koshihikari) for
preventing blood cancer.
2. Structure of dissertation
Chapter 1. General introduction
Chapter 2. Pharmaceutical properties of an invasive weed Andropogon virginicus
Chapter 3. Cytotoxic activities of momilactones against leukemia, lymphoma, and multiple
myeloma cell lines
Chapter 4. Cytotoxic mechanism of momilactones against acute promyelocytic leukemia andmultiple myeloma cell lines
Chapter 5. Effects of in vitro digestion on anti-blood cancer properties of momilactones
Chapter 6. General discussioniii

3. Materials and methods
Materials
A. virginicus’ aerial plant parts were extracted by Soxhlet extraction method, followedby liquid-liquid phase extraction to obtain total crude (T-Anvi), hexane (H-Anvi), ethyl
acetate, (E-Anvi), butanol (B-Anvi), and water (H-Anvi) extracts.
Momilactones were isolated and purified from the laboratory of Plant Physiology andBiochemistry, Hiroshima University, Japan (Quan et al., 2019b).
Biological activity
Antioxidant activities of samples were determined via antiradical (DPPH and ABTS),
reducing power, b-carotene bleaching assays (Quan et al., 2019).
Enzymatic assays including tyrosinase and ⍺-amylase inhibitory effects of A.
virginicus’ extracts were evaluated following the method presented by Quan et al. (2019).
Anticancer assays consisting of MTT, annexin V apoptosis, cell cycle, and western
blotting were conducted to screen the anticancer properties of momilactones. The
corresponding cell lines for blood cancer were included leukemia (HL60, Meg-01, andK562), lymphoma (Mino), and myeloma (KMS-11 and U266). Besides, a non-cancerous
cell line namely MeT-5A was used in this study.
In vitro digestion model
The effects of in vitro digestion on the bioaccessibility and cytotoxic activities ofmomilactones against blood cancer cell lines were investigated following the method
described by Un et al. (2022).
Phytochemical analysis
Thechemicalanalyses

were

conducted

applying

spectrophotometer,gas

chromatography-mass spectrometry (GC-MS), and liquid chromatography-electrosprayionization-tandem mass spectrometry (LC-ESI-MS/MS) methods.

iv

4. Results and discussion
In Chapter 2, we indicate that the invasive weed A. virginicus may be a promisingsource of antioxidants, anti-⍺-amylase, and anti-tyrosinase abilities, and cytotoxicity against
chronic myeloid leukemia cell lines. Besides, A. virginicus aerial parts are rich in flavonoids,
palmitic acid, phytol, and γ-sitosterol, which may play a vital role in the biological activitiesof the respective extracts.
From Chapter 3, we highlight that MB and MAB can inhibit various blood cancer celllines including leukemia, lymphoma, and multiple myeloma by inducing cell apoptosis.
Notably, the cytotoxicity of MB and MAB is close to the well-known drugs of Doxorubicinin inhibiting Meg-01 and K562. Meanwhile these compounds are stronger than the widely
applied medicines of Doxorubicin and Ibrutinib in suppressing HL-60 and U266,
respectively. Whereas these compounds display an insignificant effect on non-cancerous
cells.
In Chapter 4, MB and MAB at 5 µM promote acute promyelocytic leukemia (HL-60)
and multiple myeloma (U266) cell apoptosis by activating the phosphorylation of p-38 in
the mitogen-activated protein kinase (MAPK) pathway and regulating the relevant proteins(BCL-2 and caspase-3) in the mitochondrial pathway. Besides, these compounds may induce
G2 phase arrest in HL-60 cell cycle through the activation of p-38 and disruption of CDK1
and cyclin B1 complex. Meanwhile MB and MAB show insignificant effects on cell
apoptosis and cell cycle of non-cancerous cell line MeT-5A.
For Chapter 5, the finding shows that the bioaccessibility and cytotoxicity ofmomilactones significantly reduce through the digestive stages including oral, gastric, andintestinal phases.
A general discussion is presented in Chapter 6. Particularly, this is the first study tohighlight that A. virginicus may be a promising source of antioxidant, anti-diabetic, anti-skinv

aging, and anti-blood cancer agents. In addition, momilactones isolated from rice (O. sativa
var. Koshihikari) husks can inhibit blood cancer cell lines by regulating the proteins related
to apoptosis and cell cycle pathways. However, the cytotoxic activities of momilactones may
be significantly decreased during digestion. Therefore, forthcoming studies should be
conducted to improve the bioaccessibility of momilactones, followed by in vivo and clinicaltests to confirm their capacity to develop novel anti-blood cancer drugs. In another aspect,
the combination of momilactones from rice and isolated components from A. virginicus may
be a potential approach for developing blood cancer treatments, especially for patientscomplicated with other health problems such as oxidative stress and chronic disorders.

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ABBREVIATIONSABTS: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
APL: acute promyelocytic leukemia
ATCC: American Type Culture Collection
B-Anvi: butanol extract from Andropogon virginicus
BHT: butylated hydroxytoluene
CML: chronic myeloid leukemia
DPPH: 2,2-diphenyl-1-picrylhydrazyl
DW: dry weight
E- Anvi: ethyl acetate extract from Andropogon virginicus
GAE: gallic acid equivalent
GC-MS: gas chromatography-mass spectrometry (GC-MS)
H-Anvi: hexane extract from Andropogon virginicus
KI: Kovats index
LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry
LPI: lipid peroxidation inhibition
LRI: linear retention index
MA: momilactone A
MAB: the mixture of momilactones A and B (1:1, w/w)
MB: momilactone B
MCL: mantle cell lymphoma
MM: multiple myeloma
MS: mass spectra
RE: relative expressionvii

RE: rutin equivalent
T-Anvi: total crude extract from Andropogon virginicus
TFC: total flavonoid content
TPC: total phenolic content
UPLC-ESI-MS: ultra-performance liquid chromatography-electrospray ionization-mass
spectrometry
W-Anvi: water extract from Andropogon virginicus

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LIST OF CONTENTSACKNOWLEDGMENTS ....................................................................................................ISUMMARY ....................................................................................................................... III
ABBREVIATIONS.......................................................................................................... VII
LIST OF CONTENTS....................................................................................................... IX
LIST OF TABLES .......................................................................................................... XIV
LIST OF FIGURES ........................................................................................................ XVICHAPTER 1: GENERAL INTRODUCTION.................................................................. 1
1.1. Blood cancer .................................................................................................................. 1
1.2. Anticancer potentials of natural compounds from plants ........................................ 3
1.3. Rice ................................................................................................................................. 4
1.3.1. Rice and rice by-products ........................................................................................ 4
1.3.2. Momilactones .......................................................................................................... 5
1.3.3. Biological activities of momilactones ..................................................................... 8
1.4. Andropogon virginicus ................................................................................................ 10
1.4.1. Morphology ........................................................................................................... 11
1.4.2. Distribution ............................................................................................................ 11
1.5. Main objectives ........................................................................................................... 11
CHAPTER 2: PHARMACEUTICAL PROPERTIES OF AN INVASIVE WEED
ANDROPOGON VIRGINICUS ......................................................................................... 13
2.1. Introduction ................................................................................................................ 13
2.2. Materials and methods ............................................................................................... 15
2.2.1. Materials ................................................................................................................ 15
2.2.2. Sample preparation and extraction ........................................................................ 16
2.2.3. Total phenolic (TPC) and flavonoid (TFC) contents............................................. 16ix

2.2.4. Antioxidant activity ............................................................................................... 17
2.2.5. Enzymatic inhibitory assays .................................................................................. 18
2.2.6. Cytotoxic activity against K562 and Meg-01 cell lines ........................................ 19
2.2.7. Identification of phytochemical component .......................................................... 20
2.2.8. Data analysis .......................................................................................................... 21
2.3. Results .......................................................................................................................... 22
2.3.1. Yield and total phenolics (TPC) and flavonoids (TFC) of A. virginicus extracts . 22
2.3.2. Antioxidant activity of extracts from A. virginicus ............................................... 22
2.3.3. Tyrosinase and a-amylase inhibitory activities of A. virginicus extracts ............. 25
2.3.4. Cytotoxic activity of extracts from A. virginicus against K562 and Meg-01 cell lines......................................................................................................................................... 26
2.3.5. Correlations between total phenolics (TPC) and flavonoids (TFC) and biologicalproperties of A. virginicus extracts. ................................................................................. 27
2.3.6. GC-MS results ....................................................................................................... 28
2.3.7. HPLC-ESI-MS/MS results .................................................................................... 29
2. 4. Discussion ................................................................................................................... 32
2.5. Conclusions.................................................................................................................. 38
CHAPTER 3: CYTOTOXIC ACTIVITIES OF MOMILACTONES AGAINSTLEUKEMIA, LYMPHOMA, AND MULTIPLE MYELOMA CELL LINES............ 39
3.1. Introduction ................................................................................................................ 39
3.2. Materials and methods ............................................................................................... 40
3.2.1. Materials ................................................................................................................ 40
3.2.2. Cell viability (MTT) assay .................................................................................... 41
3.2.3. Cell apoptosis (annexin V) assay........................................................................... 42
3.3. Results .......................................................................................................................... 42x

3.3.1. Effects of momilactones A (MA) and B (MB) and their mixture (MAB) on cell
viability of blood cancer cell lines ................................................................................... 42
3.3.2. Apoptosis-inducing activities of momilactones against blood cancer cell lines ... 45
3.4. Discussion .................................................................................................................... 51
3.5. Conclusions.................................................................................................................. 54
CHAPTER 4: CYTOTOXIC MECHANISM OF MOMILACTONES A AND B
AGAINST

ACUTE

PROMYELOCYTICLEUKEMIAAND

MULTIPLE

MYELOMA CELL LINES............................................................................................... 55
4.1. Introduction ................................................................................................................ 55
4.2. Materials and methods ............................................................................................... 57
4.2.1. Materials ................................................................................................................ 57
4.2.2. Cell viability (MTT) assay .................................................................................... 57
4.2.3. Cell apoptosis (annexin V) assay........................................................................... 58
4.2.4. Cell cycle assay ..................................................................................................... 59
4.2.5. Western blotting assay ........................................................................................... 59
4.2.6. Statistical analysis.................................................................................................. 60
4.3. Results .......................................................................................................................... 60
4.3.1. Effects of momilactones on cell viability of non-cancerous (MeT-5A), acutepromyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines .................. 60
4.3.2. Apoptosis-inducing activities of momilactones against non-cancerous (MeT-5A),
acute promyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines ......... 63
4.3.3. Effects of momilactones on inducing cell cycle arrest of non-cancerous (MeT-5A),
acute promyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines ......... 66

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4.3.4. Effects of momilactones on expressions of proteins related to apoptosis induction
and cell cycle arrest of acute promyelocytic leukemia (HL-60), and multiple myeloma(U266) cell lines .............................................................................................................. 68
4.4. Discussion .................................................................................................................... 71
4.5. Conclusions.................................................................................................................. 77
CHAPTER 5: EFFECTS OF IN VITRO DIGESTION ON ANTI-BLOOD CANCERPROPERTIES OF MOMILACTONES .......................................................................... 78
5.1. Introduction ................................................................................................................ 78
5.2. Materials and methods ............................................................................................... 79
5.2.1. Materials ................................................................................................................ 79
5.2.2. In vitro digestion model ......................................................................................... 80
5.2.3. Cell viability (MTT) assay .................................................................................... 82
5.2.4. Quantification of momilactones ............................................................................ 83
5.3. Results .......................................................................................................................... 84
5.3.1. Effects of in vitro digestion on cytotoxic activity of momilactones against acutepromyelocytic leukemia (HL-60) cell line ...................................................................... 84
5.3.2. Effects of in vitro digestion on cytotoxic activity of momilactones against chronic
myeloid leukemia (Meg-01) cell line .............................................................................. 85
5.3.3. Effects of in vitro digestion on cytotoxic activity of momilactones against mantle
cell lymphoma (Mino) cell line ....................................................................................... 86
5.3.4. Effects of in vitro digestion on cytotoxic activity of momilactones against multiple
myeloma (U266) cell line ................................................................................................ 87
5.3.5. Bioaccessibility of momilactones during in vitro digestion .................................. 88
5.3.6. Correlation coefficients between quantities of momilactones and their cytotoxic
activities during in vitro digestion ................................................................................... 90xii

5.4. Discussion .................................................................................................................... 91
CHAPTER 6: GENERAL DISCUSSION ....................................................................... 94
6.1. Key findings................................................................................................................. 94
6.2. Comparison between cytotoxic activities of isolated components from rice (O. sativa
var. Koshihikari) and the invasive weed (A. virginicus)................................................. 97
6.3. Exploitation of products from rice (O. sativa) and the invasive weed (A. virginicus)
for developing blood cancer treatments .......................................................................... 98
SUPPLEMENTARY DATA ........................................................................................... 101
REFERENCES ................................................................................................................ 119

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LIST OF TABLES
Table 1. Extraction yield and total phenolic (TPC) and flavonoid (TFC) contents ............ 22
Table 2. Tyrosinase and a-amylase inhibitory activities of A. virginicus extracts ............. 25
Table 3. Pearson’s correlation coefficients between total phenolic and flavonoid contents
and biological activities of A. virginicus extracts ................................................................ 28
Table 4. Dominant phytochemicals in H-Anvi extract from A. vigirnicus identified by GCMS ....................................................................................................................................... 29
Table 5. Phytocompounds in E-Anvi extract from A. virginicus detected by HPLC-ESIMS/MS................................................................................................................................. 31
Table 6. Cytotoxic activities of momilactones A and B against non-cancerous (MeT-5A),
chronic myeloid leukemia (Meg-01 and K562), acute promyelocytic leukemia (HL-60),
mantle cell lymphoma (Mino), and multiple myeloma (U266 and KMS-11) cell lines ..... 43
Table 7. Cytotoxic activities (IC50) of momilactones A and B against MeT-5A, HL-60, andU266 cell lines (Obtained results from Chapter 3) .............................................................. 62
Table 8. List of blood cancer cell lines ............................................................................... 79
Table 9. Components of artificial digestion juices (Un et al., 2022) .................................. 80
Table 10. Effect of in vitro digestion on cytotoxic activity against HL-60 cell line ofmomilactones (IC50, µM)..................................................................................................... 85
Table 11. Effect of in vitro digestion on cytotoxic activity against Meg-01 cell line ofmomilactones (IC50, µM)..................................................................................................... 86
Table 12. Effect of in vitro digestion on cytotoxic activity against Mino cell line ofmomilactones (IC50, µM)..................................................................................................... 87
Table 13. Effect of in vitro digestion on cytotoxic activity against U266 cell line ofmomilactones (IC50, µM)..................................................................................................... 88
Table 14. Variation percentages of momilactones under digestion .................................... 89xiv

Table 15. Correlation coefficients between quantities of momilactones and their cytotoxicity
during digestion ................................................................................................................... 90
Table 16. Key findings ........................................................................................................ 95
Table 17. Cytotoxic activities of isolated components from broomsedge bluestem and rice
(Achieved results from Chapters 2 and 3) ........................................................................... 97

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LIST OF FIGURES
Figure 1. Number of worldwide cancer new cases and mortality rate in 2020 (GLOBOCAN,
2020) ..................................................................................................................................... 1
Figure 2. Number of worldwide blood cancer new cases and mortality rate in 2020 (Sung etal., 2021) ................................................................................................................................ 2
Figure 3. Anti-blood cancer substances derived from natural plants (Xie et al., 2016; Zadeh
et al., 2022; Espinoza et al., 2017)......................................................................................... 3
Figure 4. Structures of momilactones A (MA) and B (MB)................................................. 6
Figure 5. Momilactones isolated and purified from the laboratory of Plant Physiology andBiochemistry, Hiroshima University, Japan (Quan et al., 2019) ........................................... 7
Figure 6. Anticancer potentials of diterpene lactones (Kumar et al., 2004; Ji et al., 2007) 8
Figure 7. Andropogon virginicus in Higashi-Hiroshima Campus, Hiroshima University . 11
Figure 8. Anti-radical activities (ABTS and DPPH assays) of A. virginicus extracts.
Different letters (a,b,c,d) enclosed with columns (same colors) express significant
differences at p < 0.05 (one-way ANOVA). DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS,
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); T-Anvi, total crude extract; H-Anvi,hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water
extract; BHT, butylated hydroxytoluene. ............................................................................ 23
Figure 9. Inhibition on lipid peroxidation of A. virginicus extracts. Different letters (a,b,c,d)enclosed with columns indicate significant differences at p < 0.05 (one-way ANOVA). LPI,
lipid peroxidation inhibition; T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi,
ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water extract; BHT, butylatedhydroxytoluene. ................................................................................................................... 24xvi

Figure 10. Potassium ferricyanide reducing power of A. virginicus extracts. T-Anvi, total
crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol
extract; W-Anvi, water extract; BHT, butylated hydroxytoluene. ...................................... 25
Figure 11. Dose-response curves of A. virginicus extracts for cytotoxicity against (a) K562
and (b) Meg-01 cell lines. T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi, ethyl
acetate extract; B-Anvi, butanol extract; W-Anvi, water extract. ....................................... 27
Figure 12. GC-MS chromatogram of H-Anvi extract ........................................................ 29
Figure 13. HPLC chromatogram of E-Anvi extract ........................................................... 30
Figure 14. Dose-responding curves of momilactones A and B for cytotoxic activities against
non-cancerous (MeT-5A), chronic myeloid leukemia (Meg-01 and K562), acutepromyelocytic leukemia (HL-60), mantle cell lymphoma (Mino), and multiple myeloma(U266 and KMS-11) cell lines. MA, momilactone A; MB, momilactone B; MAB, themixture of momilactones A and B (1:1, w/w). .................................................................... 45
Figure 15. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against non-cancerous MeT-5A cell line
after 24 h and 48 h. .............................................................................................................. 46
Figure 16. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against chronic myeloid leukemia (CML)(a) Meg-01 and (b) K562 cell lines after 24 h and 48 h. ..................................................... 47
Figure 17. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against acute promyelocytic leukemia(APL) HL-60 cell line after 24 h and 48 h. ......................................................................... 48
Figure 18. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against mantle cell lymphoma (MCL)
Mino cell line after 24 h and 48 h. ....................................................................................... 49

xvii

Figure 19. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against multiple myeloma (MM) (a) U266
and (b) KMS-11 cell lines after 24 h and 48 h. ................................................................... 50
Figure 20. Effects of momilactones A (MA) and B (MB) and their mixture (MAB) (1:1,
w/w) on cell viability of non-cancerous (MeT-5A), acute promyelocytic leukemia (HL-60),
and multiple myeloma (U266) cell lines after 48 h. Data are expressed as mean ± standard
deviation (SD). Statistical significance was determined by t-test. *p < 0.05 versus control (0µM); **p < 0.01 versus control (0 µM); ***p < 0.001 versus control (0 µM); ns, not significant
versus control. ...................................................................................................................... 61
Figure 21. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against (a) non-cancerous MeT-5A, (b)acute promyelocytic leukemia (APL) HL-60, and (c) multiple myeloma (MM) U266 celllines after 24 h and 48 h. Statistical significance was determined by t-test. *p < 0.05 versus
control; **p < 0.01 versus control; ns, not significant versus control. ................................. 65
Figure 22. Effects of momilactone B (MB) and the mixture of momilactone A and B (MAB)(1:1, w/w) at 5 µM on the cell cycle of (a) non-cancerous (MeT-5A), (b) acute promyelocytic
leukemia (HL-60), and (c) multiple myeloma (U266) cell lines after 24 h and 48 h. Statisticalsignificance was determined by t-test. *p < 0.05 versus control; **p < 0.01 versus control;***p < 0.01 versus control; ^^p < 0.01 versus MAB-24 h; ##p < 0.01 versus MB-48 h; ns, notsignificant versus control. .................................................................................................... 67
Figure 23. Effects of momilactone B (MB) and the mixture of momilactones A and B(MAB) (1:1, w/w) on the expressions of proteins related to apoptosis induction and cell cycle
arrest of (a) acute promyelocytic leukemia (APL) HL-60, and (b) multiple myeloma (MM)U266 cell lines after 24 h and 48 h. Statistical significance was determined by t-test. *p <0.05 versus control; **p < 0.01 versus control; ***p < 0.001 versus control; †p < 0.05 versus

xviii

MB-24 h; ††p < 0.01 versus MB-24 h; †††p < 0.01 versus MB-24 h; ^p < 0.05 versus MAB24 h; ^^p < 0.01 versus MAB-24 h; ^^^p < 0.001 versus MAB-24 h; #p < 0.05 versus MB-48
h; ##p < 0.01 versus MB-48 h. ............................................................................................. 69
Figure 24. Cytotoxic mechanism of momilactones against acute promyelocytic leukemia(HL-60) and multiple myeloma (U266) cell lines. .............................................................. 75
Figure 25. Procedure for in vitro simulated digestive model ............................................. 82
Figure 26. Bioaccessibility of momilactones under digestion. MA, momilactone A; MB,momilactone B; MAB, the mixture of momilactones A and B (1:1, w/w). ........................ 89
Figure 27. Potential approach for exploiting natural compounds from rice (O. sativa) andthe invasive weed (A. virginicus) for therapeutic purposes ................................................. 99xix

SUPPLEMENTARY DATA
Figure S1. Standard curves of gallic acid and rutin. ......................................................... 101
Figure S2. Antiradical activity of A. virginicus extracts via 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. T-Anvi, total crude extract; H-Anvi,hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water
extract. ............................................................................................................................... 101
Figure S3. Antiradical activity of A. virginicus extracts via 2,2-diphenyl-1-picrylhydrazyl(DPPH) assay. T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate
extract; B-Anvi, butanol extract; W-Anvi, water extract. ................................................. 102
Figure S4. Inhibition on lipid peroxidation of A. virginicus extracts. T-Anvi, total crude
extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; WAnvi, water extract. ........................................................................................................... 103
Figure S5. Tyrosinase inhibitory activities of A. virginicus extracts. T-Anvi, total crude
extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract. 104
Figure S6. a-Amylase inhibitory activities of A. virginicus extracts. T-Anvi, total crude
extract; H-Anvi, hexane extract. ........................................................................................ 104
Figure S7. Standard curve of palmitic acid ...................................................................... 104
Figure S8. GC chromatogram of n-alkane mixture (C13–C30) ....................................... 104
Figure S9. Mass spectra (MS) of palmitic acid found in hexane extracts (H-Anvi) from A.
virginnicus analyzed by gas chromatography-mass spectrometry (GC-MS). ................... 105
Figure S10. Effects of momilactones A (MA) and B (MB) on cell viability of tested bloodcancer cell lines via MTT assay. ....................................................................................... 105
Figure S11. Effects of Bortezomib on cell viability of multiple myeloma (U266) cell lines
after 48 h. **p < 0.01 versus control (0 nM); ***p < 0.001 versus control (0 nM). ............ 105
Figure S12. Standard curves of momilactones A and B. .................................................. 106

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Figure S13. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of thestandards of momilactones A (MA) and B (MB) by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 107
Figure S14. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in oral digested sample by ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS). .......................................... 108
Figure S15. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 109
Figure S16. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 110
Figure S17. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in oral digested sample by ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS). .......................................... 111
Figure S18. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 112
Figure S19. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 113
Figure S20. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in oral digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 115

xxi

Figure S21. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 117
Figure S22. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS)................ 118

xxii

CHAPTER 1: GENERAL INTRODUCTION
1.1. Blood cancer
In human, cancer has been a serious human disease during a long history. In 2020, theincidence of worldwide cancers was reported 19 million new cases with approximately 50%
of mortality rate (Figure 1) (GLOBOCAN, 2020). Among cancer types, blood cancers, also
known as hematological cancers, achieved in 1.24 million new cases, and caused nearly 60%
of deaths in 2020 (Sung et al., 2021). Blood cancer initiates in the bone marrow, where
normal blood cells are produced. Of which, cancer-associated mutation can cause abnormal
proliferation of immature or mature of blood cells and disruption of their function.
Consequently, many human health problems occur including fever, chills, persistent fatigue,
weakness, loss of appetite, nausea, unexpected weight loss, night sweats, bone/joint pain,
abdominal discomfort, headaches, shortness of breath, frequent infections, itchy skin/skinrash, swollen lymph nodes in the neck/underarms/groin (CTCA, 2020).

Figure 1. Number of worldwide cancer new cases and mortality rate in 2020 (GLOBOCAN,
2020)
There are three main types of blood cancers including leukemia, lymphoma, andmultiple myeloma (Figure 2). In which, leukemia patients suffer from the uncontrolled

1proliferation of mature or immature white blood cells with loss of function and obstruct the
production of red blood cells and platelets. Leukemia comprises four major sub-groups basedon the affected white blood cell types and whether it is acute (acute myeloid leukemia andacute myeloid leukemia) or chronic (chronic lymphocytic leukemia and chronic myeloid
leukemia) condition (GLOBOCAN, 2020). In 2020, leukemia is the second most common
blood cancer with 474,519 new cases with 66% of deaths (Figure 2) (Sung et al., 2021). On
the other hand, lymphoma is the most common blood cancer that comprises 2 main types of
non-Hodgkin lymphoma and Hodgkin lymphoma. Non-Hodgkin lymphoma develops fromlymphocytes in the lymphatic system. While Hodgkin lymphoma is characterized by thepresence of an abnormal lymphocyte namely Reed-Sternberg cell. Lymphoma was reported
544,352 new cases with mortality rate of 48% in 2020 (Figure 2) (Sung et al., 2021). Anothermajor blood cancer type is multiple myeloma, which initiates in the plasma cells. Multiple
myeloma accounted for 176,404 new cases and 66% of deaths in 2020 (Figure 2) (Sung etal., 2021).

Blood cancers
New cases
176,404 cases(15%)
MyelomaMortality rate
474,519 cases(40%)
300000

Leukemia
Lymphoma
544,352 cases(45%)

200000311,594(45%)

Total: 688,464
259,793(38%)
117,077(17%)

100000Total cases: 1,195,275

0Leukemia Lymphoma Myeloma

Figure 2. Number of worldwide blood cancer new cases and mortality rate in 2020 (Sung etal., 2021)
In the context that blood cancer becoming serious and complicated problems over theyears, effective approaches are urgently needed to generate effective treatments for thiscancer. In this study, we focus on the exploitation of natural based products from plants with2

concern about their biomass availability which can contribute to the development ofchemotherapy against blood cancer.
1.2. Anticancer potentials of natural compounds from plants
Recent trends in developing various herbal medicines have been to exploit benefits
from plants, which are extremely abundant and well-distributed all over the world (Xu et al.,2017). Numerous compounds derived from plants exhibited antitumor properties (Zadeh etal., 2022; Espinoza et al., 2017; Kim et al., 2007; Xie et al., 2016; Zhao et al., 2018). In
which, some phytocompounds showed their potential in suppressing blood cancer cells. For
example, genistein found in Leguminosae suppressed the proliferation of human multiple
myeloma cells through the downregulation of nuclear factor‑κB and upregulation of
microRNA‑29b (Xie et al., 2016) (Figure 3). Besides, genistein impeded the proliferationand promoted cell apoptosis of human leukemia (MOLT-4) cells by inhibiting anti-apoptotic
proteins (Zadeh et al., 2022) (Figure 3). Gnetin C is another anti-cancer substance derived
from melinjo (Gnetum gnemon L. seeds) and grapes (Vitis vinifera) (Figure 3). This
compound exhibited simultaneous inhibition on the mTOR and MAPK pathways, leading to
apoptosis enhancement in acute myeloid leukemia (Espinoza et al., 2017).

Figure 3. Anti-blood cancer substances derived from natural plants (Xie et al., 2016; Zadeh
et al., 2022; Espinoza et al., 2017)3

Although numerous plant species have shown their valuable pharmaceutical andmedicinal properties, the overuse of these natural plants may lead to the loss of biodiversity(Teschke & Xuan, 2020). Therefore, the exploitation of selected plant objects for therapeutic
purpose should also concern their available resources. Among plant sources, the family of
Poaceae includes numerous plant species that have dominant biomass sources and revealed
potentials for therapeutic uses (Moreira et al., 2010). In which, rice (Oryza sativa) has been
acknowledged for their high valuable consumption during a long history. Besides, riceproduction remains an abundant source of rice by-products that should be exploited more
properly. On the other hand, Andropogon virginicus is another species of Poaceae family,
which is a valueless invasive weed with serious expansion all over the world (OEPP, 2019).
Hitherto, there has been no strategy to take the advantages of this weed for human healthpurposes. Therefore, in this study, we selected A. virginicus and rice as the main objects.
Notably, both rice and A. virginicus are monocot plants, which adapt to a wide range ofenvironmental conditions (Simpson, 2010). Thus, they can be feasibly exploited for
medicinal production and therapeutics with a dominant biomass availability. The study is
expected to contribute to the development of anti-blood cancer treatments and solve theproblems in biodiversity conservation and sustainable development.
1.3. Rice
1.3.1. Rice and rice by-products
Rice (Oryza sativa L.) is an important food crop that plays a crucial role in ensuring
food security for the whole world. Rice grain has a long use history with high beneficial
consumption as rich in nutrients (Esa & Ling, 2016; Sohail et al., 2017). In addition tovaluable use of rice grain, rice by-products have recently attracted more and more attention
from the pharmaceutical and food processing industries (Esa & Ling, 2016; Sohail et al.,4

2017). Rice by-products including bran, husk, germ, and straw has been reported containing
beneficial nutrients of proteins, vitamins, oryzanol, and phenolics etc. (Esa & Ling, 2016;
Rahaie et al., 2014).
Theoretically, rice fields provide about 70% yield of white rice as major product.
Besides, 30% yield of rice by-products such as husk and bran will be wasted without proper
exploitation (Esa & Ling, 2016). Although rice by-products have been used for animal feed,
biofuels, fertilizers, and nutrient media for mushrooms. However, the pharmaceutical andmedicinal properties of isolated components from rice by-products have broad potentials
that need to be elucidated. Additionally, there no toxicity of compounds derived from riceon human health. Therefore, bioactive compounds from rice and its by-products are apromising source to be exploited for medicinal and pharmaceutical purposes.
1.3.2. Momilactones
Momilactones were first isolated from rice husk (Kato et al., 1973). “Momilactone”
was presented by Kato et al. (1973) (Figure 4). Of which, “momi” (籾 means the rice husk,
while lactone is a group of compounds. Based on this term, every rice husk-derived lactone
can be acknowledged as a momilactone compound.
Momilactones can be found in rice bran and rice by-products (husk, leaf, straw, androot). However, very few laboratories in the world can successfully isolate and purify
momilactones from rice. Besides, momilactones are difficult to purchase from commercial
sources. Therefore, the pharmaceutical and medicinal properties of momilactones have beenleast elucidated.

5

H
H

H
HO

O

O

H
O H

O H
O

O
Momilactone A (MA)

Momilactone B (MB)Figure 4. Structures of momilactones A (MA) and B (MB)In a preceding study, MA (150 mg) and MB (100 mg) were purified from a total of
200 kg of Koshihikari rice husks (Kato et al., 1973). Notably, an optimized isolation method(Figure 5) was conducted by Quan et al. (2019d) in our laboratory of Plant Physiology andBiochemistry, Hiroshima University, Japan to obtain a greater amount of momilactones from
Koshihikari (11.7 and 8.3 mg/kg rice husk for MA and MB, respectively). Briefly, MA andMB were isolated from the ethyl acetate (EtOAc) extract of rice husks by repeated column
chromatography over silica gel. The mobile phases of the first and the second column were
hexane:EtOAc (v/v) and chloroform:methanol (v/v), respectively (Quan et al., 2019d). The
identification

andconfirmation

of

such

pure

compounds

applying

thin-layer

chromatography (TLC), high-performance liquid chromatography (HPLC), liquid
chromatography-electrosprayionization-mass

spectrometry(LC-ESI-MS),gas

chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) were
described in the previous study of Quan et al. (2019d). Additionally, a specific technique inthe preparation of samples and advanced ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS) method were improved toincrease the detection sensitivity that helps quantify MA and MB in different rice organs
with a minor amount. Those outstanding achievements may significantly contribute to the6in-depth studies aiming to exploit momilactones from rice and rice by-products forpharmaceutical purposes.
Koshihikari rice husks
Fatty acid removal
Defatted methanolic extract
Liquid-liquid phase separation Hexane and ethyl acetate
Ethyl acetate extracts
The first column chromatography

Hexane : ethyl acetate (v/v)

Mixture of MA and MB
The second column chromatography Chloroform : methanol (v/v)

O

H
O

H
HO

H

H

O
O

MA

O
O

MB

Figure 5. Momilactones isolated and purified from the laboratory of Plant Physiology andBiochemistry, Hiroshima University, Japan (Quan et al., 2019)
On the other hand, numerous diterpene lactones such as andrographolide,neoandrographolide, halimanolide, etc. were indicated having inhibitory effects against
cancer cells (Silva et al., 2011) (Figure 6). Based on that, momilactones belonging to thisgroup of compounds can be considered high potentials for anti-blood cancer properties.

7

O
O

O
HO

O
H
HO

HO

HO

H
OH

HO

O
O
OH

Neoandrographolide

Andrographolide

Breast, central nervous system, colon, lung,melanoma, ovarian, prostate, renal cancers, andhepatoma
Figure 6. Anticancer potentials of diterpene lactones (Kumar et al., 2004; Ji et al., 2007)
1.3.3. Biological activities of momilactones
Allelopathic activity
Momilactones A (MA) and B (MB) exhibit a substantial allelopathic activity against
natural enemies of plants such as problematic weeds (e.g., Echinochloa crus-galli,
Monochoria vaginalis, Solidago altissima) (Fukuta et al., 2007; Kato-Noguchi et al., 2013;
Quan et al., 2019e) and blast fungus (Magnaporthe oryzae) (Cartwright et al., 1980; Zhao etal., 2018). In which, MB displays a stronger allelopathic activity than that of MA. MA andMB were proven to inhibit the growth of barnyard grass (Fang et al., 2015) and Arabidopsis(Kato-Noguchi et al., 2013) through the related gene expression and transcription pathways.
Whereas MA and MB presented a negligible effect on rice growth in the report of (KatoNoguchi et al., 2013).
Antioxidant, antibacterial, anti-diabetes, anti-obesity, anti-skin aging, and anticancer
potentials
MA (EC50 = 783.9 μg) and MB (EC50 = 790.7 μg) exhibited antiradical activity through2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Fukuta et al., 2007). In the research of Quan8

et al. (2019a), MA, MB, and their mixture MAB (1:1, v/v) present antiradical properties
against 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations with
IC50 values of 2.84, 1.28, and 0.319 mg/mL, respectively.
MA and MB revealed antibacterial properties against 4 common strains, which cause
problems in human digestion such as diarrhea and gastroenteritis. Accordingly, MB
significantly inhibited the growths of Pseudomonus ovalis, Bacillus cereus, and Bacillus
pumilus which is more potent than MA. Meanwhile, the inhibitory activity of MB was close
to that of MA against Escherichia coli (Fukuta et al., 2007).
In previous study, MA (IC50 = 132.56 µg/mL) and MB (IC50 = 129.02 µg/mL) revealed
a potent suppression on the key enzymes linked to type 2 diabetes (α-amylase and αglucosidase) which were much stronger than the standard inhibitor γ-oryzanol (IC50 = 779.03µg/mL). Besides, MA and MB showed inhibitory activity on an important enzyme associatedwith obesity (trypsin) with the IC50 values of 921.55 and 884.03 μg/mL, respectively. In
another research, momilactones exhibited potentials for preventing skin wrinkles andfreckles since these compounds inhibited the relevant enzymes including pancreatic elastaseand tyrosinase through in vitro bioassays (Quan et al., 2019a).
Via cell viability (MTT) assay, MA and MB displayed a potent cytotoxicity on themurine P388 leukemia cells with IC50 of 0.85 and 0.07 μg/mL, respectively (Chung et al.,2005). In the research of Lee et al. (2008), MB suppressed the cell viability of human
myeloblastic leukemia (HL-60), human leukemic T cells (Jurkat), basophilic leukemia(RBL-2H3), and mouse mastocytoma (p815) cell lines at concentration of less than 1.98
μg/mL. In which, MB prevent Jurkat cells by inducing cell apoptosis through the caspaseand mitochondrial pathways (Lee et al., 2008). In other studies, the potential of MB in
inhibiting colon cancer cells (HT-29 and SW620) was indicated via MTT, lactate
dehydrogenase (LDH), and colony-forming ability assays (Kim et al., 2007). Besides, MB

9suppressed human monocytic leukemia cell line U937 by stimulating apoptosis and the cell
cycle arrest at G1 phase via the decrease in pRB phosphorylation and the upregulation of a
CDK inhibitor p21Waf1/Cip1 (Park et al., 2014). Since the potential of momilactones forpreventing some cancers was previously indicated, the cytotoxic activity and mechanism ofthese compounds against many other cancer cell lines remain least elucidated.
1.4. Andropogon virginicus
Andropogon virginicus (Figure 7) is an invasive weed that distributes globally,
threatening agriculture and economics in many countries. Until now, there has been no
efficient way to remove once the weed has invaded (OEPP, 2019). However, different fromrice, A. virginicus has hitherto received scant attention from scholars. To the best of ourknowledge, there has been no strategies to exploit the dominant biomass source of thisproblematic invasive weed for pharmaceutical purposes. In a previous study, this weed wasreported to exhibit strong allelopathic activities (Rice, 1972). Interestingly, someallelochemicals have been shown pharmaceutical properties including anticancer. Therefore,
in this study, we expected to extract such potential compounds from A. virginicus forpreventing blood cancer cell lines.

10Figure 7. Andropogon virginicus in Higashi-Hiroshima Campus, Hiroshima University
1.4.1. Morphology
A. virginicus is a C4, herbaceous, perennial plant (Figure 7). It grows densely(cespitous form) with 50–200 cm high (Campbell, 2003). A. virginicus is a hemicryptophyte
following the Raunkiaer life-form system (EPPO, 2019). The culm branches distally, withlight green to reddish brown color during growth duration (EPPO, 2019). The leaf sheath islong. The leaf surface is scabrous. The ligule is yellow to brownish and membranous, 0.2–1
mm in size, with cilia 0.2–1.3 mm long (EPPO, 2019). The leaf blade is approximately 52
cm long and 6.5 mm wide. This blade has a variety of smooth and glabrous/or sparsely todensely pubescent spreading hairs. The inflorescence is racemes with up to 4 cm longcontaining spikelet (3.5–3.8 mm long).
1.4.2. Distribution
A. virginicus is original from the USA, presenting from the Central to the North of theUSA. In the USA, A. virginicus mainly has an eastern and central native range. The weed
also appears in California, where it is not native species. In 1924, A. virginicus wasannounced as invasive weed in Hawaii. This weed has been found naturally in Australia,
New Zealand, Japan and South Korea. In other reports, A. virginicus has been found inGeorgia, Russian Federation, and France. In 1940, A. virginicus was first reported aproblematic invasive weed in Aichi Prefecture, Japan (NIES, 2017; Enomoto et al., 2007).
This invasive weed is also detected in South Korea (Lee et al., 2008).
1.5. Main objectives
Considering the potentials of rice and the invasive weed A. virginicus for therapeutic
purposes as well as their dominant biomass availability, the main objective of this study was:11

(1) To explore potential phytochemicals and pharmaceutical properties of the invasive
weed A. virginicus with focus on antioxidant, anti-blood cancer, anti-skin aging, and antidiabetes activities.
(2) To determine the potentials of momilactones from rice for preventing different
types of blood cancer.

12CHAPTER 2: PHARMACEUTICAL PROPERTIES OF AN INVASIVE WEED
ANDROPOGON VIRGINICUS
2.1. Introduction
Andropogon virginicus is a problematic invasive weed distributing widely in theworld. A. virginicus is an herbaceous, perennial, C4 plant. This weed grows densely with up
to 210 cm in height (EPPO, 2019). A. virginicus has been generally known with limited
valuable use (EPPO, 2019). Moreover, this invasive weed is seriously threatening
biodiversity with its strong invasiveness (EPPO, 2019). A. virginicus is problematicallycompeting with endangered plant species such as Tetramolopium remyi, Santalum
freycinetianum var. Lanaiense, and Schiedea nuttallii (EPPO, 2019). In addition, A.
virginicus causes negative influence on horticultural systems because of its dense and robust
growth. For example, the high competition of A. virginicus has been dealing a blow
economically to the forage and timber production in the southeastern USA (EPPO, 2019).
Besides, farmlands of Charmhaven apple in Australia have been being degraded due to its
invasion (Weeds of Australia, 2020). The weed also affects the cycle of nitrogen and watercontent in soil which diminishing the abundance of native species (Balandier et al., 2006).
Until now, there has been no efficient way to remove once the weed has invaded (Weeds ofAustralia, 2020). Despite the serious problem caused by A. virginicus, it has hitherto received
scant attention from scholars. Therefore, an effective management of this natural resource isurgently needed, considering the potential of many other plants for therapeutic use, anattractive field of current research. Numerous plant species with antioxidant, antiinflammatory, antibacterial, antiviral, anti-skin aging, and anticancer properties have been
globally known (Xu et al., 2017). However, the pharmaceutical properties of the invasive
weed A. virginicus have not been comprehensively investigated.

13

In humans, oxidative stress exists and contribute to several chronic diseases including
diabetes, aging, and cancer through the correlation with inflammatory process (Collins,1999). Thereby, an essential part of our study was to investigate the antioxidant capacities
of extracted components from A. virginicus. Among chronic disorders, diabetes is a serious
disease with various types requiring different treatment methods (Tarling et al., 2008). The
most common diabetes is type 2, which causes the elevated level in bloodstream sugar. Of
which, a-amylase is an important digestive enzyme acting on starch which involves inglucose formation. Therefore, inhibition of a-amylase is a crucial enzyme can be mentioned
as a potential approach to mitigate type 2 diabetes (Tarling et al., 2008). On the other hand,freckles caused by hyperpigmentation are attributed to the upregulation of melanin formation
through an abnormal tyrosinase activity (Demirseren et al., 2014). Accordingly, preventing
tyrosinase can be an effective solution to avoid the symptom of skin hyperpigmentation. In
blood cancer, chronic myeloid leukemia (CML) is a serious disease, caused by the unregularproliferation of myeloid cells in the bone marrow. In which, K562 and Meg-01 are typicalcell lines, which are widely applied in anti-CML studies (NIH, 2020). Therefore,
simultaneous inhibition of both K562 and Meg-01 can be a promising method in preventing
CML. Furthermore, the synergic suppression against oxidative stress, tyrosinase and aamylase enzymes, and K562 and Meg-01 cell lines can be considered a prospective approachfor developing natural products which can be applied to treat multiple human healthproblems.
In this study, we aimed to investigate the pharmaceutical properties of the invasive
weed A. virginicus targeting antioxidant, anti-skin aging, anti-diabetes, and anticancer
properties. In addition, relevant phytochemicals to biological activities of this invasive
species were identified using high-performance liquid chromatography-electrospray14

ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) and gas chromatography-mass
spectrometry (GC-MS).
2.2. Materials and methods
2.2.1. Materials
Chemicals and cell lines
Solvents comprising methanol, hexane, ethyl acetate, butanol, and chloroform (Junsei
Chemical Co., Ltd., Tokyo, Japan) and standards including gallic acid, rutin, butylatedhydroxytoluene (BHT), kojic acid, (Kanto Chemical Co., Inc., Tokyo, Japan), palmitic acid
(FUJIFILM Wako Pure Chemical Corporation) were used in this study.
Chemicals including Folin-Ciocalteu’s reagent, aluminum chloride (AlCl3), sodium
carbonate (Na2CO3), sodium hypochlorite (NaClO), potassium persulfate (K2S2O8), 2,2′azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl(DPPH), sodium acetate (CH3COONa), β-carotene, linoleic acid, tween 40, dipotassium
phosphate (K2HPO4), monopotassium phosphate (KH2PO4), trichloro acetic acid
(CCl3COOH), potassium ferricyanide (K₃[Fe(CN)₆]), ferric chloride (FeCl3), dimethyl
sulfoxide (DMSO), sodium chloride (NaCl), monosodium phosphate (NaH2PO4),hydrochloric acid (HCl) were purchased from Kanto Chemical Co., Inc., Tokyo, Japan.
Besides, L-tyrosine, tyrosinase, starch, a-amylase, iodine, Iscove's Modified Dulbecco's
Medium (IMDM), 3-(4,5-dimethylthiazolyl)2,5-diphenyl-tetrazolium bromide (MTT), andcell lysis buffer were obtained from Sigma-Aldrich, St. Louis, MO, USA.
The cell lines consisting of K562 and Meg-01 (American Type Culture Collection(ATCC), Virginia, United States) were applied in cell viability (MTT) assay.
Plant materials
A. virginicus’ aerial parts were harvested in fields located inn Higashi-HiroshimaCampus, Hiroshima University, Hiroshima, Japan in October 2019. The identification was

15based on the publication of European and Mediterranean Plant Protection Organization(EPPO, 2019), available database of the National Institute for Environmental Studies, Japan(NIES, 2020), Missouri Botanical Garden, United States (Tropicos, 2020), and the bookentitled: “Invasive Weed” published by Japan Livestock Technology Association (JLTA,
1994). The specimen (voucher number: Anvi-J2019) was kept at the Laboratory of Plant
Physiology and Biochemistry, Graduate School of Advanced Science and Engineering,
Hiroshima University, Japan.
2.2.2. Sample preparation and extraction
A. virginicus’ aerial plant parts were rinsed with 0.5% NaClO in water before placing
in an oven at 40 °C for drying. After 1-week, the samples were blended into well powder.
The obtained powder (10 g) was loaded into a Soxhlet extractor. An aliquot (400 mL) of
methanol:ethyl acetate (2:8, v/v) was used as the extraction solvent. After 5 hours, theachieved extract was filtrated and concentrated using a rotary evaporator (Nihon Buchi K.K.,
Tokyo, Japan) to retrieve the total crude extract (T-Anvi). Subsequently, T-Anvi mixed with
distilled water (50 mL) was subjected to liquid-liquid phase (1:1, v/v) extraction to obtainhexane (H-Anvi), ethyl acetate (E-Anvi), butanol (B-Anvi), and water (W-Anvi) extracts,
respectively. The dried extracts were collected by evaporation at 50 °C under vacuum.
2.2.3. Total phenolic (TPC) and flavonoid (TFC) contents
The TPC of A. virginicus’ extracts was determined applying Folin-Ciocalteu method(Elzaawely et al., 2007). The result was scanned at 765 nm and presented as milligrams ofgallic acid equivalent per one gram of dry weight (mg GAE/g DW).
The TFC of A. virginicus’ was evaluated using the aluminum chloride colorimetricmethod Tuyen et al. (2017). The result was recorded at 430 nm and expressed as milligramsof rutin equivalent per one gram of dry weight (mg RE/g DW).

16

2.2.4. Antioxidant activity
Anti-radical (ABTS and DPPH) assays
The procedures of ABTS and DPPH assays were described in the research of Quan etal. (2019f). The anti-radical capacities were observed as the discoloration of the final
solution with samples and evaluated as the decreased percentage of absorbance over thenegative control (MeOH) at 517 nm and 734 nm for DPPH and ABTS assays, respectively.
The anti-radical (ABTS and DPPH) ability was evaluated following the below
formula:Anti-radical ability (%) = (AC – AS)/AC × 100
AC: Absorbance of reaction with negative control (methanol) after subtracting control’s
blank (without radical solution), AS: Absorbance of reaction with sample/or positive control(BHT) after subtracting sample’s blank (without radical solution).
An established dose-dependent curve (linear equation) applying different
concentrations of samples was used to calculate IC50 values (required concentration
inhibiting 50% of radicals). A higher IC50 value indicates weaker inhibition.
β-Carotene bleaching assay
The reaction containing β-carotene (200 μg), chloroform (1 mL), linoleic acid (20 μL),
and tween 40 (200 mg) was generated according to the protocol of Tuyen et al. (2017). The
obtained solution was then concentrated under vacuum at 40 °C. Subsequently, 50 mL ofoxygenated water were slowly mixed and shaken to obtain a stable emulsion. After thatmethanolic sample/or control (500 µg/mL) was added to the working emulsion (1:8, v/v) ina total of 225 μL of reaction solution. Similar to anti-radical assay, methanol and BHT wereused as negative control and standard inhibitor, respectively. The absorbance was read every
15 min for a total of 180 min at 492 nm. The lipid peroxidation inhibition (LPI) was
measured as follows:

17

LPI (%) = A180/A0 × 100
A180: absorbance of reaction at the 180th min, A0: absorbance of reaction at the beginning
time point.
Reducing power assay
We followed the same protocol described in the study of Quan et al. (2019f). For
preparation, the solutions including phosphate buffer (0.2 M, pH = 6.6); potassium
ferricyanide (1 g/100 mL); trichloro acetic acid (10 g/100 mL); ferric chloride (0.01 g/10mL) were prepared in distilled water.
The mixture of 0.1 mL of methanolic sample/or control, 0.25 mL of phosphate buffer(0.2 M, pH = 6.6), and 0.25 mL of potassium ferricyanide solution (1 g/100 mL) was
incubated for 30 min at 50 °C. After that, 0.25 mL of trichloro acetic acid solution (10 g/100mL) was supplied and centrifuged for 10 min at 4000 rpm. Subsequently, 100 µL of thesupernatant was combined with 100 µL of distilled water, followed by adding 20 µL of ferricchloride solution (0.01 g/10 mL). The absorbance of obtained solution was scanned at 700
nm. BHT and methanol were tested as positive and negative controls, respectively.
Sample/or control with different concentrations (10, 62.5, 125, 250, 500 µg/mL) was
screened to establish the dose-responding curves.
2.2.5. Enzymatic inhibitory assays
Tyrosinase inhibition
The assay components included tyrosinase (500 units/mL in buffer), L-tyrosine
substrate (2 mM in distilled water) solutions. Samples/or controls were prepared in DMSO.
Firstly, potassium phosphate buffer (20 mM, pH = 6.8) was prepared. At the beginning,
20 µL of sample/or control and 20 µL of tyrosinase solution (500 units/mL in buffer) were
mixtured in 120 µL of buffer. The mixture was incubated (25 °C, 5 min). After that, theenzymatic reaction was performed by pipetting 50 µL of L-tyrosine (2 mM in distilled18

water). After 10 min of incubation at 25 °C, the reaction’s absorbance was read at 470 nm(Quan et al., 2019c). kojic acid and DMSO applied used as standard inhibitor and negative
control, respectively. The inhibitory ability and IC50 values were evaluated with the same
method with anti-radical assay.

a-Amylase inhibition
a-Amylase inhibitory activity was examined following the procedure presented by
Quan et al. (2019f). Firstly, iodine (0.25 mM in distilled water), phosphate buffer saline (0.2
M, pH = 6.9), starch substrate (0.5% in distilled water), and a-amylase (5 units/mL in buffer)solutions were prepared. Initially, the mixture of sample/or control (20 µL) and enzyme (20µL) was incubated (37 °C, 9 min). After that starch was (30 µL) added. The reaction was
continued at 37 °C for 7 min before stopping by HCl 1M (20 µL). Finally, 100 µL of iodine
was supplied. The outcome was recorded at 565 nm. Buffer:DMSO (1:1, v/v) and palmitic
acid was negative control and standard inhibitor, respectively. The inhibitory activity wascalculated as follows:% inhibition = AS/AC × 100
AS: Absorbance of reaction with sample/or inhibitor after subtracting an absorbance of the
enzymatic reaction without inhibitor, AC: Absorbance of reaction without sample/or control
and enzyme after subtracting an absorbance of the enzymatic reaction without inhibitors.
IC50 values were established in the same way with anti-radical assay to compare among
samples.
2.2.6. Cytotoxic activity against K562 and Meg-01 cell lines
The effects of A. virginicus’ extracts on tested cell viability were determined applying
MTT assay. IMDM (Sigma-Aldrich, Missouri, United States) containing Penicillin (100IU/mL), Streptomycin (100 µg/mL), L-glutamine (5 mM), and fetal bovine serum (10%)
was used as cell culture. Samples were diluted in 0.1% DMSO (in culture media). 10 µL of

19the cells (5 ´ 103) was seeded in each plate well filled with 90 µL of culture media andplaced in an incubator (Thermo Fisher Scientific, Waltham, United States) (37 °C and CO25%). After 24 h, 10 µL of sample were pipetted to each well and incubated for 40 h.
Subsequently, 10 µL of MTT solution (5 mg/mL) were supplied and continuously incubatedat 37 °C for 4 hours. Finally, 100 µL of cell lysis buffer (10% SDS in 0.01 M HCl) wereadded. The cell growth was observed using an inverted microscope (LabX, Midland,
Canada). The cell viability was examined at 595 nm. 10 µL of DMSO (0.1% in culture
media) was tested as negative control.
The inhibition percentage and IC50 value were calculated following the same method with
anti-radical assay.
2.2.7. Identification of phytochemical component
GC-MS
Phytochemical profiles of H-Anvi were analyzed applying GC-MS (JMS-T100 GCV,JEOL Ltd., Tokyo, Japan). The column (DB-5MS, 30 m × 0.25 mm I.D. × 0.25 µm film
thickness) (Agilent Technologies, J&W Scientific Products, Folsom, United States) wasconnected to GC-MS system. Sample (2 mg/mL) was automatically injected using an
autosampler. Helium with the split ratio of 5:1 was performed. The temperature program ofoven was scheduled as follows: Initial temperature was 50 °C without holding; Rushingtemperature was 10 °C/min for 30 min then maintained for 20 min; Injector and detector
temperatures were 300 °C and 320 °C, respectively. The scanned mass range (29 to 800amu) was operated. The results of chromatogram, mass spectrum, linear retention index(LRI), and Kovats index (KI) of detected compound were collected. Identification ofcompound was conducted using standard sources comprising the library of JEOL’s GC-MS
Mass Center System Version 2.65a, and the online database of National Center for
Biotechnology Information, U.S. National Library of Medicine, Bethesda MD, USA

20

(Pubchem-https://pubchem.ncbi.nlm.nih.gov), and the National Institute of Standards andTechnology, U.S. Department of Commerce (NIST-https://webbook.nist.gov). Amongdetected compounds by GC-MS, palmitic acid was the most abundant compound in H-Anvi.
Therefore, the quantity of palmitic acid was determined based on a calibration curve
established with different concentrations of palmitic acid standard (0.1, 0.5, and 1 mg/mL).
The result was expressed as milligrams per one gram of sample dry weight (mg/g DW).
HPLC-ESI-MS/MS
The phytochemical profiles of E-Anvi were identified based on the HPLC-ESIMS/MS system equipping with LTQ Orbitrap XL mass spectrometers (Thermo Fisher
Scientific, Waltham, United States) and an electrospray ionization (ESI) source. For
analysis, 3.0 µL of sample (2 mg/mL) was injected into the XBridge® Shield RP18 (5 μm,
2.1 ´ 100 mm) column, with maintained temperature at 25 °C. The gradient program withthe flow rate of 0.4 mL/min was set up as follows: 95% A and 5% B (0 – 2 min), then 30%
A and 70% B (2 – 12 min), followed by 0% A and 100% B (12 – 22 min). From 22 – 34
min, the initial condition was performed. In which, solvent A was 0.1% formic acid in waterand solvent B was pure acetonitrile. A negative FTMS mode (mass range from 100 to 700m/z) was applied for mass scanning. The online database (Pubchem, National Center for
Biotechnology Information, U.S. National Library of Medicine, Bethesda MD, USA) wasused for reference to MS/MS spectra.
2.2.8. Data analysis
Data were analyzed applying one-way ANOVA in Minitab software version 16.2.3.
Same group expresses insignificant differences (Turkey’s test, p < 0.05). Pearson’s
correlation coefficients were evaluated using the same software.

21

2.3. Results
2.3.1. Yield and total phenolics (TPC) and flavonoids (TFC) of A. virginicus extracts
From 10 g of A. virginicus dried samples, the yields of total crude (T-Anvi), ethyl
acetate (E-Anvi), hexane (H-Anvi), butanol (B-Anvi), and water (W-Anvi) extracts are 0.98
g (9.8%), 0.32 g (3.2%), 0.16 g (1.6%), 0.12 g (1.2%), and 0.11 g (1.1%), respectively (Table1). The TPC results of T-Anvi, E-Anvi, H-Anvi, B-Anvi, and W-Anvi are 24.80, 25.34, 1.26,
3.26, and 0.49 mg GAE/g DW, respectively. T-Anvi contains the highest TFC (37.40 mg
RE/g DW), followed by E-Anvi, H-Anvi, B-Anvi, and W-Anvi with TFCs are 25.44, 6.30,
4.91, and 0.25 mg RE/g DW, respectively (Table 1).
Table 1. Extraction yield and total phenolic (TPC) and flavonoid (TFC) contents
Extraction yield

TPC

TFC

(%)

(mg GAE/g DW)(mg RE/g DW)

T-Anvi9.8

24.80 ± 0.51a

37.40 ± 0.74a

H-Anvi1.6

1.26 ± 0.03c

6.30 ± 0.13c

E-Anvi3.2

25.34 ± 0.47a

25.44 ± 0.45b

B-Anvi

1.2

3.26 ± 0.06b4.91 ± 0.08d

W-Anvi1.1

0.49 ± 0.01c

0.25 ± 0.01e

Samples

Data express means ± standard deviation (SD). Different superscript letters in a column
indicate significant differences at p < 0.05. TPC, total phenolic content; TFC, total flavonoid
content; T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract;
B-Anvi, butanol extract; W-Anvi, water extract. GAE, gallic acid equivalent; RE, rutin
equivalent; DW, dry weight.
2.3.2. Antioxidant activity of extracts from A. virginicus
The antioxidant activity of A. virginicus’ extracts is determined via ABTS, DPPH, βcarotene bleaching, and reducing power assays.

22

In the ABTS assay, the strongest sample inhibiting ABTS cations is E-Anvi (IC50 =43.59 µg/mL), which is stronger than standard inhibitor BHT (IC50 = 63.51 µg/mL). The
IC50 values of T-Anvi, B-Anvi, H-Anvi, and W-Anvi are 77.71, 86.73, 323.88, and 586.31µg/mL, respectively (Figure 8).

aIC50 value (µg/mL)

600

a

400
b

200
b
c

d

f

cd

e

c

e

de

0
T-Anvi

H-AnviE-Anvi
ABTS

B-Anvi

W-AnviBHT

DPPH

Figure 8. Anti-radical activities (ABTS and DPPH assays) of A. virginicus extracts.
Different letters (a,b,c,d) enclosed with columns (same colors) express significant
differences at p < 0.05 (one-way ANOVA). DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS,
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); T-Anvi, total crude extract; H-Anvi,hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water
extract; BHT, butylated hydroxytoluene.
In the DPPH assay, E-Anvi exhibit the most potent suppression against DPPH radicals
with IC50 of 13.96 µg/mL, followed by T-Anvi, B-Anvi, H-Anvi, and W-Anvi with IC50
values of 30.54, 33.79, 126.27, and 386.91 µg/mL, respectively. Significantly, the23scavenging capacity against DPPH of E-Anvi is approximately twice stronger than that ofBHT (Figure 8).
In the case of β-carotene bleaching model, the inhibition on lipid peroxidation of TAnvi, E-Anvi, H-Anvi, B-Anvi, and W-Anvi are 90.81%, 90.85%, 92.05%, 90.17%, and81.65%, respectively (Figure 9). Almost all samples show strong inhibition on lipid
peroxidation which is close to BHT (LPI = 94.22%), except for W-Anvi.

Figure 9. Inhibition on lipid peroxidation of A. virginicus extracts. Different letters (a,b,c,d)enclosed with columns indicate significant differences at p < 0.05 (one-way ANOVA). LPI,
lipid peroxidation inhibition; T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi,
ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water extract; BHT, butylatedhydroxytoluene.
In the reducing power assay, E-Anvi displays the most potent activity (IC50 = 124.11µg/mL) which is 1.4-fold stronger than BHT (IC50 = 175.52 µg/mL). The following extracts
are T-Anvi and B-Anvi with IC50 of 257.35 and 340.62 µg/mL, respectively. H-Anvi and
W-Anvi exhibit negligible activity (Figure 10).

24

Figure 10. Potassium ferricyanide reducing power of A. virginicus extracts. T-Anvi, total
crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol
extract; W-Anvi, water extract; BHT, butylated hydroxytoluene.
2.3.3. Tyrosinase and a-amylase inhibitory activities of A. virginicus extracts
In the tyrosinase inhibitory results (Table 2), almost all samples reveal suppression
effects. Among them, E-Anvi shows the most substantial activity (IC50 = 2.58 mg/mL),followed by T-Anvi, H-Anvi, and B-Anvi (IC50 = 4.57, 6.22, and 9.40 mg/mL, respectively).
W-Anvi has no effect on the enzyme (Table 2).
In a-amylase inhibitory assay, H-Anvi and T-Anvi (IC50 values = 0.72 and 3.48
mg/mL, respectively) are the most potent extracts preventing a-amylase activity. Notably,
the inhitory effects of H-Anvi is over twice stronger than that of palmitic acid, a well-known
a-amylase inhibitor. E-Anvi and B-Anvi exhibit insignificant suppression on a-amylase(inhibition percentage = 31.93% and 17.52%, respectively at a concentration of 10 mg/mL.
a-amylase is not affected by W-Anvi (Table 2).
Table 2. Tyrosinase and a-amylase inhibitory activities of A. virginicus extracts25

Tyrosinase inhibition

a-Amylase inhibition

IC50 (mg/mL)

IC50 (mg/mL)

T-Anvi4.57 ± 0.05c

3.48 ± 0.07a

H-Anvi6.22 ± 0.08b

0.72 ± 0.01c

E-Anvi2.58 ± 0.13d

neB-Anvi9.40 ± 0.02a

ne

W-Anvina

na

Kojic acid0.02 ± 0.00e-

Palmitic acid-

1.57 ± 0.04b

Samples

Data express mean ± standard deviation (SD). Different superscript letters in a column
indicate significant differences at p < 0.05. T-Anvi, total crude extract; H-Anvi, hexane
extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water extract; na,no activity; ne, negligible effect; -, not determined.
2.3.4. Cytotoxic activity of extracts from A. virginicus against K562 and Meg-01 cell lines
The dose-response curves of A. virginicus extracts for cytotoxic effects against K562
and Meg-01 cell lines are shown in Figure 11. For K562 cell line, E-Anvi is the mostsubstantial extract inhibiting the viability of this cell line (IC50 = 112.01 µg/mL) which is
twice strong than T-Anvi (IC50 = 247.88 µg/mL). Other extracts exhibit insignificant
inhibitions on K562 cell expansion.
In the case of Meg-01 cell line, the extracts of T-Anvi, E-Anvi, and H-Anvi reveal
substantial cytotoxicity with IC50 values of 91.40, 168.94, and 198.07 µg/mL, respectively.
While B-Anvi and W-Anvi have negligible prevention against Meg-01 cell viability (Figure
11).

26

(a)

(b)
1
Meg-01 cell viability

K562 cell viability

1

0.5

0

0.5

0
62.5

125

250

62.5

Concentration (µg/mL)
T-Anvi

H-Anvi125

250

Concentration (µg/mL)
E-Anvi

B-Anvi

W-AnviFigure 11. Dose-response curves of A. virginicus extracts for cytotoxicity against (a) K562
and (b) Meg-01 cell lines. T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi, ethyl
acetate extract; B-Anvi, butanol extract; W-Anvi, water extract.
2.3.5. Correlations between total phenolics (TPC) and flavonoids (TFC) and biologicalproperties of A. virginicus extracts.
The results from Table 3 show that TPC and TFC have a strong correlation withantioxidant activity via ABTS, DPPH, and reducing power assays. On the other hand, thesecompounds might not be involved in lipid peroxidation and a-amylase inhibitory effects of
A. virginnicus extracts. In contrast, flavonoids might contribute to the anti-tyrosinasepotential of A. virginicus. Remarkably, the cytotoxicity on K562 and Meg-01 cell viability
might be mainly determined by the presence of phenolics, especially flavonoid compounds
in A. virginicus’ extracts. The results show that the extract inhibiting K562 cell viability canfeasibly suppress Meg-01 as well. Moreover, a close association between antioxidant, antityrosinase, and K562, Meg-01 cytotoxic activities was recorded, suggesting that A.
virginicus’ extracts have synergic effects against various factors involving in humandiseases.

27

Table 3. Pearson’s correlation coefficients between total phenolic and flavonoid contents
and biological activities of A. virginicus extracts
RP

Tyro

a-Amy

K562

TFC

TFC

0.952*-

ABTS

0.833* 0.674*

DPPH

0.813* 0.633* 0.993*

β-Ca

0.423

RP

0.843* 0.667* 0.987* 0.997* 0.523*

Tyro

0.479

a-Amy

-0.236 -0.043 -0.439 -0.416

0.217

K562

0.898* 0.736* 0.891* 0.914*

0.466

ABTS DPPH

β-Ca

TPC

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-0.391

0.302

-

-

0.330

0.942*

0.393-0.307

-

0.435

0.424

0.526*

0.351

0.554*

0.589* 0.549*

0.525* 0.601* 0.572* 0.970* 0.555*

Meg-01 0.787* 0.913*

0.404

0.378

*, a significance at p < 0.05; TPC, total phenolic content; TFC, total flavonoid content;
ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay; DPPH, 2,2-diphenyl1-picrylhydrazyl assay; β-Ca, β-carotene bleaching assay; RP, reducing power assay; Tyro,
tyrosinase inhibitory assay; a-Amy, a-amylase inhibitory assay; K562, K562 cytotoxic
assay; Meg-01, Meg-01 cytotoxic assay.
2.3.6. GC-MS results
Based on the GC-MS results (Figure 12), four dominant compounds are detected in HAnvi which are reported in Table 4. In which, palmitic acid is the most abundant compound(peak area accounts for 27.97%). The following dominant compound is phytol, 8-methyl-1undecene, and γ-sitosterol with peaks areas of 16.42%, 10.77%, and 7.38%, respectively.
The linear retention index (LRI) and Kovats index (KI) of detected compounds are comparedto the literature. The quantity of palmitic acid is 0.86 mg per 1 g of A. virginicus dry weightand 0.05 mg per 1 g of H-Anvi dried extract (Table 4).

28

Figure 12. GC-MS chromatogram of H-Anvi extract

Table 4. Dominant phytochemicals in H-Anvi extract from A. vigirnicus identified by GCMS
Content
Identified

RT

No.

Peak
MW Formula Classification

compound

(min)

LRI

KI

(mg/g

area (%)
DW)

1
2

Phytol15.80

296

C20H40O Diterpenoids

16.42

1835

1835

-

8-Methyl-1-undecene 16.64

168

C12H24

Alkenes

10.77

1916

1917

-

3

Palmitic acid17.02

256 C16H32O2

Fatty acids

27.97

1955

1955

0.86

4

γ-Sitosterol

28.34

414

Steroids

7.38

-

-

-

C29H50O

RT, retention time; LRI, linear retention index; KI, Kovats index; DW, dry weight; -, notdetermined.
2.3.7. HPLC-ESI-MS/MS results
The results of HPLC-ESI-MS/MS in Figure 13 indicate eight tentative compounds in
E-Anvi. Among them, five compounds can be classified as flavonoids including genistin,29

kaempferol-O-galactopyranoside,kaempferol

eupatilin,

3-O-β-d-glucopyranoside.

quercetin-3-O-β-d-glucopyranoside,The

groupsofandbenzophenones(annulatophenonoside), phenolic glycosides (dihydroferulic acid 4-O-glucuronide),
proanthocyanidins (prodelphinidin B6) are also identified (Table 5).

Figure 13. HPLC chromatogram of E-Anvi extract30

Table 5. Phytocompounds in E-Anvi extract from A. virginicus detected by HPLC-ESI-MS/MS
No.

RT[M+H]-

ChemicalTentative

Molecular

Exact

Fragment ions

Signals

(min)

(m/z)

classification

identity

formula

mass

(m/z)

1

0.68

377.086

Benzophenones

Annulatophenonoside

C18H18O9

378.3

89.024(100); 119.086(53);143.036(45); 149.099(28)Dihydroferulic acid 4-O2

5.74

371.098

Phenolic glycosides

231.158(100); 243.293(23);
C16H20O10

372.3

glucuronide

225.296(19); 121.105(14)Kaempferol-O3

6.08

447.093

Flavonoids

327.154(100); 357.141(48);
C21H20O11

448.4

galactopyranoside

429.155(5); 369.242(4)311.140(100); 341.117(6);

4

6.35

431.098

Flavonoids

Genistin

C21H20O10

432.4
283.142(2)Quercetin-3-O-β-d56.54463.088

Flavonoids

445.145(100); 343.148(92);
C21H20O12

464.4

glucopyranoside

427.169(75); 373.161(48)

Kaempferol 3-O-β-d66.67447.093

Flavonoids

285.167(100); 402.158(6);C21H20O11

glucopyranoside

448.4
428.144(5); 374.128(3)31313.135(100); 297.155(73);78.37

413.088

Proanthocyanidins

Prodelphinidin B6

C21H18O9

414.4
369.145(70); 285.154(20)

8

9.71

343.082

Flavonoids

Eupatilin

C18H16O7

344.3

311.153(100)RT, retention time.
2. 4. Discussion
Recently, the impacts of oxidative stress on many human diseases have been proposed though rarely established (Ambade & Mandrekar,
2012; Biswas et al., 2007; Cachofeiro et al., 2008; Tucker et al., 2015). Numerous studies were carried out worldwide to evaluate the contributionof antioxidant substances to several chronic disorders, for example, skin aging, cancer, diabetes, etc. However, the application on human throughclinical tests have shown failure (Sesso, 2008; Zhang et al., 2008a, 2008b). This unsuccessful result may be due to select only potent antioxidant
compounds to inhibit varuous diseases (Biswas, 2016). Therefore, in our study, the synergic effects of antioxidant, anti-tyrosinase, anti-aamylase, and cytotoxic potentials were investigated on A. virginicus, an invasive weed that was once thought to be of no use.

32

Basically, plant’s biological properties are determined by their chemical profile.
Therefore, potential phytocompounds of A. virgnicus are determined in this study. The
results of total phenolics (TPC) and flavonoids (TFC) indicate that E-Anvi contains thehighest TPC and TFC. The most dominant compounds in E-Anvi belong to the groups offlavonoids and flavonoid glycosides. Theoretically, such compounds show a higher affinity
for water or butanol fractions. Therefore, other studies tend to choose polar solvents andextraction procedures such as hexane, ethyl acetate, butanol, 50% ethanol, and water, which
are more effective in extracting flavonoid glycosides, and their solubility in ethyl acetate israther law (Tagousop et al., 2018; Thavamoney et al., 2018). In fact, the identification andcharacterization of the phytocompound composition of each plant sample is affected bynumerous factors such as plant species and extraction methods. The TPC and TFC in ethyl
acetate extracts were found to be equal to or higher than those obtained from aqueous andbutanol solutions in the case of some species including Euphorbia splendida (Kefayati et al.,2017) or Ziziphus jujube (Al-Saeedi et al., 2016). On the other hand, Soxhlet extraction
applied in this study with methanol:ethyl acetate (2:8, v/v) could collect flavonoid glycosidecomponents. This suggests that the mentioned extraction procedure may be suitable forextracting flavonoid glycosides from A. virginicus. However, further extraction and isolation
protocols should be performed to improve the purification of potential compounds from thisweed.
In antioxidants, the potent activities of phenolics and flavonoids such as radical
scavenging and metal ion chelation have been widely reported. It can be explained via their
specific structure of aromatic ring, and especially the number of hydroxyl (-OH) groups(Pandey & Mishra, 2012; Heim et al., 2002; Kumar & Pandey, 2013; Soobrattee et al., 2005).
The correlation among TPC, TFC, and antioxidant ability of A. virginicus reinforces theabove statement (Table 3). Remarkably, the strongest samples of antioxidant activities

33

include the total crude and ethyl acetate extracts, which are recorded to contain high levelsof phenolic compounds, particularly flavonoids (Table 1). Notably, the ethyl acetate extract
exhibits stronger radical scavenging capacities via ABTS, and DPPH assays and higher
reducing power than BHT, a well-known antioxidant agent (Figure 8). Hence, the HPLCESI-MS/MS method was applied to analyze the chemical profiles in this extract.
Accordingly, the ethyl acetate extract is found rich in flavonoid content, which may
determine the potent antioxidant ability of this extract (Table 5).
Besides the antioxidant activity, flavonoids in A. virginicus may be a main contributor
to anti-tyrosinase ability of the total crude and ethyl acetate extracts in this study (Tables 2,
3, and 5). Previously, flavonoids were recorded having inhibitory effects against tyrosinasethrough in silico and in vitro screenings (Şöhretoğlu et al., 2018). However, unlike theantioxidant capacities, the enzymatic reactions are much more complicated and difficult topredict, which are influenced by various features in chemical structures (Quan et al., 2019f).
For example, the rings A and C, (-OH) substituent at the position of 7, and the ring B linked
to (-OH) groups at the position of para or para and meta in the flavonoid structures lead tothe competitive inhibition against tyrosinase (Şöhretoğlu et al., 2018). In the case of anti-aamylase activity, the inhibition may depend on different factors such as the position of (OH) substituents, methoxy groups, lactone rings, or the interaction between compounds inthe mixture (Quan et al., 2019f). Via the results of a-amylase inhibitory assay (Table 2), thehexane extract from A. virginicus shows the strongest suppression. Thus, the GC-MS
analysis was performed to find out the major compounds involving in the prevention against
a-amylase in this extract. Among the most dominant compounds, palmitic acid shows thehighest peak area and accounts for 0.86 and 0.05 mg per 1 g of A. virginicus dry weight andhexane dried extract, respectively (Table 4), whilst other compounds in Table 4 were neither
successfully isolated nor be purchased, thus need further clarification. Notably, palmitic acid34

is an acknowledged a-amylase inhibitor (Anh et al., 2020; Su et al., 2013a, 2013b), thus it
was used as a positive control in this study. Remarkably, the hexane extract reveals over
twice stronger inhibitory effect than palmitic acid by comparing their IC50 values. Because
palmitic acid is a major compound (Table 4) in the plant, it is suggested that this compound
may play an important role in the biological potential of the invasive weed, beside the aamylase inhibitory effect as shown in this study. The hexane extract’s activity may bestrengthened due to the interaction between four dominant compounds consisting of palmitic
acid, γ-sitosterol, phytol, and 8-methyl-1-undecene (Table 4). In which, γ-sitosterol is also ahigh potential anti-diabetes substance, which has been affirmed through in vitro and in vivo
tests on rats in recent studies (Kumar et al., 2013). Therefore, this compound may play an
influential role in the anti-a-amylase ability of the hexane extract.
The detected flavonoids in the A. virginicus’s total crude and ethyl acetate extracts
may be also the primary agents for the cytotoxic activities against K562 and Meg-01 celllines. According to the previous studies including laboratory bioassays, epidemiologicalinvestigations, and clinical trials, flavonoids have shown important chemotherapy roles forcancer. The anticancer property of flavonoids has been comprehensively explained throughvarious biological pathways such as antioxidants, enzymatic activities, genetic factors, andcell-related mechanisms, or synergic effects of these mentioned factors (Sharma et al., 2011).
Remarkably, the antioxidant activity of flavonoids is closely associated with anticancer
regulation (Kopustinskiene et al., 2020). Meanwhile, phytoflavonoids are not harmful tonormal myeloid, peripheral blood, and epithelial cell lines (Veeramuthu et al., 2017). In our
research, we also find a strong correlation between total phenolics and flavonoids,
antioxidant and cytotoxic capacities in the ethyl acetate extract (Table 3). On the other hand,
the hexane extract’s cytotoxic activity against Meg-01 cell line may be due to the presenceof phytol and γ-sitosterol or the combined influence of the found compounds in this extract35

(Figure 11 and Table 4). Particularly, phytol is a popular anticancer agent, which has been
mentioned in numerous recent studies. The inhibition of this compound on different types ofcancer cells and relevant modes of action have been globally published (Alencar et al., 2019;de Alencar et al., 2019; Pejin et al., 2014; Sakthivel et al., 2018). Whilst γ-sitosterol has been
also known to be an antitumor agent, its specific mechanism has been reported in otherstudies (Endrini et al., 2015; Sundarraj et al., 2012; Teschke & Xuan, 2020).
In addition to finding out potential therapeutic properties and phytocompounds, thespecific direction of developing natural-based drugs is essentially considered to come up
with the most effective approach (Teschke & Xuan, 2020). For example, clinical tests
through randomized controlled trials are required before the products are registered as drugs.
One of the critical factors is that natural products must satisfy the safety requirements. In
fact, some of them have shown hepatotoxicity with high application dose in clinical trials on
humans (Teschke et al., 2016, 2018; Teschke & Xuan, 2020). Therefore, although numerous
plant products have proven their potentials for therapeutic uses, very few of them have been
officially registered and widely applied. An effective approach can be addressed such as
investigating the biological properties of folk medicines and traditional food stuffs. Ginger
shell (Alpinia zerumbet) can be an example, which is considered as a significant contributor
to human longevity in Japanese Okinawa (Teschke & Xuan, 2018, 2020). This plant species
is popularly used with even no clinical data available. However, the overuse of these natural
plants also leads to the loss of biodiversity (Teschke & Xuan, 2020). Therefore, changing
the conventional objects to other available resources, especially problematic invasive
species, is a prospective direction. The exploitation of invasive weed for medicinal purposes
can simultaneously solve the problems in biodiversity conservation and sustainable
development.

36

In general, the invasive weed A. virginicus shows the potential for antioxidant, antidiabetes, anti-skin aging, and anti-CML properties via in vitro approaches. Therefore, in vivo
tests and clinical trials should be further performed to develop novel natural-based drugs.
Additionally, the screening results by GC-MS (Table 4) and HPLC-ESI-MS/MS (Table 5)
indicate the most dominant compounds including palmitic acid, γ-sitosterol, phytol, and 8methyl-1-undecene, flavonoids, and flavonoid glycosides, which might be the main
contributors to the biological activities of this plant. Based on that, the research on
confirmation of their role in biological and pharmaceutical properties of A. virginicus should
be conducted. Furthermore, the quantification of bioactive compounds in mixtures andextracts showing the potent activity should be also carried out. Palmitic acid has beenreported as an inhibitor for anti-diabetes (Anh et al., 2020; Su et al., 2013a, 2013b), phytoland γ-sitosterol are popularly known as anticancer agents (Alencar et al., 2019; de Alencar
et al., 2019; Pejin et al., 2014; Sakthivel et al., 2018). While flavonoids and flavonoid
glycosides can be used as antioxidant agents and applied in treatment for numerous diseases(Pandey & Mishra, 2012; Heim et al., 2002; Kumar & Pandey, 2013; Sharma et al., 2011;
Soobrattee et al., 2005). The quantification of dominant and specific bioactive compounds
should use advantage analytical instruments such as LC-MS and LC-ESI-MS/MS rather thanthe estimation of total flavonoids and phenols and quantification by only GC-MS. Futurestudies focusing on isolation, purification, and quantification of bioactive compounds fromA. virginicus should be performed to explore the medicinal and pharmaceutical potential ofthis invasive species. This could help farmers in developing countries to improve theirincome from the utilization of this problematic weed as a source of antioxidants, anti-skinaging, anti-diabetes, and anticancer.

37

2.5. Conclusions
This is the first investigation of the biological properties of the invasive weed A.
virginicus including antioxidants, anti-a-amylase, and anti-tyrosinase abilities, and cytotoxicity against CML cell lines. The results show that the ethyl acetate extract from A.
virginicus exhibits the highest antioxidant activity via ABTS, DPPH, reducing power assays,and β-carotene bleaching models. In addition, this extract reveals potential anti-tyrosinase
capacity and potent cytotoxicity against K562 and Meg-01 cell lines. Meanwhile, the hexane
extract displays strong a-amylase and Meg-01 inhibitory effects. The chemical analysis
results indicate that A. virginicus aerial parts are rich in flavonoids, palmitic acid, phytol,
and γ-sitosterol, which may principally play a vital role in biological activities of therespective extracts. The finding suggests that A. virginicus is a promising source ofantioxidant, anti-diabetic, anti-tyrosinase, and antitumor agents. Further in vivo and clinicaltests should be performed to confirm and develop the natural functional products from A.
virginicus for pharmaceutical purposes.

38

CHAPTER 3: CYTOTOXIC ACTIVITIES OF MOMILACTONES AGAINSTLEUKEMIA, LYMPHOMA, AND MULTIPLE MYELOMA CELL LINES
3.1. Introduction
Hematological disorder is a serious type of cancer, causing 688,464 deaths worldwide
in 2020 (approximately 60%) (Sung et al., 2021). Among hematological cancers, lymphoma
is the most common type, which achieved in 544,352 cases and 48% of deaths (Sung et al.,2021). In which, mantle cell lymphoma is a subtype of non-Hodgkin’s lymphoma, resulting
in a short survival time of 3 to 5 years for patient (Cheah et al., 2016). Leukemia including
acute and chronic conditions is another dangerous blood cancer type that accounted for474,519 new cases with mortality rate of 66% (Sung et al., 2021). The overgrowth of whiteblood cell and the loss of their functional activities are happened in leukemia, leading toadverse problems for human health (Sung et al., 2021). Besides, multiple myeloma wasreported 176,404 new cases with 66% of deaths (Sung et al., 2021). Patients affected by thisdisease can be suffered from the disruption of bone structure and function (Hanamura, 2021).
In the context of blood cancer has been becoming serious and complicated problems over
the years without any signal of stopping, promising candidates are needed to generate
effective treatments for patients suffering from this cancer.
In the last few decades, numerous studies were carried out aiming to develop
anticancer agents from plant-based products (Anh et al., 2020; Lam et al., 2022; Quan et al.,2022; Un et al., 2022). These natural products have been reported having therapeutic
properties with less toxicity compared to synthetic drugs (Teschke & Xuan, 2020).
Interestingly, simultaneous application of herbal medicines and modern drugs has exhibitedcertain effects to the treatment (Ameade et al., 2018). Among valuable compounds isolated
from plants, diterpene lactones namely, momilactones, have been detected only in rice
(Oryza sativa) and the Hypnum moss (Hypnum plumaeforme). These compounds were39

reported that play an important role in rice responses to biotic and abiotic stresses (KatoNoguchi, 2009; Kato-Noguchi et al., 2010; Kato-Noguchi & Peters, 2013; Quan et al.,2019e; Xuan et al., 2016; Zhao et al., 2018). In other studies, momilactones have shown
potentials for preventing oxidative stress, diabetes (Quan et al., 2019a, 2019b, 2019d),
obesity (Quan et al., 2019d), and skin aging (Quan et al., 2019a). Notably, momilactone B
was reported suppressing colon cancer (Kim et al., 2007), monocytic leukemia (Park et al.,2014), leukemic T cell lines (Lee et al., 2008). However, the cytotoxic potentials ofmomilactones against chronic myeloid leukemia (CML), acute promyelocytic leukemia(APL), mantle cell lymphoma (MCL), and multiple myeloma (MM) have been remained
least elucidated.
In metabolic control of cell death, apoptosis is a natural mechanism to kill unnecessaryor unwanted cells. Accordingly, numerous studies have been targeted the activation of theapoptotic process to suppress the undisciplined invasion of cancer cells (Pfeffer & Singh,2018). In cancer therapy, apoptosis enhancement can represent the most effective nonsurgical approaches for all cases (Pfeffer & Singh, 2018). Therefore, in this study, weinvestigated, for the first time, the cytotoxic potentials of momilactones A (MA) and B (MB)and their mixture MAB (1:1, w/w) against CML (Meg-01 and K562), APL (HL-60), MCL(Mino), and MM (U266 and KMS-11) cell lines through apoptotic pathways.
3.2. Materials and methods
3.2.1. Materials
Purified momilactones A (MA) and B (MB) were obtained from previous study
conducted in our laboratory of Plant Physiology and Biochemistry, Hiroshima University,
Japan (Quan et al., 2019b). In brief, MA and MB were isolated and purified from rice husks(Oryza sativa var. Koshihikari) by column chromatography over silica gel. The identification40

and confirmation of pure MA and MB using TLC, HPLC, LC-ESI-MS, GC-MS, and 1HNMR, and 13C-NMR were presented by Quan et al. (2019b).
The tested cell lines in the current investigation included normal cell MeT-5A (CRL9444™), chronic myeloid leukemia (CML) Meg-01 (CRL-2021™) and K562 (CCL-243™),
acute promyelocytic leukemia (APL) HL-60 (CCL-240™), mantle cell lymphoma (MCL)
Mino (CRL-3000™), and multiple myeloma (MM) U266 (TIB-196™) were purchased from
American Type Culture Collection (ATCC), Virginia, United States. Another MM cell line
KMS-11 (JCRB1179) was obtained from Japanese Collection of Research Bioresource Cell
Bank.
3.2.2. Cell viability (MTT) assay
Cell culture media was prepared in the same method described in Chapter 2. The cells(10 µL) were seeded into a 96-well plate filled with culture media (90 µL) and placed in a
CO2 incubator at 37 °C. After 24 h, the cells were treated with MA, MB, and MAB (10 µL)
with different concentrations (0.5, 1, 5, and 10 µM) for 48 h. Subsequently, 10 µL of the
MTT solution (5 mg/mL, Sigma-Aldrich) was pipetted into each well. The cells were
continuously incubated for 4 h. Finally, 100 µL of cell lysis buffer (10% SDS in 0.01 M
HCl) was applied to dissolve the colored formazan crystals. Culture media instead ofmomilactones was used as the negative control. Meanwhile the well-known medicinescomprising Doxorubicin, Imatinib, and Ibrutinib were tested as the standard inhibitors. The
absorbance at 595 nm was scanned to determine the cell growth rate in value using aspectrophotometer (SpectraMAX M5, Molecular Devices, Sunnyvale, CA, USA) (Anh etal., 2020). The cytotoxic activity (% inhibition) of momilactones/or inhibitors on the tested
cell lines was as follows:
Inhibition (%) = (ANC – AS)/ANC ´ 100

41ANC: Absorbance of reaction with negative control, AS: Absorbance of reaction withmomilactone/or inhibitor.
Dose-responding curves and IC50 values of the momilactones and standard inhibitors
for cytotoxicity against tested cell lines were established. A higher IC50 indicates a weakercytotoxicity activity.
3.2.3. Cell apoptosis (annexin V) assay
The procedure was conducted following Lam et al. (2022). In brief, the cells wereseeded into a 6-well plate and cultured in a CO2 incubator for 24 h with the same condition
as mentioned in MTT assay. The cells were then treated with momilactones for 24 h and 48
h. The non-treated cells were used as a control. After that, the control and treated cells wereincubated with annexin V-conjugated fluorescein isothiocyanate (FITC) (Biolegend, San
Diego, CA, USA) and propidium iodide (PI) for 15 min at 25 °C. The intensities of annexin
V-FITC and PI, and the percentages of apoptotic cells were determined by a flow a flow
cytometer (BD, Franklin Lakes, NJ, USA).
3.3. Results
3.3.1. Effects of momilactones A (MA) and B (MB) and their mixture (MAB) on cell
viability of blood cancer cell lines
The in vitro cytotoxic activities of momilactones A (MA) and B (MB) and their
mixture (MAB) (1:1, w/w) against blood cancer cell lines, including the chronic myeloid
leukemia (CML) Meg-01 and K562, acute promyelocytic leukemia (APL) HL-60, mantle
cell lymphoma (MCL) Mino, and multiple myeloma (MM) U266 and KMS-11 cell lines are
expressed as IC50 values in Table 6. The mesothelium cell line MeT-5A was tested as the
non-cancerous control. In addition, Doxorubicin, Imatinib, and Ibrutinib were applied as thestandard inhibitors.

42

Table 6. Cytotoxic activities of momilactones A and B against non-cancerous (MeT-5A), chronic myeloid leukemia (Meg-01 and K562), acutepromyelocytic leukemia (HL-60), mantle cell lymphoma (Mino), and multiple myeloma (U266 and KMS-11) cell lines
MeT-5A

Meg-01

K562

HL-60

Mino

U266

KMS-11(% Inhibition at 10 µM)

IC50 (µM)

IC50 (µM)

IC50 (µM)

IC50 (µM)

IC50 (µM)

IC50 (µM)

MA28.52 ± 2.93a

7.19 ± 0.39a

8.18 ± 0.33a

ne

ne

ne

ne

MB

38.00 ± 2.29b

4.39 ± 0.32b5.02 ± 0.56b

4.49 ± 0.34a

5.89 ± 0.14a

5.09 ± 0.58b

4.97 ± 0.10b

MAB37.82 ± 3.64b4.30 ± 0.55b4.13 ± 0.38b4.61 ± 0.10a

5.19 ± 0.39b5.59 ± 0.17b5.36 ± 0.10a

Doxorubicin

49.23 ± 6.17c

3.34 ± 0.42c

3.13 ± 0.21c

5.22 ± 0.15b

0.64 ± 0.03d

0.24 ± 0.01c

0.46 ± 0.01c

Imatinibne0.61 ± 0.01d

0.67 ± 0.01d

ne

-

-

-

Ibrutinib

ne

-

-

-

0.85 ± 0.03c

7.97 ± 0.34a

neCompounds

Outcomes are presented as means ± standard deviations (SD). Means within a column followed by similar superscript letters (a,b,c,d) areinsignificantly different at p < 0.05. MA, momilactone A; MB, momilactone B; MAB, the mixture of MA and MB (1:1, w/w); ne, negligible
effect; -, not determined.
According to Table 6, MA, MB, and MAB exhibit a slight inhibition on the non-cancerous cell line MeT-5A with the percentages of
28.52%, 38.00%, and 37.82% which are weaker than Doxorubicin (% inhibition = 49.23%) at a concentration of 10 µM. Imatinib and Ibrutinib
are observed as having insignificant effects on MeT-5A.

43

The proliferation of typical CML cell lines including Meg-01 and K562 is remarkably
prevented by MA, MB, and MAB (IC50 = 7.19, 4.39, and 4.30 µM, respectively for Meg-01
and IC50 = 8.18, 5.02, and 4.13 µM, respectively for K562). For APL cell line HL-60, MA
shows negligible inhibition. Meanwhile, MB and MAB display a strong cytotoxicity with
IC50 values of 4.49 and 4.61 µM, respectively. Besides, Doxorubicin and Imatinib reveal a
potent suppression against Meg-01 (IC50 = 3.34 and 0.61 µM, respectively) and K562 (IC50= 3.13 and 0.67 µM, respectively). Significantly, Imatinib presents a negligible effect on
HL-60, while Doxorubicin (IC50 = 5.22 µM) exhibits a lower cytotoxic activity than MB(IC50 = 4.49 µM) and MAB (IC50 = 4.61 µM).
In the case of MCL, the expansion of Mino is negligibly diminished by MA. Whereas
MB and MAB exhibit a strong inhibition (IC50 = 5.89 and 5.19 µM, respectively). While thecell line is dramatically suppressed by the drug Doxorubicin (IC50 = 0.64 µM), followed by
Ibrutinib (IC50 = 0.85 µM).
The cytotoxic effects on MM cell lines consisting of U266 and KMS11 indicate that
all the tested samples significantly inhibit the cell growth, except for MA. Among them,
Doxorubicin displays the strongest prevention against U266 and KMS11 (IC50 = 0.24 and0.46 µM, respectively), followed by MB (IC50 = 5.09 and 4.97 µM, respectively), and MAB(IC50 = 5.59 and 5.36 µM, respectively). Remarkably, Ibrutinib shows a weaker cytotoxic
activity compared to MB and MAB when the IC50 value is 7.97 µM for U266. Meanwhile
Ibrutinib reveals negligible inhibition on KMS-11.
The dose–responding curves of MA, MB, and MAB against non-cancerous (MeT-5A),
CML (Meg-01 and K562), APL (HL-60), MCL (Mino), and MM (U266 and KMS-11) celllines are presented in Figure 14.

44

MeT-5A

Meg-01

K562

0

0.5

0
0.5
1
5
10
Concentration (µM)

0.5

0
0.5
1
Concentration (µM)

Mino

5

Cell viability

Cell viability

0

0.5

5

0.5
1
5
10
Concentration (µM)

1

0.5

0

0
0.5
1
Concentration (µM)

0

KMS-11

1

0.5

0.5

0
0.5
1
5
10
Concentration (µM)

U266

1

Cell viability

1

Cell viability

0.5

HL-60

1

Cell viability

1

Cell viability

Cell viability

1

0.5
1
5
10
Concentration (µM)

0.5
1
5
10
Concentration (µM)

Figure 14. Dose-responding curves of momilactones A and B for cytotoxic activities against
non-cancerous (MeT-5A), chronic myeloid leukemia (Meg-01 and K562), acutepromyelocytic leukemia (HL-60), mantle cell lymphoma (Mino), and multiple myeloma(U266 and KMS-11) cell lines. MA, momilactone A; MB, momilactone B; MAB, themixture of momilactones A and B (1:1, w/w).
Following the results shown in Table 6 and Figure 14, MA is the weakest compound
inhibiting tested cancer cell lines. While MB and MAB substantially suppress these cell lines
at around 5 µM. Therefore, MB and MAB at a concentration of 5 µM were selected forfurther investigation of their effects on tested cancer cell apoptosis.
3.3.2. Apoptosis-inducing activities of momilactones against blood cancer cell lines
Through annexin V assay, only MB-48 h slightly increases MeT-5A cell apoptosis.
Meanwhile MB-24 h, MAB-24 h and -48 h exhibit no effect on apoptosis of this cell line.
Particularly, MeT-5A apoptotic cells account for 3.86%, 2.87%, 2.44%, 10.3%, 4.73% in
the untreated control and the treatments of MB-24 h, MAB-24 h, MB-48 h, and MAB-48 h,
respectively (Figure 15).

45

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing
activities of momilactones
against non-cancerous cellline (MeT-5A)

Annexin V-FITC

Figure 15. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against non-cancerous MeT-5A cell line
after 24 h and 48 h.
Regarding CML cell line Meg-01, MB and MAB have insignificant influences on theapoptosis of this cell line after 24 h, whereas these compounds increase the apoptotic process
in Meg-01 after 48 h (% apoptosis = 26.2% and 21.7% for the treatments with MB and MAB,
respectively) (Figure 16). In another CML cell line K562, the tested compounds significantly
increase cell apoptosis after 24 h (7.96- and 10.28-fold for MB and MAB, respectively) and48 h (18.44-fold for both MB and MAB), compared to the untreated control (Figure 16).

46

(a)

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing
activities of momilactones
against leukemia cell line
(Meg-01)

Annexin V-FITC(b)

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing
activities of momilactones
against leukemia cell line
(K562)

Annexin V-FITC

Figure 16. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against chronic myeloid leukemia (CML)(a) Meg-01 and (b) K562 cell lines after 24 h and 48 h.

47For APL cell line, MB and MAB enhance HL-60 cell apoptosis after 24 h by 1.30 and3.26 times, respectively, over the control (Figure 17). Significantly, after 48 h, HL-60
apoptotic process significantly increases to 40.5% and 42.1% in the cell affected by MB and
MAB, respectively which are much higher than the control (% apoptosis = 0.86%) (Figure
17).

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing
activities of momilactones
against leukemia cell line
(HL-60)Annexin V-FITC

Figure 17. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against acute promyelocytic leukemia(APL) HL-60 cell line after 24 h and 48 h.
For the tested MCL cell line, MB and MAB substantially promote Mino cell apoptosis
after both 24 h- and 48 h-treatment. The percentages of apoptosis achieve in 4.06%, 35.7%,
32.3%, 51.6%, and 44.1%, respectively, in the control and the treatments comprising MB24 h, MAB-24 h, MB-48 h, and MAB-48 h, respectively (Figure 18).

48

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing activities
of momilactones against
mantlecelllymphoma(Mino)

Annexin V-FITC

Figure 18. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against mantle cell lymphoma (MCL)
Mino cell line after 24 h and 48 h.
In the case of MM cell line, the apoptotic cells of U266 are recorded increasing by
7.44%, 6.32%, 9.31%, and 9.62%, respectively, under the effects of MB-24 h, MAB-24 h,
MB-48 h, and MAB-48 h, in comparison with the non-treated control (Figure 19). Similarly,
MB and MAB remarkably activate the apoptotic process in KMS-11 (Figure 19). The
number of KMS-11 apoptotic cells account for 22.4%, 20.3%, 55.7%, 44.4% under thetreatments of MB-24 h, MAB-24 h, MB-48 h, and MAB-48 h, respectively, while thepercentage of apoptosis is 1.58% in the control.

49

(a)

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing activities
of momilactones against
multiple myeloma (U266)

Annexin V-FITC(b)

Cell count

CTRL

MB-24 hMAB-24 h

MB-48 hMAB-48 h

Apoptosis-inducing activities
of momilactones against
multiple myeloma (KMS-11)

Annexin V-FITC

Figure 19. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against multiple myeloma (MM) (a) U266
and (b) KMS-11 cell lines after 24 h and 48 h.

50Overall, MB and MAB inhibit the tested blood cancer cell lines by inducing apoptotic
process. Meanwhile, these compounds exhibit an insignificant effect on normal cells.
3.4. Discussion
In recent years, a vast number of studies have been conducted to exploit anticancer
potentials of plant-based products (Anh et al., 2020; Lam et al., 2022; Quan et al., 2022; Unet al., 2022). In these studies, the cell viability (MTT) assay was an integral part to examine
the potentials of a promising candidate. Principally, the cytotoxic activity of derived
compounds from plants is dose-dependent (Lam et al., 2022). Therefore, we investigated theeffects of momilactones in increased concentrations on selected blood cancer cell lines.
Importantly, a promising candidate should inhibit tumors with an IC50 of less than or equal
to 5 µM (Lichota & Gwozdzinski, 2018). Accordingly, MB and MAB with IC50 of around 5µM against tested blood cancer cells might be useful for developing novel medicines.
Besides, the cytotoxic ability of momilactones is compared to that of widely applied
medicines consisting of Doxorubicin, Imatinib, and Ibrutinib in our study. In which,
Doxorubicin is commonly used to treat lymphoma and MM (Thorn et al., 2011). Especially,
Doxorubicin was widely applied as a positive control in the research on cytotoxic activity
against HL-60 cell line (Rodrigues et al., 2021). Imatinib is popularly used for leukemia
patients (Moen et al., 2007). On the other hand, Ibrutinib has been announced as anti-MMagent via in vitro, in vivo, and clinical tests (Li, 2021; Richardson et al., 2018). Based on the
MTT results, the cytotoxicity of MB and MAB is close to that of Doxorubicin against Meg01 and K562. Significantly, MB and MAB are stronger than Doxorubicin in preventing HL60 cells and Ibrutinib in inhibiting U266 cells. Interestingly, momilactones are less toxic tonormal cell line MeT-5A compared to Doxorubicin, implying that they may be promising
candidates for anti-blood cancer therapy.

51

In cancer, the loss of apoptosis occurs in all cases which allow cancer cells to avoid
death, leading to their continuous proliferation and expansion (Pfeffer & Singh, 2018).
Interestingly, chain events of apoptosis can be promoted by decreasing the activities ofantiapoptotic factors and/or enhancing the activities of proapoptotic factors which cansubsequently eliminate cancer cells (Pfeffer & Singh, 2018). Accordingly, apoptosis isconsidered a major target in the development of effective anticancer therapy (Pfeffer &Singh, 2018). Through the annexin V assay, we determined, for the first time, that MB and
MAB at a concentration of 5 µM can suppress blood cancer cells by promoting cell
apoptosis. Notably, apoptosis induction is regulated by relevant protein expressions. For
example, in intrinsic pathway, an anti-apoptotic family, namely BCL-2, can inhibit apoptotic
process (Pfeffer & Singh, 2018), while a pro-apoptotic member, namely caspase, candecompose proteins, resulting in cell death (Pfeffer & Singh, 2018). Based on that, further
studies on cytotoxic mechanism of momilactones against blood cancer cells through thealteration of regulatory proteins related to the apoptotic process such as BCL-2 and caspaseshould be conducted.
In other studies, momilactone B was reported to suppress colon cancer (Kim et al.,2007), monocytic leukemia (Park et al., 2014), leukemic T cell lines (Lee et al., 2008).
However, the cytotoxic ability of momilactones was compared to that of widely applieddrugs in our research which has not been reported in other studies. Besides, this is the firstreports that MB and MAB can inhibit chronic myeloid leukemia (CML), acutepromyelocytic leukemia (APL), mantle cell lymphoma (MCL), and multiple myeloma (MM)cell lines by inducing cell apoptosis. Interestingly, the antioxidant capacity of momilactones
was also previously indicated. In which, synergistic effect of MA and MB revealed stronger
antioxidant capacity than individual compounds (Quan et al., 2019a). This may be aneffective approach in cancer treatment since the negative effects of medicines (e.g.,52

Doxorubicin) relate to oxidative stress by enhancing free radicals (Thorn et al., 2011).
Thereby, candidates simultaneously exhibiting antioxidant and cytotoxicity against tumors
may be useful for developing cancer therapies. Moreover, a correlation between chronic
disorders including diabetes, obesity, aging, and cancer through the central role ofinflammation and oxidative stress has been acknowledged (Collins, 1999). Interestingly,
momilactones have exhibited potential for anti-diabetes, anti-obesity, and anti-skin aging
activities (Quan et al., 2019a, 2019b, 2019d). Thereby, these compounds can be considered
a promising source for improving blood cancer treatments, especially for patientscomplicated with oxidative stress and chronic diseases.
MA and MB were principally found in rice husk (Kang et al., 2017; Kato-Noguchi etal., 2010; Lee et al., 1999; Minh et al., 2018), leaf (Xuan et al., 2016), root and root exudates(Kato-Noguchi, 2011; Kato-Noguchi et al., 2010). Recently, liquid chromatographyelectrospray ionization-mass spectrometry (LC-ESI-MS) method was improved todetermine MA and MB content in white rice bran (Quan et al., 2019b). Therefore, our finding
may support forthcoming strategies to take the advantages of rice bran and rice by products
as a potential source for anti-blood cancer. Notably, rice is monocot plants, which adapt toa wide range of environmental conditions (Simpson, 2010). Thus, rice organs can be feasibly
exploited for pharmaceutical purposes with an abundant biomass source.
In another concern, the bioaccessibility of bioactive compounds can be regulated
through human digestion (Un et al., 2022). Thus, future research should be conducted toclarify the effects of digestive process on the bioaccessibility and bioavailability ofmomilactones. Besides, a natural-based drug must have benefits over risks (Teschke &Xuan, 2020). Therefore, the optimized dose of momilactones should be established tosupress substantially tumors without harmful effects on normal blood cells. On the other
hand, the potential risk such as neurotoxicity and hepatotoxicity by using herbal products

53

should be seriously considered and thoroughly evaluated (Quan et al., 2020). Accordingly,
in vivo tests and clinical trials should be further conducted for the development ofmomilactones as novel anti-CML, anti-APL, anti-MCL, and anti-MM drugs.
3.5. Conclusions
This is the first study indicate that MB and MAB can inhibit chronic myeloid leukemia(CML) Meg-01 and K562, acute promyelocytic leukemia (APL) HL-60, mantle cell
lymphoma (MCL) Mino, and multiple myeloma (MM) U266 and KMS-11 cell lines with
IC50 of around 5 µM via MTT test. The cytotoxic activity of MB and MAB is close to thatof Doxorubicin in preventing Meg-01 and K562. Meanwhile, MB and MAB are stronger
than Doxorubicin and Ibrutinib in inhibiting HL-60 and U266, respectively. Throughannexin V assay, momilactones suppress the tested cancer cell lines by promoting their cell
apoptosis. Significantly, these compounds exhibit negligible effects on non-cancerous MeT5A cell line. The finding implies that momilactones may be a promising source for thedevelopment of novel anti-CML, anti-APL, anti-MCL, and anti-MM medicines.

54

CHAPTER 4: CYTOTOXIC MECHANISM OF MOMILACTONES A AND B
AGAINST ACUTE PROMYELOCYTIC LEUKEMIA AND MULTIPLE
MYELOMA CELL LINES
4.1. Introduction
Blood cancer is a serious human disorder, accounting for over 1.2 million cases
annually in the world (Sung et al., 2021). Among blood cancer types, the incidence ofworldwide leukemia was reported 474,519 new cases with 311,594 deaths in 2020 (Sung etal., 2021). Of which, acute promyelocytic leukemia (APL) regularly gets aggravation duringchemotherapy and has a poor prognosis with a high level of early death because of bleedingfrom coagulopathy. On the other hand, multiple myeloma (MM) was achieved in 176,404
cases with 117,007 deaths in 2020 (Sung et al., 2021). In MM patients, the accumulated cells
in bone marrow can lead to bone lesions with the disruption of structure and function(Hanamura, 2021). APL and MM have been becoming serious and complicated problems
over the years without any signal of stopping. Therefore, pharmaceutical and medicinalcandidates are urgently needed to develop effective treatments for patients suffering fromthese cancers.
In anticancer studies, numerous strategies have been conducted to promote theapoptotic process, which is a natural mechanism for cell death to control or eliminate theundisciplined expansion of tumor (Pfeffer & Singh, 2018). Enhanced apoptosis is one of themost effective approaches for developing specific anticancer therapy and represents the mostsuccessful non-surgical treatment for all cancer cases (Pfeffer & Singh, 2018). Anotherpotential target in anticancer research is the cell cycle, which strictly regulates cell division
through multiple control mechanisms (Matthews et al., 2022). Cancer-associated mutations
lead to abnormal regulation that prevents cells from exiting the cell cycle, followed bycontinuous cell division (Matthews et al., 2022). Therefore, inducing cell cycle arrest is a55

promising method for inhibiting tumor’s proliferation and expansion. Interestingly, both
apoptotic and cell cycle processes can be regulated by regulatory proteins (Otto & Sicinski,
2017; Pfeffer & Singh, 2018). Therefore, substances with synergistic effects on apoptosis
induction and cell cycle arrest through mediating the activities of relevant protein may be
excellent candidates for developing efficient cancer therapies.
In recent years, a vast number of studies have been conducted considering the
anticancer potentials of plant-based products (Anh et al., 2020; Lam et al., 2022; Quan et al.,2022; Un et al., 2022) which have exhibited benefits for therapeutic purposes with lesstoxicity than synthetic medicines (Teschke & Xuan, 2020c). Reality also shows that thesimultaneous use of herbal remedies and modern medicine has brought certain effectivenessto the treatment (Ameade et al., 2018). Among valuable plant-derived analytes,
momilactones, diterpene lactones, have been found only in rice (Oryza sativa) and the
Hypnum moss (Hypnum plumaeforme). These compounds were first known as phytoalexins
which principally play a role in the defense system of rice against pathogens (Zhao et al.,2018). Recently, momilactones have exhibited antioxidant, anticancer (leukemia (Park et al.,2014), lymphoma (Lee et al., 2008), and colon cancer (Kim et al., 2007), anti-diabetes (Quanet al., 2019b, 2019d), anti-obesity (Quan et al., 2019d), and anti-skin aging properties (Quanet al., 2019a). Hitherto, the mechanism of cytotoxic and anticancer actions of momilactones
has not been scrutinized comprehensively. The limitation of in-depth studies about
anticancer activity of momilactones may be due to the confined availability on the market aswell as the difficulty in isolation and purification (Quan et al., 2019b). Our laboratory is oneof the few laboratories in the world can purify momilactones from natural sources. In
preceding reports, we successfully established a method to achieve a remarkable amount ofmomilactones A and B from rice by-products (Quan et al., 2019b).

56

The aforementioned rationales prompted us to investigate the cytotoxic mechanism ofmomilactones A (MA) and B (MB) and their mixture MAB (1:1, w/w) on HL-60 (a typicalcell line isolated from APL patients) and U266 (a well-known cell line derived from MM
patients) through apoptotic and cell cycle pathways, and the expressions of relevant
regulatory proteins.
4.2. Materials and methods
4.2.1. Materials
Momilactones A (MA) and B (MB) were previously isolated and purified from ricehusk in our laboratory of Plant Physiology and Biochemistry, Hiroshima University, Japan(Quan et al., 2019b). Briefly, MA and MB were isolated from the ethyl acetate (EtOAc)
extract of rice husks (Oryza sativa var. Koshihikari) by column chromatography over silica
gel with the mobile phase of hexane:EtOAc (8:2). The identification and confirmation ofsuch pure compounds applying TLC, HPLC, LC-ESI-MS, GC-MS, and 1H-NMR, and 13CNMR were described in the previous study of Quan et al. (2019b).
The cell lines including non-cancerous MeT-5A (CRL-9444™), acute promyelocytic
leukemia HL-60 (CCL-240™), and multiple myeloma U266 (number: TIB-196™) werepurchased from ATCC (Manassas, VA, USA).
4.2.2. Cell viability (MTT) assay
In this assay, culture media was prepared by adding fetal bovine serum (10%), Lglutamine (5 mM), penicillin (100 IU/mL), and streptomycin (100 µg/mL) to IMDM
(Sigma-Aldrich, Missouri, United States). The cells (5 × 103 cells/well) were seeded into a
96-well plate filled with 90 µL of culture media and placed in a CO2 incubator at 37 °C.
After 24 h, the cells were treated with MA, MB, and MAB (10 µL) with different
concentrations (0.5, 1, 5, and 10 µM) for 48 h. Subsequently, 10 µL of the MTT solution (5mg/mL, Sigma-Aldrich) was pipetted into each well. The cells were continuously incubated57

for 4 h. Finally, 100 µL of cell lysis buffer (10% SDS in 0.01 M HCl) was applied to dissolve
the colored formazan crystals. Culture media instead of momilactones was used as thenegative control. Meanwhile the drugs consisting of Doxorubicin, Ibrutinib, all-trans retinoic
acid (ATRA), arsenic trioxide (ATO), and Bortezomib were tested as the standard inhibitors.
The absorbance at 595 nm was scanned to determine the cell growth rate in value using aspectrophotometer (SpectraMAX M5, Molecular Devices, Sunnyvale, CA, USA) (Anh etal., 2020). All tests were performed with 3 replications. The cytotoxic activity (% inhibition)of momilactones/or inhibitors on the tested cell lines was as follows:
Inhibition (%) = (ANC – AS)/ANC ´ 100
ANC: Absorbance of reaction with negative control, AS: Absorbance of reaction withmomilactone/or inhibitor.
Dose-responding curves and IC50 values of the momilactones and standard inhibitors forcytotoxicity against tested cell lines were established. A lower IC50 indicates a stronger
cytotoxicity activity.
4.2.3. Cell apoptosis (annexin V) assay
The procedure was conducted in triplicate following Lam et al. (2022). In brief, thecells (5 × 105 cells/well) were seeded into a 6-well plate filled with 1.5 mL of culture media
and cultured in a CO2 incubator for 24 h with the same condition as mentioned in MTT
assay. The cells were then treated with momilactones at a concentration of 5 µM for 24 h
and 48 h. The non-treated cells were used as a control. Harvested cells were washed twicewith cold phosphate-buffered saline (PBS). After that, the control and treated cells wereincubated with annexin V-conjugated fluorescein isothiocyanate (FITC) (Biolegend, San
Diego, CA, USA) and propidium iodide (PI) for 15 min at 25 °C. The obtained cells weredissolved in 450 µL of PBS. The solution was filtered by a nylon membrane to prevent cellclumping and kept on ice until analysis. The intensities of annexin V-FITC and PI, and the58percentages of apoptotic cells were instantly determined by a flow a flow cytometer (BD,Franklin Lakes, NJ, USA).
4.2.4. Cell cycle assay
The cells (5 × 105 cells/well) were cultured and treated in triplicate following the same
methods with apoptosis assay. The FxCycle PI/RNase staining solution was applied
according to the manufacturer’s instructions (Calbiochem, Darmstadt, Germany). The cell
cycle distribution at each phase of G1, S, and G2 was determined based on the cell’s DNA
content. The percentages of cells in different phases of the cell cycle were quantified by aflow cytometer (BD, Franklin Lakes, NJ, USA). In brief, the collected cells were washedwith ice-cold PBS. Subsequently, ice-cold PBS in pure ethanol was added to disperse thecells. The obtained solution was stored at 4 °C for 24 h for fixing. For analysis, the cells
were incubated with 10 mg/ml RNase A (Sigma-Aldrich) for 5 min on ice. The following
step was conducted by adding 1 mg/mL PI (in PBS). After incubating for 10 min at room
temperature, the cells were dissolved in 450 µL of PBS. The solution was then filtered using
a nylon membrane to remove cell clumping before analysis. Subsequently, the flow
cytometric measurement was immediately performed.
4.2.5. Western blotting assay
The cells (5 × 105 cells/well) were cultured and treated in triplicate following the same
methods with apoptosis and cell cycle assays. The cell lysates were conducted by rinsing
cells with PBS, followed by adding two times of 2x loading buffer (4% SDS, 10% 2mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl, pH 6.8). The
extracted protein (200 pg) was subjected to sodium dodecyl sulfate (SDS)-polyacrylamidegels applying 10% acrylamide and subsequently transferred to a polyvinylidene fluoride
membrane (Takara Bio, Shiga, Japan) by electroblotting. The membrane was blocked using
3% skim milk in PBS-0.05% Tween 20 (PBS-T) at 25 °C for 1 h. The incubation with each59

antibody (2 µg/mL) against anti-rabbit total p-38/MAPK, phosphorylated p-38/MAPK,
BCL-2, procaspase-3, cleaved caspase-3, CDK1/cdc2, cyclin B1, and GAPDH (BioLegend,
California, United States) in blocking buffer was conducted overnight at 4 °C. The following
step was performed by washing the membrane with PBS-T in triplicate. The collected
membrane was incubated with a secondary antibody of horseradish peroxidase-labeled goatanti-rabbit IgG (20 ng/mL) (IBL, Gunma, Japan) at 37 °C for 1 h. Protein bands werevisualized with the use of the LAS-4000 image analyzer (GE Healthcare, Tokyo, Japan).
The relative expression (RE) was calculated by normalizing the intensity of targeted proteinsto the intensity of the housekeeping protein GAPDH.
4.2.6. Statistical analysis
Data are displayed as mean ± standard deviation (SD) (n = 3). Student’s t-test or oneway ANOVA was used to compare groups. Differences were considered significant at p <0.05 (Minitab 16.0 software, Minitab Inc., State College, United States).
4.3. Results
4.3.1. Effects of momilactones on cell viability of non-cancerous (MeT-5A), acutepromyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines
The cytotoxic activities of momilactones A (MA) and B (MB) and their mixture(MAB) (1:1, w/w) in increased concentrations against the cell viability of non-cancerous
MeT-5A, acute promyelocytic leukemia (APL) HL-60, and multiple myeloma (MM) U266
cell lines after 48 h of treatments are displayed in Figure 20. In addition, the cytotoxicity ofmomilactones is compared with that of well-known inhibitors including Doxorubicin,
Ibrutinib, all-trans retinoic acid (ATRA), arsenic trioxide (ATO), and Bortezomib.

60

MeT-5A

U266

ns

100

*

*

120

ns
*

ns

**

** *** ** *80nsCell viability (%)

Cell viability (%)

120***** *** ***

60

***

*** ***
***

40
20

ns

100

0

ns

ns

ns

** *

80

**

*** ***

**

60
40

***

20

***

*** ************ ***

***
***

0
0

0.5

1
5
Concentration (µM)

10

0

0.5

1
5
Concentration (µM)

10

HL-60
Cell viability (%)

120

**

**

* ns ns

100

** *

80

*
**

ns
**
***

***

***

60

***
***

***

40
***

20

***

***
***

*** ***

***

0
0

0.5

1

5

10

Concentration (µM)

Figure 20. Effects of momilactones A (MA) and B (MB) and their mixture (MAB) (1:1,
w/w) on cell viability of non-cancerous (MeT-5A), acute promyelocytic leukemia (HL-60),
and multiple myeloma (U266) cell lines after 48 h. Data are expressed as mean ± standard
deviation (SD). Statistical significance was determined by t-test. *p < 0.05 versus control (0µM); **p < 0.01 versus control (0 µM); ***p < 0.001 versus control (0 µM); ATRA, all-transretinoic acid; ATO, arsenic trioxide; ns, not significant versus control (0 µM).
According to Figure 20 and Table 7, MA, MB, and MAB exhibit a minor inhibition
on normal cell line MeT-5A with percentages of 28.52%, 38.00%, and 37.82%, respectively
which are lower than Doxorubicin (inhibition percentage = 49.23%) at a concentration of 10µM (Figure 20).
The APL cell line HL-60 is least inhibited by MA with a percentage of 30.25% at 10µM (Figure 20). Meanwhile, MB and MAB display a potent cytotoxic capacity against HL60 (IC50 = 4.49 and 4.61 µM, respectively) which is more substantial than Doxorubicin and
ATO (IC50 = 5.22 µM and 13.80, respectively) (Table 7 and Figure 20). The inhibitory
effects of MB and MAB on HL-60 are close to that of ATRA (IC50 = 3.99 µM) (Table 7 andFigure 20).

61

In the case of MM cell line, MA exhibits the lowest effect on U266 cell proliferation(inhibition percentage = 40.97%) at 10 µM (Figure 20). On the other hand, Doxorubicin
reveals the strongest prevention against U266 (IC50 = 0.24 µM), followed by MB and MAB(IC50 = 5.09 and 5.59 µM, respectively) (Table 7 and Figure 20). Notably, MB and MAB
show higher cytotoxicity against U266 cells than Ibrutinib (IC50 = 7.97 µM) (Table 7 andFigure 20). Whereas the widely applied drug Bortezomib for MM shows an outstanding
cytotoxicity on U266 with an IC50 value of 7.97 nM (Supplementary Data, Figure S11).
Table 7. Cytotoxic activities (IC50) of momilactones A and B against MeT-5A, HL-60, andU266 cell lines (Obtained results from Chapter 3)
MeT-5A

HL-60

U266

(% Inhibition at 10 µM)

IC50 (µM)

IC50 (µM)

MA28.52 ± 2.93a

-

-

MB

38.00 ± 2.29b

4.49 ± 0.34b

5.09 ± 0.58b

MAB37.82 ± 3.64b4.61 ± 0.10b5.59 ± 0.17b

Doxorubicin

49.23 ± 6.17c

5.22 ± 0.15b

0.24 ± 0.01c

Ibrutinib--

7.97 ± 0.34a

ATRA

-

3.99 ± 0.16b

-

ATO

-

13.80 ± 1.77a-

Compounds

Outcome is presented as mean ± standard deviation (SD). Means within a column followedby similar superscript letters (a,b,c,d) are insignificantly different at p < 0.05 (one-way
ANOVA). MA, momilactone A; MB, momilactone B; MAB, the mixture of MA and MB(1:1, w/w); ATRA, all-trans retinoic acid; ATO, arsenic trioxide; -, not determined.
In general, MA is the weakest compound inhibiting tested cancer cell lines. While MB
and MAB substantially suppress these cell lines at around 5 µM. Therefore, MB and MAB

62

at a concentration of 5 µM were selected for further investigation of their cytotoxic
mechanism.
4.3.2. Apoptosis-inducing activities of momilactones against non-cancerous (MeT-5A),
acute promyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines
In this assay, the annexin V method was applied to evaluate the effects of MB and
MAB at 5 µM on the cell apoptosis of non-cancerous MeT-5A, acute promyelocytic
leukemia (APL) HL-60, and multiple myeloma (MM) U266 cell lines (Figure 21).

63

(a)
MeT-5A

MB-24 hMAB-24 hNormal cellline MeT-5A

ns

8

ns

4

ns

*

h

h

24

24

48
h
M
A
B48
h

MAB-48 hMB-

MB-48 h

M
B-

C

on
tr
ol

0

M
A
B-

Control% Apoptosis

Cell count12

Annexin V-FITC

(b)
HL-60

10

h
48

h

h
24

M
A

MAB-48 h

B-

48

h
24

M
B-

on
tr
ol
C
MB-48 h

ns

ns

0

M
A

APL cell line
HL-60

20

M
B-

MAB-24 h30

B-

MB-24 h% Apoptosis

Cell countControl

**

**

40

Annexin V-FITC

(c)
U266
25

*

MB-24 hMAB-24 h

MM cell line
U266

*

15

*

*

10
5

MAB-48 h

h
24

B24
h
M
B48
h
M
A
B48
h

MB-48 h

M
A

C

on
tr
ol

0

M
B-

Control% Apoptosis

Cell count20

Annexin V-FITC

64

Figure 21. Apoptosis-inducing effects of momilactone B (MB) and the mixture ofmomilactone A and B (MAB) (1:1, w/w) at 5 µM against (a) non-cancerous MeT-5A, (b)acute promyelocytic leukemia (APL) HL-60, and (c) multiple myeloma (MM) U266 celllines after 24 h and 48 h. Statistical significance was determined by t-test. *p < 0.05 versus
control; **p < 0.01 versus control; ns, not significant versus control.
The effects of MB and MAB at 5 µM on cell apoptosis of normal cells (MeT-5A) arepresented in Figure 21a. The results obtain that after 24 h, MB and MAB reveal a mild
decrease in cell apoptosis of MeT-5A (% apoptosis = 2.87% and 2.44%, respectively), while
apoptotic cells in the non-treated control account for 3.86% (Figure 21a). After 48 h, a slightincrease in cell apoptosis of MeT-5A is recorded under the influences of MB and MAB(2.67- and 1.23-fold, respectively) compared to the non-treated control (Figure 21a).
Regarding APL cell line, MB and MAB reveal an insignificant increase in theapoptotic process of HL-60 after 24 h (1.30- and 3.26-fold, respectively over the control)(Figure 21b). Remarkably, after 48 h, the number of HL-60 apoptotic cells is dramaticallyenhanced to 40.50% and 42.10% under MB and MAB effects, respectively which are muchhigher than the control (% apoptosis = 0.86%) (Figure 21b).
For the tested MM cell line, MB and MAB remarkably promote U266 cell apoptosis
after 24 h and 48 h. Apoptotic cells account for 4.06%, 16.90%, 22.70%, 18.0%, and 20.5%
in the control and the treatments of MB-24 h, MAB-24 h, MB-48 h, and MAB-48 h,
respectively (Figure 21c).
Overall, MB and MAB promote apoptosis in cancer cells (HL-60 and U266) andexhibit a minor effect on normal cells (MeT-5A).

65

4.3.3. Effects of momilactones on inducing cell cycle arrest of non-cancerous (MeT-5A),
acute promyelocytic leukemia (HL-60), and multiple myeloma (U266) cell lines
The effects of MB and MAB at 5 µM on the cell cycle of MeT-5A, HL-60, and U266
cell lines after 24 h and 48 h are displayed in Figure 22.
(a)
MeT-5A
ns

60

ns
ns

40

Control

MB-24 hMAB-24 h% Cells

Cell count

ns

ns
ns

20

Normal cellline MeT-5A

**

ns

ns

**

*
*

*

ns

**

**

0
Control

MB-48 hMB-24 hMAB-48 h

Sub-G1

DNA content

MAB-24 h
G1S

MB-48 hMAB-48 h

G2

(b)
HL-60

Control

MB-24 hMAB-24 h% Cells

Cell count

60

*

**

40

*

**

**
**

**
***

20

APL cell line
HL-60

*

ns

**,^^
***

**

*,##

ns

ns

0
Control

MB-48 hMB-24 hMAB-48 h

Sub-G1

MAB-24 h
G1S

MB-48 hMAB-48 h

G2

DNA content

(c)
U266
60

ns
ns

ns
*

Control

MB-24 hMAB-24 h***% Cells

Cell count

40
**

20
*

MM cell line
U266

*

ns
**

*

*
**

**

*

0
Control

MB-48 hDNA content

MAB-48 h

MB-24 hSub-G1

MAB-24 h
G1S

MB-48 hMAB-48 hG266

Figure 22. Effects of momilactone B (MB) and the mixture of momilactone A and B (MAB)(1:1, w/w) at 5 µM on the cell cycle of (a) non-cancerous (MeT-5A), (b) acute promyelocytic
leukemia (HL-60), and (c) multiple myeloma (U266) cell lines after 24 h and 48 h. Statisticalsignificance was determined by t-test. *p < 0.05 versus control; **p < 0.01 versus control;***p < 0.01 versus control; ^^p < 0.01 versus MAB-24 h; ##p < 0.01 versus MB-48 h; ns, notsignificant versus control.
In Figure 22a, MB and MAB reveal a negligible impact on sub-G1 phase of normal
cell (MeT-5A) after 24 h. After 48 h, the cell percentages of sub-G1 are increased by 2.59
and 2.65 times under the effects of MB and MAB, respectively after 48 h, compared to theuntreated control. The outcomes reveal that MeT-5A cell death is slightly elevated in thetreatment with MB and MAB for 48 h which is consistent with the apoptosis (annexin V)
results. Whereas the cell counts have insignificant changes in G1 and G2 phases after 24 h
and 48 h. While the decreased cell numbers are recorded in S phase of the cells affected by
MB and MAB. This finding implies that MB and MAB have an insignificant effect on thecell cycle of MeT-5A (Figure 22a).
For HL-60, MB- and MAB-treated cells are significantly accumulated in G2 phase of
the cell cycle. Particularly, the cell counts are increased from 13.9% in G2 phase of thecontrol cells to 30.1% and 52.8%, respectively in the cells affected by MB and MAB after
24 h (Figure 22b). After 48 h, the percentages of HL-60 cells in G2 phase are 29.2% and27.7% under the effects of MB and MAB, respectively (Figure 22b). The results indicatethat MB and MAB remarkably arrest the cell cycle of HL-60 at G2 phase, accompanied by
the reduced percentages of cells in G1 and S phases.
In the case of U266 cells, there is no remarkable difference in the cell number of G1
phase among the control and the treatments with MB and MAB (Figure 22c). Meanwhile
the cell counts are reduced in S and G2 phases which may be caused by the increased cell67

deaths in sub-G1 phase under the effects of MB and MAB after 24 h and 48 h (Figure 22c).
Generally, MB and MAB have negligible impacts on U266 cell cycle (Figure 22c).
Generally, MB and MAB show a negligible effect on the cell cycle of normal cells(MeT-5A) and MM cells (U266). Whereas these compounds activate G2 arrest in the cell
cycle of APL cells (HL-60).
4.3.4. Effects of momilactones on expressions of proteins related to apoptosis induction
and cell cycle arrest of acute promyelocytic leukemia (HL-60), and multiple myeloma(U266) cell lines
Based on the results of apoptosis induction and G2 phase arrest in the cell cycle of HL60 treated with MB and MAB, the expressions of relevant proteins to apoptosis (total p-38,
phosphorylated p-38, BCL-2, procaspase-3, and cleaved caspase-3) and G2 phase (total p38, phosphorylated p-38, CDK1, and cyclin B1) were evaluated (Figure 23). Meanwhile,
MB and MAB promote the apoptotic process in U266, but fail to activate cell cycle arrest.
Therefore, the expressions of regulatory proteins in the apoptotic pathways comprising total
p-38, phosphorylated p-38, BCL-2, procaspase-3, and cleaved caspase-3 were examined(Figure 23).

68

(a) HL-60

**

**,†

0.8
0.4

4
2
***

**

**,†

**,††

**

h
8

h

B4
M
A

M
A

B2

4

h

Co
nt
ro
l

h
B4

M
A

M
A

B2

8

h

h
M
B48

4

h

M
B24

Phosphorylated p-38/Total p-38

Cyclin B1

**

1
0

Co
nt
ro
l

Total p-38***,†††

30

Phosphorylated p-38**,^^,##

5

h

GAPDH

**

M
B48

BCL-2

1.2

M
B24

MAB
24h 48h

Mitochondrial pathway

6**

Relative expression

MB
Ctrl 24h 48h

Relative expression

Mitogen-activated protein kinase(MAPK) pathway

BCL-2/GAPDH
Cleaved caspase-3/Procaspase-3

CDK1

G2 phase of cell cycle pathway

Procaspase-3
Relative expression

1.6

Cleaved caspase-3
GAPDH***,†††

1.2

**

**

0.8

***,†

***,^^^,##**,††**,†††***,^^,##

0.4
0Control

MB-24 h MB-48 h MAB-24 h MAB-48 h

Cyclin B1/GAPDHCDK1/GAPDH(b) U266
Mitogen-activated protein kinase(MAPK) pathway

0.5
0***,^^**,††***,††

0.8

***
**

0.4

**

**

**,†

h
B4

8

h
4
M
A

h
M
B48

B2

h

M
B24

Co
nt
ro
l

h
8

4

B4
M
A

h

h

0

Phosphorylated p-38/Total p-38
GAPDH

Relative expression

1

B2

Cleaved caspase-3**,†††

1.5

M
B48

Procaspase-3

1.2**,^,#

hTotal p-38

Mitochondrial pathway

**

2

M
B24

Phosphorylated p-38

2.5
Relative expression

GAPDH

***

M
A

BCL-2

M
A

MAB
24h 48h

Co
nt
ro
l

MB
Ctrl 24h 48h

BCL-2/GAPDH
Cleaved caspase-3/Procaspase-3

Figure 23. Effects of momilactone B (MB) and the mixture of momilactones A and B(MAB) (1:1, w/w) on the expressions of proteins related to apoptosis induction and cell cycle
arrest of (a) acute promyelocytic leukemia (APL) HL-60, and (b) multiple myeloma (MM)U266 cell lines after 24 h and 48 h. Statistical significance was determined by t-test. *p <0.05 versus control; **p < 0.01 versus control; ***p < 0.001 versus control; †p < 0.05 versus
MB-24 h; ††p < 0.01 versus MB-24 h; †††p < 0.01 versus MB-24 h; ^p < 0.05 versus MAB24 h; ^^p < 0.01 versus MAB-24 h; ^^^p < 0.001 versus MAB-24 h; #p < 0.05 versus MB-48
h; ##p < 0.01 versus MB-48 h.

69

According to Figure 23a, expression of phosphorylated p-38/total p-38 is increased in
APL (HL-60) cells affected by MB and MAB with the relative expression (RE) values in thecontrol and the treatments including MB-24 h, MB-48 h, MAB-24 h, and MAB-48 h are
0.48, 1.15, 0.96, 0.82, and 0.85, respectively. Meanwhile, the protein bands of BCL-2 aredramatically degraded under the effects of MB (the RE values in 24 h- and 48 h-treatment
are 1.92- and 2.32-fold, respectively, lower than the control) and MAB (the RE values in 24
h- and 48 h-treatment are 2.16- and 2.67-fold, respectively, lower than the control) (Figure23a). Besides, the RE values of cleaved caspase-3/procaspase-3 are substantially enhanced
in HL-60 treated with MB (RE = 0.56 and 0.77 after 24 h and 48 h, respectively) and MAB(RE = 2.97 and 5.17 after 24 h and 48 h, respectively), while the value in the untreatedcontrol is 0.17 (Figure 23a). In the cell cycle pathway, cyclin B1 and CDK1 expressions are
remarkably impeded in HL-60 affected by MB and MAB. The RE values in the control andthe treatments comprising MB-24 h, MB-48 h, MAB-24 h, and MAB-48 h are 1.17, 0.80,
0.69, 1.39, 0.79, respectively, for cyclin B1. While the values are 1.55, 1.10, 0.75, 0.68, 0.40,
respectively, for CDK1 (Figure 23a).
For MM cell line U266, MB and MAB remarkably elevate the expression ofphosphorylated p-38/total p-38 (RE = 1.00, 2.47, 2.35, 1.47, and 1.88 in the control and thetreatments of MB-24 h, MB-48 h, MAB-24 h, and MAB-48 h, respectively) (Figure 23b).
Besides, MB and MAB inhibit BCL-2 expression after 24 h by 2.37 and 2.74 times,
respectively, compared to the control. Meanwhile, after 48 h, MB and MAB decrease theexpression of BCL-2 by 4.41- and 2.88-fold, respectively, over the control (Figure 23b). The
expression of cleaved caspase-3/procaspase-3 in U266 treated with MB after 24 h and 48 h
is increased by 5.33 and 8.83 times, respectively, over the control. While in the treatment
with MAB, the RE values of caspase-3/procaspase-3 after 24 h and 48 h are 6.92 and 9.08
times, respectively, higher than the control (Figure 23b).

70

4.4. Discussion
In anticancer research, the cell viability (MTT) assay is an indispensable initial step in
exploring antitumor candidates (Anh et al., 2020; Lam et al., 2022; Quan et al., 2022; Un etal., 2022). Principally, the cytotoxicity of natural compounds is dose-dependent (Anh et al.,2020; Lam et al., 2022; Quan et al., 2022; Un et al., 2022). Therefore, we examined theactivities of momilactones in increased concentrations against APL (HL-60) and MM(U266) cell lines, compared with non-cancerous (MeT-5A) cell lines. The drugs including
Bortezomib, all-trans retinoic acid (ATRA), arsenic trioxide (ATO), Doxorubicin, andIbrutinib were tested as reference suppressors (Table 1, Figure 1, and Supplementary
Materials, Figure S1). In which, Bortezomib is a famous medicine for MM treatment(Rajkumar, 2022). ATRA and ATO show their effectiveness in APL therapies. (Sanz et a.,2019). Doxorubicin is commonly used to treat lymphoma and MM (Thorn et al., 2011).
Besides, Doxorubicin is commonly used to treat lymphoma and MM (Thorn et al., 2011).
Especially, Doxorubicin was widely applied as a positive control in the research on cytotoxic
activity against HL-60 cell line (Rodrigues et al., 2021). Ibrutinib has been announced asanti-MM agent via in vitro, in vivo, and clinical tests (Li, 2021; Richardson et al., 2018).
However, Ibrutinib was proven that selectively inhibits FLT3-ITD mutant cell lines among
acute myeloid leukemia types, which do not include HL-60 (Wu et al., 2016). In fact,
Ibrutinib revealed mild cytotoxicity on HL-60 in preceding study (IC50 = 41.94 µM) (Li etal., 2017). Therefore, the drug is not considered a standard inhibitor against this cell line.
Following the results shown in Figure 20, MB and MAB may be ideal candidates fordeveloping novel anticancer drugs since their cytotoxic abilities (IC50 of around 5 µM) arestronger than Doxorubicin in preventing HL-60 cells and Ibrutinib in inhibiting U266 cells(Figure 20). Moreover, the candidate must have a high cytotoxic selectivity against tumors
without damaging normal cells (Indrayanto et al., 2021). Interestingly, these compounds are71less toxic to non-cancerous cell MeT-5A than Doxorubicin (Figure 20). Based on that, weselected MB and MAB to clarify their cytotoxic mechanism in suppressing HL-60 and U266
cancer cells.
Via apoptosis, cell cycle, and western blotting analyses (Figures 21, 22, and 23), MB
and MAB may inhibit tested cancer cells through apoptosis induction and cell cycle arrestby regulating relevant protein expressions. Among targeted proteins, p-38 group serves as
an important signaling mediator in the mitogen-activated protein kinase (MAPK) pathway,
which contributes to many biological processes including inflammation, cell cycle,
apoptosis, development, differentiation, senescence, and tumor formation in specific cells(Zarubin & Han, 2005). Remarkably, previous studies focusing on the regulation of p-38
were conducted to overcome the drug resistance and improve the suppressive effects on MM
cell lines consisting of MM.1S, RPMI8226, and U266 (Hideshima et al., 2003). The
activation of p-38 can be determined by the fold increase in the expression of phosphorylated
p-38/total p-38 (Rodrigues et al., 2021). Accordingly, the upregulated phosphorylation of p38 observed from western blotting outcomes may cause apoptosis enhancement of HL-60
and U266 treated with MB and MAB (Figure 23). On the other hand, the elevated expressionof p-38 can destabilize cdc25b and cdc25c which subsequently disrupt CDK1/cyclin B1
complex (Ferenbach & Bonventre, 2016). This disruption may lead to HL-60 cell cycle arrest
at G2 phase in this study. Regarding apoptosis, an anti-apoptotic member, namely BCL-2,
suppresses apoptotic process by sequestering the preforms of fatal cysteine proteases orblocking the release of mitochondrial cell death factors into the cytoplasm (Tsujimoto,1998). Significantly, elevated expression of BCL-2 has been detected in more than half of
all cancer cases (Pfeffer & Singh, 2018). Thus, apoptosis promotion by inhibiting BCL-2
activities could be a promising approach to eliminating tumors. Interestingly, numerous
plant-based products have shown their role in activating cancer cell apoptosis through BCL-

72

2 pathway, for example, curcumin from Curcuma longa or graviola from Annona muricata(Pfeffer & Singh, 2018), suggesting a great potential in the development of anticancer
medicines. In this study, MB and MAB extremely impede the expression of BCL-2 in HL60 and U266 cell lines after 48 h (Figure 23) which may motivate the apoptotic process inthese cell lines. In another concern, many traditional medicines suppress cancer cells
primarily depending on the BCL-2/BAX mechanism (Yip & Reed, 2008). Disruption of thissignaling pathway can cause intrinsic resistance to drugs (Pfeffer & Singh, 2018). Therefore,
substances targeting multiple factors can enhance the effectiveness of cancer treatment. In
the present study, the effects of MB and MAB on the activation of a pro-apoptotic factor,
namely caspase-3, were also determined (Figure 23). During the apoptotic process,procaspase-3 is converted to the active form caspase-3, which decomposes proteins,
resulting in cell death (Pfeffer & Singh, 2018). Importantly, elevated procaspase-3 has been
observed in various cancer cases (e.g., acute myeloid leukemia), involving in poor prognosis(Boudreau et al., 2019; Testa & Riccioni, 2007). Through the regulations of procaspse-3 andcleaved caspase-3 expressions (Figure 23), it may be conjectured that MB and MAB
stimulate the conversion of procaspase-3 to caspase-3 in both tested cancer cell lines HL-60
and U266. This may cause the promotion of proteolysis in these cancer cells which
subsequently kills them. In the cell cycle pathway, the activation of the CDK1/cyclin B1
complex plays a major role in the transition from G2- to M-phase. Thereby, inhibited
CDK1/cyclin B1 activity leads to G2 phase arrest (Wang et al., 2000). Several anticancer
studies were conducted focusing on G2/M phase arrest through this mechanism. For
example, genistein arrested G2/M phase in the cell cycle of colon cancer cells (Zhang et al.,2013) and breast cancer (Choi et al., 1998). In our study, the expressions of CDK1 and cyclin
B1 in HL-60 are significantly impeded by MB and MAB (Figure 23a). The finding indicatesthat MB and MAB may disrupt the interaction of CDK1/cyclin B1 complex, causing HL-60

73

cell cycle arrest at G2 phase, followed by mitosis inhibition. In other studies, the potentialof MB in inhibiting colon cancer cells (HT-29 and SW620) was shown via MTT, lactate
dehydrogenase (LDH), and colony-forming ability assays (Kim et al., 2007). Besides, MB
suppressed human monocytic leukemia cell line U937 by stimulating apoptosis and the cell
cycle arrest at G1 phase via the decrease in pRB phosphorylation and the upregulation of a
CDK inhibitor p21Waf1/Cip1 (Park et al., 2014). Lee et al. (2008) announced that MB prevented
human leukemic T cells (Jurkat) by inducing apoptosis through the mitochondrial pathways.
In addition, the inhibitory effect of MB on HL-60 cell viability was previously demonstrated(Lee et al., 2008), but the cytotoxic mechanism has not been elucidated. Furthermore, to thebest of our knowledge, the present study is the first to clarify the induction of MB and MAB
on apoptotic and cell cycle arrest pathways of HL-60 and U266 cells through the regulationof relevant proteins (Figure 24). Additionally, the cytotoxic potential of momilactones ishighlighted by comparing with that of acknowledged standard suppressors in our study
which has not been reported elsewhere. Forthcoming trials to develop momilactones as novelanti-APL and anti-MM drugs should also include the comparison between momilactones
and well-known medicines. For example, in the case of APL therapies, all-trans retinoic acid
(ATRA) and arsenic trioxide (ATO) are widely used (Sanz et al., 2019). While for MM
treatments, proteasome inhibitors (PI) (e.g., Bortezomib, Carfilzomib, and Ixazomib),immunomodulatory agents (IMiD) (e.g., Lenalidomide, Pomalidomide, and Thalidomide),
monoclonal antibodies (e.g., Daratumumab and Elotuzumab), and targeted B cell maturation
agent (BCMA) therapies are commonly applied (Rajkumar, 2022). Moreover, thecombination use of momilactones and these drugs should be investigated aiming to enhancethe efficiency of targeted therapies, reduce the negative side effects, and overcome drug
resistance (Jaaks et al., 2022; Quan et al., 2020; Rajkumar, 2022; Sanz et al., 2019). On theother hand, the impacts of momilactones on normal cells should be comprehensively

74interpreted since they might be affected by the same course of event with tumors (TaşkınTok & Gowder, 2014). In this study, momilactones slightly promote cell apoptosis of normal
mesothelial cell line (MeT-5A) but exhibit no effects on the cell cycle. Forthcoming studies
should focus on the alterations of relevant proteins to the apoptotic process in MeT-5A cells
treated with momilactones. Additionally, other sensitive cells to drugs including bonemarrow, gonads (sex organs), gastrointestinal tract, and skin (hair follicle cells) should bealso included to clearly understand the toxicity or adverse effects of momilactones (TaşkınTok & Gowder, 2014). This is an integral requirement to minimize failure in later stages ofdrug development. Furthermore, this may help establish potential drug combinations as wellas effective therapies (Taşkın-Tok & Gowder, 2014).

Inhibition

Cytotoxicity against HL-60

Stimulation

Cytotoxicity against U266

p-38

CDK1/Cyclin B1

BCL-2

Cell cycle

Mitochondria

Caspase-3

Cell death© Quan Juno 2022

Figure 24. Cytotoxic mechanism of momilactones against acute promyelocytic leukemia(HL-60) and multiple myeloma (U266) cell lines.
In addition to the above-mentioned anticancer potentials, the antioxidant capacity ofmomilactones was previously reported. It was noteworthy that the synergistic effect of MA
and MB revealed stronger antioxidant capacity than individual compounds (Quan et al.,2019a). This may address the complications in cancer progression and treatment due to the75side effects of drugs associated with oxidative stress (Thorn et al., 2011). Thus, substances
simultaneously revealing antioxidant and cytotoxic properties may be excellent candidates
for the development of effective cancer therapies. Moreover, a correlation between chronic
disorders including diabetes, obesity, aging, and cancer through the central role ofinflammation and oxidative stress has been acknowledged (Collins, 1999). Interestingly,
momilactones have recently exhibited potential for anti-diabetes, anti-obesity, and anti-skinaging activities (Quan et al., 2019a, 2019b, Quan, 2019d). Thereby, these compounds canbe considered a promising source for improving blood cancer treatments, especially forpatients complicated with oxidative stress and chronic diseases.
MA and MB were principally found in rice husk (Kato-Noguchi et al., 2010), leaf
(Xuan et al., 2016), and root (Kato-Noguchi et al., 2010). Recently, a specific sample
preparation technique and advanced ultra-performance liquid chromatography-electrosprayionization-mass spectrometry (UPLC-ESI-MS) method were improved to increase thedetection sensitivity that help quantify MA and MB in different rice organs with a minor
amount (e.g., in rice bran) (Quan et al., 2019b). Those outstanding achievements may
support prospective strategies to take the advantages of momilactones from rice and rice byproducts for pharmaceutical purposes. Notably, rice is monocot plant, which adapt to a wide
range of environmental conditions (Simpson, 2010). Thus, rice organs can be feasibly
exploited for medicinal production and therapeutics with an abundant biomass availability.
In addition to momilactones, a number of 47 momilactone-like molecules haveacknowledged (Zhao et al., 2018), suggesting an abundant source for further investigationsof their cytotoxic potentials against blood cancer cells. In another concern, the biologicalactivity and bioaccessibility of substances can be affected by human digestion (Un et al.,2022). Accordingly, next studies should be conducted to investigate the bioaccessibility andbioavailability of momilactones during the digestive stages. Moreover, a natural-based

76

product must satisfy the requirements of benefits outweighing risks (Teschke & Xuan,
2020c). Therefore, the effective concentration of momilactones should be established to
exhibit the strongest cytotoxicity against tumors without harmful effects on normal cells. On
the other hand, the potential risks such as neurotoxicity and hepatotoxicity by using herbal
products should be seriously considered and thoroughly evaluated (Quan et al., 2020).
Overall, MB and MAB are promising candidates, which are highly recommended forthe next steps of developing novel anti-APL and anti-MM medicines. In vivo tests are
required to confirm their possibilities for proposing prospective and appropriate clinical
trials.
4.5. Conclusions
In this report, we interpret, for the first time, the cytotoxic mechanism of momilactones
A (MA) and B (MB) and their mixture (MAB) against acute promyelocytic leukemia (APL)
HL-60 and multiple myeloma (MM) U266 cell lines. Remarkably, the cytotoxicity of MB
and MAB is more potent than that of Doxorubicin and Ibrutinib in preventing HL-60 andU266. MB and MAB may induce HL-60 and U266 cell apoptosis via the mitogen-activatedprotein kinase (p-38) and mitochondrial (BCL-2 and caspase-3) signaling pathways. In
addition, HL-60 cell cycle is arrested at G2 phase by MB and MAB through the regulationsof related protein (p-38, CDK1, and cyclin B1) expressions. Significantly, momilactones
reveal an insignificant effect on the normal cell line MeT-5A. It can be concluded thatmomilactones are promising candidates for developing novel anti-APL and anti-MM
medicines. Moreover, momilactones and momilactone-like compounds are expected asprospective natural sources for future production. However, the dose-effectiveness,
bioaccessibility, and bioavailability of these analytes need validation via in vivo tests before
considering further clinical trials.

77

CHAPTER 5: EFFECTS OF IN VITRO DIGESTION ON ANTI-BLOOD CANCERPROPERTIES OF MOMILACTONES
5.1. Introduction
Blood cancer is a dangerous human disease, causing about 1.2 million cases annually(Sung et al., 2021). In which, lymphoma accounting for more than half million cases per
year is the most common type with 48% of death (Sung et al., 2021). Mantle cell lymphoma
is a subtype of lymphoma, resulting in a short survival time of 3 to 5 years for patient (Cheahet al., 2016). On the other hand, leukemia comprising chronic and acute conditions is thesecond most common blood cancer type with over 400,000 new cases every year. However,leukemia results a high mortality rate of 66% (Sung et al., 2021). In leukemia, the disruptionof white blood cell’s functional activities and their overgrowth are occurred, causing adverse
problems for human health (Sung et al., 2021). Another blood cancer type is multiple
myeloma with the fewest new cases of 176,404 annually. However, this disorder results in
66% of deaths (Sung et al., 2021). Additionally, patients suffering from multiple myeloma
can go through with the loss of bone structure and function (Hanamura, 2021). Recently,
blood cancer has been causing serious and complicated problems for patients. Therefore,
novel anti-blood cancer agents are urgently needed, considering the potential of plant-based
products for therapeutic use, an attractive field of current research.
In the development of anticancer therapy, numerous oral anticancer substances havebeen registered as drugs, and many others are under improvement. Orally administered
medications have been attracted much attention because of its convenience and simple
application, especially in palliative conditions (Liu et al., 1997; Payne, 1992). Regarding
oral administration, numerous phytocompounds exhibited potential biological activities,
however they severe conditions of gastrointestinal digestion, leading to the reduction in theirbioaccessibility and bioavailability (Krzyzanowska et al., 2010; Un et al., 2022; Choonara

78

et al., 2014). Therefore, a thorough understanding of the effects of human digestion on
bioaccessibility and bioavailability of interested candidates is required to develop them forpharmaceutical purposes (Krzyzanowska et al., 2010). However, in vivo and clinical studies
are expensive, challenging, and required ethical constraints (Kong & Singh, 2008a, 2008b).
Notably, an effective protocol of in vitro digestion (oral, gastric, and intestinal stages) has
been widely developed to simulate human digestive process (Kong & Singh, 2008a, 2008b).
The models are low-priced, useful, flexible, and no ethical concerns regarding clinical trials(Kong & Singh, 2008a, 2008b). Accordingly, in vitro simulated digestion method is simple
and convenient to investigate structural modifications, digestibility, and the release of target
products.
Based on the above-mentioned rationales, the objectives of this study were (1) toelucidate the cytotoxic potentials of momilactones against chronic myeloid leukemia (CML)
Meg-01, acute promyelocytic leukemia (APL) HL-60, mantle cell lymphoma (MCL) Mino,and multiple myeloma (U266) cell lines; and (2) to clarify the effects of in vitro simulated
digestion (oral, gastric, and intestinal stages) on cytotoxic activity and bioaccessibility ofmomilactones.
5.2. Materials and methods
5.2.1. Materials
Momilactones were purified and confirmed from the previous study conducted in ourlaboratory of Plant Physiology and Biochemistry, Hiroshima University, Japan (Quan et al.,2019b).
The tested blood cancer cell lines in this study were obtained from American Type
Culture Collection (ATCC) and presented in Table 8.
Table 8. List of blood cancer cell lines
No.

Cell line

ATCC number

Cell type79

1

Meg-01(CRL-2021™)

Chronic myeloid leukemia (CML)

2

HL-60

(CCL-240™)

Acute promyelocytic leukemia (APL)

3

Mino

(CRL-3000™)

Mantle cell lymphoma (MCL)

4

U266

(TIB-196™)

Multiple myeloma (MM)5.2.2. In vitro digestion model
The model was conducted following the protocol described by Un et al. (2022).
Accordingly, the artificial saliva, gastric, and intestinal juices were prepared with the major
components and enzymes which are presented in Table 9.
Table 9. Components of artificial digestion juices (Un et al., 2022)

Artificial saliva

Gastric juice

Major components

Volume/amountKCl (89.6 mg/mL)

10.0 mL

KSCN (20 mg/mL)

10.0 mL

NaH2PO4 (88.8 mg/mL)

10.0 mL

Na2PO4 (57 mg/mL)

10.0 mL

NaCl (175.3 mg/mL)

1.7 mLurea (25 mg/mL)

8.0 mL

α-Amylase

145.0 mg

Uric acid15.0 mg

Mucin50.0 mg

NaCl (175.3 mg/mL)

15.7 mL

NaH2PO4 (88.8 mg/mL)

3.0 mLKCl (89.6 mg/mL)

9.2 mLCaCl2.2H2O (22.2 mg/mL)

18.0 mL

NH4Cl (30.6 mg/mL)

10.0 mLpH

6.5 ± 0.1

1.0 ± 0.180Intestinal juice

HCl (37%, g/g)

8.3 mL

Glucose (65 mg/mL)

10.0 mL

Glucuronic acid (2 mg/mL)

10.0 mL

Urea (25 mg/mL)

3.4 mL

BSA

1.0 g

Pepsin

1.0 g

Mucin

3.0 g

NaCl (175.3 mg/mL)

40.0 mL

NaHCO3 (84.7 mg/mL)

40.0 mL

KH2PO4 (8 mg/mL)

10.0 mL

KCl (89.6 mg/mL)

6.3 mLMgCl2 (5 mg/mL)

10.0 mL

HCl (37%, g/g)

0.18 mL

Urea (25 mg/mL)

4.0 mLCaCl2.2H2O (22.2 mg/mL)

9.0 mLBSA

1.0 g

Pancreatin

3.0 g

Lipase0.5 g

7.8 ± 0.2

In the first step, 1 mL of sample (1.0 mg/mL) was added to 3 mL of artificial saliva.
The combination was incubated for 5 min at 37 ℃ under shaking. After that, an equivalent
volume of ethyl acetate to samples was supplied for stopping enzymatic reaction andextracting oral digested samples in upper layer. The extraction was repeated for five times.
The simulated digestion in stomach was carried out by providing 4 mL of gastric juice tooral digested samples. The mixture was then incubated under shaking for 1 hour at 37 ℃.
The gastric digested samples were obtained by the same method of extracting oral digested

81samples. For intestinal phase, 4 mL of intestinal juice was mixed with gastric digested
samples before incubating under the same condition for 1 hour. The intestinal digested
samples were achieved by the same extraction method with ethyl acetate as above
description. The schematic representation of in vitro digestion is summarized in Figure 25.
(1mg/mL) of MA, MB, and MAB
mixture (1:1, w/w)
Oral stage
1 mL sample + 3 mL artificial saliva
pH = 6.5 ± 0.1

Gastric stage
Oral sample + 4 mL gastric juice
pH = 1.0 ± 0.1

Intestinal stage
Gastric sample + 4 mL intestinal juice
pH = 7.8 ± 0.2

Undigested samples

incubating for 5 min at 37 ℃
under shaking
Add EtOAc to samples and collect upper
layer. Repeat the extraction for five times,
followed by evaporation to get dried extracts
incubating for 1 h at 37 ℃
under shaking
Add EtOAc to samples and collect upper
layer. Repeat the extraction for five times,
followed by evaporation to get dried extracts
incubating for 1 h at 37 ℃
under shaking
Add EtOAc to samples and collect upper
layer. Repeat the extraction for five times,
followed by evaporation to get dried extracts

Prepared digestive juices

Oral digested samples

Gastric digested samples

Intestinal digested samples

Sample incubation

Figure 25. Procedure for in vitro simulated digestive model
5.2.3. Cell viability (MTT) assay
In this assay, culture media was prepared by adding fetal bovine serum (10%), Lglutamine (5 mM), penicillin (100 IU/mL), and streptomycin (100 µg/mL) to IMDM

82

(Sigma-Aldrich, Missouri, United States). The cells (10 µL) were seeded into a 96-well platefilled with culture media (90 µL) and placed in a CO2 incubator at 37 °C. After 24 h, thecells were treated with undigested and digested (oral, gastric, and intestinal phases) samples(10 µL) with different concentrations (0.5, 1, 5, and 10 µM) for 48 h. Subsequently, 10 µLof the MTT solution (5 mg/mL, Sigma-Aldrich) was pipetted into each well. The cells were
continuously incubated for 4 h. Finally, 100 µL of cell lysis buffer (10% SDS in 0.01 M
HCl) was applied to dissolve the colored formazan crystals. Culture media instead ofmomilactones was used as the negative control. Meanwhile the well-known medicinescomprising Doxorubicin, Imatinib, and Ibrutinib were tested as the standard inhibitors. The
absorbance at 595 nm was scanned to determine the cell growth rate in value using aspectrophotometer (SpectraMAX M5, Molecular Devices, Sunnyvale, CA, USA) (Anh etal., 2020). The cytotoxic activity (% inhibition) of samples/or inhibitors on the tested celllines was as follows:
Inhibition (%) = (ANC – AS)/ANC ´ 100
ANC: Absorbance of reaction with negative control, AS: Absorbance of reaction withsample/or inhibitor.
Dose-responding curves and IC50 values of the momilactones and standard inhibitors
for cytotoxicity against tested cell lines were established. A lower IC50 indicates a stronger
cytotoxicity activity.
5.2.4. Quantification of momilactones
Momilactones A and B in rice extract were quantified based on the method describedby Quan et al. (2019b). In particular, the UPLC-ESI-MS system included LTQ Orbitrap XL
mass spectrometers (Thermo Fisher Scientific, Waltham, United States) and an electrosprayionization (ESI) source. For analysis, a methanolic sample (10 µg/ml) with a volume of 3.0µL was injected into the ZORBAX Eclipse Plus C18 (1.8 μm, 2.1 × 50 mm) column (Agilent

83Technologies, Santa Clara, California, United States). The column temperature wasmaintained at 25 °C. The gradient model with 2 solvents was set up as follows: solvent A
was 0.1% trifluoroacetic acid in water, solvent B was 0.1% trifluoroacetic acid in
acetonitrile. The gradient program was: 50% B during 0–5 min, then adjusted to 100% B
during 5–10 min, subsequently maintained for 0.1 min, and another 5 min for equilibration.
The flow rate was 300 μL/min. The operation was run in 15.1 min. A positive FTMS modewith a range of 100 to 800 m/z was applied for mass scanning. The presence of MA and MB
in rice samples was confirmed by comparing their extracted ion chromatograms (EIC) andmass spectra of samples with those of standard momilactones. The calibration curves of MA
and MB were established by applying different concentrations of momilactone standards(0.5, 1, 5, 10 µg/ml) to determine the momilactone content in rice seedlings. The peak areasof MA and MB detected in samples were used to calculate the amount of each compound bystandard curves. The result was expressed as micrograms per gram of sample dry weight(µg/g DW). The bioaccessibility of momilactones during digestion was calculated following
the below formula:
Bioaccessibility = (A – B)/A ´ 100
A: quantified amount before digestion; B: quantified amount after digestion.
5.3. Results
5.3.1. Effects of in vitro digestion on cytotoxic activity of momilactones against acutepromyelocytic leukemia (HL-60) cell line
The cytotoxic activity of momilactones A and B and their mixture (1:1, w/w) before
and after digestion against acute promyelocytic leukemia (APL) HL-60 cell line is presented
in Table 10. Accordingly, undigested MA and digested MA present insignificant effects on
this cell line. In contrast, the cytotoxicity of MB and MAB before digestion against HL-60(IC50 = 4.30 and 3.22 µM, respectively) is stronger than that of the well-known drug84

Doxorubicin (IC50 = 5.22 µM). However, after digestion, the inhibitory effects of MB and
MAB are significantly reduced. Particularly, the IC50 values of MB and MAB in oral phaseare 28.94 and 47.82 µM, respectively. In gastric phase, the IC50 values of MB and MAB are
12.94 and 34.63 µM, respectively. Meanwhile, these values are 10.36 and 39.84 µM,
respectively in intestinal phase Table 10.
Table 10. Effect of in vitro digestion on cytotoxic activity against HL-60 cell line ofmomilactones (IC50, µM)
Compound

U

O

G

I

MA

ne

ne

ne

ne

MB

4.30 ± 0.33g

28.94 ± 1.40d

12.94 ± 0.87e

10.36 ± 1.44e

MAB

3.22 ± 0.02g

47.82 ± 0.47a

34.63 ± 0.73c

39.84 ± 2.00b

Doxorubicin

5.22 ± 0.15f

-

-

-

U, undigested sample; O, oral digestion; G, gastric digestion; I, intestinal digestion; MA,
momilactone A; MB, momilactone B; MAB, the mixture of momilactones A and B (1:1,w/w); ne, negligible effect; -, not determined. Data are expressed as mean ± SD (n = 3).
Different superscript letters indicate significant differences by Tukey’s test (p < 0.05).
5.3.2. Effects of in vitro digestion on cytotoxic activity of momilactones against chronic
myeloid leukemia (Meg-01) cell line
The cytotoxic activity of momilactones against chronic myeloid leukemia (CML)
Meg-01 cell line under the effects of digestion including oral, gastric, and intestinal stages
is described in Table 11. The results show that MA before and after digestion reveals
negligible impacts on Meg-01. In undigested samples, MB exhibits the most potent
suppression against Meg-01 (IC50 = 6.87 µM), followed by MAB (IC50 = 18.19 µM). During
oral and gastric stages, the cytotoxic activity of MB is remarkably decreased (IC50 = 69.35
and 80.73 µM in oral and gastric phases, respectively). Notably, MB reveals negligible85effects on Meg-01 in intestinal stage. On the other hand, MAB show no impact on Meg-01
after digestion (Table 11).
Table 11. Effect of in vitro digestion on cytotoxic activity against Meg-01 cell line ofmomilactones (IC50, µM)
Compound

U

O

G

I

MA

ne

ne

ne

ne

MB

6.87 ± 0.42d

69.35 ± 1.40b

80.73 ± 5.96a-

MAB

18.19 ± 0.97c

-

-

-

Doxorubicin

3.34 ± 0.42e

-

-

-

Imatinib

0.61 ± 0.01f

-

-

-

U, undigested sample; O, oral digestion; G, gastric digestion; I, intestinal digestion; MA,
momilactone A; MB, momilactone B, MAB, the mixture of momilactones A and B (1:1,w/w); ne, negligible effect; -, not determined. Data are expressed as mean ± SD (n = 3).
Different superscript letters indicate significant differences by Tukey’s test (p < 0.05).
5.3.3. Effects of in vitro digestion on cytotoxic activity of momilactones against mantle cell
lymphoma (Mino) cell line
In the case of multiple cell lymphoma (MCL), MB shows the most substantial
inhibition on Mino cell viability (IC50 = 1.07 µM) which is close to the widely applied drugs
including Doxorubicin (IC50 = 0.64 µM) and Ibrutinib (IC50 = 0.85 µM) (Table 12). The
second most potent sample in suppressing Mino is MAB (IC50 = 7.31 µM), followed by MA(IC50 = 23.85) (Table 12). However, the activity of MA, MB and MAB is substantiallyreduced during digestion. In which, MA displays insignificant effects on Mino cell
expansion. On the other hand, the cytotoxicity of MB decreased by 15.14, 16.52, and 33.78
times through the oral, gastric, and intestinal stages, respectively, compared to theundigested stage (Table 12). Meanwhile, the cytotoxicity of MAB reduced by 3.94, 2.21,

86

5.37 times via the oral, gastric, and intestinal phases, respectively, compared to undigested
MAB (Table 12).
Table 12. Effect of in vitro digestion on cytotoxic activity against Mino cell line ofmomilactones (IC50, µM)
Compound

U

O

G

I

MA

23.85 ± 0.75c

ne

ne

ne

MB

1.07 ± 0.03f

16.20 ± 1.32d

17.68 ± 0.15d

36.14 ± 0.76a

MAB

7.31 ± 0.12e

28.77 ± 1.63b

16.16 ± 1.84d

39.22 ± 2.38a

Doxorubicin

0.64 ± 0.03h

-

-

-

Ibrutinib

0.85 ± 0.03g

-

-

-

U, undigested sample; O, oral digestion; G, gastric digestion; I, intestinal digestion; MA,
momilactone A; MB, momilactone B; MAB, the mixture of momilactones A and B (1:1,w/w); ne, negligible effect; -, not determined. Data are expressed as mean ± SD (n = 3).
Different superscript letters indicate significant differences by Tukey’s test (p < 0.05).
5.3.4. Effects of in vitro digestion on cytotoxic activity of momilactones against multiple
myeloma (U266) cell line
The cytotoxic activity of MA, MB, and MAB before and after digestion against
multiple myeloma (MM) U266 cell line is presented in Table 13. The outcomes show that
MB and MAB reveal potent suppression against U266 cell proliferation (IC50 = 3.42 and3.70 µM, respectively) which is over twice stronger than Ibrutinib (IC50 = 7.97 µM) (Table
13). Doxorubicin is the strongest sample inhibiting U266 with IC50 value of 0.24 µM,
whereas MA is the weakest sample with IC50 value of 20.09 µM (Table 13). However,digestion significantly diminished the activity of MA. Besides, the cytotoxicity of MB is
dramatically impeded under the effects of oral, gastric, and intestinal phases with IC50 valuesof 23.41, 7.60, 16.30 µM, respectively (Table 13). Meanwhile, the activity of MAB during

87

oral, gastric, and intestinal stages is reduced by 5.30, 3.65, and 7.68, respectively, compared
to undigested sample (Table 13).
Table 13. Effect of in vitro digestion on cytotoxic activity against U266 cell line ofmomilactones (IC50, µM)
Compound

U

O

G

I

MA

20.09 ± 1.28c

ne

ne

ne

MB

3.42 ± 0.34g

23.41 ± 1.31b

7.60 ± 0.57f

16.30 ± 0.86d

MAB3.70 ± 0.10g

19.62 ± 0.26c

13.50 ± 0.78e

28.42 ± 1.63a

Doxorubicin

0.24 ± 0.01h

-

-

-

Ibrutinib

7.97 ± 0.34f

-

-

-

U, undigested sample; O, oral digestion; G, gastric digestion; I, intestinal digestion; MA,
momilactone A; MB, momilactone B; MAB, the mixture of momilactones A and B (1:1,w/w); ne, negligible effect; -, not determined. Data are expressed as mean ± SD (n = 3).
Different superscript letters indicate significant differences by Tukey’s test (p < 0.05).
5.3.5. Bioaccessibility of momilactones during in vitro digestion
The digestive stages including oral, gastric, and intestinal phases dramaticallydecreased the bioaccessibility of MA, MB, and MAB (Figure 26).

88

1

mg/mg

0.8

0.6

0.4

0.2

0
Undigested

Oral

Gastric

MA

MB

Intestinal

MAB

Figure 26. Bioaccessibility of momilactones under digestion. MA, momilactone A; MB,momilactone B; MAB, the mixture of momilactones A and B (1:1, w/w).
According to the results shown in Figure 26 and Table 14, the bioaccessibility of MA,
MB, and MAB is decreased by 58.28%, 48.83%, and 32.59%, respectively, under the effectsof oral digestive stage, compared to undigested samples. Meanwhile, in gastric phase, thequantities of MA, MB, and MAB is reduced by 75.14%, 28.44%, and 29.03%, respectively,
over undigested samples (Figure 26 and Table 14). Through intestinal digestion, the contentsof MA, MB, and MAB are impeded by 72.37%, 53.91%, and 47.13%, respectively,
compared to undigested samples (Figure 26 and Table 14).
Table 14. Variation percentages of momilactones under digestion
Variation (%)
Compound

MA

O

G

I

-58.28-75.14-72.3789

MB-48.83-28.44-53.91

MAB-32.59-29.03-47.13MA, momilactone A; MB, momilactone B; MAB, the mixture of momilactones A and B(1:1, w/w); O, oral phase; G, gastric phase; I, intestinal phase.
5.3.6. Correlation coefficients between quantities of momilactones and their cytotoxic
activities during in vitro digestion
Based on the correlation coefficient results, the reduced cytotoxic activities ofmomilactones strongly correlated with their decreased quantities under the effects ofdigestion. Concretely, the correlation value between MA content and inhibitory activities
against Mino and U266 is 0.97 (Table 15). On the other hand, the reduced amounts of MB
during digestion closely associate with the decreased cytotoxic activity of this compounds
against HL-60, Meg-01, Mino, and U266 cell lines with the correlation levels of 0.85, 0.91,
0.90, and 0.97, respectively (Table 15). Besides, the impeded content of MAB may cause
the weakened cytotoxicity of this sample under the effects of digestion (Correlation levels =0.91, 0.91, 0.97, and 0.95 for HL-60, Meg-01, Mino, and U266, respectively) (Table 15).
Table 15. Correlation coefficients between quantities of momilactones and their cytotoxicity
during digestion
Quantity during digestion
Cytotoxicity
MA

MB

MAB

HL-60

-

0.845

0.914

Meg-01

-

0.907

0.912

Mino

0.970

0.896

0.965

U266

0.969

0.971

0.953

-, not determined; MA, momilactone A; MB, momilactone B; MAB, the mixture ofmomilactones A and B (1:1, w/w).

90

5.4. Discussion
In earlier research, momilactone B was reported to suppress colon cancer (Kim et al.,2007), monocytic leukemia (Park et al., 2014), leukemic T cell lines (Lee et al., 2008).
However, the cytotoxic potentials of momilactones were not previously compared to that ofanticancer medicines. In the present study, we indicate, for the first time, that momilactone
B (MB) and the mixture of momilactones A and B (MAB) (1:1, w/w) may prevent the cellproliferation of acute promyelocytic leukemia (APL) HL-60, chronic myeloid leukemia(CML) Meg-01, mantle cell lymphoma (MCL) Mino, and multiple myeloma (MM) U266
cell lines. Besides, the well-known drugs of Doxorubicin, Imatinib, and Ibrutinib were tested
as standard inhibitors. Doxorubicin is commonly used to treat lymphoma and MM (Thorn etal., 2011). Especially, Doxorubicin was widely applied as a positive control in the researchon cytotoxic activity against HL-60 cell line (Rodrigues et al., 2021). Ibrutinib has beenannounced as anti-MM agent via in vitro, in vivo, and clinical tests (Li, 2021; Richardson etal., 2018). The findings from our research imply that the cytotoxic activity of MB and MAB
is close to that of Doxorubicin against Meg-01 and Mino. Notably, MB and MAB arestronger than Doxorubicin and Ibrutinib in suppressing HL-60 and U266, respectively.
Besides, the applicability of momilactones for the development of novel anti-blood cancer
drugs, considering oral application was investigated in the current research. Particularly, theeffects of digestive processes including oral, gastric, and intestinal phases on bioaccessibility
and cytotoxic activities of momilactones against selected blood cancer cell lines areindicated in our research which has not been reported in any other studies.
Under digestion, the stability of compound structures and functional groups can beaffected that may involve in the regulations of associated biological properties. In which, pH
level may play an important role in altering the bioaccessibility of bioactivity of target
compounds (Hodgkinson et al., 2018; Pérez-Vicente et al., 2002; Un et al., 2022; Wootton-

91

Beard et al., 2011). For example, anthocyanin compounds are mostly converted to non-red
forms and/or degraded from the pomegranate juice due to pH shift during digestion. Vitamin
C in the pomegranate juice was also significantly degraded under the changes in pH of eachdigestive stages (Pérez-Vicente et al., 2002). In the research of (Un et al., 2022), the changes
in pH conditions in simulated digestive model may cause the possible formation ordegradation of bioactive compounds isolated from Sargassum spp., leading to the variationin their associated biological activities (Un et al., 2022). In line with that research, thecytotoxicity of momilactones reduce through digestion which is consistent with the reductionof momilactone contents in digested samples. The variations of momilactone contents andassociated cytotoxic activities may be affected by different pH and enzyme constituents.
Notably, MB content significantly reduces after the oral phase, which may be due to ⍺amylase activity. MB amount increases during the gastric phase compared to the previous
phase, suggesting that acidic environment (pH = 1.0) together with pepsin activity may leadto the re-formation of MB. With the increasing pH in the intestinal phase and lipase effects,
the quantity of MB again decreases. Further studies should be conducted to validate thisparadigmatic research.
To develop momilactones as novel oral administrated medicines, advanced solutions
such as capsule technology and nanotechnology are highly recommended in further
anticancer research. In which, capsule method has been revealed positive impacts on
bioaccessibility and bioavailability of target bioactive components during digestion.
Moreover, this approach can ensure the compounds’ targets as well as control their release(Grgić et al., 2020). Besides, capsule can address the undesirable odors, tastes as well as
contribute to better organoleptic properties (Grgić et al., 2020). Nanotechnology is anotherpromising approach that can increase the effectiveness of orally administrated medicines.
Particularly, nanocarriers can help enhance solubility, stability, and epithelium permeability92

of target products, since then increase their bioaccessibility and bioavailability (Hsu et al.,2019). Furthermore, our study indicates that cytotoxic activities of momilactones are variedamong digestive stages against different blood cancer cell lines. The finding may lead tofurther effective approaches of capsule- or nano-technology to enhance bioaccessibility andbioavailability of momilactones under digestion effect against specific targeted blood cancer
cell line.
In another concern, a natural-based drug must satisfy many strict requirements
including benefits over risks to be acknowledged as novel drugs (Teschke & Xuan, 2020c).
Therefore, the optimized dose of momilactones should be established to suppress
substantially tumors without harmful effects on normal blood cells. On the other hand, thepotential risk such as neurotoxicity and hepatotoxicity by using herbal products should beseriously considered and thoroughly evaluated (Quan et al., 2020). Accordingly, in vivo tests
and clinical trials are required for the development of momilactones as novel anti-bloodcancer drugs.

93

CHAPTER 6: GENERAL DISCUSSION
6.1. Key findings
In this study, we indicated, for the first time, that the problematic invasive weed A.
virginicus, which was once thought to be of no use, may be a promising source ofantioxidant, anti-diabetic, anti-skin aging, and anti-blood cancer agents (Table 16). The
findings are expected to support both crop protection and human health concerns.
In addition, momilactone B (MB) and the mixture of momilactones A and B (MAB)(1:1, w/w) isolated from rice (O. sativa) significantly inhibit the viability of different bloodcancer cell lines including chronic myeloid leukemia (CML), acute promyelocytic leukemia(APL), mantle cell lymphoma (MCL), and multiple myeloma (MM). The cytotoxic activityof MB and MAB against CML cell lines is close to that of the well-known drug Doxorubicin.
Meanwhile MB and MAB are more potent than Doxorubicin and Ibrutinib in suppressing
APL and MM cell lines, respectively. Besides, MB and MAB prevent the tested blood cancer
cell lines by promoting their cell apoptosis. Whereas these compounds reveal insignificant
effects on the non-cancerous cell line. The findings were not presented in any other studies.
Another novel finding of this study is to scrutinize that MB and MAB promote MM
cell apoptosis by regulating relevant protein expressions (Table 16). While these compounds
altering the expressions of regulatory proteins related to apoptosis activation and G2 phasearrest of the cell cycle that cause the inhibition of APL cells (Table 16).
Besides, this is the first study to investigate the cytotoxic activity on blood cancer celllines and bioaccessibility of momilactones through in vitro simulated human digestion.
Accordingly, the cytotoxicity of momilactones may reduce under the effects of digestion
which is consistent with the reduction of momilactone contents in digested samples. Further
studies should be conducted applying advanced methods such as capsule- and nano-94

technology to enhance the bioaccessibility and bioavailability of momilactones fordeveloping novel anti-blood cancer drugs.
Table 16. Key findings
ChapterThis study•

Previous study

This is the first report describing•

There has been no conductedthat A. virginicus is a potential

research on pharmaceuticalnatural source of antioxidants,

properties of A. virginicus

Chapter 2tyrosinaseanda-amylase

inhibitors,

andanti-chronic

myeloid leukemia (CML) agents••

Chapter 3

MB

and

MAB

revealThe cytotoxic activity ofinsignificant effects on non-momilactones on normal cells

cancerous cell line (MeT-5A).

was not indicated.

MB and MAB inhibit the blood•

MB inhibits the cell viability

cancer cell lines comprisingof human cancer cell lineschronic myelogenous leukemia

including colon cancer (HT-(Meg-0129

andK562),

acuteand

SW620),

acutepromyelocytic leukemia (HL-60),

promyelocytic leukemia (HL-

mantle cell lymphoma (Mino);60), and leukemic T cells

multiple myeloma (U266 and(Jurkat and RBL-2H3) (Kim

KMS-11) by promoting cellet al., 2007; Lee et al., 2008).

apoptosis.
•••

MB induces apoptosis and G1

The cytotoxic activity of MB andphase arrest in the cell cycle

MAB against chronic myeloidof U937 cells (Park et al.,leukemia (CML) cells is close to2014).

95

that

of

well-known

drug

•

Doxorubicin. While MB and
MAB are stronger than the drugs

MB promotes Jurkat cell
apoptosis (Lee et al., 2008).

•

The

cytotoxicity

ofof Doxorubicin and Ibrutinib in

momilactones

preventing acute promyelocytic

compared to that of widely

leukemia (APL) and multiple

appliedmyelomamedicines.

(MM)

cells,

wasanti-bloodnotcancerrespectively.
••

Chapter 4

MB and MAB show negligible

MB induces apoptosis and G1

effects on cell apoptosis and cellarrest of the cell cycle incycle of non-cancerous cell line

monocytic leukemia U937

MeT-5A.

cells through downregulation

MB and MAB at 5 µM inhibitof pRB phosphorylation and

HL-60

induction

cells

through

the

of

the

CDK

regulations of relevant proteins to

inhibitor p21Waf1/cip1 (Park etapoptosis-inducing factors (p-38,

al., 2014).

BCL-2, and caspase-3) and cell

•

•

•

MB promotes Jurkat cell

cycle arrest at G2 phase (p-38,

apoptosis via caspase and

CDK1, and cyclin B1).

mitochondria pathways (LeeMB and MAB at 5 µM enhance

et al., 2008).

U266 cell apoptosis by altering p38,

BCL-2,

andcaspase-3

signaling pathways.

96

•

Chapter 5•

The cytotoxicity of momilactones

There has been no research on

may reduce through the digestive

cytotoxic activities againststages, which is in line with theblood cancer cell lines andreduction

bioaccessibilityofmomilactone

contents in digested samples.

momilactones

of
under

the

effects of human digestion.

6.2. Comparison between cytotoxic activities of isolated components from rice (O. sativa
var. Koshihikari) and the invasive weed (A. virginicus)
Based on the cytotoxic results against chronic myeloid leukemia (CML) cell lines,
momilactones from rice exhibit much stronger inhibitory activities on Meg-01 and K562 cell
viability than extracts from A. virginicus (Table 17). Therefore, momilactones were selected
to investigate their cytotoxic mechanisms and bioaccessibility during digestion in our study.
However, further studies should be conducted focusing on the isolation and purification of
the responsible compounds for the anti-blood cancer activity of A. virginicus, followed byindicating their cytotoxic mechanisms and bioaccessibility under digestive processes.
Table 17. Cytotoxic activities of isolated components from broomsedge bluestem and rice
(Achieved results from Chapters 2 and 3)
Original plant

Meg-01

K562

IC50 (µg/mL)

IC50 (µg/mL)

T-Anvi91.40c247.88a

H-Anvi198.07a-

E-Anvi168.94b112.01b

MA

2.26d

2.57c

SampleBroomsedge bluestem(A. virginicus)

Rice

97

(O. sativa)

MB

1.45f

1.66d

MAB

1.39f

1.33e

Doxorubicin

1.82e

1.70d

Imatinib

0.33g

0.33f

Standard inhibitor
Different superscript letters indicate significant differences. T-Anvi, total crude extract; HAnvi, hexane extract; E-Anvi, ethyl acetate extract; MA, momilactone A; MB, momilactone
B; MAB, the mixture of momilactones A and B (1:1, w/w).
6.3. Exploitation of products from rice (O. sativa) and the invasive weed (A. virginicus)
for developing blood cancer treatments
In this study, we indicated the potentials of momilactones isolated from rice andextracts from A. virginicus for anti-blood cancer properties. In which, momilactones
significantly inhibit tested blood cancer cell lines by regulating the related proteins toapoptosis and cell cycle pathways. On the other hand, the impacts of momilactones on
normal blood cells including red blood cells (erythrocytes), white blood cells (monocytes,
lymphocytes, neutrophils, eosinophils, basophils, and macrophages), and platelets(thrombocytes) should be comprehensively interpreted since they might be affected by thesame course of event with tumors (Taşkın-Tok & Gowder, 2014). Forthcoming studies
should focus on the effects of momilactones on relevant proteins to cell apoptosis and cell
cycle pathway of normal blood cells treated with momilactones. This is an integral
requirement to minimize failure in later stages of drug development. Furthermore, this mayhelp establish potential drug combinations as well as effective therapies (Taşkın-Tok &Gowder, 2014).
Through in vitro digestion assay, cytotoxic activities of momilactones significantly
decrease during digestion. Therefore, forthcoming studies should be conducted to improve98

the bioaccessibility of momilactones, followed by in vivo and clinical tests to confirm theircapacities in the development of novel anti-blood cancer drugs.
In another aspect, the application of anti-cancer medicines (e.g., Doxorubicin) may
lead to oxidative stress by increasing free radicals (Thorn et al., 2011) and a high risk ofgetting other chronic disorders such as diabetes, obesity, and skin diseases (Libby et al.,2009). Moreover, a relationship between oxidative stress, inflammation, cancer, and chronic
problems including diabetes, obesity, and skin disorders which may eventually cause anincrease in morbidity and mortality (Figure 27). Accordingly, momilactones isolated fromrice with potentials for antioxidant (Quan et al., 2019a), anti-diabetes, anti-obesity, and antiskin aging (Quan et al., 2019a, 2019b, 2019c), and anti-blood cancer activities (Chapters 3
and 4), and products extracted from A. virginicus with potentials for antioxidant, antidiabetes, anti-skin aging, and anti-blood cancer properties (Chapter 2) can be considered apromising source for improving blood cancer treatments, especially for patients complicatedwith oxidative stress and chronic diseases.
CancerDiabetes
Momilactones
from O. sativa

Inflammation

Synergic
inhibition

Morbidity
and mortality

Constituentsfrom A. virginicus

Oxidative stressObesity

Skin problems: Inhibition: Stimulation

Figure 27. Potential approach for exploiting natural compounds from rice (O. sativa) andthe invasive weed (A. virginicus) for therapeutic purposes
On the other hand, the interactions between compounds may be more important than
individual compounds in determining biological activity (Quan et al., 2019f). Therefore, thecombination of momilactones from rice and isolated components from A. virginicus may be99

a promising approach for developing blood cancer treatments avoiding the risks of getting
oxidative stress and other chronic disorders. The results from our study may lead to further
strategies to take advantage of this problematic invasive weed and rice by-products forpharmaceutical purposes.
Additionally, both A. virginicus and rice are monocot plants, which adapt to a wide
range of environmental conditions (Simpson, 2010). Thus, they can be feasibly exploited forpharmaceutical purposes with an abundant biomass availability. For momilactones, inaddition to rice source, the Hypnum moss (Hypnum plumaeforme) should be paid moreconsiderations as a promising source of momilactones. On the other hand, research on
synthetic momilactones may be an effective approach for the development of medicinal
production and therapeutics. Besides, a number of 47 momilactone-like molecules werereported (Zhao et al., 2018), suggesting an abundant source for further investigation of theircytotoxic potentials against blood cancer cells. Moreover, a natural-based product mustsatisfy the requirements of benefits outweighing risks (Teschke & Xuan, 2020c). Therefore,
the effective concentration of isolated momilactones from rice and components from A.
virginicus should be established to exhibit the strongest cytotoxicity against tumors without
harmful effects on normal blood cells. In contrast, the potential risk such as neurotoxicity
and hepatotoxicity by using derived products from plants should be seriously considered andthoroughly evaluated (Quan et al., 2020). Thus, in vivo tests and clinical trials are highly
recommended in forthcoming studies.
Overall, the study is expected to support the sustainable development goals (SDGs) of
ensuring healthy lives and life on land, ending poverty and hunger in the world, especiallyin the rice-producing countries as well as the countries suffering from the problematic
invasive A. virginicus.

100

SUPPLEMENTARY DATA

Gallic acidRutin

y = 0.0052x + 0.0645
R² = 0.9969

0.6
0.4
0.2
0
0

20

40
60
80
Concentration (µg/mL)

AbsorbanceAbsorbance

0.8

1.2
1
0.8
0.6
0.4
0.2
0

100

y = 0.0107x + 0.051
R² = 1

0

20

40
60
80
Concentration (µg/mL)

100

Figure S1. Standard curves of gallic acid and rutin.
T-Em

H-Em
80

y = 0.3392x + 23.644
R² = 0.9959

80% Inhibition% Inhibition

100
60
40
20
0

y = 0.1248x + 9.5893
R² = 0.9912

60
40
20
0

0

50

100
150
200
Concentration (µg/mL)

250

0

200
400
Concentration (µg/mL)

E-Em

B-Em
y = 0.353x + 19.369
R² = 0.9944

80% Inhibition% Inhibition

100
60
40
20
0
0

50

100
150
200
Concentration (µg/mL)

600

250

120
100
80
60
40
20
0

y = 0.8307x + 13.779
R² = 0.9966

0

50
100
Concentration (µg/mL)

150

W-Em
% Inhibition100
y = 0.0687x + 9.6979
R² = 0.996

80
60
40
20
0
0

500
1000
Concentration (µg/mL)

1500

Figure S2. Antiradical activity of A. virginicus extracts via 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. T-Anvi, total crude extract; H-Anvi,101

hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water
extract.
T-Em
100

y = 1.8571x - 6.7242
R² = 0.9997

80% Inhibition% Inhibition

100

H-Em

60
40
20

y = 0.3698x + 2.5923
R² = 0.991

80
60
40
20

0

0
0

20
40
Concentration (µg/mL)

60

0

50

100
150
200
Concentration (µg/mL)

E-Em

B-Em

120

80

% Inhibition

80% Inhibitiony = 1.5255x + 21.503
R² = 0.8252

100
60
40
20
0

y = 1.3984x + 2.7394
R² = 0.998

60
40
20
0

0

20
40
Concentration (µg/mL)

60

0

20
40
Concentration (µg/mL)

60

W-Em
100
% Inhibition250

y = 0.0802x + 14.714
R² = 0.9705

80
60
40
20
0
0

200

400
600
800 1000
Concentration (µg/mL)

1200

Figure S3. Antiradical activity of A. virginicus extracts via 2,2-diphenyl-1picrylhydrazyl (DPPH) assay. T-Anvi, total crude extract; H-Anvi, hexane extract; E-Anvi,
ethyl acetate extract; B-Anvi, butanol extract; W-Anvi, water extract.

102

Absorbance

0.6

0.4

0.2

0
0

15

T-Em

30

45

H-Em

60

75 90 105 120 135 150 165 180
Time (min)

E-Em

B-Em

W-Em

Control

Figure S4. Inhibition on lipid peroxidation of A. virginicus extracts. T-Anvi, total
crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol
extract; W-Anvi, water extract.

T-Em

H-Em
80

y = 0.0119x - 4.277
R² = 0.9791

40% Inhibition% Inhibition

60

20
0

y = 0.0063x + 13.124
R² = 0.9599

60
40
20
0

0

1000

2000 3000 4000 5000
Concentration (µg/mL)

6000

0

2000

4000 6000 8000 10000 12000
Concentration (µg/mL)

E-Em

B-Em
100

y = 0.0098x + 24.575
R² = 0.8943

60% Inhibition% Inhibition

80

40
20
0

y = 0.0028x + 23.985
R² = 0.9924

80
60
40
20
0

0

1000

2000 3000 4000 5000
Concentration (µg/mL)

6000

0

5000

10000 15000 20000
Concentration (µg/mL)

103

25000

Figure S5. Tyrosinase inhibitory activities of A. virginicus extracts. T-Anvi, total
crude extract; H-Anvi, hexane extract; E-Anvi, ethyl acetate extract; B-Anvi, butanol extract.
H-Em

T-Em
100
y = 0.0047x + 29.498
R² = 0.7984

80
60% Inhibition% Inhibition

100

40
20

y = 0.0278x + 25.507
R² = 0.8593

80
60
40
20
0

0
0

5000
10000
Concentration (µg/mL)

0

15000

500
1000
1500
2000
Concentration (µg/mL)

2500

Figure S6. a-Amylase inhibitory activities of A. virginicus extracts. T-Anvi, total
crude extract; H-Anvi, hexane extract.
Palmitic acid
6000000
y = 5393x - 319967
R² = 0.9989

Area intensity

5000000
4000000
3000000
2000000
1000000
0
0

200

400
600
800
Concentration (µg/mL)

1000

1200

Figure S7. Standard curve of palmitic acid
TIC; / EI+(eiFi) / Alkanes
Hex
6

x10

Intensity (11955935)
13.21

15.44

12.00

17.47

19.32
20.19

10

8

21.82

24.76
24.06

25.45

10.72

6

4

9.37

2

0
0

5.0

10.0

15.0

20.0

25.0

30.0

Time[min]

35.0

40.0

Carbon Time (min)
C13
9.37
C14
10.72
C15
12.00
C16
13.21
C17
14.35
C18
15.44
C19
16.48
C20
17.47
C21
18.42
C22
19.32
C23
20.19
C24
21.03
C25
21.82
C26
22.60
C27
23.34
C28
24.06
C29
24.76
C30
25.45

Figure S8. GC chromatogram of n-alkane mixture (C13–C30)

104

Figure S9. Mass spectra (MS) of palmitic acid found in hexane extracts (H-Anvi) fromA. virginnicus analyzed by gas chromatography-mass spectrometry (GC-MS).

Figure S10. Effects of momilactones A (MA) and B (MB) on cell viability of tested
blood cancer cell lines via MTT assay.
Bortezomib
IC50 = 7.97 nM

Cell viability (%)

100
**

80

**

60
40
20

***

***

1
5
10
Concentration (nM)

20

0
0

Figure S11. Effects of Bortezomib on cell viability of multiple myeloma (U266) celllines after 48 h. **p < 0.01 versus control (0 nM); ***p < 0.001 versus control (0 nM).

105

Momilactone A
50000000

40000000
Peak areay = 4,435.81x + 1,224,644.84
R² = 0.99

40000000
Peak area

Momilactone B

30000000
20000000
10000000

y = 2,958.15x - 85,548.68
R² = 1.00

30000000
20000000
100000000002000 4000 6000 8000 10000
Concentration (ng/µL)

02000 4000 6000 8000 10000
Concentration (ng/µL)

Figure S12. Standard curves of momilactones A and B.
(a)
MA

MB

106

(b)

MA

MB
Figure S13. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of thestandards of momilactones A (MA) and B (MB) by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

107

(a)

(b)

Figure S14. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in oral digested sample by ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS).

108

(a)

(b)

Figure S15. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

109

(a)

(b)

Figure S16. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

110

(a)

(b)

Figure S17. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in oral digested sample by ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS).

111

(a)

(b)

Figure S18. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

112

(a)

(b)

Figure S19. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone B (MB) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

113

(a)
MA

MB

(b)

MA

114

MB
Figure S20. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in oral digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

(a)

115

(b)

MA

MB

116

Figure S21. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in gastric digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).
(a)

(b)

MA

117

MB
Figure S22. (a) Extracted ion chromatograms (EIC) and (b) mass spectra (MS) of
momilactone A (MA) and B (MB) in intestinal digested sample by ultra-performance liquid
chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS).

118

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