Results and Discussion
A total of 224 articles from PubMed, ScienceDirect, and Scopus databases were downloaded to the EndNote database. Eighty-two articles were excluded, and further analysis of the titles and
abstracts of the remaining 142 articles led to the exclusion of 19 articles (excluded based on title and abstract). Finally, 123 articles were included in the analysis. The flow diagram of the
study inclusion and exclusion is presented in [Fig. 1 ]. Antiproliferative activities of plant extracts or active compounds are summarized in [Table 1 ] and results of clinical studies of some herbal formulations are summarized in [Table 2 ]. Mechanisms of antiproliferative
activities including in vivo studies in animals are provided in the Supporting Information. The included articles involve 68 herbs, isolated compounds, and/or synthetic analogs, 9
herbal formulations, and 199 compounds that are commonly found in several plant species. The most investigated plant was Atractylodes lancea (Thunb.) DC. (Compositae) (n = 17), followed
by Curcuma longa L. (Zingiberaceae) (n = 15), Garcinia hanburyi Hook.f. (Clusiaceae) (n = 6), Artemisia annua L. (Compositae) (n = 5), Zingiber officinale Roscoe
(Zingiberaceae) (n = 5), Andrographis paniculata (Burm.f.) Nees (Acanthaceae) (n = 4), Capsicum spp. (Solanaceae) (n = 3), Derris indica (Lamk.) Benn. (Leguminosae)
(n = 3), Piper longum L. (Piperaceae) (n = 3), and Tripterygium wilfordii Hook. f. (Celastraceae) (n = 3). Other plants were reported in one or two research articles.
Pra-Sa-Prao-Yai was the most investigated formulation (n = 2). Resveratrol (n = 5) and capsaicin (n = 3) derived from several plants was the most investigated compounds for anticancer activity
against CCA. Most studies reported the antiproliferative activities using different in vitro tests (n = 108), including MTT, SRB, WST-1, Hoechst, neutral red, acridine orange/ethidium
bromide, cell counting kit-8, crystal violet, PrestoBlue, calcein-AM, tryphan blue, cell titer 96 aqueous, IncuCyte zoom, morphological examination, flow cytometry, and clonogenic assays.
In vivo evaluation of anti-CCA activity in animal models [xenograft mouse model, OV/DMN-induced CCA hamster model, and allograft hamster model] was reported in 26 articles. Mechanisms
or targets of action at the molecular or cellular level were reported in 95 studies. Others involved studies on antioxidative (n = 3) and immunomodulatory activities (n = 2), as well as their
inhibitory activities on cell migration (n = 22) and cell invasion (n = 17), pharmacokinetic studies (n = 2), clinical studies (safety and/or efficacy) (n = 3), development of nanoformulations
(n = 2), and synergizing effects on chemotherapeutic drugs (n = 5).
Fig. 1 Flow chart of the article selection process.
Table 1 Plants/isolated compounds/symthetc compounds (underlined) under investigation and available antiproliferative activity against CCA cell lines.
References
Plants/Active compounds (Family)
Antiproliferative activity
SI = selectivity index
[5 ]
Crude ethanol extracts:
Amomum testaceum Ridl. (Zingiberaceae), Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav.
(Apiaceae), Angelica sinensis (Oliv.) Diels. (Apiaceae), Anethum graveolens L. (Apiaceae), Artemisia annua L. (Compositae), Asclepias curassavica
L. (Apocynaceae), Atractylodes lancea (Thunb.) DC. (Compositae), Cuminum cyminum L. (Apiaceae), Curcuma longa L. (Zingiberaceae), Dioscorea membranacea
Pierre ex Prain & Burkill (Dioscoreaceae), Dracaena loureirin Gagnep. (Asparagaceae), Foeniculum vulgare Mill. (Apiaceae), Kaempferia galanga L. (Zingiberaceae),
Ligusticum sinense Oliv. (Apiaceae), Mammea siamensis Kosterm. (Guttiferae), Mesua ferrea L. (Calophyllaceae), Mimusops elengi L.
(Sapotaceae), Myristica fragrans Houtt. (Myristicaceae), Nigella sativa L. (Ranunculaceae), Piper chaba Hunt. (Piperaceae), Piper
interruptum Opiz. (Piperaceae), Piper sarmentosum Roxb. (Piperaceae), Plumbago indica L. (Plumbaginaceae), Smilax corbularia Kunth (Smilacaceae),
Syzygium aromaticum (L.) Merr. & L. M.Perry (Myrtaceae), Zingiber officinale Roscoe (Zingiberaceae), Zingiber ligulatum Roxb. (Zingiberaceae), Ben-ja-Kul 1
formulation, Ben-ja-Kul 2 formulation, Pra-Sa-Prao-Yhai formulation, Tein-5 formulation
A. lancea (Thunb.) DC. (Compositae): most potent and selective against CL6 cells (IC50 = 24.09 µg/mL, SI = 8.6); five others with promising activity (< 50% cell
survival at 50 µg/mL) = K. galanga L. (Zingiberaceae), Z. officinal Roscoe (Zingiberaceae), P. chaba Hunt. (Piperaceae), M. ferrea L. (Calophyllaceae),
and Pra-Sa-Prao-Yhai formulation (IC50 s of 37.36, 34.26, 40.74, 48.23, 44.12 µg/mL, respectively)
[33 ]
Cardiospermum halicacabum L. (Sapindaceae), Gomphrena celosioides Mart. (Amaranthaceae), Scoparia dulcis L. (Plantaginaceae) (ethanolic
extracts)
S. dulcis L. (Plantaginaceae): most potent (56 – 75% growth inhibition on KKU-100 and KKI-213 cells at 250 µg/mL for 72 h)
[17 ]
Andrographis paniculata (Burm.f.) Nees (Acanthaceae)/Semisynthetic andrographolide analog (19-triphenylmethyl ether andrographolide, AG 050)
Excellent activity against KKU-M213 cells (IC50 = 3.33 µM)
[18 ]
Andrographis paniculata (Burm.f.) Nees (Acanthaceae)/14-deoxy-11,12-didehydroandrographolide analogs
Analogs 5a, 5b: most potent and selective against KKU-M213 cells (IC50 = 3.37, 3.08 µM); KKU-100 = 2.93, 3.27 µM
[19 ]
Andrographis paniculata (Burm.f.) Nees (Acanthaceae)/Andrographolide
Significant activity against KKU-100 cells (IC50 ~ 120 µM)
[34 ]
Aesculus hippocastanum L. (Sapindaceae)/
β -escin
IC50 Mz-ChA1 cells: 34.21 µM (24 h), 28.48 µM (48 h), 22.1 µM (72 h); SK-ChA1 cells: 59.04 µM (24 h), 41.69 µM (48 h), 33.3 µM (72 h); QBC939 cells: 63.3 µM (24 h),
44.36 µM (48 h), 34.06 µM (72 h)
[35 ]
Anthocyanin complex [from cobs of purple way corn Zeamays, certina Kulesh, and petals of blue butterfly pes Clitoria ternatea L. (Leguminosae)]
IC50 for KKU-213 cells = 620 µ g/mL
[36 ]
Arachis hypogaea L. (Leguminosae)/Peanut testa extract, KK4 and ICG15042
Potent activity against KKU-M214 cells (KK4: IC50 = 38.28 µg/mL; ICG15042: IC50 = 43.91 µg/mL) and KKU-100 cells: (KK4: IC50 = 78.40 µg/mL;
ICG15042: IC50 = 82.77 µg/mL) at 72 h
[37 ]
Artemisia annua L. (Compositae)/Artemisinins
Potent activity against CL6 cells: IC50 = 339 µM (artemisinin), 131 µM (artesunate), 354 µM (β -artemeter), 75 µM (dihydroartemisinin)
[21 ]
Atalantia monophylla DC. (Rutaceae)/7 new benzoyltyramines, atalantums A – G (1 – 7) and 5 known compounds
Compound 5 : most potent activity against KKU-M156 (IC50 = 1.97 µM), 4.7-fold higher than ellipticine standard.1 : potent activity against
KKU-M214 (IC50 = 3.06 µM), comparable with 5-FU.2, 4, 11 : more potent activity against KKU-M213 than ellipticine (IC50 = 2.36, 5.63,
2.71 µM).1, 5, 7 : activity against KKU-M214 (IC50 = 3.06, 8.44, 7.37 µM, respectively).
[22 ]
Atalantia monophylla DC. (Rutaceae)/limonophyllines A – C (1, 4, 5), limonoids (2, 3), acridone alkaloids (6 – 16)
Compounds 12, 14, 16 : activity against KKU-M156 cells (IC50 = 3.39 – 4.1 µg/mL)
[38 ]
Atractylodes lancea (Thunb.) DC. (Compositae)/Atractylodin
IC50 = 216.8 µM for CL6 cells
[39 ]
Atractylodes lancea (Thunb.) DC. (Compositae)/Atractylodin (AT) and Atractylodin-loaded PLGA nanoparticle (ALNPs)
IC50 for CL6 cells, ALNPs vs. AT: 29.28 vs. 56.36 µM (24 h), 35.06 vs. 37.66 µM (48 h), 50.74 vs. 52.02 µM (72 h) µg/mL; IC50 for HuCC-T1 cells: ALNPs vs. AT:
47.68 vs. 53.66 µg/mL (24 h), 66.09 vs. 59.74 µM (48 h), 71.3 vs. 76.15 µg/mL (72 h)
[40 ]
Atractylodes lancea (Thunb.) DC. (Compositae)/Atractylodin (AT) Atractylodin-loaded PLGA nanoparticle (ALNPs)
IC50 for CL-6 cells: ALNPs vs. AT: 15 vs. 43 (24 h), 23 vs. 40 (48 h), 43 vs. 40 (72 h) µg/mL; IC50 for HuCC-T1 cells: ALNPs-1 vs. AT: 9 vs. 65 (24 h), 16 vs.
42 (48 h), 39 vs. 65 (72 h) µg/mL
[41 ]
Atractylodes lancea (Thunb.) DC. (Compositae)/Atractylodin and β -eudesmol
IC50 for CL6 cells: atractylodin = 41.66 µg/mL, β -eudesmol = 39.33 µg/mL
[42 ]
Atractylodes lancea (Thunb.) DC. (Compositae)/
β -eudesmol
IC50 for CL6 cells = 166 µM
[43 ]
Caesalpinia mimosoides Lam. (Leguminosae) ethylacetate extract/Gallic acid (natural: nGA, commercial: cGA)
IC50 : nGA = 120 µM (M213 cells) and 124 µM (M214 cells cGA), 119 µM (M213 cells), and 147 µM (M214 cells)
[44 ]
Clausena harmandiana (Pierre) Pierre ex Guillaumin (Rutaceae) hexane, ethyl acetate, methanol extracts/isolated and purified 12 azarbazoles and coumarins
7-hydroxy-heptaphylline and nordentatin: potent activity against KKU-OCA17 cells (IC50 = 88.7, 46.1 µM, respectively and KKU-214 cells (IC50 = 43.7, 39.1 µM,
respectively)
[45 ]
Corilagin (natural plant polyphenol tannic acid)
IC50 for QBC9939 and MZ-Cha-1 cells = 39.73 and 36.88 µM, respectively
[46 ]
Cratoxylum formosum (Jack) Benth. & Hook. f. ex Dyer (Hypericaceae) aqueous and ethanolic Dyer leaf extract
Potent activity (IC50 for the aqueuous extract = 11.3 µg/mL, ethanol extract = 12.1 µg/mL)
[24 ]
Curcuma longa L. (Zingiberaceae)/Curcumin
IC50 = 5 – 17 µM (sensitive) for KKU-100, KKU-214, and KKU-OCA17 cells
[25 ]
Curcuma longa L. (Zingiberaceae)/Curcumin
IC50 = 5.9 µM for KKU-214 cells
[26 ]
Curcuma longa L. (Zingiberaceae)/Curcumin
Activity against CCLP-1 cells (10, 48, and 56% growth inhibition) and SG-231 cells (13, 25, and 50%) at 7.5, 10, and 15 mM, repectively
[27 ]
Curcuma longa L. (Zingiberaceae) New allylated mono-carbonyl curcumin analogs (MACs)
Compound 6c : potent activity (IC50 for HuCCA cells = 8.7 µM, QBC-939 cells = 9.3 µM, and RBE = 8.9 µM)
[31 ]
Derris indica (Lamk.) Benn. (Leguminosae)/Candidione
Potent activity against KKU-M156 cells: IC50 = 6 µg/mL (17 µM) and 4.24 µg/mL (12.03 µM) at 8 and 24 h, respectively; KKU-M213 cells: IC50 = 5.7 µg/mL
(16.17 µM) and 5.74 µg/mL (15.28 µM) at 8 and 24 h, respectively
[47 ]
Derris indica (Lamk.) Benn. (Leguminosae) ethylacetate extract/
Pinnatin: potent activity against KKU-100 cells (IC50 = 6.0 µg/mL), Emax of 88 – 90%50 = 1.3 µg/mL), but with moderate efficacy (Emax of 50.7%)
[48 ]
Derris indica (Lamk.) Benn. (Leguminosae) hexane extract/isolated Derrivanone (1) and Derrischalcone + 14 known compounds
Potent activity against KKU-M156 cells: Chalcones 2, 3, 4: IC50 = 7.0, 0.73, 0.59 µg/mL, respectively; Flavanones 14, 15, 16: IC50 = 0.59, 7.8,
2.4 µg/mL, respectively
[49 ]
Derris malaccensis (Benth.) Prain (Leguminosae)/Pomiferin (prenylated isoflavonoid)
IC50 for HuCCA-1 cells = 0.9 µg/mL
[50 ]
Derris malaccensis (Benth.) Prain (Leguminosae)/Pomiferin-4′-O-methyl ether, and a new prenylated chalcone,
2′,4′-dihydroxy-4-methoxy-3′-(2-hydroxy-3-methylbut-3-enyl)chalcone, 4 known flavonoids
Compounds 2 and 3 : potent activity against HuCCA-1 cells (IC50 = 4.8 and 3.8 µg/mL, respectively) Compounds 1, 4, 5, 6 : weak activity against HuCCT-1
cells (IC50 = 10.5, 14.0, 24.0, and 25.0 µg/mL, respectively)
[8 ]
Dioscorea membranacea Pierre ex Prain & Burkill (Dioscoreaceae) ethanol extract/7 isolated compounds
Crude extract: weak but selective activity against KKU-M156 cells (IC50 = 30.49 µg/mL); Compound 5 : selective activity against KKU-M156 cells
(IC50 = 3.46 µM); Compounds 1 – 3 : no activity against KKU-156 cells (IC50 = 4100 µM)
[6 ]
Garcinia hanburyi Hook.f. (Clusiaceae) ethyl acetate and methanol extracts & fractions
Ethyl acetate extracts from bark (VR12874) and fruits (VR11626): potent activity (IC50 = 1.84 – 2.49 and 1.69 – 4.41 µg/mL); VR12876 and VR12879: weak activity; VR12880:
no activity
[51 ]
Garcinia hanburyi Hook.f. (Clusiaceae)/4 caged xanthones: isomorellin, isomorellinol, forbesione gambogic acid
IC50 : Isomorellin: KKU-100 cells = 0.11 µM, KKU-156 cells = 0.12 µM; Isomorellinol: KKU-100 cells = 2.2 µM, KKU-M156 cells = 0.43 µM; Forbesione: KKU-100 cells = 0.15 µM,
KKU-M156 cells = 0.02 µM; Gambogic acid: KKU-100 cells = 2.64 µM, KKU-M156 cells = 0.03 µM)
[52 ]
Garcinia hanburyi Hook.f. (Clusiaceae)/isomorellin
IC50 for KKU-100 cells vs. KKU-M156 cells: 6.2 vs. 1.9 µM (24 h), 5.1 vs. 1.7 µM (48 h), 3.5 vs. 1.5 µM (72 h)
[53 ]
Holothuria scabra Jaeger (sea cucumber)/Scabraside D (sulfated triterpene glycoside)
Significant activity against CL6 cells (IC50 = 12.8 µg/mL at 24 h)
[9 ]
Kaempferia galanga L. (Zingiberaceae) ethanol extract/Ethyl-p-methoxycinnamate (EPMC)
Extract and EPMC: moderate activity against CL6 cells (IC50 = 64.2, 49.19 µg/mL; SI = 2.2, 2.09)
[10 ]
Kaempferia galanga L. (Zingiberaceae) ethanol extract/Ethyl-p-methoxycinnamate (EPMC)
Moderate activity against CL6 cells: extract IC50 for CL6 cells = 78.41 µg/mL, SI = 4.44; EPMC: IC50 = 100.76 µg/mL, SI = 2.2; moderate activity against HuCCT1
cells: extract IC50 = 66.03 µg/mL, SI = 6.04; EPMC IC50 = 156.6 µg/mL, SI = 2.23
[54 ]
Kaempferia parviflora Wall. ex Baker (Zingiberaceae) (crude ethanol extract)/5,7,4- trimethoxyflavone (KP.8.10)
Flavonoid component in K. parviflora Wall. Ex Baker extract (KP.8.10): potent activity against HuCCA1 cells (IC50 = 46.1 µg/mL) and RMCCA-1 cells
(IC50 = 62 µg/mL)
[20 ]
Mylabris phalerata (Pallas) or Mylabris cichorii (Laeus)/Cantharidin, Norcantharidin
Cantharidin: most sensitive (IC50 : RBE cells = 2 µM, QBC939 cells = 3 µM, HCCC9810 cells = 3 µM)
[55 ]
Phenformin and Quercetin and Myricetin (from several plant species)
Quercetin: enhancement of activity of phenformin against KKU-256 cells (IC50 = 1363 µM)
[56 ]
Phomopsis archeri B. Sutton (fungus )/phomoarcherins A – C (sesquiterpenes), kampanol A, R-mevalonolactone, ergosterol, ergosterol peroxide
Compounds 1 – 4 : IC50 = 0.1 – 19.6 µg/mL (KKU-100, KKU-M139, KKU-M156, KKU-M213, and KKU-M214 cells)
[57 ]
Pinellia ternata (Thunb.) Makino (Araceae/Banxia: polysaccharide (PTPA)
Sk-ChA-1 cells: most sensitive (IC50 : SNU-245, CL-6, Sk-ChA-1, and MZ-ChA-1 cells = 194, 76.9, 57.2, and 29.2 mg/mL, respectively)
[28 ]
Piper longum L. (Piperaceae)/Piperlongumine
IC50 for KKU-055, KKU-213, KKU-214, KKU-139, KKU-100, MMNK1, and NIH3T3 cells = 4.2, 5.2, 6.2, 8.8, 15.9, 5.7 and, 12.7 µM, respectively
[29 ]
Piper longum L. (Piperaceae)/Piperlongumine
IC50 for HuCCT-1–1 cells = 24.8 and 4.2 µ M at 24 and 48 h, respectively
[7 ]
Piper nigrum L. (Piperaceae)/Piperine, Piperine-free Piper nigrum (black pepper) dichloroqmethane extract (PFPE)
PFPE: most potent and selective, especially on KKU-M213 cells (IC50 = 13.70 µg/mL) and TFK-1 cells (IC50 = 15.30 µg/mL)
[58 ]
Pistacia atlantica Desf. (Anacardiaceae)/Mastic gum resin
Activity against KMBC cells: IC50 = 15.34 µg/mL
[32 ]
Plumbago indica L. (Plumbaginaceae)/Plumbagin
IC50 for CL6 cells = 24.00 µM, SI = 2.28 (low)
[30 ]
Reseda luteola L. (Resedaceae)/Luteolin
Potent activity against KKU-M156 cells (IC50 = 10.5 and 8.7 µM at 24 and 48 h, respectively)
[16 ]
Rhinacanthus nasutus (L.) Kurz (Acanthaceae)/Rhinacanthin-C
Potent activity against KKU-M256 cells (IC50 = 1.50 µM)
[59 ]
Tanacetum parthenium L. (Compositae)/Parthenolide
IC50 for SCK cells = 10 µM
[23 ]
Tiliacora triandra (Colebr.) Diels (Menispermaceae)/Tiliacorinine
Significant activity against KKU-M055, KKU-100, KKU-M213, and KKU-M214 cells (IC50 = 4.5 – 7 µM)
[13 ]
Trichosanthes cucumerina L. (Cucurbitaceae)/Cucurbitacin B (CuB) (natural tetracyclic triterpene)
Potent activity against KKU-213: IC50 = 0.048 µM (24 h), 0.036 µM (48 h), and 0.032 µM (72 h) µM; KKU-214: IC50 = 0.088 µM (24 h), 0.053 µM (48 h), and 0.04 µM
(72 h)
[14 ]
Tripterygium wilfordii Hook. f. (Celastraceae)/Triptolide
Potent activity against HaLCCA-1.1, HaLcca-2, HaTCCA-1.1 cells (IC50 = 0.05 mg/mL for all cells)
[15 ]
Tripterygium wilfordii Hook. f. (Celastraceae)/Triptolide
IC50 for HuCCT1, QBC939, and FRH0201 cells = 12.6, 20.5, 18.5 nM at 48 h, respectively
[11 ]
Zingiber officinale Roscoe (ginger) (Zingiberaceae) ethanol extract
Promising activity against CL6 cells (IC50 for each assay = 10.95, 53.15 µg/mL, SI = 18.09, 3.19)
Table 2 Clinical studies of potential herbs and herbal formulations for CCA.
Ref
Plants/Active compounds
Methodology
Key findings
[124 ]
Atractylodes lancea (Thunb.) DC. (Compositae) ethanol standardized extract (CMC capsule formulation)
Clinical study: Phase I study, 48 healthy participants.Group 1 : single oral dose of 1000 mg of A. lancea or placebo (20 : 4 participants). Group
2 : daily oral doses of 1000 mg A. lancea or placebo daily for 21 days (20 : 4 participants).
Well tolerated in both groups.
[137 ]
Atractylodes lancea (Thunb.) DC. (Compositae) ethanol standardized extract (CMC formulation)/β -eudesmol and atractylodin
Antiproliferation of PBMCs against CCA (CL6) (flow cytometry-based NY cytotoxic assay).
Immunomodulatory activity of A. lancea (Thunb.) DC. and compounds in complement with the direct action on apoptosis induction. Atractylodin: significant inhibition of IL6,
TNF-α; A. lancea at a single dose: suppression of IFNγ and IL10, increase of B cells, increase of NK, CD4+, CD8+ cells, and a trend of increased antiproliferation
activity of PBMCs at 24 h. A. lancea (Thunb.) DC. at multiple dosing: suppression of all cytokine production, increase of CD4+ and CD8+, increase of antiproliferative activity
of PBMCs at 24 h (terminated at 48 h of dosing).
[155 ]
PHY906 formulation
Clinical study: open-label phase I trial (800 mg BID on days 1 – 4 + escalating doses of capecitabine (1000, 1250, 1500, 1750 mg/m2 ), orally twice daily on days 1 – 7 of
a 14-day cycle (7/7 schedule) in CCA (n = 1), pancreatic cancer (n = 15), colon cancer (n = 6), esophageal cancer (n = 1), unknown primary cancer (n = 1).
Well-tolerated at MTD of 1500 mg/m2 BID administered in a 7/7 schedule, in combination with PHY906 800 mg BID on days 1 – 4; partial response (n = 1), stable disease
> 6 weeks (n = 13).
The potential role of herbs/herbal medicines for CCA control has been one of the focuses in CCA research, as seen by a relatively large number of research articles published during the years
2000 to 2021. Evidence-based knowledge is provided by scientific support from in vitro, in vivo , and clinical studies in a total of 68 herbs, 9 herbal formulations, and 199 isolated
compounds or synthetic analogs. The plants that were investigated the most were A. lancea (Thunb.) DC. and C. longa L. Other plants with more than three research articles
published on antiproliferative activities included G. hanburyi Hook.f., A. annua L., Z. officinale Roscoe, and A. paniculata (Burm.f.) Nees. The previously reported
studies of various potential herbs (extracts or isolated compounds/synthelic analogs) for CCA focused on their antiproliferative activities against CCA cell lines or antitumor activities in
animal models, activities on cell invasion and migration, and underlying mechanisms or targets of their actions [5 ], [6 ], [7 ], [8 ], [9 ], [10 ], [11 ], [12 ], [13 ], [14 ], [15 ], [16 ], [17 ], [18 ], [19 ], [20 ], [21 ], [22 ], [23 ], [24 ], [25 ], [26 ], [27 ], [28 ], [29 ], [30 ], [31 ], [32 ], [33 ], [34 ], [35 ], [36 ], [37 ], [38 ], [39 ], [40 ], [41 ], [42 ], [43 ], [44 ], [45 ], [46 ], [47 ], [48 ], [49 ], [50 ], [51 ], [52 ], [53 ], [54 ], [55 ], [56 ], [57 ], [58 ], [59 ], [60 ], [61 ], [62 ], [63 ], [64 ], [65 ], [66 ], [67 ], [68 ], [69 ], [70 ], [71 ], [72 ], [73 ], [74 ], [75 ], [76 ], [77 ], [78 ], [79 ], [80 ], [81 ], [82 ], [83 ], [84 ], [85 ], [86 ], [87 ], [88 ], [89 ], [90 ], [91 ], [92 ], [93 ], [94 ], [95 ], [96 ], [97 ], [98 ], [99 ], [100 ], [101 ], [102 ], [103 ], [104 ], [105 ], [106 ], [107 ], [108 ], [109 ], [110 ], [111 ], [112 ], [113 ], [114 ], [115 ], [116 ], [117 ], [118 ], [119 ], [120 ], [121 ], [122 ], [123 ], [124 ], [125 ], [126 ], [127 ], [128 ], [129 ], [130 ], [131 ], [132 ], [133 ], [134 ], [135 ], [136 ], [137 ], [138 ], [139 ], [140 ], [141 ], [142 ], [143 ], [144 ], [145 ], [146 ], [147 ], [148 ], [149 ], [150 ], [151 ], [152 ], [153 ], [154 ], [155 ]. None of these herbs/isolated compounds/synthetic analogs, except
A. lancea (Thunb.) DC., has undergone the full process of nonclinical, clinical, and pharmaceutical development to deliver final products for clinical use. The IC50
(concentration that inhibits cell growth by 50%) values indicating the potency of activities were not reported for most herbs/isolated compounds/synthetic analogs/herbal formulations
investigated. The potency of activity of the antiproliferative activity against human CCA cells was classified according to the IC50 as (i) weak activity (IC50
> 100 µg/mL for the herbal extract and > 100 µM for the isolated compounds/synthetic analogs), (ii) moderate activity (IC50 10 – 100 µg/mL for the herbal extract and
10 – 100 µM for the isolated compounds/synthetic analogs), and (iii) relatively potent (IC50 < 10 µg/mL for the herbal extract and < 10 µM for the isolated compounds/synthetic
analogs). Based on available published data, the antiproliferative activities of the extracts of A. lancea (Thunb.) DC., G. hanburyi Hook.f., and Piper nigrum L.
(Piperaceae) are classified as potent [5 ], [6 ], [7 ], while those of Dioscorea membranacea Pierre ex
Prain & Burkill (Dioscoreaceae), Kaempferia galanga L. (Zingiberaceae), Mesua ferrea L. (Calophyllaceae), Piper chaba Hunt. (Piperaceae), Z. officinale Roscoe,
and Pra-Sa-Prao-Yhai formulation are classified as moderate [5 ], [8 ], [9 ], [10 ], [11 ], and that of sho-saiko-to is classified as weak activity [12 ]. For the isolated compounds/synthetic analogs, those
with the most potent activity are cucurbitacin B and triptolide [from T. wilfordii Hook. f.: IC50 < 1 µM] [13 ], [14 ], [15 ], followed by rhinacanthin C [from Rhinacanthus nasutus (L.) Kurz (Acanthaceae): IC50 = 1.5 µM] [16 ], compounds from D. membranacea Pierre ex Prain & Burkill: IC50 = 1 – 2 µM [8 ], andrographolide and analogs [from
A. paniculata (Burm.f.) Nees: IC50 = 3 µM] [17 ], [18 ], [19 ], cantharidin and
norcantharidin [from Mylabris phalerata (Pallas): IC50 = 2 – 3 µM] [20 ], isolated/synthetic compounds from Atalantia monophylla DC.
(Rutaceae): IC50 = 3 – 5 µM [21 ], [22 ], tiliacorinine [from Tiliacora triandra (Colebr.) Diels (Menispermaceae):
IC50 = 4 – 7 µM] [23 ]. Curcumin and analogs (Zingiberaceae): IC50 3 – 17 µM [24 ], [25 ], [26 ], [27 ], piperlongumine [from P. longum L.: IC50 = 4 – 15 µM] [28 ], [29 ], luteolin [from Reseda luteola L. (Resedaceae): IC50 = 10 µM] [30 ], candidione [from Derris
indica (Lamk.) Benn.: IC50 = 12 – 17 µM] [31 ], and plumbagin [from Plumbago indica L. (Plumbaginaceae): IC50 = 24 µM] [32 ] showed moderated to potent activities ([Table 1 ]). Possible molecular mechanisms of these herbs and/or isolated compounds/synthetic
analogs on CCA cells involve induction of apoptosis, autophagy, and cell cycle arrest (at G0 /G1 , G1 , G1 /S, or G2 /M phases) through
suppression of proinflammatory cytokines and growth factors (IL6, EGF, VEGF, etc.) [10 ], [13 ], [23 ], [27 ], [28 ], [29 ], [30 ], [32 ], [34 ], [36 ], [41 ], [43 ], [46 ], [51 ], [52 ], [53 ], [57 ], [58 ], [59 ], [60 ], [61 ], [62 ], [63 ], [64 ], [65 ], [66 ], [67 ], [68 ], [69 ], [70 ], [71 ], [72 ], [73 ], [74 ], [75 ], [76 ], [77 ], [78 ], [79 ], [80 ], [81 ], [82 ], [83 ], [84 ], [85 ], suppression of expression of cell surface receptors (Vegfr2 , EGFR peroxisome proliferator-activated
receptor gamma, DR4 and DR5, and TRAIL) [69 ], [86 ], [87 ], [88 ], and
deregulation of intracellular pathways (JAK/STAT3, RAS/MAPK, PI3K/AKT, GSKβ /β -catenin, NFκ B/AMPK, ERK, p38/MAPK, HO1, ROS/JNK, EGFR, VEGF, COX2, FAK, MMP2, MMP9, ICAM1,
caspase-3, -8, and − 9, TR1, MDR1, MRP1, 2, and 3, TRAF1, XIAP, p21, p53, p65, and CHOP dependent) ([Fig. 2 ]) [7 ], [11 ], [16 ], [19 ], [25 ], [26 ], [30 ], [31 ], [37 ], [38 ], [42 ], [45 ], [49 ], [54 ], [66 ], [70 ], [78 ], [79 ], [89 ], [90 ], [91 ], [92 ], [93 ], [94 ], [95 ], [96 ], [97 ], [98 ], [99 ], [100 ], [101 ], [102 ], [103 ], [104 ], [105 ], [106 ], [107 ], [108 ], [109 ], [110 ], [111 ], [112 ], [113 ], [114 ], [115 ], [116 ], [117 ], [118 ], [119 ], [120 ].
Fig. 2 Proposed molecular targets and signaling pathways of potential herbs and isolated compounds/synthetic analogs on human CCA.
The most advanced development of a potential herb as a chemotherapeutic agent for CCA is A. lancea (Thunb.) DC. A series of studies on the research and development of A. lancea
(Thunb.) DC. was systematically conducted by our research group [121 ]. A. lancea (Thunb.) DC. is a medicinal plant growing in tropical and subtropical
zones of East Asia such as China and Japan. Its dried rhizome is commonly used in Chinese (“Cang Zhu”), Japanese campo (“So-jutsu”), and Thai (“Khod-Kha-Mao”) traditional medicines for fever,
colds, flu, sore throat, rheumatic diseases, digestive disorders, night blindness, influenza, rheumatic diseases, digestive disorders, night blindness, and cancers. Modern pharmacological
studies also support the broad pharmacological effects of A. lancea (Thunb.) DC. in various diseases [122 ]. Phytochemical investigations reveal a series of
sesquiterpenoids, monoterpenes, polyacetylenes, phenolic acids, and steroids from A. lancea (Thunb.) DC. rhizomes [123 ]. The major constituents are AT
(14%), BE (6%), atractylon (2%), and HS (1%). The potential of A. lancea (Thunb.) DC. and the two major compounds AT and BE for treatment and control of CCA has extensively been
evaluated both in vitro (human CCA cell lines) and in vivo (xenograft mouse model and OV/DMN-induced CCA hamster model) [121 ], [124 ], [125 ]. Results confirm anti-CCA potential and safety profiles of both the crude A. lancea (Thunb.) DC. extract, as well as
AT and BE and the finished product [capsule pharmaceutical formulation of the standardized A. lancea (Thunb.) DC. extract] [126 ], [127 ]. A. lancea (Thunb.) DC. and both compounds exhibit potent and selective antiproliferative activities against CCA cells. The IC50 values range from 20 to
30 µg/mL, with a selectivity index of 3 – 5 [128 ], [129 ]. The potencies of activity of A. lancea (Thunb.) DC. and both
compounds on CCA cell growth is about 3- to 4-fold of the standard drug 5-FU. Furthermore, A. lancea (Thunb.) DC. extract, AT, and BE inhibit CCA cell invasion and migration and
formation of new blood vessels [86 ], [128 ], [130 ], [131 ], [132 ], suggesting a potential role as an antimetastasis and antiangiogenesis agent for CCA. The potential anticancer and antiangiogenesis properties of
A. lancea (Thunb.) DC. extract and its major constituents have been demonstrated in various types of cancer, e.g., murine blastoma cells HeLa (human cervical cells), SGC-7901 (human
gastric cancer cells), BEL-7402 (human liver cancer cells), H33, S180, HL-60, leukemic cells, and gastric cancer [131 ], [132 ], [133 ], [134 ], [135 ]. The underlying mechanisms of the antiproliferative effects of A. lancea
(Thunb.) DC., AT, and BE against CCA cells mainly involve the induction of cell cycle arrest (at G1 phase) and apoptosis through activation or suppression of molecular
targets/signaling pathways involved in CCA pathogenesis. These include the activation of caspase-3/7 and suppression of HO1 production, activation of STAT1/2 and JAK/STAT signaling cascades,
suppression of NFκ B, and suppression of cytoprotective enzymes and key growth regulatory transcription factors [38 ], [41 ], [42 ], [62 ], [98 ], [99 ], [100 ].
The first-in-human starting dose was estimated from the MRSD (maximum recommended starting dose) from toxicology testing in animals [136 ], which was 2400 mg for a
person weighing 60 kg. Despite the concern of bleeding (antiplatelet aggregation) and adverse effect on the nervous system previously reported in vitro and in animals [123 ], results of phase I clinical trials using 1 g A. lancea (Thunb.) DC. (about 50% of the estimated maximum dose in humans) confirmed the safety profile in
healthy Thai subjects [124 ]. The pharmacokinetics of AT was investigated in healthy Thai subjects following a single (1 g) or daily (1 g for 21 days)
administration of the capsule formulation of the standardized A. lancea (Thunb.) DC. extract [124 ]. AT was rapidly absorbed but with low systemic
bioavailability and a short residence time (within 8 h). The immunostimulatory activity of the standardized A. lancea (Thunb.) DC. extract was linked with suppression of the production
of TNF-α and IL6 cytokines, which are involved in the pathogenesis and severity of CCA [137 ]. A phase II dose-finding study is underway to confirm
efficacy, tolerability, and immunomodulatory activity of A. lancea (Thunb.) DC. in patients with advanced-stage CCA. It is noted for the toxic effect of AT and BE on zebrafish embryo
development [86 ]. Although the results may imply similar toxicity in humans, considering the much more sensitivity of the zebrafish model compared with mammalian
cells and rodent models, high intensity of the effect would not be expected in humans. Further studies are needed to confirm this finding.
Apart from A. lancea (Thunb.) DC., C. longa L., G. hanburyi Hook.f., A. annua L., Z. officinale Roscoe, and A. paniculata (Burm.f.) Nees are among
the herbs that have been of research interest for anti-CCA development. Curcumin is a major component of C. longa or turmeric. It is a dietary constituent with tumor-suppressing
potential by inhibiting multiple molecular targets/signaling pathways involved in carcinogenesis, including CCA. Curcumin and synthetic analogs exhibit potent antiprolifertive activities
against human CCA cells with IC50 values of 3 – 17 µM [24 ], [27 ]. However, clinical uses of curcumin in CCA and other types
of cancer may be limited due to its low systemic bioavailability [138 ]. It inhibits cell migration and induces cell cycle arrest at the G2 /M phase
[66 ]. The action of curcumin in CCA involves multiple molecular targets/signaling pathways, including transcription factors (NFκ B, STAT3, and AP1.16),
peroxisome proliferator-activated receptor, AKT activation pathway, B-cell lymphoma 2, B-cell lymphoma-extra large, cell survival proteins (cIAP1, cIAP2, and surviving 15), and Notch1
signaling [25 ], [26 ], [66 ], [83 ], [106 ], [107 ], [109 ], [110 ].
The anticancer potentials of G. hanburyi Hook.f. extract and isolated compounds/synthetic analogs have been well demonstrated in various types of cancer [139 ]. G. hanburyi Hook.f. and its isolated caged xanthones (gambogic acid, forbesione, isomorellin, and isomorellinol, etc.) from the resin and fruits have been used widely in
Thai traditional medicine [51 ]. Gambogic acid was shown to have a favorable safety profile in a phase II a trial in patients with advanced malignant tumors, i.e.,
lung, gastrointestinal, liver, breast, and renal adenocarcinoma [140 ]. Neverthess, no clinical study was conducted in patients with advanced-stage CCA. The
antiproliferative activity of both the extract (IC50 = 2 – 3 µg/mL) and isolated compounds/synthetic analogs (IC50 = 0.03 – 3 µM) is considered potent [6 ], [51 ]. The extract and caged xanthones induce apoptosis via the mitochondrial pathway [51 ] and induction
of G0 /G1 -phase cell cycle arrest through p53 and NFκ B signaling pathways [52 ]. Combinations of isomorellin or forbesione with
doxorubicin exhibited a significant synergistic effect on CCA cells through suppression of MRP1, activation of NFκ B, enhancement of Bcl2-like protein 4 (Bax)/Bcl2, activation of
caspase-9 and caspase-3, and suppression of the expression of survivin, procaspase-9, and procaspase-3 [112 ]. The combination of forbesione with 5-FU strongly
suppressed the expression of Bcl2 and procaspase-3 while enhancing the expression of p53, Bax, Apaf-1, caspase-9 and caspase-3 compared with single-drug treatment [111 ]. The safety profile of gambogic acid in humans together with its potent antiproliferative activity against CCA make this compound a strong candidate for further development as a
CCA chemotherapeutic agent. In addition, gambonic acid is available in the parenteral formulation, which is suitable for CCA patients.
The sesquiterpene lactones artemisinin and derivatives (artemether, artesunate, arteether, and dihydroartemisinin) derived from A. annua L. constitute a unique class of antimalarial
drugs with significant potential for drug repurposing for a wide range of diseases, including cancer [141 ]. The antiproliferative activities of artemisinins
against CCA cells are relatively weak (IC50 = 75 – 377 µM) [37 ]. The mechanisms of their action against CCA have been reported to involve multiple
critical biological targets/signaling pathways of CCA pathogenesis, i.e., DAPK1, BECLIN1, Bcl2, PI3KC3, and MCL-1 [61 ], [96 ], [97 ]. The anti-CCA activities have been shown to be through induction of both apoptosis and autophagy-dependent caspase-independent cell death and cell cycle arrest at
phases S, G0 /G1 , and G2 /M.
Z. officinale Roscoe, or ginger, is a popular spice used globally, especially in most Asian countries. It has been used as a pain relief for arthritis, muscle soreness, chest pain, low
back pain, stomach pain, and menstrual pain. The rhizomes contain over 400 different compounds. The phenolic compounds gingerol and shogaol are found in higher quantities than others. Evidence
from in vitro , animal, and epidemiological studies suggest that ginger and its active constituents suppress the growth and induce apoptosis of a variety of cancer types, including skin,
ovarian, colon, breast, cervical, oral, renal, prostate, gastric, pancreatic, liver, and brain cancer. The active ingredients of ginger, mainly, 6-gingerol and 6-shogaol, target several
cellular molecules that contribute to tumorigenesis, cell survival, cell proliferation, invasion, and angiogenesis (NFκ B, STAT3, Rb, MAPK, PI3k/Akt Ca2+ signals, Akt, ERK,
cIAP1, cyclin A, cyclin D1, Cdk, cathepsin D, caspase-3/7, survivin, cIAP1, XIAP, Bcl2, MMP9, ER stress, and eIF2α ) [142 ]. In vitro studies showed
that ginger has promising antiprolifertive and antioxidant activities against human CCA cells by inducing programmed cell death [11 ], [84 ]. The ethanolic extract of ginger exhibits significant tumor growth inhibition, prolongs survival time, and increases survival rate in CCA-xenografted mice and OV/DMN-induced CCA
in hamsters. In the xenograft model, the crude extract of ginger produced significant anti-CCA activity compared with cisplatin and the untreated control. The extract at medium (1 g/kg body
weight) and high (2 g/kg body weight) dose levels (oral daily dose for 30 days) significantly inhibited tumor growth to about 55.6 and 51.1% of the untreated control, respectively, while
cisplatin inhibited tumor growth to 60% of the control [84 ]. Interestingly, significant reduction of lung metastasis was observed in the xenografted mice treated
with the crude extract of ginger and cisplatin compared with the untreated control. In OV/DMN-induced CCA hamsters, promising anti-CCA activity of the crude extract of ginger was observed at
all dose levels, particularly at the highest oral dose level of 5 g/kg body weight for 30 days [143 ]. The median survival rate and survival time were
significantly prolonged (about two times) in hamsters treated with the extract at all dose levels compared with 5-FU-treated and untreated control groups during the 4 – 6 months observation
period. At week 36, all hamsters except those treated with the highest ginger dose died (1 hamster died, 80% survival rate). The untreated control animals started to die as early as 14
weeks.
A. paniculata (Burm.f.) Nees is an important herbal medicine widely used in several Asian countries, including China, India, and Thailand, for the treatment of respiratory infection,
inflammation, immunostimulation, hepatoprotective, cardioprotective, cold, fever, bacterial dysentery, diarrhea, and hypoglycemic and anticancer activities [144 ], [145 ], [146 ], [147 ], [148 ], [149 ]. Recently, the Ministry of Public Health of Thailand has approved A. paniculata (Burm.f.) Nees for the treatment of COVID-19 [150 ].
A. paniculata (Burm.f.) Nees and its active compound andrographolide have been shown to inhibit cancer cell migration and invasion, including CCA. Due to their low potencies of
activity and requirement of a large dose [151 ], a number of andrographolide analogs, particularly C19 triphenylmethyl ether substitution (AG050) and its
nanoencapsulated formulation, have recently been developed with improved activities against CCA (IC50 = 3 µM) [17 ], [18 ].
These analogs and nanoformulation exhibit potent activity against CCA cells. The inhibitory effect on CCA cell proliferation is through induction of apoptosis and cell cycle arrest at the
Go /G1 and G2 /M phases through downregulation of cyclin D1, Bcl2, and caspase-3, while the upregulation of proapoptotic protein Bax and cleavage of poly
(ADP-ribose) polymerase occurs [60 ]. Andrographolide was also shown to inhibit CCA cell invasion and migration via suppression of claudin 1 through the activation
of p38 MAPK signaling [19 ]. The long history of use and relatively safe profile [152 ] together with evidence of the potency of
antiproliferative activity against human CCA cells make A. paniculata (Burm.f.) Nees extract or andrographolide a candidate as a repurposed drug for CCA.
Resveratrol and capsaicin are among other reported compounds derived from several plant species that have been investigated for anti-CCA activities [73 ], [75 ], [76 ], [77 ]. Resveratrol is a polyphenol found naturally in red wine, grapes, mulberries, cranberries,
and peanuts. The compound exhibits cancer chemopreventive activity through inhibition of tumor initiation, promotion, and progression. In CCA cell lines, resveratrol was shown to interfere
with cell cycle progression, resulting in arresting different phases of the cell cycle (Go /G1 , S, and G2 phases) to induce apoptosis via the
mitochondrial-dependent pathway (caspase-dependent and -independent) [75 ], to stimulate autophagy, and to suppress IL6 by CAFs secretory product [76 ]. It also produces the chemosensitizing effect of 5-FU on CCA growth inhibition [73 ]. Capsaicin, found in hot red chilli peppers
[Capsicum spp. (Solanaceae)], possesses several pharmacological activities, i.e., analgesic, anti-inflammation, and antiproliferative effects, on different gastrointestinal cancer
cells [154 ]. The anti-CCA activity of capsaicin was shown to be associated with the induction of apoptosis and attenuation of the GLI1 and GLI2 targets of the
Hedgehog signaling pathway (role in carcinogenesis) [101 ], [102 ]. The use of capsaicin as a food supplement to inhibit Hedgehog
signaling might therefore be of additional therapeutic benefit in patients with CCA. In the xenograft mouse model, a combination of capsaicin with 5-FU was synergistic and significantly
suppressed tumor growth compared with 5-FU alone. Further investigation revealed that the autophagy induced by 5-FU was inhibited by capsaicin. The mechanism of action was shown to be through
the inhibition of 5-FU-induced autophagy by activating the PI3K/AKT/mTOR signaling pathway [103 ].
Herbs constitute a promising source of medicine for CCA control. The anti-CCA potential of several herbs and isolated compound/synthetic analogs have been demonstrated in different
experimental models in conjunction with their underlying mechanisms of action at the molecular and cellular levels. As herbal medicines usually contain several pharmacologically active
compounds, their multi-ingredient characteristics may make the evaluation of clinically useful products more complex than synthetic drugs. With regard to the therapeutic aspect, however, using
the whole herbal extract would be expected to provide more therapeutic benefit compared to synthetic drugs concerning efficacy (synergistic action) and tolerability (buffering effect). The
limitation of the current study includes only articles published in English were included in the analysis and the number of the reported articles may therefore be underestimated. Comparison of
the potencies of antiproliferative activities of the investigated plants/isolated or synthetic compounds/herbal formulations was made based on only available data on the IC50
values, which were not reported in some studies. Some reported the antiproliferative activity potencies as the percentage of inhibitory effects on cell growth at specified concentrations. In
addition, different CCA cell lines and assay methods for assessment of antiproliferative activities were used in different studies.
In conclusion, a number of plants, isolated compounds, synthetic analogs, and herbal formulations have been demonstrated for their potential to control CCA. However, only A. lancea
(Thunb.) DC. was fully developed based on the reverse pharmacology approach. Future research should be geared toward the full development of the candidate herbs until delivery of final
products that are safe and effective for CCA control. Other targets of their action should be further investigated. Research targeting inflammatory, proliferative, and angiogenesis processes,
development, and progression has been an extensive area. Blocking the generation of an inflammatory infiltrate by interfering with critical molecules of the adhesion process is an attractive
strategy to control CCA.
