Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Traditional Chinese Medical Sciences

journal homepage: www.elsevier.com/locate/jtcms

Preclinical anticancer activity of Typhonium flagelliforme (Lodd.) Blume

and its potential mechanism: A systematic review
Kwok Wen Ng a, *, Suk Fei Tan b, Shu Ying Looi a, Faiza Naimat c, Hazrina Hamid d
a
Faculty of Pharmacy, Quest International University, 30250 Ipoh, Perak, Malaysia
b
School of Pharmacy, Management and Science University, 40100 Shah Alam, Selangor, Malaysia
c
Faculty of Pharmacy, Universiti Teknologi MARA, Cawangan Selangor, 40450 Shah Alam, Selangor, Malaysia
d
Faculty of Pharmacy, Lincoln University College, 47301 Petaling Jaya, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Objective: To assess the potential of Typhonium flagelliforme (Lodd.) Blume (T. flagelliforme, Bian Yan Li
Received 23 January 2023 Tou Jian), a traditional Chinese medicinal plant, as an anticancer agent in a systematic review of pre-
Received in revised form clinical research.
22 September 2023
Methods: Seven databases, namely, Scopus, PubMed, Web of Science, ScienceDirect, LILACS, EBSCO
Accepted 22 September 2023
Available online xxx
Medline, and Mendeley were thoroughly searched from their inception up until September 8, 2023. Peer-
reviewed English language studies that conducted in vitro and in vivo investigations of T. flagelliforme
extracts, fractions, or isolated compounds were included. Clinical trials and non-original peer-reviewed
Keywords:
Typhonium flagelliforme
reports were excluded. The effectiveness of T. flagelliforme on various cancer cells, tumor sizes, and
Shui Ban Xia mechanisms was qualitatively assessed. The quality of evidence was assessed using the ToxRTool by three
Rodent tuber independent raters, and their consistency was verified.
Cancer Results: The search included 27 studies: twenty two in vitro, four in vivo, and one involving both. T.
Systematic review flagelliforme extracts were shown to be effective against leukemic, breast, colorectal, and lung cancers.
Most studies had “Reliable with Restrictions” scores. T. flagelliforme induced apoptosis by halting the cell
cycle, activating caspase-3/-9, cleaving PARP, fragmenting DNA, reducing survivin, decreasing ROS,
suppressing COX-2 and HSP70, and inhibiting the NF-kB pathway. When combined with interferon, T.
flagelliforme exerts antiangiogenic effects.
Conclusion: Although T. flagelliforme shows promising activity against cancer, its efficacy as a standalone
anticancer treatment remains uncertain. It appears to be better suited as complementary or combined
therapy. The lack of conclusive evidence could be attributed to suboptimal study design, incomplete
reporting, and inadequate inclusion of proper positive controls and statistical analyses across multiple
articles.
© 2023 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Study inclusion and exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Search strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Study selection and data collection process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Quality of assessment of included study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. Quality assessment of articles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

* Corresponding author.
E-mail address: kwokwen.ng@gmail.com (K.W. Ng).
Peer review under responsibility of Beijing University of Chinese Medicine.

https://doi.org/10.1016/j.jtcms.2023.09.009
2095-7548/© 2023 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: K.W. Ng, S.F. Tan, S.Y. Looi et al., Preclinical anticancer activity of Typhonium flagelliforme (Lodd.) Blume and its potential
mechanism: A systematic review, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/j.jtcms.2023.09.009
K.W. Ng, S.F. Tan, S.Y. Looi et al. Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

3.2. Cytotoxicity evaluation of T. flagelliforme extracts and fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.3. Anticancer compounds from T. flagelliforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.4. In vivo evaluation of T. flagelliforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.5. Antiproliferative mechanism of T. flagelliforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction scientific evidence. This review will help gather information and
draw conclusions on the effects and efficacy of T. flagelliforme in
According to the International Agency for Research on Cancer, cancer therapy.
there were 19.1 million new cancer cases worldwide in 2020, with
the burden expected to increase by approximately 50 percent to
2. Methods
28.4 million cases by 2040. Despite advances in diagnostic testing
and treatment technologies, cancer is responsible for approxi-
2.1. Study inclusion and exclusion criteria
mately 23% of all deaths. It is the second largest cause of death in
both industrialized and developing countries.1
Articles meeting specific inclusion criteria in their titles and
The need to search for and develop new anticancer drugs is
abstracts were selected for an in-depth review of the primary
driven by the severe side effects of some chemotherapies, low ef-
literature: (1) the study reported on the use of T. flagelliforme alone,
ficacy of chemotherapy due to cancer heterogeneity, and develop-
either in the form of extracts, active fractions, or isolated com-
ment of resistance to therapy. Approximately 74% of all cancer
pounds; (2) the articles were written in English language only to
therapies are initially developed from natural products. Although
prevent translation errors and ensure reliability; (3) they were
natural product extracts have fallen out of favor in drug discovery,
peer-reviewed research articles; and (4) the study outcomes were
they still serve as an eminent reservoir of diverse chemicals,
reported on the preclinical in vitro and in vivo anticancer activities
including alkaloids, coumarins, quassinoids, terpenoids, lignans,
of the T. flagelliforme extract.
and coumarins, that can act against a wide range of oncotargets.2,3
Studies were excluded if they met any of the following criteria:
Typhonium flagelliforme (Lodd.) Blume (T. flagelliforme),
(1) articles that solely referred to the plant's vernacular names
commonly known as “Bian Yan Li Tou Jian” in traditional Chinese
without explicitly stating the scientific names of T. flagelliforme or
medicine or “rodent tuber” in Malaysia, is a tropical plant from the
Arum divaricatum; (2) studies that failed to disclose the levels of the
Araceae (Arum) family. The plant thrives on damp, soft, and shaded
anticancer assay endpoints; (3) studies that solely reported on the
terrain throughout Southeast Asia, China, and the Indian subcon-
chemistry of T. flagelliforme and did not include biological testing,
tinent. Traditionally, it has been used to alleviate cough, relieve
such as phytochemical isolation, or in silico or compound identifi-
asthma, and treat cancers. A multitude of studies have demon-
cation; (4) articles that investigated the combined effect or
strated the usefulness of T. flagelliforme in the treatment of
formulation of T. flagelliforme with other substances; (5) conference
numerous ailments, including cancer.
abstracts without full text were excluded due to the possibility of
There is a clear need for a systematic review of the medicinal
inadequate information.
properties of T. flagelliforme, as the current literature on the topic is
limited and outdated. During our initial literature search, we
discovered four review papers on T. flagelliforme, the most recent of 2.2. Search strategy
which was published in 2022 as a proceeding paper.4 However, this
review did not summarize the anticancer mechanism of T. flag- Two key questions were created using the guidelines from the
elliforme and did not conduct a quality assessment of the included Preferred Reporting Items for Systematic Reviews and Meta-
articles. Another study published in 2021 focused on the impact of Analyses (PRISMA).8 The questions were: a) “Does T. flagelliforme
T. flagelliforme on breast cancer, but it was written in Bahasa have a substantial preclinical anticancer effect?”, and; b) “What is
Indonesia and limited to a specific type of cancer.5 An article the anticancer mechanism of T. flagelliforme?” Variation of key-
reviewed the phytochemical constituents and pharmacological words of “T. flagelliforme” or “A. divaricatum” and “cancer” were
activities of the Typhonium genus in 2019, but it did not specifically utilized in the search. Seven databases, namely Scopus, Web of
focus on the anticancer properties of T. flagelliforme.6 The most Science, PubMed, EBSCO, ScienceDirect, Lilacs, and Mendeley, were
relevant review on the topic was published eight years ago in 2013, searched from inception until September 8, 2023. As T. flagelliforme
highlighting the need for an updated and comprehensive exami- was previously mistakenly referred to as Typhonium divaricatum,
nation of the current research on T. flagelliforme.7 Hence, we con- the common name “rodent tuber” or “keladi tikus” was sometimes
ducted this structured and systematic review to gather referred to the wrong species, we did not include the common
comprehensive and up-to-date information on the in vitro and name in our search string (Supplementary Data 1).9 We also
in vivo anticancer activities of T. flagelliforme. The articles were manually examined the references of retrieved publications and
critically appraised during the review process using specific criteria conducted citation snowballing to locate additional studies that
to generate a solid, reliable, and objective assessment of the met the inclusion criteria.

2
K.W. Ng, S.F. Tan, S.Y. Looi et al.
2.3. Study selection and data collection process
Two reviewers (KWN and SFT) initially assessed the study titles
and abstracts based on established inclusion and exclusion criteria.
The full texts of the relevant studies were retrieved and indepen-
dently evaluated. The references of the selected publications were
examined to identify other studies which may have been over-
looked during the database search. When KWN and SFT disagreed,
a third reviewer (FN) was consulted. The data presented in these
studies were carefully extracted and included in evidence tables.
The extracted data included the author, year of publication,
research type, nature of the extract, portion of the plant utilized,
type of cancer examined, biochemical models employed, com-
pound identification, and results.
2.4. Quality of assessment of included study
Three reviewers (KWN, SFT, and FN) utilized ToxRTool,10 a
spreadsheet-based tool developed by the European Commission's
Joint Research Center (Brussels, Belgium), to analyze the quality of
the selected articles. The rubrics used to assess the studies
comprised 21 criteria for in vivo studies and 18 criteria for in vitro
studies, with a focus on five groups of evaluation criteria: identi-
fication of test materials, characterization of the test system,
research design, outcome documentation, and study design and
data plausibility. Each criterion was given a score of either “1” if the
criterion was fulfilled or “0” if not fulfilled (Supplementary Data 2
and 3). Total scores were then computed, and the quality of evi-
dence was assigned as Category 1, 2, or 3.10
Category 1 studies (criteria fulfillment score for in vivo studies

18 and in vitro
15) had the highest quality evidence and were
deemed to be reliable without any restrictions. Category 2 studies
(in vivo 13e17 and in vitro 11e14), were reliable but had some
limitations, while Category 3 studies (in vivo < 13 and in vitro < 11)
were considered to be of the lowest quality, unreliable, and typi-
cally not used as a primary reference.10 Instead, they may be used as
supporting materials.
2.5. Statistical analysis
Inter-rater reliability was assessed using the Intraclass Correla-
tion Coefficient (ICC), following the guidelines proposed by Koo and
Li.11 Three raters independently scored the quality of the articles
using ToxRTool. The ICC was calculated separately from the com-
bined score of each article. A two-way mixed-effects model with
absolute agreement was used to calculate the ICC. The resulting ICC
estimates were interpreted based on the following criteria: An ICC
value of less than 0.5 indicated poor reliability, whereas a value
between 0.5 and 0.75 suggested moderate reliability. If the value
fell between 0.75 and 0.9, it indicated good reliability, and if the
value was greater than 0.9, it suggested excellent reliability.11 In
addition to ICC, the average assessment score in each group using
ToxRTool by the three assessors and standard deviation (SD) for
each item were also calculated. All statistical analyses were con-
ducted using SPSS version 28.0 (IBM Corp., Armonk, NY).
3. Results
By cross-referencing the citations from the previous review
papers, we identified 15 preclinical in vitro and in vivo studies that
investigated the potential of Typhonium flagelliformes for anticancer
activity. Screening of the databases yielded 240 studies: 31 from
Scopus, 33 from Web of Science, 12 from PubMed, 69 from Men-
deley, 38 from ScienceDirect, 55 from EBSCO MEDLINE, 0 from LI-
LACS, and two from a manual search after examining the references
3
Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx
of all retrieved publications. Of these, 90 were rejected because of
duplication. Additionally, one article was rejected because of non-
accessible to abstract. Upon further examination of the title and
abstracts, we removed another 100 papers because 80 did not
contain reference to T. flagelliforme or cancer in the abstract and
title, six were review articles, four were non-full text conference
abstracts, one was a letter to the editor, and nine were articles
written in a language other than English. The full texts of the
remaining 49 articles were searched and screened, and 17 articles
were excluded because they were not related to in vitro or in vivo
anticancer studies. Two other papers were rejected because T.
flagelliforme extracts were combined with other plant extracts
without testing the individual extract, one article only reported the
in silico results, one article reported the cytotoxic results but did not
clearly describe the experimental method, and one article was
rejected because its full text was written in a foreign language. Of
these, 15 papers were cited in previous review articles, and this
review encompassed an additional 12 papers that had not been
previously included. Finally, twenty two in vitro anticancer studies,
four in vivo studies, and one combination in vitro and in vivo study
were chosen for the final analysis (Fig. 1). When categorized by
treatment agents, nine studies employed entire plant extracts or
fractions, ten used only tuber or root extracts, three used leaves,
three used leaves and tubers, and two did not specify which part of
the plant was used. Table 1 summarizes the significant findings of
the review.
3.1. Quality assessment of articles
The current study used the ToxRTool to objectively assess the
quality of T. flagelliforme preclinical anticancer studies. Nonetheless,
the evaluations were based solely on published text, and we did not
contact the authors for further clarification if there was any missing
information or components.
An inter-rater agreement analysis was conducted to determine
the consistency among the three raters in assessing the quality of
the articles. The ICC was calculated for each study using a combined
score of 21 questions for the in vivo study and 18 questions for the
in vitro study. The average ICC values for the in vitro and in vivo
studies were 0.988 (95% confidence interval [CI] ¼ 0.972e0.995)
and 0.967 (95% CI ¼ 0.852e0.996), respectively. These results
indicate a remarkable degree of agreement among the raters in
their assessment of article quality.
The average ToxRTool reliability evaluation scores for the indi-
vidual in vitro and in vivo preclinical studies on T. flagelliforme are
presented in Table 2. Based on their original category score, seven
in vitro studies were judged to be “reliable without restriction,” six
were determined to be “reliable with restriction,” and ten were
determined to be “not reliable.” One out of the five in vivo in-
vestigations was deemed “reliable without limitations,” while the
other four were classified as “reliable with limitations.”
The assessment of Category I in vitro studies focused primarily
on the identification of the test substance. A thorough evaluation
was conducted to determine whether the plant material used in the
study was properly authenticated and whether a voucher specimen
was deposited, given the potential for confusion between T. flag-
elliforme and T. divaricatum. Notwithstanding the possibility that
some studies may have undergone authentication without explicit
documentation, the present evaluation relied on a declaration of
the authentication process, and did not seek further confirmation
from the authors. Moreover, studies that provided a precise iden-
tification of the plant source, such as the supplier, location, or name,
were assigned additional weight. Broader geographic regions, such
as states or countries, were not considered specific locations.
Additionally, it was imperative that the plant parts used in the
K.W. Ng, S.F. Tan, S.Y. Looi et al.
Fig. 1. Flow diagram for the systematic search for anticancer articles of Typhonium flagelliforme (Lodd.) Blume.
study were clearly indicated, or that the study identified the
bioactive constituents.
In Category II, the evaluation criteria included providing
detailed information about the test system employed, which
involved specifying the cells or tissue used and the source of the
test materials, as well as disclosing information on cell passage (for
cell lines), characterization, and cultivation conditions. The
maximum score attainable in this section was three. Some studies
failed to indicate the origin of the test system,12,22,37 such as the
laboratory or scientist responsible for providing the materials.
None of the studies provided information on cell line passages in
the text or conducted cell line authentication, even when the cells
were obtained from a laboratory or scientist. However, most studies
provided detailed information on culture conditions and pretest
preparations for other in vitro assays.
Category III involved the rigorous evaluation of various param-
eters to ensure the accuracy and reliability of the study. These pa-
rameters included the cell number, type of solvent and vehicle
used, total sample volume applied, dosage, duration of treatment,
and use of positive and negative controls. Positive controls were
only acceptable if they were known to influence the desired
outcome, such as cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-
2e5-diphenyltetrazolium bromide (MTT) assay, whereas negative
controls were limited to a blank medium with the vehicle. The use
of cell-free wells as a negative control in the MTT assay was deemed
inappropriate by the raters.30 However, some articles lacked evi-
dence or failed to report the results of positive and negative
controls.13,15,16,18,22,23,25,27,29,35,37 The number of replications
4
Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx
performed was also required to be specified in the manuscript and
marks were awarded accordingly. While distinguishing between
technical and biological replications was complex, a study was
awarded a mark if the number of replications was stated in the text.
In Category IV, the authors were expected to provide a
comprehensive description of the endpoint determination method,
including all endpoints, and to present the results for all study
endpoints described in the methods section. The results for the
positive and negative controls, as well as other results, were ex-
pected to be presented alongside the test sample results. However,
some antiproliferative studies could not be evaluated, as they only
reported the inhibition value.12,13,15,20,21 The raters did not conduct
any statistical calculations but awarded marks based on the au-
thors' results if they were described in the text.
The fifth and final category, Category V, assessed the adequacy
and suitability of the test system for detecting the claimed effect.
Most studies utilized the MTT assay to determine cytotoxicity or
cell viability. However, for studies aimed at determining half
maximal inhibitory concentration (IC50), marks were awarded only
if the concentration range used produced a curve that crossed the
50% inhibition line. If not, the determination of IC50 was considered
invalid, as it was based on an assumption.22,30 In one study, the
linearity analysis was insufficient to detect the IC50, as evidenced by
the significant difference in IC50 values between the reported IC50
value and the result curve.35 In cases where a study had not been
repeated or if the method of administration was unsuitable, such as
the use of an inappropriate vehicle or the absence of any observed
positive results, the study was deemed inconclusive. If the results
K.W. Ng, S.F. Tan, S.Y. Looi et al. Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

Table 1
Summarization of the primary results of the publications included.

Study ID Isolated compound/ Part of Mode Approaches Biochemical system Main Finding
plant extract/fractions plant
used

Choo 200112 Hexane and fractions T In vitro MTT** assay P388 cells Poor cytotoxicity activity of the
from open column hexane fraction; nine chemicals
chromatography. identified lacked cytotoxic activity.
Choo 200113 Methanol extracts, n- R, T, S, L In vitro MTT assay P388 cells Nonpolar fractions displayed
hexane, chloroform, cytotoxicity on P388 cell lines.
and n-butanol fractions
Lai 200514 Methanol extracts, n- WP In vitro Methylene blue staining NCI-H23, T47D, HepG2 Mature plants and plantlets showed
hexane, chloroform, n- cells cytotoxicity on NCI-H23 (ED50: 1.2
butanol fractions e5.2 mg/mL) and (ED50: 1.5e18.7 mg/
mL) respectively, while exhibiting
mild cytotoxicity on T-47D (mature
plant, ED50: 12.2e54.6 mg/mL;
plantlets, ED50: 39.1‒100 mg/mL). No
cytotoxic effects were observed on
HepG2 cells.
Lai 200815 Hexane, DCM, and WP In vitro MTT assay, microscopic NCI-H23, BALB/c 3T3 Fractions rich in hexadecanoic acid, 1-
MeOH extracts observation, TUNEL assay cells hexadecene, phytol, and a phytol
derivative suppressed NCI-H23 cell
line proliferation via apoptosis
induction, without affecting normal
BALB/c 3T3 cell line.
Mohan 200816 Hexane, DCM, ethyl L, T In vitro MTT assay CEMs cells T. flagelliforme's DCM and ethyl
acetate, and methanol acetate extracts exhibited
extracts antiproliferative effects against CEMs
cells, with IC50 values of 10.8 and
5.8 mg/mL for leaves and 6.5 and
8.2 mg/mL for tuber, respectively.
Lai 201017 Isolated compounds WP In vitro Photocytotoxicity (MTT), NCI-H23, HS578T cells The isolated bioactive pheophorbide
TUNEL, microscopic compounds did not exhibit equal
evaluation cytotoxic activity as the upstream
fraction upon individual evaluation.
The compounds have phototoxicity
effect. The authors suggested a
potential synergistic effect within the
compound-rich fraction.
Mohan 201018 Sequential extraction L,T In vitro and In vivo MTT assay, AO/PI cell WEHI-3 cells, male T. flagelliforme's DCM extracts (IC50
with hexane, DCM, viability assay, BALB/c mice inoculated 24.0 ± 5.2 mg/mL) induced apoptosis
ethyl acetate and histopathology with WEHI-3 cells on WEHI-3 cells. It reduced immature
methanol evaluation, TUNEL assay cells in peripheral blood, and splenic
tumor size in leukemic mice.
Mohan 201019 Linoleic acid rich WP In vitro MTT assay, Annexin V, CEMs cell Linoleic acid-rich fraction strongly
fraction from vacuum DNA laddering, inhibited human T4-lymphoblastoid
liquid chromatography colorimetric caspase cell (IC50 3 ± 0.08 mg/mL) via
assay, immunoblot assay, mitochondrial-dependent apoptotic
flow cytometry pathway evidenced by G0/G1 cell
cycle arrest, high DNA fragmentation,
increased caspase-3 and caspase-9,
cytochrome c accumulation, PARP
fragmentation, decreased Bcl-2, and
absence of FasL.
Mohan 201120 DCM extract and VLC L,T In vitro MTT assay, TUNEL, CEMs, PBL cells T. flagelliforme fractions selectively
fractions microscopic observation induced cytotoxicity in CEMs cell,
with evidence of apoptosis and DNA
fragmentation via cytological
evaluation, AO/PI assay, SEM, and
TEM. GC-MS analysis identified
linoleic acid, hexadecanoic acid, and
9-hexadecanoic acid in the active
fraction.
Majid 201421 Extracts T In vitro Colourimetry assay, KB, ORL-48, normal T. flagelliforme induced early
TUNEL, DNA mouse fibroblast cells apoptosis after 72 h exposure to
fragmentation analysis 100 mg/mL of the extracts. No effect
on normal fibroblasts was reported.
Weak apoptosis activity was observed
via TUNEL assay. No significant
cytotoxic effect was observed on KB
cells (IC50 95.67 ± 1.15 mg/mL) or ORL
48 cells.
(continued on next page)

5
K.W. Ng, S.F. Tan, S.Y. Looi et al. Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

Table 1 (continued )

Study ID Isolated compound/ Part of Mode Approaches Biochemical system Main Finding
plant extract/fractions plant
used

Nobakht Methanol: DCM (1:1) WP In vitro MTT assay MCF-7 cells Older ex-vitro plant extracts have
201422 extracts better cytotoxicity activity against
MCF7 comparing to young ex vitro
and in vitro growth plant.
Purwaningsih Extract WP In vitro MTT assay Hela, MCF7 cells T. flagelliforme's ethanolic extract is
201423 more cytotoxic against MCF-7 cells
than HeLa cells.
Chodidjah T In vivo Immunohistochemistry, 3 months female CH3 Administration of T. flagelliforme
201424 physical examination mice with syrup decreased the expression of the
subcutaneous tumour Her2neu receptor and the BCL2
inoculation, protein in mice but did not decrease
breast cancer volume.
Khiru 201525 Methanolic extract L In vitro MTT assay MDA-MB-231, The plant extract exerted a cytotoxic
CHO cells effect on both cancerous MDA-MB-
231 and non-cancerous CHO cells.
Nurrochmad 96% ethanolic extracts T In vivo MTT assay, microscopic Cyclophosphamide- T. flagelliforme ethanolic extract
201526 observation, ELISA, flow treated Wistar rats reduced immunosuppression in
cytometry. cyclophosphamide treated rats by
enhancing lymphocyte proliferation,
phagocytic activity, CD8þ T cell
proliferation, and cytokine levels,
including TNF-a and IL-1a.
Purwaningsih Extracts WP In vitro Immunohistochemistry Hela, T47D, Vero cells T. flagelliforme extract reduced
201627 telomerase expression (stronger in
Hela cells compared to T47D and Vero
cells).
Setiawati Ethyl acetate extracts L In vitro MTT assay, microscopic WiDr colon cancer cells T. flagelliforme showed anticancer
201628 observation, activity and induced apoptosis on
immunohistochemistry WiDr cells through inhibition of COX-
2 expression.
Purwaningsih 80% ethanol extract WP In vitro Immunohistochemistry Raji, Vero cells T. flagelliforme reduced telomerase
201729 expression in Raji cells treated at ½
IC50 and 1 IC50 dose more than in
normal Vero cells.
Sianipar 201930 Ethanol extract T In vitro MTT assay MCF-7 cells Gamma mutated plant has higher
MCF7 cytotoxicity than non-mutated
probably due to new compounds
(detected using GC-MS).
Maysarah 95% ethanol extract NA In vivo Survival rate, tumor Female Sprague T. flagelliforme extract (50 mg/kg)
202031 latency period, eDawley rats induced reduced tumor latency period in rats
percentage of tumor breast cancer (1 dose at 12 weeks compared to the control
occurrence, number and 15 mg DMBA) group at 9 weeks. tumor incidence,
size of tumors, the number, and size were significantly
weight of tumors, lower than those in the control group.
fluorescence intensity of
autofluorosphore,
histological evaluation
Priosoeryanto Ethanolic extracts L In vitro Cell count (trypan blue), CSCC. MCM-IPB-B3, T. flagelliforme and canine interferons
202032 chicken chorioallantois FSCC, CAM (natural and recombinant)
membrane (CAM). synergistically reduced CSCC and
FSCC cell proliferation in MCM-IPB-
B3. Also, they inhibited rabbit
endothelial cell proliferation and
blood vessel formation in CAM assay.
Putra 202033 Hexane and DCM T In vitro Apoptosis assay, Breast cancer stem cells T. flagelliforme inhibited breast cancer
fraction microscopic observation, stem cell proliferation and apoptosis
immunohistochemistry, by lowering survivin's expression
flow cytometry level and elevating caspase-9 and
caspase-3 levels.
Sianipar 202034 Ethanol, ethyl acetate T In vitro Presto Blue assay MCF-7, CV1 cells Ethanol and ethyl acetate fractions of
and water fractions of TF have cytotoxic effects on MCF-7,
gamma ray irradiated but not on normal CV1 cells. No
plant results for other fractions on normal
cells were reported.
Sianipar 202135 96% ethanol mutant T In vitro MTT assay MCF-7 cells Nano emulsified mutant extract of the
plant extracts and T. flagelliforme has cytotoxic activity
nanoemulsified against MCF7 and contains
extracts hexadecanoic acid and n-
hexadecanoic acid as revealed using
GC-MS analysis.

6
K.W. Ng, S.F. Tan, S.Y. Looi et al. Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

Table 1 (continued )

Study ID Isolated compound/ Part of Mode Approaches Biochemical system Main Finding
plant extract/fractions plant
used

Dharmana Ethanolic extract T In vivo ELISA, TUNEL, 10 weeks C3H mice T. flagelliforme increased tumor cell
202336 microscopic observation allografted with death at 200 mg/kg without affecting
adenocarcinoma TNF-a levels compared to the control
mamma group.
Susanto 202237 96% ethanol of gamma NA In vitro Cell count MCF-7 cells Gamma irradiated T. flagelliforme
irradiated dried plant dried plant showed lesser
cytotoxicity, but effect was not
significant.
Ibrahim 202238 Hexane and DCM T In vitro Magnetic cell sorting, Breast cancer stem cells T. flagelliforme decreased breast
fraction immunocytochemistry cancer stem cell populations by
increasing HSP-70 expression in
tumor-associated macrophages.

Notes: AO/PI: acridine orange and propidium iodide; CSCC: canine squamous cell carcinoma; DCM: dichloromethane; DMBA: dimethylolbutanoic acid; ED50: median effective
dose; ELISA: enzyme linked immunosorbent assay; FSCC: feline squamous cell carcinoma; GC-MS: gas chromatography-mass spectrometry; IC50: half maximal inhibitory
concentration; L: leaves; MeOH: Methanol; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NA: not available; R: root; S: stem; SEM: scanning electron
microscope; T: tuber; TEM: transmission electron microscope; TNF-a: tumor necrosis factor-a; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; WP:
whole plant.

did not indicate any repetition and were not described elsewhere,
no marks were granted.
In the evaluation of in vivo studies, most of the assessment
criteria were similar to those used in the in vitro evaluation. How-
ever, additional information is required, such as a clear description
of the type of animal used, including the species, sex, and number
of animals per group, as well as the administration conditions,
including volume, dosage, and duration. Studies that failed to
report the number of animals per group or only tested one con-
centration without justification were downgraded.31 Moreover, any
study that did not provide accurate reference were also down-
graded as we could not validate the method based on the reference
provided in the text.31 In the Chodidjah et al study,24 the authen-
tication of the plant was not properly described. Despite these
shortcomings, all five studies were able fulfilled most of the 21
criteria outlined by the ToxRTool.

Table 2
ToxRTool reliability assessment scores for individual in vitro and in vivo studies.

No Study Group I Group II Group III Group IV Group V Total score* Category#

In vitro
1 Ref. 12 3.00 (0.00) 2.00 (0.00) 3.00 (0.00) 0.67 (0.47) 0.00 (0.00) 9.00 (0.00) 3
2 Ref. 13 3.00 (0.00) 3.00 (0.00) 3.00 (0.00) 0.67 (0.47) 0.00 (0.00) 10.00 (0.00) 3
3 Ref. 14 3.00 (0.00) 3.00 (0.00) 5.33 (0.47) 3.00 (0.00) 2.00 (0.00) 16.33 (0.47) 1
4 Ref. 15 4.00 (0.00) 3.00 (0.00) 4.67 (0.47) 1.67 (0.47) 1.33 (0.47) 14.67 (0.94) 1
5 Ref. 16 3.33 (0.47) 3.00 (0.00) 3.00 (0.00) 1.00 (0.00) 1.33 (0.94) 11.67 (0.47) 2
6 Ref. 17 4.00 (0.00) 3.00 (0.00) 5.67 (0.47) 2.00 (0.00) 2.00 (0.00) 16.67 (0.47) 1
7 Ref. 19 3.00 (0.00) 3.00 (0.00) 6.00 (0.00) 3.00 (0.00) 2.00 (0.00) 17.00 (0.00) 1
8 Ref. 18D 3.00 (0.00) 3.00 (0.00) 4.00 (0.00) 2.00 (0.00) 1.00 (0.00) 13.00 (0.00) 2
9 Ref. 22 3.00 (0.00) 1.33 (0.47) 4.00 (0.00) 1.33 (0.47) 0.00 (0.00) 10.00 (0.82) 3
10 Ref. 20 3.33 (0.47) 2.67 (0.47) 5.67 (0.47) 2.00 (0.00) 1.33 (0.47) 15.00 (0.00) 1
11 Ref. 23 2.00 (0.00) 3.00 (0.00) 2.00 (0.00) 1.67 (0.47) 0.00 (0.00) 9.00 (0.00) 3
12 Ref. 25 3.00 (0.00) 3.00 (0.00) 5.00 (0.00) 3.00 (0.00) 0.67 (0.47) 14.67 (0.47) 1
13 Ref. 21 3.33 (0.47) 2.67 (0.47) 5.33 (0.47) 1.67 (0.47) 0.67 (0.47) 13.67 (0.47) 2
14 Ref. 28 4.00 (0.00) 2.00 (0.00) 5.33 (0.47) 3.00 0.00 1.33 (0.47) 15.67 (0.47) 1
15 Ref. 27 3.00 (0.00) 2.67 (0.47) 1.00 (0.00) 1.67 (0.47) 0.00 (0.00) 8.67 (0.47) 3
16 Ref. 29 2.00 (0.00) 3.00 (0.00) 1.33 (0.47) 1.67 (0.47) 0.00 (0.00) 8.33 (0.47) 3
17 Ref. 30 2.00 (0.00) 2.00 (0.00) 4.67 (0.47) 2.67 (0.47) 0.00 (0.00) 11.67 0.47 2
18 Ref. 33 2.67 (0.47) 2.67 (0.47) 5.33 (0.94) 2.00 (0.82) 1.00 (0.82) 13.67 (1.25) 2
19 Ref. 34 2.00 (0.00) 3.00 (0.00) 4.00 (0.82) 1.00 (0.82) 0.00 (0.00) 10.33 (0.47) 3
20 Ref. 32 3.00 (0.00) 1.00 (0.00) 3.00 (0.00) 1.00 (0.00) 0.00 (0.00) 8.00 (0.00) 3
21 Ref. 35 3.00 (0.00) 2.00 (0.00) 4.00 (0.00) 1.00 (0.00) 0.00 (0.00) 10.33 (0.47) 3
22 Ref. 38 4.00 (0.00) 3.00 (0.00) 3.00 (0.00) 3.00 (0.00) 1.00 (0.00) 14.00 (0.00) 2
23 Ref. 37 2.00 (0.00) 1.00 (0.00) 3.00 (0.00) 0.00 (0.00) 0.00 (0.00) 6.00 (0.00) 3
In vivo
1 Ref. 18D 3.00 (0.00) 4.00 (0.00) 7.00 (0.00) 3.00 (0.00) 2.00 (0.00) 19.00 (0.00) 1
2 Ref. 31 0.33 (0.47) 5.00 (0.00) 5.00 (0.00) 3.00 (0.00) 1.33 (0.47) 15.33 (0.00) 2
3 Ref. 24 2.00 (0.00) 4.00 (0.00) 6.67 (0.470) 3.00 (0.00) 1.67 (0.47) 17.33 (0.94) 2
4 Ref. 26 4.00 (0.00) 2.67 (0.47) 6.00 (0.00) 2.67 (0.47) 2.00 (0.00) 17.33 (0.94) 2
5 Ref. 36 3.00 (0.00) 3.00 (0.00) 4.67 (0.58) 2.00 (0.00) 0.00 (0.00) 12.67 (0.58) 2

Notes: * Values shown are the aggregated score for 5 groups of a total of 18 criteria for in vitro study and 21 criteria for in vivo study. The values were presented as mean
(standard deviation) from three raters. Group I: identification of the test substance (max. marks ¼ 4), Group II: characterization of the test system (in vitro max ¼ 3, in vivo
max ¼ 5), Group III: research design description (in vitro max ¼ 6, in vivo max ¼ 7), Group IV: study outcome documentation (max ¼ 3), and Group V: study design and data
plausibility (max ¼ 2). # Quality of evidence: Category 1, reliable without restriction; Category 2, reliable with restriction; and Category 3, not reliable. D Regarding Ref. 18, both
in vitro and in vivo studies were performed.

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K.W. Ng, S.F. Tan, S.Y. Looi et al. Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx

3.2. Cytotoxicity evaluation of T. flagelliforme extracts and fractions octadecanoic, 9-octadecenoic, and 9,12-octadecadienoic acids, and
dodecane, tridecane, tetradecane, pentadecane, hexadecane, hep-
A comprehensive review revealed that 22 cytotoxic studies have tadecane, octadecane, nonadecane, and eicosane. The authors
been undertaken since 2001, using T. flagelliforme extracts, frac- pursued the isolation of the compounds and tested them, but none
tions, or compounds (Table 3). The most thorough research has of the reported compounds demonstrated or were known to
been conducted using breast tissue cell lines. Extraction of the plant exhibit cytotoxic behavior.
using mild polarity solvents such as chloroform and ethyl acetate
and polar solvent such as ethanol or methanol exert selective
cytotoxicity toward established MCF-7, T-47D, MDA-MB-23, and
HS578T breast cancer cell lines.14,17,22,25 Similar lethal effects on
breast cancer cells were seen in ex vitro grown normal T. flag-
elliforme and mutant variations induced by gamma
irradiation.14,22,30,34,35,37 Agung et al33 discovered that the n-hex-
ane and dichloromethane fractions of the plant's tuber mildly
suppressed the growth of CD24/CD44þ breast cancer cells isolated
from MCF7. The toxicity of T. flagelliforme after 68 h of exposure was
not acute, with an IC50 of 50.89 mg/mL; however, signs of early
apoptosis were observed. High levels of CD44 expression have been
linked to cancer progression, whereas low levels of CD24 expres-
sion have been linked to nondifferentiated cells. Breast cancer cells
with CD44þ/CD24- subpopulation have highly invasive properties.
Several investigations found that T. flagelliforme fraction sup-
pressed the proliferation of human and murine leukemia, colo-
rectal, and lung cancer cell lines specifically by triggering
apoptosis.14,15,17‒20,27,28 Choo et al12 observed similar activities in T.
flagelliforme's nonpolar fraction (n-hexane); however, further
isolation and investigation of the individual compounds from the
same fractions did not produce similar cytotoxic reactions,
implying that the cytotoxic effect of the fraction was due to a
synergistic or additive effect. In addition, the tuber extract caused
signs of early apoptosis morphological changes to the cervical
carcinoma KB and ORL 48 cells but no evidence of cytotoxicity on
both cells.21 No cytotoxic activities were reported on non-
cancerous cells CV1.

3.3. Anticancer compounds from T. flagelliforme

Fig. 2. Chemical structures of pheophorbides. R-pheophorbide-a: R1 ¼ COOCH3; R2 ¼
Choo et al12 evaluated a cytotoxic hexane extract and identified H; R3 ¼ H; S-pheophorbide-a: R1 ¼ H; R2 ¼ COOCH3; R3 ¼ H; pyropheophorbide-a: R1
the compounds in the extract as methyl esters of hexadecanoic, ¼ H; R2 ¼ H; R3 ¼ H and methyl pyropheophorbide-a: R1 ¼ COOCH3; R2 ¼ H; R3 ¼ CH3.

Table 3
Cancer cell lines tested using extracts, fractions, and bioactive compounds of Typhonium flagelliforme (Lodd.) Blume.

Cell lines Cell types, tissue Organism Tumor type Ref.

P388 Monocyte, macrophage Mouse Lymphoma 12,13

NCI-H23 Epithelial, lung Human Non-small cell lung cancer 14,15,17
T-47D Epithelial, breast Human Infiltrating ductal adenocarcinoma 14,27
HepG2 Liver, epithelial-like Human Hepatocellular carcinoma 14
BALB/c 3T3 Fibroblast, embryo Mouse e 15
CEMs T4-lymphoblastoid cell line Human e 16,19,20
HS578T Epithelial, breast Human Carcinosarcoma 17
WEHI-3 Monocyte, peripheral blood Mouse Leukemic 18
PBL Lymphocytes, blood Human e 20
KB Epithelial, cervix Human Papilloma 21
ORL 48 Squamous, oral Human Gingival squamous cell carcinoma 21
MCF 7 Epithelial, breast Human Infiltrating ductal adenocarcinoma 22,23,30,35,37
Hela Epithelial, cervix Human Cervical adenocarcinoma 23,27
MDA-MB-231 Epithelial, breast Human Invasive adenocarcinoma 25
CHO Epithelial, ovary Hamster Normal 25
WiDR Epithelial, colon Human Colorectal adenocarcinoma 28
Vero Epithelial, kidney Monkey Normal 29
Raji Lymphocyte, lymph Human e 29
CV-1 Kidney Monkey Normal 34
Breast cancer stem cell Stem cell, breast CD24/CD44þ isolated from MCF7 cells 33,38
CSCC Squamous cell, unknown tissue Dog Squamous cell carcinoma 32
MCM-IPB-B3 Unknown cell type, breast Dog Mixed benign breast tumor cell 32
FSCC Squamous cell, unknown tissue Cat Squamous cell carcinoma 32

Notes: CSCC: canine squamous cell carcinoma; FSCC: feline squamous cell carcinoma.

8
K.W. Ng, S.F. Tan, S.Y. Looi et al.
Similar compounds, hexadecanoic acid, 1-hexadecene, phytol,
non-fatty acids, and a derivative of phytol, were also present in the
active fractions identified by Lai et al15 using GC-MS and NMR.
Further bioactivity-guided fractionation led to the identification
and isolation of four known cyclic-tetrapyrrolic photosensitizers
resulting from the metabolism of chlorophyll: the epimer
R-pheophorbide-a and the less stable S-pheophorbide-a,
pyropheophorbide-a, and methyl pyropheophorbide-a (Fig. 2).
Both epimers pheophorbide-a, and pyropheophorbide-a were
found to have antiproliferative activity against NCI-H23 cells at an
IC50 between 5 and 19 mg/mL. However, the effects of
pheophorbide-a and pyropheophorbide-a were not as pronounced
as those in the compound-rich fraction. The scientists speculated
that this could be due to the chemicals' synergistic effect when they
coexist in the fractions.17 These chemicals were photosensitive and
capable of inhibiting cancer cell growth when triggered by light,
with an IC50 ranging from 0.3 to 0.8 mg/mL on NCI-H23 cells and
0.8e2.6 mg/mL on HS578Tcells. The antiproliferative effect was at
least 15e30 times stronger than that observed under non-
irradiated conditions. Pheophorbide-a localizes to the mitochon-
dria of cancer cells and depolarizes the mitochondrial membrane
potential via the rapid production of oxygen radicals. This causes
cytochrome c to be produced, initiating the mitochondrial-
mediated apoptosis pathway in malignant carcinoma cells.39
Fatima et al40 hypothesised that K-ras could be a potential target
of pheophorbides. Using in silico methods, the authors showed that
pheophorbide compounds can bind to the K-ras SOS-binding site,
potentially inhibiting the interaction between K-ras and the
nucleotide exchange protein SOS.
To increase the heterogeneity of bioactive compounds in T.
flagelliforme, Sianipar et al irradiated the in vitro culture of the plant
with 6 Gy of gamma irradiation.30,41 The somoclonal variation,
which was genetically distinct from non-irradiated (control) plants,
exhibited three times higher antiproliferative activity on MCF7 cell
lines than the mother plant, likely owing to new anticancer com-
pounds in the leaves and tubers.30 The obtained GC-MS spectra
revealed the extracts contained hexadecanoic acid methyl ester,
octadecadienoic acid, phytol, gamma-sitosterol, eicosane, ger-
anylgeraniol, squalene, octacosane and 7-pentadecyne. However,
scientists did not evaluate the anticancer effects of these chemicals
in silo. The two bioactive compounds found in T. flagelliforme, 1-
hexadecane and hexadecanoic acid, are capable of binding to the
switch II region of the K-ras, which eventually prevents the release
of growth signals in cancerous cells.40
3.4. In vivo evaluation of T. flagelliforme
Rats given 7,12-Dimethylbenz[a]anthracene (DMBA) and fed the
T. flagelliforme extract (50 mg/kg body weight) had a longer breast
cancer latency period (12 weeks) than those in the control group
(DMBA only). The proportion of tumor occurrence (33% vs. 100%),
number of tumors (0.5 vs. 2.83 per rat), tumor weight (0.26 vs.
5.34 g) and tumor size (0.39 cm2 vs. 2.4 cm2) were all considerably
lower in the experimental group than in the control group. Histo-
logical evaluation of the tumor revealed that T. flagelliforme-fed rat
tumors had a higher proportion of well-differentiated cells and
fewer mitotic cells than the control group.31 The effect of T. flag-
elliforme syrup (200 or 400 mg/kg/day for 25 days) on subcutane-
ously grown breast cancer in 20e25 g body weight female CH3
mice was most likely related to a reduction in the expression of the
Her2neu receptor and the BCL2 protein.24 However, the authors
found no evidence of reduction in breast tumor size, contradicting
Maysarah's findings.31 In another study, T. flagelliforme could also
act as an immunopotentiator. The ethanolic extract of T. flag-
elliforme (250 mg/kg/day administered for 7 days) promoted
9
Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx
lymphocyte proliferation, thereby significantly improving the
phagocytic activity of macrophages in cyclophosphamide-treated
rats. The authors also reported that CD8þ T cell proliferation was
stimulated in this study.26
The extracts also reduced the suppressive effects of cyclophos-
phamide on tumor necrosis factor-a and interleukin-1a cytokines.
These observations agreed with an investigation where BALB/c
leukemia mice orally fed with dichloromethane extract of TF tuber
showed decreased counts of immature granulocytes and mono-
cytes in peripheral blood and apoptosis induction in the neoplastic
splenocytes of leukemic mice.18 The effect of T. flagelliforme on tu-
mor necrosis factor-a, however, contradicted with Chodidjah's
where no significant different between the treated and untreated
groups were observed.36
3.5. Antiproliferative mechanism of T. flagelliforme
T. flagelliforme can induce apoptosis in malignant cells by
causing mitochondrial malfunction, cytochrome c release, and
activation of caspase-3 and caspase-9, culminating in DNA frag-
mentation. This was observed in TF fractions rich in fatty acids,
including linoleic, hexadecanoic, and 9-hexadecanoic acids.19,20
These results corroborate previous findings of reduced survivin
expression in breast cancer stem cells treated with T. flagelliforme
extract.33 The authors proposed that the T. flagelliforme tuber
extract contains high levels of linoleic acid and conjugated linoleic
acid. These compounds can bind to the peroxisome proliferator-
activated receptor of breast cancer stem cells and the retinoid X
receptor, forming a complex at the peroxisome proliferator-
activated receptor response element in the proline oxidase pro-
moter area. This, in turn, stimulated the creation of reactive oxygen
species and suppressed nuclear factor kappa B signaling and
reduced surviving levels. This decrease in survivin levels resulted in
an increase in caspase-9 and caspase-3 levels, culminating in
apoptosis (Fig. 3).33
T. flagelliforme can also elicit an apoptotic event in cancerous
cells through the disruption of telomerase activity.27,29 Continuous
cell proliferation is one of the hallmarks of cancer, and almost al-
ways coincides with telomerase reactivation. Therefore, telomerase
is a viable cancer treatment target because it is essential for the
immortalization of a fraction of malignant cells, such as cancer stem
cells. The T. flagelliforme extract reduced telomerase production in
both T47D and HeLa cells, with a more pronounced effect in the
latter.27 However, further studies are required to completely un-
derstand the inhibitory action of T. flagelliforme on human telo-
merase in normal and malignant cells. The underlying mechanism
and the actual effect on telomere length in cancerous cells warrant
further investigation.
T. flagelliforme has also been found to interact with biochemical
substances secreted into the microtumor environment, such as
COX-2,28 and HSP70,38 sensitizing cancer cell death and tumor
angiogenesis.32 Limiting COX-2 transcriptional activity may have
chemopreventive properties against tumor formation. In WiDr
colon cancer cells, T. flagelliforme significantly reduced COX-2
transcriptional activity in a dose- and time-dependent manner.28
In a separate study,42 the same ethyl acetate extract of T. flag-
elliforme afforded an antioxidant compound, isovitexin. Isovitexin
promotes apoptosis, inhibits proliferation, and inhibits migration
via diverse signaling pathways, such as hypoxia-inducible factor 1
(HIF-1) and transferrin.43 The presence of this compound could
potentially contribute to the proapoptotic action the ethyl acetate
fraction of T. flagelliforme. T. flagelliforme may induce HSP-70
expression in tumor-associated macrophages (TAMs), which,
when secreted, can bind to TAMs receptors and potentially convert
TAMs from type 2 macrophages to activated type 1 macrophages.
K.W. Ng, S.F. Tan, S.Y. Looi et al.
Fig. 3. TF-induced apoptosis of cancer cell.
Notes: TF: Typhonium flagelliforme (Lodd.) Blume. a tested on WiDr cell lines; b tested on T47D breast cancer and Hela cells lines; c tested on CD44þ/CD24e MCF7 cells; d,e tested on
human T4 lymphoblastoid cell lines.
These activated M1 macrophages release TNF-a, which could bind
to tumor necrosis factor receptor-1 on breast cancer stem cells,
potentially leading to apoptosis through an extrinsic apoptotic
pathway.38
Interferon is a potent anti-angiogenic cytokine. The effects of
IFN on the vasculature are related to the suppression of endothelial
cell proliferation and migration through the downregulation of
basic fibroblast growth factor or vascular endothelial growth factor.
Using natural and recombinant canine IFNs generated in-house,
researchers from Indonesia demonstrated that, when combined
with an ethanol extract of T. flagelliforme, they synergistically
reduced rabbit endothelial cell proliferation and blood vessel
development in the chorioallantoic membrane of embryonic
eggs.32 The concoctions also significantly limited the proliferation
of canine squamous cell carcinoma.
4. Discussion
This systematic review represents a significant advancement in
the evaluation of the medicinal properties of T. flagelliforme and is a
valuable contribution to existing literature. Unlike previous re-
views,4‒7 our study used objective tools and multiple independent
raters to increase the reliability and validity of the findings. To
minimize potential heterogeneity in the assessments due to the
raters' experience and interpretation of the criteria, an inter-rater
reliability analysis was conducted. Moreover, our study provides
novel mechanistic insight into the ability of T. flagelliforme to kill
cancer cells, which combines observations from multiple sources.
10
Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx
Nevertheless, it should be noted that the studies included in this
review primarily concentrated on the potential for tumor reduction
and did not encompass the safety and toxicity testing of T. flag-
elliforme. Therefore, this review does not imply that the plants are
safe and effective for consumption.
In addition, this review primarily focuses on the anticancer ef-
fects of T. flagelliforme extracts, with the goal of linking these
properties to bioactive constituents. This approach differed from
Khalivulla's review,6 who investigated compounds from the
Typhonium genus more broadly. While there may have been some
overlap between our study and that of Khalivulla in terms of
compounds identified with potential anticancer properties, our
study reported only compounds that were tested, isolated, and
reported by researchers in the literature. However, the actual
anticancer properties of the individual compounds require further
review and evaluation.
In this review, we aimed to analyze the reported mechanisms of
the antitumor action of T. flagelliforme; thus, we only included
studies with in vitro and very limited in vivo analyses, which do not
necessarily reflect the effect of T. flagelliforme on whole organisms.
Some studies were not included in the review because of article
language or limited access to articles. Using ToxRTool as an evalu-
ation tool, our findings revealed that although numerous preclini-
cal in vitro studies have investigated the anticancer properties of T.
flagelliforme, there is considerable variability in the quality of these
studies, which may be attributed to suboptimal experimental
design and incomplete reporting. Most of these studies lacked
proper usage of positive controls and statistical analysis in their
K.W. Ng, S.F. Tan, S.Y. Looi et al.
interpretation of the results. Preclinical anticancer evaluations
were also homogeneous, as several articles were from investigators
in similar laboratories or institutions. This could lead to inaccurate
conclusions and misleading decision making regarding the anti-
cancer properties of T. flagelliforme. The lack of well-conducted
studies on T. flagelliforme underscores the necessity for further in-
depth research to fully comprehend its potential in cancer therapy.
5. Conclusions
While most current studies have focused on the ability of T.
flagelliforme to induce apoptosis in cancer cells, they have also
emphasized the role of T. flagelliforme in immune system regulation
and tumor angiogenesis as potential cancer therapeutic strategies.
Despite a growing body of scientific data, such as the discovery,
isolation, and structural elucidation of the pharmacological prin-
ciples responsible for the many therapeutic effects of T. flag-
elliforme, additional studies are required to ensure their safety in
humans. Although we believe that T. flagelliforme has additional
indications and uses, anticancer monotherapy is unlikely to be one
such option. T. flagelliforme and their bioactive components may be
suitable as adjuvants or in combination with established anticancer
agents. In light of this observation, it is imperative to exercise
caution and refrain from recommending the use of T. flagelliforme as
an anticancer therapy without further valid and rigorous clinical
studies to substantiate its safety, efficacy, and optimal modes of
administration in conjunction with established anticancer agents.
Future research should delve into elucidating the precise mecha-
nisms by which T. flagelliforme and its bioactive components
interact with established anticancer agents, aiming to identify
synergistic pathways and optimize their combined therapeutic
potential.
Funding
There was no funding assistance for this research.
CRediT authorship contribution statement
Kwok Wen Ng: Conceptualization, data curation, investigation,
supervision, visualization, formal analysis, writing e original draft,
and writing e review & editing. Suk Fei Tan: Data curation,
investigation, formal analysis, and writing e review & editing. Shu
Ying Looi: Validation and writing e review & editing. Faiza Nai-
mat: Formal analysis, validation, and writing e review & editing.
Hazrina Hamid: Validation.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgement
The authors thank the Universiti Teknologi MARA library (Shah
Alam, Malaysia) for providing access to the Scopus and Web of
Science databases.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jtcms.2023.09.009.
11
Journal of Traditional Chinese Medical Sciences xxx (xxxx) xxx
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