Cytotoxic Labdane Diterpenes, Norlabdane Diterpenes and Bis-Labdanic Diterpenes from the Zingiberaceae: A Systematic Review

Over the years, labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes with cytotoxic activities have been identified across various families in the plant kingdom including the Zingiberaceae. The present review discusses the distribution of these labdane-type diterpenes within the Zingiberaceae; their extraction, isolation, and characterization from the respective Zingiberaceae species; the structural similarities and differences within each group and between the different groups of the labdane-type diterpenes; and their cytotoxic activities against breast, cervical, liver, colorectal, pancreatic, lung and prostate cancer cell lines. The review will also provide insight into how the cytotoxic activities of the labdane-type diterpenes are influenced by their structural features.


Introduction
Cancer is a group of diseases characterized by uncontrollable cell growth. The World Health Organization (WHO) estimated in 2019 that cancer was the number one or number two cause of death among populations under the age of 70 in countries of all income levels [1]. In 2020, the total number of estimated cases was 19.3 million as compared to 14.1 and 10.9 million estimated cases in 2012 and 2002, respectively, and according to GLOBOCAN, this figure is projected to continue rising and reach an estimate of 23.4 million cases in the year 2040. The number of estimated deaths due to cancer in the years above also revealed an upward trend; 6.7 million, 8.2 million, and 10.0 million deaths were reported, respectively [1][2][3][4][5][6].
The causes of cancer can be grouped into two categories, namely internal factors and external factors. Internal factors include genetic mutations, weakened immune system and abnormal hormone levels, while external factors include smoking, alcohol abuse, diet, sedentary lifestyle, lack of physical exercise, lack of social contact, chronic stress, pollutants, and radiation [7][8][9][10][11][12][13].
Different types of treatment are available depending on the type of cancer and how advanced it is. Surgery, chemotherapy, radiation therapy, and proton therapy are conventional treatment strategies. Photodynamic therapy, photothermal therapy, gene therapy, nanoparticle-drug therapy, targeted therapy, and immunotherapy on the other hand are methods which have emerged as recently developed treatment strategies. A disadvantage of the currently available treatments, such as surgery, is the pain it causes and the potential damage to nearby tissues. Certain chemotherapy drugs can cause unintentional damage to other otherwise healthy organs. Parts of the body most commonly affected by chemotherapy drugs include bone marrow, hair follicles, and the reproductive system. There are also risks of rejection or infection from stem-cell or bone marrow transplant, and the whole process might take a long time. In addition, the costs of these treatments can be prohibitive, especially for patients from developing countries [14,15]. Table 3. Cytotoxic labdane-type diterpenes from the Zingiberaceae.

Phytochemical Investigation
The chemical structures of the cytotoxic labdane-type diterpenes and their names in relation to the corresponding Zingiberaceae species are illustrated in Figure S1 and presented in Table 3, respectively. Table S1 summarizes the techniques which were used to extract, isolate and characterize the cytotoxic labdane-type diterpenes from the respective Zingiberaceae species.

Distribution of the Cytotoxic Labdane-Type Diterpenes within the Zingiberaceae
A total of forty-four cytotoxic labdane-type diterpenes have been identified in the Zingiberaceae till the present. These compounds were obtained from thirteen different species from the following five genera: Alpinia (A. calcarata Rosc. and A. intermedia  (Table 3). Among these, thirty-four compounds were isolated and characterized from the genus Hedychium: three compounds each from Alpinia and Roscoea and two compounds each from Amomum and Curcuma (

Phytochemical Investigation
The chemical structures of the cytotoxic labdane-type diterpenes and their names in relation to the corresponding Zingiberaceae species are illustrated in Figure S1 and presented in Table 3, respectively. Table S1 summarizes the techniques which were used to extract, isolate and characterize the cytotoxic labdane-type diterpenes from the respective Zingiberaceae species.

Distribution of the Cytotoxic Labdane-Type Diterpenes within the Zingiberaceae
A total of forty-four cytotoxic labdane-type diterpenes have been identified in the Zingiberaceae till the present. These compounds were obtained from thirteen different species from the following five genera: Alpinia (A. calcarata Rosc. and A. intermedia  (Table 3). Among these, thirty-four compounds were isolated and characterized from the genus Hedychium: three compounds each from Alpinia and Roscoea and two compounds each from Amomum and Curcuma (

Phytochemical Investigation
The chemical structures of the cytotoxic labdane-type diterpenes and their names in relation to the corresponding Zingiberaceae species are illustrated in Figure S1 and presented in Table 3, respectively. Table S1 summarizes the techniques which were used to extract, isolate and characterize the cytotoxic labdane-type diterpenes from the respective Zingiberaceae species.  Table 3). Among these, thirty-four compounds were isolated and characterized from the genus Hedychium: three compounds each from Alpinia and Roscoea and two compounds each from Amomum and Curcuma (Table 3).

Extraction, Isolation, and Characterisation of the Cytotoxic Labdane-Type Diterpenes
The labdane-type diterpenes were extracted from either the rhizomes, roots, leaves, or the aerial parts of the different Zingiberaceae species using the solvent extraction method. The extraction of the plant materials was carried out either at room temperature or under reflux using organic solvents or aqueous alcohol (Table S1).
The resulting crude solvent extracts were either used directly or further partitioned with organic solvents and subjected to various chromatographic techniques, mainly silica gel column chromatography, preparative TLC, and preparative HPLC, to isolate and purify the labdane-type diterpenes (Table S1).
The structures of these labdane-type diterpenes were subsequently elucidated by a combination of various spectroscopic techniques such as infrared spectroscopy (IR), ultraviolet-visible spectroscopy (UV-Vis), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) (Table S1).

Structures of the Cytotoxic Labdane-Type Diterpenes
Among the forty-four cytotoxic labdane-type diterpenes identified through this search, forty are characterized as labdane diterpenes (3-27, 29-37, 39-44), two as norlabdane diterpenes (28,38) and two as bis-labdanic diterpenes (1, 2) ( Table 3 and Figure S1). The following sections will discuss the structural similarities and differences within each group of the labdane-type diterpenes and between the different groups of the labdane-type diterpenes.

Labdane Diterpenes
All of the labdane diterpenes in the present study consist of twenty to twenty-four carbon atoms. Their basic framework is constructed from two substructures: a decalin moiety (C-1-C-10) which consists of two fused cyclohexane rings and a branched sixmembered carbon side chain (C-11-C-16; with C-13 attached to C-16) which is connected to position C-9 of the decalin moiety [47]. The three tertiary methyl groups (C-18, C-19, and C-20) are bonded to positions C-4 and C-10 of the decalin moiety ( Figure S2).
The C-17 group which is bonded to position C-8 of the labdane framework varied between the structures. There are four different types. A majority of the labdane diterpenes  Figure S1).
Variations were also observed in the structure of the decalin moiety. In compounds 18, 22, 23, 39, 41, and 42, there is a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of their decalin moieties, while in compounds 19, 37, and 40, there is a hydroxyl group at position C-7 in addition to the double bond between C-7 and C-8 and the carbonyl group at C-6. As for compounds 8, 14, 24, 32-36, 43, and 44, either position C-6 or C-7 bears a hydroxyl group without the presence of a double bond between positions C-7 and C-8 of their decalin moieties. Compound 4 on the other hand is the only labdane diterpene which bears a carbonyl group at position C-3 of its decalin moiety. As for compounds 3, 5-7, 9-13, 15-17, 20, 21, 25-27, and 29-31, there are either no double bonds within their decalin moieties, and neither are any of the carbon atoms in their decalin moieties occupied by oxygenated functional groups ( Figure S1).
It is also evident from this review that there are two distinct types of the C-11-C-16 side chain. In  (7), an unsaturated oxane (15), or an unsaturated 1,2 dioxepane (25) ring. As for compounds 5, 8, 9, 26, and 31, none of their side chains have a ring; instead, they have a combination of one of the following functional groups: carboxylic acid, aldehyde, hydroxymethyl, hydroxyl and ether ( Figure S1).
The relative stereochemistry of the group of atoms at C-4, C-5, C-9, and C-10 for all of the labdane diterpenes are essentially the same on the basis of their biogenetic pathway. The C-18 methyl group has an equatorial orientation about position C-4 while the C-19 and C-20 methyl groups, along with the H-5 and H-9 atoms, each is axially oriented about positions C-4, C-10, C-5, and C-9, respectively ( Figure S1).

Norlabdane Diterpenes
In general, the basic framework and the relative stereochemistry for these compounds are similar to those of the labdane diterpenes, with the only difference being in the nature of the side chain at position C-9 of the decalin moiety. For compounds 28 and 38, instead of having a branched, six-membered carbon side chain, these compounds have either a three-membered (38) or a four-membered (28) aliphatic chain, in turn resulting in these compounds having fewer than 20 carbon atoms in their respective structures ( Figure S1).
A closer look at the structures of the individual norlabdane diterpenes revealed major differences. Their decalin moieties are notably different. For compound 38, there is a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of its decalin moiety. In contrast, the aforementioned functional groups are absent in the structure of compound 28. The side chain at position C-9 of compound 28 is a butenone chain (-CH=CH-C(O)-CH 3 ) while that of compound 38 is a propenoic acid chain (-CH=CH-COOH). In compound 28, its C-17 group is an exomethylene group, while in compound 38, it is a methyl group instead ( Figure S1).

Bis-Labdanic Diterpenes
The two bis-labdanic diterpenes (1 and 2) which were identified in A. calcarata through this study are a pair of stereoisomers. Their structures are identical to one another with the exception of the orientation of the H-9 and H-9 atoms at the corresponding C-9 and C-9 positions of their respective decalin moieties. It is interesting to note that compounds 1 and 2 are non-symmetrical bis-labdanic diterpenes as each compound is constructed from two different monomers. Both monomers in each compound are linked together via an ether linkage ( Figure S1).

Cytotoxic Activities
All forty-four labdane-type diterpenes included in this review were assayed against various cell lines from different types of cancers (Tables 4-10). The assays were conducted using either one of the following methods: MTT, REMA, or SRB. Eight studies performed Pharmaceuticals 2022, 15, 1517 24 of 58 cytotoxic assays against breast cancer cell lines, eight against cervical cancer cell lines, seven against lung cancer cell lines, five against liver cancer cell lines, five against colorectal cancer cell lines, and one each against pancreatic and prostate cancer cell lines. The cytotoxic activities of the included labdane-type diterpenes from the Zingiberaceae were analyzed using two parameters, namely the IC 50 and GI 50 values. IC 50 is the half-maximal inhibitory concentration, which represents the concentration of a compound where the biological process is reduced by half, whereas GI 50 is the half-maximal inhibition of cell proliferation, which represents the concentration of a compound where the total cell growth is reduced by 50%. The labdane-type diterpenes with IC 50 values or GI 50 values < 1 µM were considered potent, between 1 and 10 µM were considered strong, between 10 and 30 µM were considered moderate, and between 30 and 100 µM were considered weak (Tables 4-10). Labdane-type diterpenes whose IC 50 values or GI 50 values were >100 µM were considered inactive and, therefore, were not included in Tables 4-10. However, if the IC 50 value or GI 50 value of a labdane-type diterpene was greater than 100 µM but comparable to the IC 50 value or GI 50 value of the positive control, its potency was included in Tables 4-10 and is further discussed. Structure-activity relationship (SAR) studies were only discussed wherever possible for the active labdane-type diterpenes, i.e., for the labdane-type diterpenes which exhibited cytotoxic activities with IC 50 values < 100 µM against a particular cell line.

Breast Cancer Cell Lines
The studies included in this review carried out cytotoxic assays against the following breast cancer cell lines: MCF-7, T-47D, and MDA-MB-231 (Tables 4-6).

MCF-7 Cell Line
In 2014, Luo and his coworkers isolated and characterized three new labdane diterpenes (amomax A-B and 4) and two known labdane diterpenes (ottensinin and 5) from the dichloromethane soluble fraction of the aqueous ethanolic extract of the roots of A. maximum. When this group of researchers examined the cytotoxicity of these labdane diterpenes against the MCF-7 cell line using the MTT assay, they found compound 4 to be two times more effective (IC 50 = 29.80 µM) compared to 5-fluorouracil (IC 50 = 59.96 µM), the synthetic positive control which was used in their study [32].
According to an investigation carried out by Abas et al. back in 2005, compound 6, which they had identified in the ethyl-acetate-soluble fraction of the acetone extract of the rhizomes of C. manga, was found to be a potent inhibitor of the cytotoxic activity of the MCF-7 cell line with an IC 50 value of 0.59 µM [33]. Demethoxycurcumin (IC 50 = 18.51 µM), bisdemethoxycurcumin (IC 50 = 33.78 µM), and curcumin (IC 50 = 10.44 µM) were also isolated alongside compound 6 and assessed for their potential in inhibiting the cytotoxic activity of the same cell line. These compounds were not as cytotoxic as compound 6 [33]. A new labdane diterpene glucoside, curcumanggoside, along with compounds 26 and 31, was also identified in the rhizomes of C. mangga. However, these labdane-type diterpenes were not evaluated for their cytotoxicity against the MCF-7 cell line. A separate study conducted ten years later reported a weaker cytotoxicity (IC 50 = 39.71 µM) of compound 6 which was obtained from a different source: the rhizomes of H. ellipticum [39]. The difference in the IC 50 values of compound 6 between both studies could be attributed to the differences in the assay procedures performed by the respective group of researchers. Abas et al. investigated the cytotoxicity of compound 6 using the MTT assay, while Songsri and Nuntawong employed the REMA technique instead [33,39]. Furthermore, MCF-7 is a cell line that is composed of different phenotypes with varying chromosomal numbers that can affect the cells' gene and receptor expressions. Therefore, the variation within the MCF-7 cell lines could also have contributed to the difference in the IC 50 values in both of the studies [48].
The hexane and dichloromethane extracts of the rhizomes of H. ellipticum, in a phytochemical investigation carried out by Songsri and Nuntawong, yielded ten compounds in  50 value comparable with that of doxorubicin (IC 50 = 1.22 µM), the cytotoxicity of the rest of the compounds was considered to be either moderate (29) or weak (6, 20, 31) [39]. The rather weak cytotoxicity of compounds 20 and 31 upon comparison to compounds 6, 16, and 29 implied that the presence of an α,β-unsaturated γ-lactone ring in the side chain played an essential role in mediating the cytotoxic activity against the tested cell line. When the cytotoxicity of compounds 16 and 29 was compared to compound 6, the greater activity of compounds 16 and 29 led to the assumption that the potency of both these compounds could have resulted from the presence of a trans double bond which is conjugated to the α,β-unsaturated γ-lactone ring in their respective side chains, in contrast to compound 6 in which the trans double bond is absent. The variation in the α,β-unsaturated γ-lactone rings of compounds 16 and 29 could further explain the significant difference in the cytotoxicity between both of these labdane diterpenes. The carbonyl group being at position C-15 of the α,β-unsaturated γ-lactone ring in compound 16, instead of position C-16 as in compound 29, in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 and the absence of it in compound 29, could have increased the cytotoxicity of compound 16 to three folds of that of compound 29. In an investigation by Reddy and his coworkers, the chloroform extract which they had prepared from the rhizomes of H. spicatum exhibited a rather mild cytotoxic activity (IC 50 = 58.08 µg/mL) against the MCF-7 cell line [44]. Phytochemical investigation of the chloroform extract followed by the in vitro cytotoxic evaluation of the secondary metabolites isolated and characterized from the extract provided justification that the two new labdane diterpenes (40 and 41) along with the known labdane diterpene (32), although reported to be weakly cytotoxic against the MCF-7 cell line, could have been responsible for the extract's cytotoxicity. Reddy et al. concluded that upon comparing the activity of compound 40 with that of compounds 32 and 41, the presence of an α,β-unsaturated γ-lactone ring in the side chain along with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety of compound 40 enhanced its cytotoxicity against the tested cell line. They further deduced that even with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety, the cytotoxicity in compound 41 was decreased due to the absence of the α,β-unsaturated γ-lactone ring. The remaining labdane diterpenes, hedychilactone B and hedychilactone C-which were also identified in the chloroform extract-were found to be inactive [44].
A year ago, Singamaneni et al. prepared a chloroform fraction from the methanol extract of the rhizomes of R. purpurea and assessed its cytotoxicity against the MCF-7 cell line. The results obtained from their MTT assay revealed that the chloroform fraction was moderately cytotoxic (IC 50 = 46.64 µg/mL) and slightly stronger in its activity compared to the methanol extract (IC 50 = 48.96 µg/mL) itself. Purification of the chloroform fraction led to the isolation and characterization of three weakly cytotoxic labdane diterpenes, 14, 43, and 44, with the latter two compounds being identified for the first time in the plant kingdom [46].  [37].
Compared to the strong cytotoxic activity against the MDA-MB-231 cell line, compound 20 reported less than half of that activity against the T-47D cell line, while compound 17 reported almost equal cytotoxicity against the T-47D cell line, albeit to a slightly lesser degree [37]. The difference in the reported IC 50 values of compounds 17 and 20 against both the MDA-MB-231 and T-47D cell lines could be attributed to the fact that the two cell lines belong to different subtypes of breast cancer: basal-type and luminal-type, respectively. These subtypes of breast cancer are associated with different gene expressions and, generally, patients afflicted with basal-type breast cancers have a worse prognosis when compared to patients suffering from luminal-type breast cancers [49].
Recently, in 2021, Soumya et al. used the MTT assay to evaluate the various solvent extracts (petroleum ether, chloroform, acetone, and methanol) of the rhizomes of C. mutablis for their cytotoxicity against the MDA-MB-231 cell line [34]. All of the extracts exhibited a dose-dependent reduction in cell survival percentage, with the petroleum ether extract demonstrating the maximum cytotoxicity of 5.6 µg/mL. The bioassay-guided isolation of the petroleum extract led to the identification of a novel labdane diterpene (7) which was found to be responsible for the activity of the petroleum ether extract. Compound 7 (IC 50 = 7.71 µM) was strongly cytotoxic and four-fold more potent compared to that of the positive control, curcumin (IC 50 = 31.22 µM), a diarylheptanoid [34,37].

Cervical Cancer Cell Lines
The cervical cancer cell lines KB, SMMC-7721, SGC-7901, and HeLa were used to perform the cytotoxic assays in the studies included in this review. KB, SMMC-772,1 and SGC-7901 were originally attributed as oral, liver, and gastric cancer cell lines in the studies obtained through our search. However, these cell lines have recently been proven to have been misidentified and are actually derivatives of the HeLa cell line [50][51][52][53]. Hence, in the current review, the results related to these three cell lines are included alongside the HeLa cell line as cervical cancer cell lines (Tables 7-10). Since the KB, SMMC-7721, and SGC-7901 cell lines have been identified as cell lines that have been contaminated by HeLa cells, the cell lines have almost identical STR profiles to the HeLa cell line. However, there also exist some differences in the STR profiling of the contaminated cell lines compared to that of the Hela cells, and mutations in the microsatellites have been observed to affect certain biological functions [54,55]. These differences might have led to the discrepancies in the reported IC 50 values between the cell lines.

KB Cell Lines
In 2002, Kong et al., isolated and characterized two novel stereoisomeric bis-labdanic diterpenes (1 and 2) from the ethyl acetate fraction of the methanol extract of the rhizomes of A. calcarata. Based on the results obtained from their MTT assay, both compounds 1 (IC 50 = 0.34 µM) and 2 (IC 50 = 0.24 µM) can be regarded as potent inhibitors of the cytotoxic activity of the KB cell lines due to their IC 50 values being significantly comparable to the positive control, adriamycin (IC 50 = 0.15 µM) [30].
In addition to the MCF-7 cell line, Songsri and Nuntawong also assessed the cytotoxic potentials of the ten labdane-type diterpenes which they had identified in the hexane and dichloromethane extracts of the rhizomes of H. ellipticum against the KB cell line. Only six of them were active. For compound 16 (IC 50 = 2.75 µM), although its cytotoxic activity was strong and almost comparable to ellipticine (IC 50 = 1.23 µM), it was found to be slightly weaker than that of doxorubicin (IC 50 = 0.22 µM) [39]. As for the cytotoxic activity of compounds 6, 27, 29, 30, and 31, these compounds can be regarded as being either moderate (29) or weak (6, 27, 30, 31) [39]. The same structural features of compounds 16 and 29, which could have given rise to their potency against the MCF-7 cell line as discussed in Section MCF-7 Cell Line, may also be responsible for their potency against the KB cell line. Although compound 30 is almost identical in structure to compound 29, the presence of a methoxyl group at position C-15 of its α,β-unsaturated γ-lactone ring could have considerably suppressed its activity by five-fold in comparison to compound 29.
Chimnoi In a separate study, Zhao et al. isolated and characterized three new labdane diterpenes (hedylongnoids A-B and 36) together with three known ones (32, 34, and 35) from the rhizomes of H. longipetalum [42]. The SRB assay was used to determine the cytotoxic potentials of these isolates against the SGC-7901 cell line, and among them, compounds 32, 35, and 36 were moderately cytotoxic, with the former two exhibiting similar cytotoxic strength [42]. As for compound 34, it was only weakly cytotoxic against the SGC-7901 cell line. The remaining compounds were considered to be inactive [42]. Despite compounds 34 and 35 being structurally similar to one another, the presence of the carbonyl group at position C-15 along with the hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 35 may have increased its potency against the SGC-7901 cell line by two-fold in comparison to compound 34, whose carbonyl group is at position C-16 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring. The preliminary screening of the ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of H. coronarium by Zhan and his coworkers using the SRB method revealed it to be cytotoxic against the HeLa cell line [35]. Subsequent purification of the crude ethyl acetate fraction in order to determine the secondary metabolites responsible for its cytotoxic activity led to the isolation and characterization of ten labdane diterpenes (8)(9)(10)(11)(12)(13)(14)(15)(16)(17), with compounds 8 and 9 being identified as new. Only six of these labdane diterpenes were found to be cytotoxic, two of them moderately (15 and 16) and four of them weakly (8, 10, 13, 14) [35]. A significant difference in the potencies was observed between compounds 10 and 13 even though both compounds were almost structurally identical to each other. The additional presence of a methoxyl group at position C-15 in the γ-lactone ring of compound 10 could have suppressed its cytotoxicity by two and a half folds compared to compound 13. Instead, in compound 16, when a trans double bond is conjugated to an α,β-unsaturated γ-lactone ring bearing a hydroxyl group at position C-16, this structural feature could have notably increased its cytotoxicity by almost four-fold compared to compound 10.
Similarly, the SRB method was used to compare the cytotoxic potentials of the six labdane diterpenes from the rhizomes of H. longipetalum against the HeLa cell line [42]. In this investigation, the rhizomes yielded four cytotoxic labdane diterpenes (32,(34)(35)(36). Compounds 32 and 35 were found to moderately inhibit the cytotoxic activity of the HeLa cell lines, while the remaining two compounds were less effective inhibitors [42]. The same structural features of compounds 34 and 35, which could have given rise to their potencies against the SGC-7901 cell line as discussed in Section SMMC-7721 and SGC-7901 Cell Lines, may also be responsible for their potencies against the HeLa cell line.
Suresh and his coworkers in 2010 also assessed the growth-inhibitory properties of the labdane diterpenes, which they had identified in the rhizomes of H. coronarium against the HeLa cell line. The results from their SRB assay showed that only compounds 18-24 weakly inhibited the growth of the HeLa cell line, while the remaining labdane diterpenes were found to be inactive (GI 50 > 100 µM) [36].

Liver Cancer Cell Lines
Cytotoxicity assays of compounds performed on HepG2 and HCC-S102 liver cancer cell lines are reported in this review (Tables 11 and 12). identical to each other. The additional presence of a methoxyl group at position C-15 in the γ-lactone ring of compound 10 could have suppressed its cytotoxicity by two and a half folds compared to compound 13. Instead, in compound 16, when a trans double bond is conjugated to an α,β-unsaturated γ-lactone ring bearing a hydroxyl group at position C-16, this structural feature could have notably increased its cytotoxicity by almost fourfold compared to compound 10.
Similarly, the SRB method was used to compare the cytotoxic potentials of the six labdane diterpenes from the rhizomes of H. longipetalum against the HeLa cell line [42]. In this investigation, the rhizomes yielded four cytotoxic labdane diterpenes (32,(34)(35)(36). Compounds 32 and 35 were found to moderately inhibit the cytotoxic activity of the HeLa cell lines, while the remaining two compounds were less effective inhibitors [42]. The same structural features of compounds 34 and 35, which could have given rise to their potencies against the SGC-7901 cell line as discussed in Section SMMC-7721 and SGC-7901 Cell Lines, may also be responsible for their potencies against the HeLa cell line.
Suresh and his coworkers in 2010 also assessed the growth-inhibitory properties of the labdane diterpenes, which they had identified in the rhizomes of H. coronarium against the HeLa cell line. The results from their SRB assay showed that only compounds 18-24 weakly inhibited the growth of the HeLa cell line, while the remaining labdane diterpenes were found to be inactive (GI50 > 100 µM) [36].

Liver Cancer Cell Lines
Cytotoxicity assays of compounds performed on HepG2 and HCC-S102 liver cancer cell lines are reported in this review (Tables 11 and 12).         In an investigation conducted by Chen et al. in 2017, the leaves of A. intermedia were initially extracted with 95% ethanol, following which the aqueous ethanolic extract was partitioned between hexane and water. When the aqueous ethanolic extract and the hexane fraction were evaluated for their cytotoxicity against the HepG2 cell line using the IC 50 : 67. 56 Weak MTT [37] HepG2 Cell Line In an investigation conducted by Chen et al. in 2017, the leaves of A. intermedia were initially extracted with 95% ethanol, following which the aqueous ethanolic extract was partitioned between hexane and water. When the aqueous ethanolic extract and the hexane fraction were evaluated for their cytotoxicity against the HepG2 cell line using the MTT assay, the hexane fraction (IC 50 = 66.50 µg/mL) was found to be more cytotoxic compared to the aqueous ethanolic extract (IC 50 > 100 µg/mL). The phytochemical investigation of the hexane fraction yielded two new labdane diterpenes (3 and intermedin B) and a known labdane diterpene (27). Intermedin B is a C-12 epimer of 3, and both these labdane diterpenes were structurally related to 27 . When Chen and his coworkers assessed the cytotoxic potential of compounds 3 and 27 towards the HepG2 cell line, they discovered that compound 3 (IC 50 = 119.82 µM) exhibited a cytotoxic activity almost identical to that of the natural anticancer compound, andrographolide (IC 50 = 111.94 µM), a labdane diterpene previously isolated and characterized from Andrographis paniculata (Burm. fil.) Nees. As for compound 27, its cytotoxicity was almost three-fold weaker compared to compound 3 and andrographolide [31].
When Lou et al. evaluated compound 5 from the roots of A. maximum for its cytotoxicity towards the HepG2 cell line, the MTT assay results revealed that although compound 5 had only an IC 50 value of 30.1 µM, it possessed stronger cytotoxic activity against the cancer cell when compared to the synthetic positive control, 5-fluorouracil, which had an IC 50 value of more than double (IC 50 = 76.11 µM) that of compound 5 [32].
Abas et al. also investigated the cytotoxicity of compound 6 from the rhizomes of C. manga against the HepG2 cell line, and it was regarded as moderately cytotoxic compared to its ability in inhibiting the activity of the MCF-7 cell line. Demethoxycurcumin (IC 50 > 50 µM), bisdemethoxycurcumin (IC 50 > 50 µM), and curcumin (IC 50 = 33.53 µM) which were also identified in the rhizomes were only considered to be weakly cytotoxic against the HepG2 cell line [33].
The ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of H. coronarium prepared by Zhan and his coworkers was also found to be cytotoxic against the HepG2 cell line in addition to the HeLa cell line [35]. The ten labdane diterpenes (8-17) which were isolated and characterized from the active extract, were investigated for their cytotoxicity. Taxol, a tetracyclic diterpenoid, was used as the positive control [35]. Compounds 15, 16, and 17 were highly effective (IC 50 < 10 µM) in inhibiting the cytotoxic activity of the HepG2 cells, with the former two compounds being almost equally potent inhibitors (IC 50 = 0.9 µM and 1.1 µM, respectively) and relatively comparable to taxol (IC 50 = 0.9 µM). The remaining compounds exhibited moderate cytotoxic activities. Compounds 13 (IC 50 = 10.6 µM) and 14 (IC 50 = 10.7 µM) possessed almost identical cytotoxicity, while the cytotoxic activity of compounds 8, 9, 10, 11, and 12 increased in the following order: 12 > 11 > 10 > 8 > 9 [35]. Although compounds 10, 11, and 13 each possessed a γ-lactone ring in their respective side chains, it is evident that the presence of either a methoxyl or ethoxyl group at position C-15 of the γ-lactone ring in compounds 10 and 11 caused a slight decrease in their respective activities compared to the activity of compound 13 in which those oxygenated functional groups are absent from position C-15 of its γ-lactone ring. Upon further comparison of the activity of compound 13 with that of compound 17, although they are structurally similar to one another, the presence of a hydroxyl group at position C-14 of the γ-lactone ring in compound 17 slightly increased its activity compared to compound 13 in which the hydroxyl group is absent from position C-14 of its γ-lactone ring. The variation in the α,β-unsaturated γ-lactone rings of compounds 12 and 16 could explain the significant difference in the cytotoxicities between both of these labdane diterpenes. The carbonyl group being at position C-15 instead of position C-16, in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16, could have increased its cytotoxicity to almost twelve folds compared to that of compound 12 whose carbonyl group is at position C-16 instead of position C-15 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring. It is noteworthy to mention that the unsaturated oxane ring in compound 15 could have attributed to its remarkable potency when compared to the potencies of the other compounds in which their side chains bear either a furan ring, a γ-lactone ring, an α,β-unsaturated γ-lactone ring or oxygenated functional groups. There was a difference in the cytotoxic inhibiting potential for compound 17 according to an earlier study conducted by Chimnoi and his coworkers, who also isolated this labdane diterpene from the same plant, the rhizomes of H. coronarium [37]. In contrast to the results obtained by Zhan

HCC-S102 Cell Line
In addition to the HepG2 cell line, Chimnoi et al. further assessed the cytotoxicity of compounds 17 and 20 against the HCC-S102 cell line. Interestingly, although compound 17 was moderately cytotoxic towards the HepG2 cell line, yet it was only weakly cytotoxic towards the HCC-S102 cell line with almost twice the IC 50 value with that recorded against the HepG2 cell line [37]. HepG2 is a hepatoblastoma cell line, while HCC-S102 is a hepatocellular carcinoma cell line [56,57]. The immature liver precursor cells of the hepatoblastoma cell line could have contributed to the differences in the IC 50 values and resulted in its susceptibility to the cytotoxic activity of compound 17. Compound 20 once again was weakly cytotoxic [37].

Colorectal Cancer Cell Lines
Cytotoxic assays against the following colorectal cancer cell lines HCT-116, HT-29, COLO 205, and DLD-1 are included in this review (Tables 13-16).               HCT-116 Cell Line There were only two recent studies which evaluated the cytotoxicity of the labdanetype diterpenes from the Zingiberaceae against this cell line.
The first study by Soumya et al. in 2021 revealed compound 7 to be strongly cytotoxic. Compound 7 was isolated from the petroleum ether extract of the rhizomes of C. mutablis which demonstrated a slightly stronger cytotoxicity (IC50 = 5.4 µg/mL) against the HCT-116 cell line compared to the chloroform (IC50 = 5.5 µg/mL) and acetone (IC50 = 6.0 µg/mL) extracts and a far more potent cytotoxicity compared to the methanol (IC50 = 79.5 µg/mL) extract. The IC50 value of compound 7 was reported to be 3.21 µM, which was about seven times lower when compared to the positive control curcumin (IC50 = 21.45 µM) [34]. Compound 7 exerted its cytotoxicity towards the cancer cells by the induction of apoptosis and the suppression of genes related to cell survival and migration, while also being safe for normal cells at the same concentration [34]. Based on the investigation conducted by Soumya and her coworkers, compound 7 was identified to be 2.5-fold more effective in inhibiting the cytotoxic activity of the HCT-116 cell line compared to the MDA-MB-231 cell line [34].
In another study conducted the same year, Singamaneni et al. investigated the cytotoxic potentials of the labdane diterpenes (14, 43, and 44) from the chloroform fraction of the methanol extract of the rhizomes R. purpurea against the HCT-116 cell line. Compound 43 was found to moderately inhibit the cytotoxic activity of the cells while compounds 14 and 44 were only weakly cytotoxic [46]. Compounds 14 and 43 both have a hydroxyl group at position C-7 of their respective decalin moieties and a side chain IC 50 : 35.34 ± 6.61 Weak MTT [38] HCT-116 Cell Line There were only two recent studies which evaluated the cytotoxicity of the labdanetype diterpenes from the Zingiberaceae against this cell line.
The first study by Soumya et al. in 2021 revealed compound 7 to be strongly cytotoxic. Compound 7 was isolated from the petroleum ether extract of the rhizomes of C. mutablis which demonstrated a slightly stronger cytotoxicity (IC 50 = 5.4 µg/mL) against the HCT-116 cell line compared to the chloroform (IC 50 = 5.5 µg/mL) and acetone (IC 50 = 6.0 µg/mL) extracts and a far more potent cytotoxicity compared to the methanol (IC 50 = 79.5 µg/mL) extract. The IC 50 value of compound 7 was reported to be 3.21 µM, which was about seven times lower when compared to the positive control curcumin (IC 50 = 21.45 µM) [34]. Compound 7 exerted its cytotoxicity towards the cancer cells by the induction of apoptosis and the suppression of genes related to cell survival and migration, while also being safe for normal cells at the same concentration [34]. Based on the investigation conducted by Soumya and her coworkers, compound 7 was identified to be 2.5-fold more effective in inhibiting the cytotoxic activity of the HCT-116 cell line compared to the MDA-MB-231 cell line [34].
In another study conducted the same year, Singamaneni et al. investigated the cytotoxic potentials of the labdane diterpenes (14, 43, and 44) from the chloroform fraction of the methanol extract of the rhizomes R. purpurea against the HCT-116 cell line. Compound 43 was found to moderately inhibit the cytotoxic activity of the cells while compounds 14 and 44 were only weakly cytotoxic [46]. Compounds 14 and 43 both have a hydroxyl group at position C-7 of their respective decalin moieties and a side chain bearing a furan ring. However, in compound 14, the presence of a trans double bond conjugated to the furan ring and the absence of it in compound 43 could be a reason for the two folds decrease or more in the cytotoxic activity of compound 14 against the HCT-116 cell line when compared to that of compound 43.

HT-29 Cell Line
In 2012, Zhan and his group of researchers examined the cytotoxic potentials of ten compounds, 8-17, isolated and characterized from the ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of H. coronarium against the HT-29 cell line in addition to the HepG2 and HeLa cell lines [35]. Compounds 10 and 12 were the most cytotoxic and exhibited equal potentials in inhibiting the cytotoxic activity of the HT-29 cell line. Both compounds recorded the lowest IC 50 value of 7.8 µM followed by compounds 17 (IC 50 = 8.8 µM) and 11 (IC 50 = 9.8 µM). Compounds 8, 9, and 13-16 on the other hand were moderately cytotoxic with relatively similar inhibitory strengths [35]. In general, the compounds with side chains that bear either a furan ring, a γ-lactone ring, an α,βunsaturated γ-lactone ring, or an unsaturated oxane ring were slightly more effective in inhibiting the cytotoxic activity of the HT-29 cell line compared to the compounds whose side chain does not bear a ring. Although compounds 10, 11, and 13 each possessed a γ-lactone ring in their respective side chains, it is evident that the presence of either a methoxyl or ethoxyl group at position C-15 of the γ-lactone ring in compounds 10 and 11 resulted in an increase in their respective activities compared to the activity of compound 13 whose γ-lactone ring does not bear either one of those oxygenated functional groups. Upon further comparison of the activity of compound 13 with that of compound 17, although they are structurally similar to one another, the presence of a hydroxyl group at position C-14 of the γ-lactone ring in compound 17 increased its activity compared to compound 13 whose γ-lactone ring does not bear a hydroxyl group at its position C-14. The variation in the α,β-unsaturated γ-lactone rings of compounds 12 and 16 could explain the difference in the cytotoxicities between both of these labdane diterpenes. The carbonyl group being at position C-15 instead of position C-16 in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 could have decreased its cytotoxicity compared to that of compound 12 whose carbonyl group is at position C-16 instead of position C-15 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring.

COLO 205 Cell Line
Reddy et al. in their investigation identified the chloroform extract which they had prepared from the rhizomes of H. spicatum to be slightly more cytotoxic against the COLO 205 cell line (IC 50 = 43.14 µg/mL) compared to the MCF-7 cell line (IC 50 = 58.08 µg/mL). Once again, it would be reasonable to postulate that compounds 32, 40, and 41 could have contributed to the cytotoxic activity of the chloroform extract against the COLO 205 cell line like they did with regard to the MCF-7 cell line [44]. The same structural features of compounds 32, 40, and 41 which could have given rise to their potency against the MCF-7 cell line as discussed in Section MCF-7 Cell Line may also be responsible for their potency against the COLO 205 cell line [44].  (14 and 26), and a known norlabdane diterpene (28). Among these four compounds, only compounds 14 and 26 exhibited cytotoxic activity against the DLD-1 cell line [38]. The remaining two compounds were inactive. The inactivity of the norlabdane diterpenes led to an assumption that a sixmembered carbon side chain bonded to the decalin moiety is a prerequisite for the cytotoxic activity against the DLD-1 cell line. With regard to compound 14, its cytotoxic potential towards the DLD-1 cell line, according to the study conducted by Chen et al., was found to be almost three times weaker compared to its cytotoxic potential against the HT-29 cell line from an earlier study by Zhan et al. [35,38]. Both cell lines express the p53 gene; however, the DLD-1 cells have a mutation at position 241 of the gene, while the HT-29 cells have a mutation at position 273 [58][59][60]. The mutation in the DLD-1 cells results in a phenylalanine replacing a serine, while in the HT-29 cells, an arginine is replaced by a histidine. The change in the p53 tumor antigen could have also contributed to the differences in the reported IC 50 values.

Pancreatic Cancer Cell Lines
A limited number of pancreatic cancer cell lines were used to study the cytotoxic activities of the labdane-type diterpenes isolated and characterized from the Zingiberaceae, with BxPC-3 being the only type of cell line to date (Table 17). Among all of the compounds identified through this review, only compounds 14, 43, and 44 which were isolated and characterized from the chloroform fraction of the methanol extract of the rhizomes of R. purpurea were recently tested for their ability in inhibiting the cytotoxic activity of the BxPC-3 cell line, and according to Singamaneni et al. who conducted the assay, only compound 14 was found to be moderately cytotoxic [46]. Based on the large difference in the cytotoxic activities between compounds 14 and 44, it would be reasonable to conclude that the furan ring in the side chain of compound 14 could have promoted its cytotoxic activity compared to the α,β-unsaturated γ-lactone ring in the side chain of compound 44. Although compounds 14 and 43 are structurally similar to one another, the greater activity of compound 14 in comparison to compound 43 could be due to the presence of the trans double bond which is conjugated to the furan ring in compound 14, and the absence of it in compound 43. Singamaneni and his coworkers identified compound 14 to be more effective in inhibiting the cytotoxic activity of the BxPC-3 cell line compared to those of MCF-7 and HCT-116 [46]. the rhizomes of R. purpurea were recently tested for their ability in inhibiting the cytotoxic activity of the BxPC-3 cell line, and according to Singamaneni et al. who conducted the assay, only compound 14 was found to be moderately cytotoxic [46]. Based on the large difference in the cytotoxic activities between compounds 14 and 44, it would be reasonable to conclude that the furan ring in the side chain of compound 14 could have promoted its cytotoxic activity compared to the α,β-unsaturated γ-lactone ring in the side chain of compound 44. Although compounds 14 and 43 are structurally similar to one another, the greater activity of compound 14 in comparison to compound 43 could be due to the presence of the trans double bond which is conjugated to the furan ring in compound 14, and the absence of it in compound 43. Singamaneni and his coworkers identified compound 14 to be more effective in inhibiting the cytotoxic activity of the BxPC-3 cell line compared to those of MCF-7 and HCT-116 [46].  The studies included in this review tested the cytotoxicities of the compounds against lung cancer cell lines, namely the A-549 and NCI-H187 lung cancer cell lines (Tables 18  and 19).

Lung Cancer Cell Lines
The studies included in this review tested the cytotoxicities of the compounds against lung cancer cell lines, namely the A-549 and NCI-H187 lung cancer cell lines (Tables 18 and 19). The studies included in this review tested the cytotoxicities of the compounds against lung cancer cell lines, namely the A-549 and NCI-H187 lung cancer cell lines (Tables 18  and 19).            A-549 Cell Line The chloroform fraction (IC50 = 21.35 µg/mL) of the methanol extract of the rhizomes of R. purpurea was reported to be slightly more cytotoxic against the A-549 cell line compared to the methanol extract (IC50 = 25.71 µg/mL) itself [46]. According to the results obtained from the MTT assay of the labdane diterpenes isolated and characterized from the chloroform fraction, it can be inferred that compound 43 which exhibited moderate cytotoxicity could have mainly been responsible for the cytotoxic potential of the chloroform fraction [46]. A comparison between the structures of compounds 43 and 44 revealed that the presence of a furan ring in the side chain would be preferred for cytotoxic activity against the A549 cell line rather than the presence of an α,β-unsaturated γ-lactone ring conjugated with a trans double bond. Similarly, when the structures of compounds 14 and 43 were compared, a trans double bond conjugated to the furan ring in the side chain was found to weaken the activity of compound 14 by 4.5-folds compared to the activity of compound 43.
In another investigation by Chimnoi et al., their findings enabled us to conclude that compounds 17, 20, and 25 from the rhizomes of H. coronarium demonstrated cytotoxicity against the A-549 cell line while the new labdane diterpene, 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial, was inactive. Although compound 17 was only moderate in activity, it was found to be almost five times stronger in inhibiting the cytotoxic activity of the A-549 cell line compared to compounds 20 and 25 which exhibited similar inhibiting strengths one another [37].
In 2009, Reddy et al. reinvestigated the chemical constituents of the rhizomes of H. spicatum which they had originally investigated a year earlier [43]. In the current investigation, Reddy and his coworkers extracted the dried rhizomes with a mixture of dichloromethane: methanol (50:50 v/v) instead of using chloroform as they did in their earlier phytochemical investigation. They evaluated the cytotoxic potential of the extract IC 50 : 2.46 Strong REMA [41] A-549 Cell Line The chloroform fraction (IC 50 = 21.35 µg/mL) of the methanol extract of the rhizomes of R. purpurea was reported to be slightly more cytotoxic against the A-549 cell line compared to the methanol extract (IC 50 = 25.71 µg/mL) itself [46]. According to the results obtained from the MTT assay of the labdane diterpenes isolated and characterized from the chloroform fraction, it can be inferred that compound 43 which exhibited moderate cytotoxicity could have mainly been responsible for the cytotoxic potential of the chloroform fraction [46]. A comparison between the structures of compounds 43 and 44 revealed that the presence of a furan ring in the side chain would be preferred for cytotoxic activity against the A549 cell line rather than the presence of an α,β-unsaturated γ-lactone ring conjugated with a trans double bond. Similarly, when the structures of compounds 14 and 43 were compared, a trans double bond conjugated to the furan ring in the side chain was found to weaken the activity of compound 14 by 4.5-folds compared to the activity of compound 43.
In another investigation by Chimnoi et al., their findings enabled us to conclude that compounds 17, 20, and 25 from the rhizomes of H. coronarium demonstrated cytotoxicity against the A-549 cell line while the new labdane diterpene, 7β-hydroxy-(E)-labda-8(17),12diene-15,16-dial, was inactive. Although compound 17 was only moderate in activity, it was found to be almost five times stronger in inhibiting the cytotoxic activity of the A-549 cell line compared to compounds 20 and 25 which exhibited similar inhibiting strengths one another [37].
In 2009, Reddy et al. reinvestigated the chemical constituents of the rhizomes of H. spicatum which they had originally investigated a year earlier [43]. In the current investigation, Reddy and his coworkers extracted the dried rhizomes with a mixture of dichloromethane: methanol (50:50 v/v) instead of using chloroform as they did in their earlier phytochemical investigation. They evaluated the cytotoxic potential of the extract against the A-549 cell line using the MTT assay and found it to be mildly cytotoxic (IC 50 = 63.21 µg/mL). Upon further investigation of the active extract, the purification of it followed by spectroscopic characterization of the secondary metabolites obtained from it led to the identification of a new labdane diterpene (37) a new norlabdane diterpene (38), and two known labdane diterpenes (27 and 39). All four compounds, however, were only weakly cytotoxic with compound 37 being the most cytotoxic among the four [43]. It would be reasonable to conclude that although both compounds 27 and 37 have a trans double bond which is conjugated to a furan ring in their respective side chains, the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety of compound 37 could have contributed to its greater cytotoxicity against the tested cell line compared to compound 27 in which there is no double bond between positions C-7 and C-8 and no carbonyl group at position C-6 of its decalin moiety. Even with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 in the respective decalin moieties of compounds 37 and 39, the cytotoxicity of compound 39 was weaker than that of compound 37 due to the absence of a hydroxyl group at position C-7 in the decalin moiety of compound 39. The variation in the type of labdane-type diterpenes which were identified between the previous and present studies could have been due to the following factors: collection site (different country or town), altitude, climate (humidity, rainfall, and temperature), season, soil pH, plant maturity, genotype, variety, harvesting time, postharvest handling conditions and storage, method of extraction and isolation and purification techniques [21,[61][62][63][64].
The phytochemical investigation of the ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of H. forrestii yielded two new (hedyforrestin D and 15-ethoxyhedyforrestin D) and three known (32, 33 and yunnancoronarin C) labdane diterpenes [40]. Upon evaluation of their cytotoxic potentials against the A-549 cell line using the MTT assay, only compounds 32 and 33 were reported to demonstrate the ability to inhibit the cytotoxic activity of this cell line. Among these two labdane diterpenes, the cytotoxicity of compound 33 was found to be potent (IC 50 = 0.92 µM) and comparable to the cytotoxicity of the positive control, cisplatin (IC 50 = 0.77 µM) [40]. Although both compounds have identical decalin moieties to one another, the differences in their respective side chains could have resulted in the significant difference in their cytotoxicities. The presence of a furan ring in the side chain of compound 32 may have decreased its potency by twelve-fold compared to compound 33 which bears an α,β-unsaturated γ-lactone ring in its side chain. As for hedyforrestin D (IC 50 > 300.81 µM), 15-ethoxy-hedyforrestin D (IC 50 > 277.40 µM), and yunnancoronarin C (IC 50 > 300.81 µM), they were considered to be inactive [40].
In addition to the MCF-7 and HeLa cell lines, Suresh and his coworkers also measured the growth-inhibitory activities of the cytotoxic labdane diterpenes (10, 18-24) from the hexane extract of the rhizomes of H. coronarium against the A-549 cell line [36]. According to the results from their SRB assay, only compounds 20-22 strongly inhibited the growth of the A-549 cell line, with compound 21 (GI 50 = 4.80 µM) being the most effective growth inhibitor among the three. As for compounds 20 and 22, both were found to possess rather similar growth-inhibitory properties (GI 50 = 8.00 µM and 7.40 µM, respectively). The remaining labdane diterpenes were reported to moderately inhibit the growth of the A-549 cell line in the following order: 24 > 18 > 23 > 19 [36].

NCI-H187 Cell Line
Songsri and Nuntawong in their investigation revealed that the NCI-H187 cell line in comparison to the MCF-7 and KB cell lines was more susceptible to the cytotoxic activity of the labdane-type diterpenes which they had isolated and characterized from the hexane and dichloromethane extracts of the rhizomes of H. ellipticum [39]. Among the ten labdane-type diterpenes, the NCI-H187 cells were resistant to only two of them, zerumin A and (E)-14,15,16-trinorlabda-8(17),11-dien-13-oic acid. The IC 50 values for both these compounds were above 100 µM. Compound 29 (IC 50 = 0.40 µM) was regarded as potent. Its cytotoxic activity was comparable to doxorubicin (IC 50 = 0.10 µM) but found to be four and a halffolds stronger than that of ellipticine (IC 50 = 1.79 µM). Compounds 16 (IC 50 = 2.18 µM) and 30 (IC 50 = 2.72 µM) were almost equally cytotoxic toward the NCI-H187 cells. Both labdane diterpenes were considered to be comparable to ellipticine (IC 50 = 1.79 µM) in terms of their cytotoxicity [39]. The remaining diterpenes were either moderately (6) or weakly (20, 27, 28, 31) cytotoxic. The rather weak cytotoxicity of compounds 20, 27, 28, and 31 upon comparison to compounds 6, 16, 29, and 30 implied that the presence of an α,β-unsaturated γ-lactone ring in the side chain played an essential role in facilitating the cytotoxic activity against the tested cell line. When the cytotoxicity of compounds 16, 29, and 30 was compared to that of compound 6, the greater activity of compounds 16, 29, and 30 led to the assumption that the potency of these three compounds could have resulted from the presence of a trans double bond which was conjugated to the α,βunsaturated γ-lactone ring in their respective side chains in contrast to compound 6 in which the trans double bond was absent. The variation in the α,β-unsaturated γ-lactone rings of compounds 16, 29 and 30 could further explain the significant difference in the cytotoxicities between these labdane diterpenes. The substitution of position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 with a hydroxyl group, as well as the substitution of position C-15 of the α,β-unsaturated γ-lactone ring in compound 30 with a methoxyl group, has lowered their cytotoxicities by almost six-to seven-fold compared to compound 29, in which none of its carbon atoms in its α,β-unsaturated γ-lactone ring were substituted with an oxygenated functional group.
In 2010, Kumrit et al. screened the hexane and dichloromethane extracts prepared from the rhizomes of H. gardnerianum for their cytotoxicity against the NCI-H187 cell line. Preliminary results of their REMA assay revealed that both extracts exhibited significant cytotoxicity [41]. The extracts were subsequently purified, and the isolates were characterized in order to identify the secondary metabolites which could have been responsible for the respective extract's cytotoxicity. A total of seven cytotoxic labdane diterpenes (14, 27, 29, 32, 33, 34, and 35) were identified. The most cytotoxic labdane diterpene was compound 29 (IC 50 = 0.40 µM) followed by compounds 35 (IC 50 = 2.46 µM) and 34 (IC 50 = 3.10 µM). Interestingly, the cytotoxicity of compound 29 was almost 4.5-fold more potent than that of ellipticine (1.79uM). The remaining compounds (14, 27, 32, and 33) only weakly inhibited the cytotoxic activity of the NCI-H187 cells [41]. It was evident that although compounds 14, 27, and 32 had a trans double bond conjugated to a furan ring in their respective side chains, the presence of a hydroxyl group bonded to either position C-6 or C-7 of the decalin moieties of compounds 14 and 32 tends to have slightly increased the activity of these compounds compared to that of compound 27 in which its decalin moiety does not bear a hydroxyl group at the corresponding positions. With regards to the activity of compounds 29 and 33-35, although all four compounds have a trans double bond which is conjugated to an α,β-unsaturated γ-lactone ring in their respective side chains, the variation in the α,β-unsaturated γ-lactone rings in addition to the variation in the decalin moieties could have contributed to the differences in the extent of their cytotoxic activities. For example, the presence of a hydroxyl group at positions C-6 and C-7 in the respective decalin moieties of compounds 33 and 34 resulted in a significant decrease in their activities in contrast to compound 29 whose decalin moiety does not bear a hydroxyl group at either one of the corresponding positions. Similarly, when the activities of compounds 34 and 35 were compared to one another, although both compounds bore a hydroxyl group at position C-7 of their respective decalin moieties, the presence of a hydroxyl group bonded to the α,β-unsaturated γ-lactone ring in compound 35 slightly increased its activity compared to that of compound 34 whose α,β-unsaturated γ-lactone ring does not bear a hydroxyl group.

Prostate Cancer Cell Lines
DU145 is the only prostate cancer cell line reported in a study included in this review to evaluate the cytotoxicity of the labdane-type diterpenes isolated from the Zingiberaceae (Table 20). Till present, there has been only a single report on the cytotoxic potential of either labdane diterpenes, norlabdane diterpenes, or bis-labdanic diterpenes towards this particular prostate cancer cell line. Compound 6, isolated and characterized from the rhizomes of C. manga, exhibited a moderate cytotoxicity against the DU145 cell line, and its IC 50 value was measured to be 11.21 µM [33]. Compound 6 was once again, like in the cases of the MCF-7 and HepG2 cell lines, found to be more cytotoxic compared to the curcuminoids isolated alongside it. Demethoxycurcumin (IC 50 = 21.70 µM), bisdemethoxycurcumin (IC 50 = 30.10 µM), and curcumin (IC 50 = 20.60 µM) were less effective in inhibiting the cytotoxic activity of the DU145 cells compared to compound 6 [33]. The reported IC 50 value of compound 6 was lower than the compound's IC 50 value against the HepG2 liver cancer cell line (IC 50 = 25.33 µM), but higher than the compound's IC 50 value against the MCF-7 breast cancer cell line (IC 50 = 0.59 µM), within the same study [33]. Therefore, we could conclude that compound 6 is the most cytotoxic towards the MCF-7 breast cancer cells, followed by the DU145 prostate cancer cells and then finally the HepG2 liver cancer cells [33]. against the HepG2 liver cancer cell line (IC50 = 25.33 µM), but higher than the compound's IC50 value against the MCF-7 breast cancer cell line (IC50 = 0.59 µM), within the same study [33]. Therefore, we could conclude that compound 6 is the most cytotoxic towards the MCF-7 breast cancer cells, followed by the DU145 prostate cancer cells and then finally the HepG2 liver cancer cells [33].

Strengths and Limitations
The results in this review were summarized from 17 studies that reported on the cytotoxicity of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes which were isolated and characterized from the Zingiberaceae against various types of cancer cell lines. This systematic review provides a comprehensive overview of the relevant studies from 2000 to 2022. This review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).
In regards to the limitations, the studies included in this review are limited to the in vitro cytotoxic studies against the various types of cancer cell lines. Furthermore, the types of cancer cell lines included in the review are also limited and do not include every type of cancer. The several types of cancer cell lines included in this review have a very limited number of studies carried out on them. There is also a lack of consistency in the reporting of the cytotoxicities by the studies included. The cytotoxicities of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes were reported in both half-maximal inhibitory concentration and half-maximal growth inhibition. Furthermore, the studies included in this review applied different assays to measure the cytotoxic activities of the labdane-type diterpenes. The positive controls which were tested alongside the labdanetype diterpenes in the different studies were also different. Certain studies do not include the IC50 values for the positive control. Therefore, comparing the effectiveness of the labdane-type diterpenes in inhibiting the cytotoxic activity of a given cell line to the positive control between different studies cannot be carried out conclusively.

Strengths and Limitations
The results in this review were summarized from 17 studies that reported on the cytotoxicity of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes which were isolated and characterized from the Zingiberaceae against various types of cancer cell lines. This systematic review provides a comprehensive overview of the relevant studies from 2000 to 2022. This review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).
In regards to the limitations, the studies included in this review are limited to the in vitro cytotoxic studies against the various types of cancer cell lines. Furthermore, the types of cancer cell lines included in the review are also limited and do not include every type of cancer. The several types of cancer cell lines included in this review have a very limited number of studies carried out on them. There is also a lack of consistency in the reporting of the cytotoxicities by the studies included. The cytotoxicities of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes were reported in both halfmaximal inhibitory concentration and half-maximal growth inhibition. Furthermore, the studies included in this review applied different assays to measure the cytotoxic activities of the labdane-type diterpenes. The positive controls which were tested alongside the labdane-type diterpenes in the different studies were also different. Certain studies do not include the IC 50 values for the positive control. Therefore, comparing the effectiveness of the labdane-type diterpenes in inhibiting the cytotoxic activity of a given cell line to the positive control between different studies cannot be carried out conclusively.
As a systematic review, this review only serves to provide an overview of the existing literature and does not aim to assess the quality of the studies.

Future Research
Due to the various side effects of therapies currently used in the treatment of cancer, there continues to be a need for the search for alternatives. Several types of cancers, such as pancreatic, cervical, and prostate cancers, have little to no studies conducted to evaluate the cytotoxicities of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes from the Zingiberaceae. Future studies should focus on the effects of the labdane diterpenes, norlabdane diterpenes, and bislabdanic diterpenes against these cancer types that currently have fewer studies conducted, and therefore fewer data points to consider. There is also a lack of studies on the mechanism of action of the cytotoxicities against the tested cell lines. Further studies of the cytotoxicities against cancer cells should also investigate the mechanism of action in addition to the half-maximal inhibitory concentration and half-maximal growth inhibition of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes.

Conclusions
In the present review, forty-four labdane-type diterpenes from five different genera of the Zingiberaceae, namely Alpinia, Amomum, Curcuma, Hedychium and Roscoea which exhibited cytotoxicity with IC 50 < 100 µM were discussed. The subclasses of the labdane-type diterpenes which demonstrated cytotoxic activity were the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes. The genus Hedychium was the richest source of active compounds.
The cytotoxicity of the labdane-type diterpenes was established on various types of cancer cell lines, in particular the most common types of cancers such as breast cancer The present review thus provides an insight into the potential of the Zingiberaceae as a source of natural anticancer drugs bearing the labdane skeleton.