Strategies for the discovery of potential anticancer agents from plants collected from Southeast Asian tropical rainforests as a case study.

Covering up to early 2023The present review summarizes recent accomplishments made as part of a multidisciplinary, multi-institutional anticancer drug discovery project, wherein samples comprising higher plants were collected primarily from Southeast Asia, and also from Central America, and the West Indies. In the introductory paragraphs, a short perspective is provided on the current importance of plants in the discovery of cancer therapeutic agents, and the contributions of other groups working towards this objective are mentioned. For our own investigations, following their collection, tropical plants have been subjected to solvent extraction and biological evaluation for their antitumor potential. Several examples of purified plant lead bioactive compounds were obtained and characterized, and found to exhibit diverse structures, including those of the alkaloid, cardiac glycoside, coumarin, cucurbitacin, cyclobenzofuran (rocaglate), flavonoid, lignan, and terpenoid types. In order to maximize the efficiency of work on drug discovery from tropical plant species, strategies to optimize various research components have been developed, including those for the plant collections and taxonomic identification, in accordance with the requirements of contemporary international treaties and with a focus on species conservation. A major component of this aspect of the work is the development of collaborative research agreements with representatives of the source countries of tropical rainforest plants. The phytochemical aspects have included the preparation of plant extracts for initial screening and the selection of promising extracts for activity-guided fractionation. In an attempt to facilitate this process, a TOCSY-based NMR procedure has been applied for the determination of bioactive rocaglate derivatives in samples of Aglaia species (Meliaceae) collected for the project. Preliminary in vitro and in vivo mechanistic studies carried out by the authors are described for two tropical plant-derived bioactive lead compounds, corchorusoside C and (+)-betulin, including work conducted with a zebrafish (Danio rerio) model. In the concluding remarks, a number of lessons are summarized that our group has learned as a result of working on anticancer drug discovery using tropical plants, which we hope will be of interest to future workers.

Cytotoxic constituents of Glycosmis ovoidea collected in Vietnam.

Compounds derived from natural sources have been major contributors to the area of cancer chemotherapy for decades. As part of an ongoing effort to discover anticancer drug leads from tropical plants, a large-scale collection of Glycosmis ovoidea Pierre (Rutaceae), was made at Nui Chua National Park, Vietnam. Activity-guided fractionation of the chloroform-soluble fractions led to the isolation of nine coumarins, including the new compound, 1-(7-methoxy-2-oxo-2H-chromen-8-yl)-3-methyl-1-oxobut-2-en-2-yl (S)-2-methylbutanoate (1). An close analogue of 1, namely, kincuongin (2), was deemed as non-cytotoxic (IC50 > 10 µM) against five different cancer cell lines. However, co-administration of kimcuongin (2) showed an approximately 100 times potentiation of the MCF-7 breast cancer cell cytotoxicity of the previously reported flavonoid, 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (10). To provide a mechanistic basis for the cancer cell line inhibition enhancement observed, an initial in silico study on compound 10 indicated that it interacts with isoforms of the NF-κB complex. In a confirmatory western blot experiment conducted, kimcuongin (2) was found to potentiate the effects of flavone 10 in inhibiting both NF-κB and PARP-1. In vivo investigations using a zebrafish (Danio rerio) model showed that compounds 2, 3, 5, and 6 did not exhibit any discernible toxicity at concentrations up to 50 µM.

Bioactive small-molecule constituents of Lao plants.

Laos has a rich plant diversity, and medicinal plants are used extensively in Lao traditional medicine for the treatment of a variety of human diseases. However, only a relatively small number of these plants have been investigated for their major components with potential antitumor, anti-infective, and other types of bioactivities. These species include Asparagus cochinchinensis, Diospyros quaesita, Gongronema napalense, Marsypopetalum modestum, Nauclea orientalis, Rourea minor, Stemona pierrei, and Stemona tuberosa. Thus far, the bioactive compounds isolated from these Lao plants include alkaloids, glycerol esters, phenolic compounds such as lignans and stilbenoids, steroids, and triterpenoids. Of these, the norlignan, nyasol (1b), the triterpenes, pyracrenic acid [3ß-O-trans-caffeoylbetulinic acid (3)] and betulinic acid (3b), and the dimeric thiopyridine, dipyrithione (5), were found to show both cancer cell cytotoxicity and anti-infective activity. The present review focuses on examples of promising lead compounds isolated from Lao plants, with their possible development as potential therapeutic agents being discussed. It is hoped that this contribution will provide useful information on higher plants growing in Laos to help stimulate future discoveries of potential agents for the treatment of cancer, infections, and other diseases.

A pentamethoxylated flavone from Glycosmis ovoidea promotes apoptosis through the intrinsic pathway and inhibits migration of MCF-7 breast cancer cells.

The rare flavone 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF) has been isolated from several plant species, and its cytotoxic activity has been reported against many types of cancer cells. In this study, PMF was purified from Glycomis ovoidea collected in Vietnam, and its antiproliferative effects and underlying mechanism of action were investigated against MCF-7 cells. PMF inhibited growth in MCF-7 > MCF-10A > MDA-MB-231 cells after 72 hr treatment, with IC50 values of 1.5, 1.9, and 8.6 µg/ml, respectively. Further experiments conducted with this compound in MCF-7 cells, showed the loss of mitochondrial membrane potential, reactive oxygen species overproduction, upregulation of BAX, cytochrome c, caspase-3 and PARP-1 and down-regulation of BCL-2 proteins as well as an increase in caspase-3/-7 activity, suggesting induction of the apoptotic intrinsic pathway. Furthermore, PMF increased cell cycle arrest in the G1 phase, which correlated with increments in the p53 and p21 levels. Additionally, MCF-7 cell migration was inhibited, which could be related to NF-κB p65 downregulation. Finally, PMF did not show toxicity in vivo in a zebrafish (Danio rerio) model. In conclusion, PMF induces cell death in MCF-7 cells through regulation of the BCL-2 protein family and may be proposed as a lead as a potential alternative for breast cancer therapy.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

A new non-cytotoxic [(+)-17ß-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na+/K+-ATPase. Compound 3 docks deeply in the Na+/K+-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na+/K+-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na+/K+-ATPase. Thus, 3 was found to inhibit Na+/K+-ATPase, but 1 did not. In addition, the cytotoxic and Na+/K+-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

Structurally Modified Cyclopenta[b]benzofuran Analogues Isolated from Aglaia perviridis.

Four new cyclopenta[b]benzofuran derivatives based on an unprecedented carbon skeleton (1-4), with a dihydrofuran ring fused to dioxanyl and aryl rings, along with a new structural analogue (5) of 5‴-episilvestrol (episilvestrol, 7), were isolated from an aqueous extract of a large-scale re-collection of the roots of Aglaia perviridis collected in Vietnam. Compound 5 demonstrated mutarotation in solution due to the presence of a hydroxy group at C-2‴, leading to the isolation of a racemic mixture, despite being purified on a chiral-phase HPLC column. Silvestrol (6) and episilvestrol (7) were isolated from the most potently cytotoxic chloroform subfraction of the roots. All new structures were elucidated using 1D and 2D NMR, HRESIMS, IR, UV, and ECD spectroscopic data. Of the five newly isolated compounds, only compound 5 exhibited cytotoxic activity against a human colon cancer (HT-29) and human prostate cancer cell line (PC-3), with IC50 values of 2.3 µM in both cases. The isolated compounds (1-5) double the number of dioxanyl ring-containing rocaglate analogues reported to date from Aglaia species and present additional information on the structural requirements for cancer cell line cytotoxicity within this compound class.

Highly sweet compounds of plant origin: From ethnobotanical observations to wide utilization.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnobotanical studies have been of very great importance in recognizing plants that contain substances that modulate the heterodimer T1R2-T1R3 sweet taste receptor, inclusive of Stevia rebaudiana (Asteraceae) and Siraitia grosvenorii (Cucurbitaceae). AIM OF THE REVIEW: In addition to reviewing relevant ethnobotanical literature, inclusive of original field work conducted, the authors have provided a progress report on the ultimate regulatory acceptance of highly sweet ent-kaurane (steviol) diterpene glycosides from S. rebaudiana leaves ("stevia") and cucurbitane triterpene glycosides (mogrosides) from the fruits of S. grosvenorii (popularly known as "monk fruit"). Despite their relatively high prices relative to that of sucrose, the steviol glycosides and mogrosides are of current great interest for further more extensive utilization on the market as sweet-tasting non-caloric food additives, due to increases in the rates of obesity and diabetes all over the world. Recent phytochemical work on the sweet principles of these two species is highlighted, including the important "next-generation" sweetener, rebaudioside M, from S. rebaudiana. RESULTS: Initial observations on the ethnobotany of both S. rebaudiana and S. grosvenorii have proved crucial to indicating the presence of their sweet-tasting principles to the wider scientific community. CONCLUSIONS: Ethnobotanical observations have been pivotal in enabling the discovery of many sweet-tasting plant constituents, with those of S. rebaudiana and S. grosvenorii both being examples. Extractives prepared from these species are now commercially used widely in the U.S. as additives for the sweetening of foods and beverages.

Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives.

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Revisiting the linkage between ethnomedical use and development of new medicines: A novel plant collection strategy towards the discovery of anticancer agents.

The Vietnam-Laos International Cooperative Biodiversity Group (ICBG) based at the University of Illinois at Chicago (UIC) catalyzed a country-wide network of medicinal plant preserves (MPP) and medicinal biodiversity preserves (MBP) now established in ten provinces of the Lao People's Democratic Republic (Lao PDR), which are relied upon as protected sources of ethnomedicines for local villagers and traditional healers. In collaboration with the Lao PDR's Institute of Traditional Medicine (ITM), our ongoing P01 Program Project (Ohio State University) examined the anticancer bioprospecting potential for two of the most exhaustively inventoried of these sites: the Bolikhamxay MPP and the Xiengkhouang MBP. Guided by prior voucher specimens sourced from these preserves with an overwhelming emphasis on plants employed in traditional medicine, 201 distinct samples from 96 species were collected along with proper herbarium documentation. Aliquots of these plant samples were extracted in azeotropic ethanol and evaporated to dryness for initial biological evaluation. In six samples from six different species (2.99% of the collected samples, 6.25% of taxa) it was observed that extracts exhibited notable cytotoxicity against HT-29 colon adenocarcinoma cells. The wisdom behind the utilization of HT-29 cells in this preliminary biological screen is discussed. Furthermore, comparison of screening results based on longstanding considerations and ideological underpinnings of ethnobotanical vs. "random" biodiversity-based collection approaches is detailed herein. The results of this interdisciplinary study support the hypothesis that, by privileging the initial sample set in terms of human safety and pharmacological activity, ethnobotanically driven collection for biological screening efforts can produce leads unprecedented by the strict traditional usages of plants.

Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa.

Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

Isolation of Bioactive Rotenoids and Isoflavonoids from the Fruits of Millettia caerulea.

Three new rotenoids (1-3), two new isoflavonoids (4 and 5), and six known analogues (6-11) were isolated from an n-hexane partition of a methanol extract of the fruits of Millettia caerulea, with the structures of the new compounds elucidated by analysis of their spectroscopic data. The relative configurations of the rotenoids were determined by interpretation of their NMR spectroscopic data, and their absolute configurations were established using electronic circular dichroism spectra and specific rotation values. All compounds isolated were evaluated for their cell growth inhibitory activity against the HT-29 human colon cancer cell line, and the known compounds, (-)-3-hydroxyrotenone (6) and (-)-rotenone (7), were found to be potently active. When tested in an NF-κB inhibition assay, compound 6 showed activity. This compound, along with the new compound, (-)-caeruleanone D (1), and the known compound, ichthynone (8), exhibited K-Ras inhibitory potency. Further bioactivity studies showed that the new compounds, (-)-3-deoxycaeruleanone D (2) and (-)-3-hydroxycaeruleanone A (3), and the known compounds 8 and 11 induced quinone reductase in murine Hepa 1c1c7 cells.

New Bioactive Lupane Triterpene Coumaroyl Esters Isolated from Buxus cochinchinensis.

Five new lupane triterpene coumaroyl esters (1-5), together with betulin (6) and a known Buxus alkaloid, N-3-benzoyldihydrocyclomicrophylline F (7), were isolated from a CHCl3-soluble partition of a methanol extract of Buxus cochinchinensis Pierre ex Gagnep. (Buxaceae) collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29). The structures of the new compounds (1-5) were determined on the basis of spectroscopic data interpretation. In addition to their cytotoxicity against HT-29 cells and nuclear factor-kappa B (p65) inhibitory activity in an enzyme-linked immunosorbent assay, all isolates as well as two semisynthetic compounds derived from betulin and 5, respectively, were also evaluated for their in vitro antiplasmodial activities against the drug-resistant Dd2 strain of Plasmodium falciparum and antifungal effects on the growth of the pathogenic yeast Candida albicans. The new lupane triterpene coumaroyl esters (1-5), along with a betulin derivative and the known Buxus alkaloid, were found to show significant in vitro antimalarial activities, with IC50 values ranging from 0.26 to 2.07 µM.

Sphenostylisins A-K: bioactive modified isoflavonoid constituents of the root bark of Sphenostylis marginata ssp. erecta.

Sphenostylisins A-C (1-3), three complex dimeric compounds representing two novel carbon skeletons, along with an additional eight new compounds, sphenostylisins D-K (4-11), were isolated from the active chloroform-soluble extract of the root bark of Sphenostylis marginata ssp. erecta using a bioactivity-guided isolation approach. The structures were elucidated by means of detailed spectroscopic analysis, including NMR and HRESIMS analysis, and tandem MS fragmentation was utilized to further support the structures of 1-3. The absolute configuration of sphenostylisin C (3) was established by electronic circular dichroism analysis. Plausible biogenetic relationships between the modified isoflavonoids 1-11 are proposed, and a cyclization reaction of 9 was conducted to support one of the biogenetic proposals made. All of these pure isolates were evaluated against a panel of in vitro bioassays, and among the results obtained, sphenostylisin A (1) was found to be a very potent NF-κB inhibitor (IC50 = 6 nM).

Lipidated steroid saponins from Dioscorea villosa (wild yam).

Two groups of lipidated steroid saponins including seven new compounds (2, 3, 5, and 7-10) were isolated from the widely used botanical, wild yam (Dioscorea villosa), employing a fractionation protocol of metabolomic mining. This methodology recently led to the isolation of 14 diarylheptanoids from the same plant. Together with these lipidated steroid saponins, they establish additional new markers for D. villosa. The lipidation of steroids with analog long-chain fatty acids containing different degrees of unsaturation generates an entire series of compounds which are difficult to purify and analyze. The structures of the two series of lipidated steroid saponins (series A and B) were established by a combination of 1D and 2D NMR as well as GC-MS after chemical modification. Series A was determined to be a mixture of lipidated spirostanol glycosides (1-5), while series B (6-10) was proved to be a mixture of five lipidated clionasterol glucosides. The latter group represents the first derivatives of clionasterol to be found in D. villosa. The discovery of this specific structural type of aliphatic esters of steroid saponins expands the characterization of the secondary metabolome of D. villosa. It may also inspire biological studies which take into account the lipophilic character and significantly altered physiochemical characteristics of these otherwise relatively polar phytoconstituents.

Bioactive constituents of the stem bark of Mitrephora glabra.

Bioactivity-guided fractionation of the stem bark of Mitrephora glabra yielded nine compounds, comprising three ent-kaurenoids (1-3), five polyacetylenic acids/esters (4-8), and one aporphine alkaloid, liriodenine (9). The structures of the six new compounds (1-3, 5, 7, and 8) were determined by spectroscopic data interpretation. All compounds were evaluated for their inhibitory activities against a panel of cancer cell lines and a battery of microorganisms.

Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus.

Tumors associated with Kaposi's sarcoma-associated herpesvirus infection include Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Virtually all of the tumor cells in these cancers are latently infected and dependent on the virus for survival. Latent viral proteins maintain the viral genome and are required for tumorigenesis. Current prevention and treatment strategies are limited because they fail to specifically target the latent form of the virus, which can persist for the lifetime of the host. Thus, targeting latent viral proteins may prove to be an important therapeutic modality for existing tumors as well as in tumor prevention by reducing latent virus load. Here, we describe a novel fluorescence-based screening assay to monitor the maintenance of the Kaposi's sarcoma-associated herpesvirus genome in B lymphocyte cell lines and to identify compounds that induce its loss, resulting in tumor cell death.

Alvaradoins E-N, antitumor and cytotoxic anthracenone C-glycosides from the leaves of Alvaradoa haitiensis.

Bioactivity-directed fractionation of an extract of the leaves of Alvaradoa haitiensis, using the KB (human oral epidermoid carcinoma) cell line, led to the isolation and identification of 10 new anthracenone C-glycosides, alvaradoins E-N (1-10), along with the known compound chrysophanol (11). The cytotoxicity of all compounds was evaluated, and preliminary structure-activity relationships are suggested. The most potent compounds in the in vitro assays (1 and 2) were evaluated in vivo versus the P388 (murine lymphocytic leukemia) model, and alvaradoin E (1) showed antileukemic activity (125% T/C) at a dose of 0.2 mg kg-1 per injection when administered intraperitoneally.

Cytotoxic constituents from the fruiting branches of Callicarpa americana collected in southern Florida.

Bioassay-guided fractionation of the combined fruits, leaves, and twigs (fruiting branches) of Callicarpa americana, collected from a plot in a forested area in southern Florida, led to the isolation of six new clerodane diterpenes (1-6) and eight known compounds. The structures of 1-6 [12(S),16xi-dihydroxycleroda-3,13-dien-15,16-olide (1), 12(S)-hydroxy-16xi-methoxycleroda-3,13-dien-15,16-olide (2), 12(S)-hydroxycleroda-3,13-dien-15,16-olide (3), 16xi-hydroxycleroda-3,11(E),13-trien-15,16-olide (4), 3beta,12(S)-dihydroxycleroda-4(18),13-dien-15,16-olide (5), and 12(S)-hydroxycleroda-3,13-dien-16,15-olide (6)] were elucidated by interpretation of spectroscopic data and chemical methods. The absolute configuration at C-12 in 1 and 3 was ascertained using the Mosher ester technique. The cytotoxicity of all isolates was tested against a panel of human cancer cell lines, and compounds 1, 4, and 6, and the known compounds genkwanin, 16xi-hydroxycleroda-3,13-dien-15,16-olide, and 2-formyl-16xi-hydroxy-3-A-norcleroda-2,13-dien-15,16-olide were active (ED50 <5 microg/mL). However, 1 was found to be inactive against human cancer cells implanted in mice using a hollow-fiber tumor model.