Soloxolone para-methylanilide effectively suppresses aggressive phenotype of glioblastoma cells including TGF-ß1-induced glial-mesenchymal transition in vitro and inhibits growth of U87 glioblastoma xenografts in mice.

Soloxolone amides are semisynthetic triterpenoids that can cross the blood-brain barrier and inhibit glioblastoma growth both in vitro and in vivo. Here we investigate the impact of these compounds on processes associated with glioblastoma invasiveness and therapy resistance. Screening of soloxolone amides against glioblastoma cells revealed the ability of compound 7 (soloxolone para-methylanilide) to inhibit transforming growth factor-beta 1 (TGF-ß1)-induced glial-mesenchymal transition Compound 7 inhibited morphological changes, wound healing, transwell migration, and expression of mesenchymal markers (N-cadherin, fibronectin, Slug) in TGF-ß1-induced U87 and U118 glioblastoma cells, while restoring their adhesiveness. Confocal microscopy and molecular docking showed that 7 reduced SMAD2/3 nuclear translocation probably by direct interaction with the TGF-ß type I and type II receptors (TßRI/II). In addition, 7 suppressed stemness of glioblastoma cells as evidenced by inhibition of colony forming ability, spheroid growth, and aldehyde dehydrogenase (ALDH) activity. Furthermore, 7 exhibited a synergistic effect with temozolomide (TMZ) on glioblastoma cell viability. Using N-acetyl-L-cysteine (NAC) and flow cytometry analysis of Annexin V-FITC-, propidium iodide-, and DCFDA-stained cells, 7 was found to synergize the cytotoxicity of TMZ by inducing ROS-dependent apoptosis. Further in vivo studies showed that 7, alone or in combination with TMZ, effectively suppressed the growth of U87 xenograft tumors in mice. Thus, 7 demonstrated promising potential as a component of combination therapy for glioblastoma, reducing its invasiveness and increasing its sensitivity to chemotherapy.

Isolation of C-29 oxygenated oleanane triterpenoids and a (+)-muurolene type sesquiterpenoid from the fruiting bodies of Fuscoporia torulosa and their bioactivities.

Basidiomycetes with a wide variety of skeletons of secondary metabolites can be expected to be the source of new interesting biological compounds. During our research on basidiomycetes, two new C-29 oxygenated oleanane-type triterpenes (1 and 2) and torulosacid (3), a muurolene type sesquiterpenoid with a five-membered ether ring along with nine known compounds (4-12), were isolated from the MeOH extract of the fruiting bodies of Fuscoporia torulosa. The structures of 1-3 were determined by NMR and HREIMS analysis. Further studies on the stereochemistry of 3 were conducted using X-ray crystallographic analysis and comparison of experimental and calculated ECD spectra. In the antimicrobial assay of isolates, 1, 7, and 9 showed growth inhibitory activity against methicillin-resistant Staphylococcus aureus and other gram-positive strains. Isolation of oleanane type triterpenes from fungi including basidiomycetes, is a unique report that could lead to further isolation of new compounds and the discovery of unique biosynthetic enzymes.

Manniosides G-J, New Ursane- and Lupane-Type Saponins from Schefflera mannii (Hook.f.) Harms.

Four previously unreported triterpenoid saponins named 3ß-hydroxy-23-oxours-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (mannioside G) (1), 23-O-acetyl-3ß-hydroxyurs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (mannioside H) (2), ursolic acid 28-O-[α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl] ester (mannioside I) (3), and 3ß-hydroxy-23-oxolup-20(29)-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (mannioside J) (4) were isolated as minor constituents from the EtOAc soluble fraction of the MeOH extract of the leaves of Schefflera mannii along with the known compounds 23-hydroxyursolic acid 28-O-ß-D-glucopyranosyl ester (5), ursolic acid 28-O-ß-D-glucopyranosyl ester (6), pulsatimmoside B (7) betulinic acid 28-O-[α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl] ester (8), 23-hydroxy-3-oxo-urs-12-en-28-oic acid (9), hederagenin (10), ursolic acid (11), betulinic acid (12), and lupeol (13). Their structures were elucidated by a combination of 1D and 2D NMR analysis and mass spectrometry. The MeOH extract, the EtOAc and n-BuOH fractions, and some of the isolated compounds were evaluated for their antibacterial activity against four bacteria: Staphylococcus aureus ATCC1026, Staphylococcus epidermidis ATCC 35984, Escherichia coli ATCC10536, and Klepsiella pnemoniae ATCC13882. They were also screened for their antioxidant properties, but no significant results were obtained.

Exploring the effect and mechanism of Hippophae rhamnoides L. triterpenoid acids on improving NAFLD based on network pharmacology and experimental validation in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides L.) is a traditional Chinese medicinal and possesses a rich medical history in terms of treating gastric disorders, sputum and cough and liver injuries in oriental medicinal system. By reason of the complicated chemical constituents, the material basis and potential pharmacological mechanism of sea buckthorn acting on Non-alcoholic fatty liver disease (NAFLD) has not been clearly elucidated. AIM OF THE STUDY: To explore the pharmacological efficacy and underlying mechanism of sea buckthorn triterpenoid acid enrichment (STE) in the treatment of NAFLD. MATERIALS AND METHODS: The approaches of Network pharmacology and experiment validation in vitro and in vivo were applied in this study. Firstly, targets of triterpenoid acid compounds and NAFLD were collected from databases. The crucial targets were screened by the construction of protein-protein interaction (PPI) network. Furthermore, the potential signaling pathways and targets affected by STE was predicted by GO together with KEGG enrichment analysis. Finally, the experiment validation was carried out through high-fat feeding NAFLD mice and lipid accumulation HepG2 cell model. Lipids and liver related biochemical indicators were determined, Oil Red O and H&E staining were employed to observe fat accumulation. In addition, the expression levels of proteins of key target and signal pathway anticipated in network pharmacology were detected to elaborated its action mechanism. RESULTS: A total of 180 intersecting potential targets for enhancing NAFLD with STE were eventually identified. 6 key targets including AKT1, TNF, IL6, INS, JUN, STAT3 and TP53 were further identified and the AMPK-SREBP1 pathway was enriched. Animal experiment result showed that STE treatment could significantly reduce the levels of TG, TC, LDL-C, ALT and AST, increase the levels of HDL-C in serum, and improve lipid accumulation of epididymal fat and liver. The results of the lipid accumulation cell model indicated that STE and key compound oleanolic acid could diminish intracellular lipid levels of TG, TC, LDL-C and number of lipid droplets. Western blot results showed that the above beneficial effects could be achieved by regulating the expression of p-AMPK/AMPK, SREBP1, FAS, ACC, SCD protein. CONCLUSION: This study confirmed the effect of STE on improving NAFLD and the potential action mechanism was involved in the regulation of the AMPK-SREBP1 pathway.

Triterpenoid saponins from the roots of Panax notoginseng with protective effects against APAP-induced liver injury.

Five previously undescribed protopanaxatriol-type saponins, notoginsenosides Ta-Te (1-5), together with eighteen known triterpenoid saponins (6-23) were isolated from the roots of Panax notoginseng. The structures of new compounds were determined by HRESIMS and NMR spectroscopic analyses and chemical methods. Compounds 1 and 2 were the first examples of ginsenosides featuring a 6-deoxy-ß-d-glucose moiety from Panax species. Compounds 1-4, 7, 10, 12, 21-22 showed protective effects on L02 cells against the injury of acetaminophen (APAP). Among them, notoginsenoside R1 (12), ginsenoside Rg1 (21), and ginsenoside Re (22) were the most potent ones, with cell viabilities >80%. Moreover, compounds 12 and 22 remarkably alleviated APAP-induced liver injury in mice. These saponins are potential hepatoprotective agents.

Myxaoside A, a new triterpenoid saponin from Cordia myxa L. (Boraginaceae).

The phytochemical study of Cordia myxa L. led to the isolation, through chromatographic techniques, of a new triterpenoid saponin, 3-O-[α-L-rhamnopyranosyl-(1→3)-(6-O-acetyl-ß-D-glucopyranosyl)]-22ß-hydroxyolean-12-ene (3) namely Myxaoside A, together with three known compounds, Soyasaponine I (1), oleanolic acid (2), and 3-O-acetyl-oleanolic acid (4). All structures were established, based on 1 & 2D-NMR spectroscopic analysis and comparison with previous published reports. Compound 1-4 were evaluated for their antibacterial activity on various strains of bacteria including Salmonella typhi, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Vibrio cholerae. It appears that compounds 1 and 3 were active on all the tested microbial species, while compounds 2 and 4, shown no significant effect on S. aureus and K. pneumoniae at low concentrations 6.5 mg/mL and 3.0 mg/mL.

Saponins from Dolichos lablab seeds with anti-inflammatory activity.

As one of a traditional Chinese medicine with dual applications in both medicinal treatment and dietary consumption, the mature seeds of D. lablab were reported to be rich in saponins and have a good effect on inflammatory related diseases. However, the substance basis for its anti-inflammatory activity remains unclear. Thus, a comprehensive phytochemical investigation on triterpenoid saponins from D. lablab seeds was carried out, resulting in the isolation and identification of twenty-one new triterpenoid saponins including dolilabsaponins A1-A4, B, C, D1-D3, E-M, N1, N2 and O (1-21) along with thirteen known analogs (22-34). Notably, the known saponins, 31, 32, and 34 were obtained from Leguminosae family for the first time. The 1H and 13C NMR data of saponins 24 and 28 were firstly reported here. Additionally, lipopolysaccharide (LPS)-stimulated RAW264.7 cells model was utilized to assess inhibitory activities of compounds 1-34 on nitric oxide (NO) production. The results revealed that compounds 1-3, 9, 10, 13-15, 18, 22, 23 and 28-34 significantly suppressed the elevation of NO levels in LPS-induced RAW264.7 cells at the concentration of 30 µM, exhibiting a concentration-dependent manner at 3, 10, and 30 µM. The results suggested that compounds 1-3, 9, 10, 13-15, 18, 22, 23, and 28-34 possessed potential anti-inflammatory activity. Further western blot assay demonstrated that 1, 9, 10, 13, 14, and 18 suppressed inflammatory response via down-regulated the expression levels of inflammatory factors, tumor necrosis factor-alpha and interleukin-6.

Optimization of saponin extraction from the leaves of Panax notoginseng and Panax quinquefolium and evaluation of their antioxidant, antihypertensive, hypoglycemic and anti-inflammatory activities.

Panax notoginseng and Panax quinquefolium are important economic plants that utilize dried roots for medicinal and food dual purposes; there is still insufficient research of their stems and leaves, which also contain triterpenoid saponins. The extraction process was developed with a total saponin content of 12.30 ± 0.34% and 12.19 ± 0.64% for P. notoginseng leaves (PNL) and P. quinquefolium leaves (PQL) extracts, respectively. PNL and PQL saponin extracts showed good antioxidant, antihypertensive, hypoglycemic, and anti-inflammatory properties in vitro and RAW264.7 cells. A total of 699 metabolites were identified in PNL and PQL saponin extracts, with the majority being triterpenoid saponins, flavonoids and amino acids. Fourteen ginsenosides, 18 flavonoids or alkaloids, and 16 amino acids were enriched in both saponin extracts. Overall, the utilization of saponins from medicinal plants PNL and PQL has been developed to facilitate systematic research in the functional food and natural product industries.

Saponin components in Polygala tenuifolia as potential candidate drugs for treating dementia.

Objective: This study aims to elucidate the intervention effects of saponin components from Polygala tenuifolia Willd (Polygalaceae) on dementia, providing experimental evidence and new insights for the research and application of saponins in the field of dementia. Materials and Methods: This review is based on a search of the PubMed, NCBI, and Google Scholar databases from their inception to 13 May 2024, using terms such as "P. tenuifolia," "P. tenuifolia and saponins," "toxicity," "dementia," "Alzheimer's disease," "Parkinson's disease dementia," and "vascular dementia." The article summarizes the saponin components of P. tenuifolia, including tenuigenin, tenuifolin, polygalasaponins XXXII, and onjisaponin B, as well as the pathophysiological mechanisms of dementia. Importantly, it highlights the potential mechanisms by which the active components of P. tenuifolia prevent and treat diseases and relevant clinical studies. Results: The saponin components of P. tenuifolia can reduce ß-amyloid accumulation, exhibit antioxidant effects, regulate neurotransmitters, improve synaptic function, possess anti-inflammatory properties, inhibit neuronal apoptosis, and modulate autophagy. Therefore, P. tenuifolia may play a role in the prevention and treatment of dementia. Conclusion: The saponin components of P. tenuifolia have shown certain therapeutic effects on dementia. They can prevent and treat dementia through various mechanisms.

Triterpenoid and trichothecenes from a rhizospheric soil-derived Paramyrothecium sp. KMU22107.

A new phthalide derivative named paramlyktone (1) and a new arborinane-type triterpenoid named paramyrpenoid (2), together with ten previously described trichothecenes derivatives (3-12) were isolated and identified from a rhizospheric soil-derived Paramyrothecium sp. KMU22107 associated with Delphinium yunnanense. Their structural elucidation was achieved by the comprehensive analysis of spectroscopic data and comparison with literature values. Notably, paramyrpenoid (2) was the first example of an arborinane-type triterpenoid with a double bond at Δ12(13) and an additional methyl motif at C-8. This was the first report of arborinane-type triterpenoids from a fungus belonging to Paramyrothecium genus. In pharmacological studies, paramyrpenoid (2) demonstrated significant cytotoxic activity against the HL-60, SW480, A-549, MDA-MB-231 and SMMC-7721 cell lines, with IC50 values from 2.0 to 16.1 µM. Compounds 1 and 2 were also evaluated for anti-inflammatory, anti-acetylcholinesterase (AChE), and protein tyrosine phosphatase 1B (PTP1B) inhibitory activities in vitro.

Two new cucurbitane-type triterpenoid saponins from the fruit of Citrullus colocynthis.

Two new cucurbitane-type triterpenoid saponins, 2,20ß,22ß-trihydroxy-16α,23(R)-epoxycucurbita-1,5,24-triene-3,11-dione 2-O-ß-D-glucopyranoside (1), 2,20ß,22α-trihydroxy-16α,23(S)-epoxycucurbita-1,5,11,24-tetraene-3-one 2-O-ß-D-glucopyranoside (2) were isolated from the fruit of Citrullus colocynthis (L.) Schrad. Their structures were elucidated by mass spectrometry, IR, 1D, and 2D NMR spectroscopy, etc. Besides, both of the compounds showed significant hepatoprotective activities at 10 µM against paracetamol-induced HepG2 cell damage.

Mode of molecular interaction of triterpenoid saponin ginsenoside Rh2 with membrane lipids in liquid-disordered phases.

Ginsenoside Rh2 (Rh2) is a ginseng saponin comprising a triterpene core and one unit of glucose and has attracted much attention due to its diverse biological activities. In the present study, we used small-angle X-ray diffraction, solid-state NMR, fluorescence microscopy, and MD simulations to investigate the molecular interaction of Rh2 with membrane lipids in the liquid-disordered (Ld) phase mainly composed of palmitoyloleoylphosphatidylcholine compared with those in liquid-ordered (Lo) phase mainly composed of sphingomyelin and cholesterol. The electron density profiles determined by X-ray diffraction patterns indicated that Rh2 tends to be present in the shallow interior of the bilayer in the Ld phase, while Rh2 accumulation was significantly smaller in the Lo phase. Order parameters at intermediate depths in the bilayer leaflet obtained from 2H NMR spectra and MD simulations indicated that Rh2 reduces the order of the acyl chains of lipids in the Ld phase. The dihydroxy group and glucose moiety at both ends of the hydrophobic triterpene core of Rh2 cause tilting of the molecular axis relative to the membrane normal, which may enhance membrane permeability by loosening the packing of lipid acyl chains. These features of Rh2 are distinct from steroidal saponins such as digitonin and dioscin, which exert strong membrane-disrupting activity.

Synthesis and Investigation of Biological Activity of New Betulonic Acid Derivatives Containing 1,2,3-Triazole Fragments.

The results of this study showed that the compounds synthesized by the authors have significant potential due to their antibacterial and cytotoxic properties. The apparent antibacterial activity demonstrated by the compounds suggests that they are active antimicrobial agents against common microbial pathogens that cause various socially significant infectious diseases. Compound 6 showed pronounced antimicrobial activity against the Gram-positive test strain Staphylococcus aureus ATCC 6538, and compound 7 demonstrated pronounced antimicrobial activity against the Gram-negative test strain Escherichia coli ATCC 25922 (MIC = 6.3 µg/mL). This allowed us to consider these compounds to have great potential.

Genome-Wide Identification of the CYP716 Gene Family in Platycodon grandiflorus (Jacq.) A. DC. and Its Role in the Regulation of Triterpenoid Saponin Biosynthesis.

The main type of saponins occurring in the root of Platycodon grandiflorus (Jacq.) A. DC. are oleanolic acid glycosides. The CYP716 gene family plays a major role in catalyzing the conversion of ß-amyrin into oleanolic acid. However, studies on the CYP716 genes in P. grandiflorus are limited, and its evolutionary history remains poorly understood. In this study, 22 PgCYP716 genes were identified, distributed among seven subfamilies. Cis-acting elements of the PgCYP716 promoters were mainly involved in plant hormone regulation and responses to abiotic stresses. PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716BWv3 genes were upregulated in the root and during saponin accumulation, as shown by RNA-seq analysis, suggesting that these four genes play an important role in saponin synthesis. The results of subcellular localization indicated that these four genes encoded membrane proteins. Furthermore, the catalytic activity of these four genes was proved in the yeast, which catalyzed the conversion of ß-amyrin into oleanolic acid. We found that the content of ß-amyrin, platycodin D, platycoside E, platycodin D3, and total saponins increased significantly when either of the four genes was over expressed in the transgenic hair root. In addition, the expression of PgSS, PgGPPS2, PgHMGS, and PgSE was also upregulated while these four genes were overexpressed. These data support that these four PgCYP716 enzymes oxidize ß-amyrin to produce oleanolic acid, ultimately promoting saponin accumulation by activating the expression of upstream pathway genes. Our results enhanced the understanding of the functional variation among the PgCYP716 gene family involved in triterpenoid biosynthesis and provided a theoretical foundation for improving saponin content and enriching the saponin biosynthetic pathway in P. grandiflorus.

Five anti-inflammatory compounds from the resins of Liquidambar orientalis.

Five undescribed compounds, including a triterpenoid (1), three phenylpropanoids [(±)-2 and 4], and an aromatic compound (3), as well as six known analogues (5-10), were isolated from the resins of Liquidambar orientalis Mill. Their structures, including absolute configurations, were determined by using spectroscopic and computational methods, and the five new compounds displayed anti-inflammatory activities in LPS-induced RAW264.7 cells.

Insecticidal Triterpenes in Meliaceae III: Plant Species, Molecules, and Activities in Munronia-Xylocarpus.

Plants of the Meliaceae family have long attracted researchers' interest due to their various insecticidal activities, with triterpenes being the main active ingredients. In this paper, we discuss 93 triterpenoids with insecticidal activity from 37 insecticidal plant species of 15 genera (Munronia, Neobeguea, Pseudocedrela, Nymania, Quivisia, Ruagea, Dysoxylum, Soymida, Lansium, Sandoricum, Walsura, Trichilia, Swietenia, Turraea, and Xylocarpus) in the family Meliaceae. Among these genera, Trichilia deserves further research, with twelve species possessing insecticidal activity. The 93 insecticidal molecules included 27 ring-seco limonoids (comprising 1 ring A-seco group chemical, 1 ring B-seco group chemical, 5 ring D-seco group chemicals, 14 rings A,B-seco group chemicals, 5 rings B,D-seco group chemicals, and 1 rings A,B,D-seco group chemical), 22 ring-intact limonoids (comprising 5 cedrelone-class chemicals, 6 trichilin-class chemicals, 7 havanensin-class chemicals, 2 azadirone-class chemicals, 1 vilasinin-class chemical, and 1 other chemical), 33 2,30-linkage chemicals (comprising 25 mexicanolide-class chemicals and 8 phragmalin-class chemicals), 3 1,n-linkage-group chemicals, 3 onoceranoid-type triterpenoids, 2 apotirucallane-type terpenoids, 2 kokosanolide-type tetranortriterpenoids, and 1 cycloartane triterpene. In particular, 59 molecules showed antifeedant activity, 30 molecules exhibited poisonous effects, and 9 molecules possessed growth regulatory activity. Particularly, khayasin, beddomei lactone, 3ß,24,25-trihydroxycycloartane, humilinolides A-E and methyl-2-hydroxy-3ß-isobutyroxy-1-oxomeliac-8(30)-enate showed excellent insecticidal activities, which were comparable to that of azadirachtin and thus deserved more attention. Moreover, it was noteworthy that various chemicals (such as 12α-diacetoxywalsuranolide, 11ß,12α-diacetoxycedrelone, 1α,7α,12α-triacetoxy-4α-carbomethoxy-11ß-hydroxy-14ß,15ß-epoxyhavanensin, and 11-epi-21-hydroxytoonacilide, etc.) from Turraea showed excellent insecticidal activity. Specially, the insecticidal activity of khayasin from Neobeguea against the coconut leaf beetle were similar to that of rotenone. Therefore, it was a promising candidate insecticide for the control of the coconut leaf beetle.

Synthesis and comparative analysis of the cytotoxicity and mitochondrial effects of triphenylphosphonium and F16 maslinic and corosolic acid hybrid derivatives.

The cytotoxic profile and antiproliferative and mitochondrial effects of triterpene acid conjugates with mitochondriotropic lipophilic triphenylphosphonium (TPP+) and F16 cations were evaluated. Maslinic and corosolic acids chosen as the investigation objects were synthesized from commercially available oleanolic and ursolic acids. Study of the cytotoxic activity of TPP+ and F16 triterpenoid derivatives against six tumor cell lines demonstrated a comparable synergistic effect in the anticancer activity, which was most pronounced in the case of MCF-7 mammary adenocarcinoma cells and Jurkat and THP-1 leukemia cells. The corosolic and maslinic acid hybrid derivatives caused changes in the progression of tumor cell cycle phases when present in much lower doses than their natural triterpene acid precursors. The treatment of tumor cell lines with the conjugates resulted in the cell cycle arrest in the G1 phase and increase in the cell population in the subG1 phase. The cationic derivatives of the acids were markedly superior to their precursors as inducers of hyperproduction of reactive oxygen species and more effectively decreased the mitochondrial potential in isolated rat liver mitochondria. We concluded that the observed cytotoxic effect of TPP+ and F16 triterpenoid conjugates is attributable to the ability of these compounds to initiate mitochondrial dysfunctions. Their cytotoxicity, antiproliferative action, and mitochondrial effects depend little on the type of cationic groups used.

Seven New Noroleanane Triterpenoids from Mastic and their NO Inhibitory and Cytotoxic Activities.

Mastic is a natural resin produced by Pistacia lentiscus L. (Anacardiaceae) with high medicinal value and have been traditionally used as Uighur imported medicine for centuries. In this study, 16 triterpenoids including seven new norleanane triterpenoids (1-7), along with nine known oleanane triterpenoids (8-16), were isolated from the mastic. Their chemical structures were determined on the basis of extensive spectroscopic analyses (including IR, UV, ESI-HR-MS and NMR spectroscopy) and single-crystal X-ray diffraction. Compounds 4-7, 11, 14 and 16 showed strong inhibitory NO production in LPS-induced RAW264.7 cells with IC50 values 7.44-9.76 µM, respectively (positive control dexamethasone, 9.93 ± 1.17 µM). Furthermore, compounds 3 and 12 significantly inhibited the growth of SW480 cells, compound 3 showed the most pronounced inhibitory effect with an  IC50 of 2.30 ± 0.38 µM.

Unraveling the role of cytochrome P450 enzymes in oleanane triterpenoid biosynthesis in arjuna tree.

Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated ß-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed ß-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.