Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica.

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO2-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

New Dipterocarpol-Based Molecules with α-Glucosidase Inhibitory and Hypoglycemic Activity.

Dammarane triterpenoids are affordable and bioactive natural metabolites with great structural potential, which makes them attractive sources for drug development. The aim of the study was to investigate the potency of new dipterocarpol derivatives for the treatment of diabetes. Two dammaranes (dipterocarpol and its 20(24)-diene derivative) were modified by a Claisen-Schmidt aldol condensation to afford C2(E)-arylidenes in good yields. The majority of the synthesized compounds exhibited an excellent-to-moderate inhibitory effect toward α-glucosidase (from S. saccharomyces), among them eight compounds showed IC50 values less than 10 µM. 3-Oxo-dammarane-2(E)-benzylidenes (holding p-hydroxy- 3 l and p-carbonyl- 3 m substituents) demonstrated the most potent α-glucosidase inhibition with IC50 0.753 and 0.204 µM, being 232- and 857-times more active than acarbose (IC50 174.90 µM), and a high level of NO inhibition in Raw 264.7 cells with IC50 of 1.75 and 4.57 µM, respectively. An in vivo testing of compound 3 m (in a dose of 20 mg/kg) on a model of streptozotocin-induced T1DM in rats showed a pronounced hypoglycemic activity, the ability to reduce effectively the processes of lipid peroxidation in liver tissue and decrease the excretion of glucose and pyruvic acid in the urine. Compound 3 m reduced the death of diabetic rats and preserved their motor activity.

DSC and FTIR study on the interaction between pentacyclic triterpenoid lupeol and DPPC membrane.

Natural products are a great resource for physiologically active substances. It is widely recognized that a major percentage of current medications are derived from natural compounds or their synthetic analogues. Triterpenoids are widespread in nature and can prevent cancer formation and progression. Despite considerable interest in these triterpenoids, their interactions with lipid bilayers still need to be thoroughly investigated. The aim of this study is to examine the interactions of lupeol, a pentacyclic triterpenoid, with model membranes composed of 1,2­dipalmitoyl­sn­glycerol­3­phosphocholine (DPPC) by using non-invasive techniques such as differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. The DSC study demonstrated that the incorporation of lupeol into DPPC membranes shifts the Lß'-to-Pß' and Pß'-to-Lα phase transitions toward lower values, and a loss of main phase transition cooperativity is observed. The FTIR spectra indicated that the increasing concentration (10 mol%) of lupeol causes an increase in the molecular packing and membrane fluidity. In addition, it is found that lupeol's OH group preferentially interacts with the head group region of the DPPC lipid bilayer. These findings provide detailed information on the effect of lupeol on the DPPC head group and the conformation and dynamics of the hydrophobic chains. In conclusion, the effect of lupeol on the structural features of the DPPC membrane, specifically phase transition and lipid packing, has implications for understanding its biological function and its applications in biotechnology and medicine.

Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A.

Wilforlide A is one of the main active constituents produced in trace amounts in Tripterygium wilfordii Hook F, which has excellent anti-inflammatory and immune suppressive effects. Despite the seeming structural simplicity of the compound, the biosynthetic pathway of wilforlide A remains unknown. Gene-specific expression analysis and genome mining were used to identify the gene candidates, and their functions were studied in vitro and in vivo. A modularized two-step (M2S) technique and CRISPR-Cas9 methods were used to construct engineering yeast. Here, we identified a cytochrome P450, TwCYP82AS1, that catalyses C-22 hydroxylation during wilforlide A biosynthesis. We also found that TwCYP712K1 to K3 can further oxidize the C-29 carboxylation of oleanane-type triterpenes in addition to friedelane-type triterpenes. Reconstitution of the biosynthetic pathway in engineered yeast increased the precursor supply, and combining TwCYP82AS1 and TwCYP712Ks produced abrusgenic acid, which was briefly acidified to achieve the semisynthesis of wilforlide A. Our work presents an alternative metabolic engineering approach for obtaining wilforlide A without relying on extraction from plants.

Combined transcriptome and metabolome analysis identifies triterpenoid-induced defense responses in Myzus persicae Sülzer-infested peach.

The defense response of peach (Prunus persica) to insect attack involves changes in gene expression and metabolites. Piercing/sucking insects such as green peach aphid cause direct damage by obtaining phloem nutrients and indirect damage by spreading plant viruses. To investigate the response of peach trees to aphids, the leaf transcriptome and metabolome of two genotypes with different sensitivities to green peach aphid (GPA, Myzus persicae) were studied. The transcriptome analysis of infected peach leaves showed two different response patterns. The gene expression of aphid-susceptible peach plants infected by aphids was more similar to that of the control plants, while the gene expression of aphid-resistant peach plants infected by aphids showed strongly induced changes in gene expression compared with the response in the control plants. Furthermore, gene transcripts in defense-related pathways, including plant-pathogen interaction, MAPK signaling, and several metabolic pathways, were more strongly enriched upon aphid infestation. Untargeted secondary metabolite profiling confirmed that aphid treatment induced larger changes in aphid-resistant peaches than in aphid-susceptible peaches. Consistent with transcriptomic alterations, nine triterpenoids showed extremely significant GPA-induced accumulation in aphid-resistant peaches, whereas triterpenoid abundance remained predominantly unchanged or undetected in aphid- susceptible peaches. Furthermore, some types of transcription factors (including WRKYs, ERFs, NACs, etc.) were more strongly induced upon GPA infestation in aphid-resistant peaches but not in aphid-susceptible peaches. Aphid feeding-dependent transcriptome and metabolite profiles provide the foundation for understanding the molecular mechanisms underlying the response of peach to aphid infestation. These results suggested that accumulation of specialized triterpenoids and the corresponding pathway transcripts may play a key role in peach GPA resistance.

Time-dependent proteomics and drug response in expanding cancer cells.

Cancer cell line is commonly used for discovery and development of anti-cancer drugs. It is generally considered that drug response remains constant for a certain cell line due to the identity of genetics thus protein patterns. Here, we demonstrated that cancer cells continued dividing even after reaching confluence, in that the proteomics was changed continuously and dramatically with strong relevance to cell division, cell adhesion and cell metabolism, indicating time-dependent intrinsically reprogramming of cells during expansion. Of note, the inhibition effect of most anti-cancer drugs was strikingly attenuated in culture cells along with cell expansion, with the strongest change at the third day when cells were still expanding. Profiling of an FDA-approved drug library revealed that attenuation of response with cell expansion is common for most drugs, an exception was TAK165 that was a selective inhibitor of mitochondrial respiratory chain complex I. Finally, we screened a panel of natural products and identified four pentacyclic triterpenes as selective inhibitors of cancer cells under prolonged growth. Taken together, our findings underscore that caution should be taken in evaluation of anti-cancer drugs using culture cells, and provide agents selectively targeting overgrowth cancer cells.

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.

BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

In silico homology modeling of dengue virus non-structural 4B (NS4B) protein and its molecular docking studies using triterpenoids.

BACKGROUND: Dengue fever has become a significant worldwide health concern, because of its high morbidity rate and the potential for an increase in mortality rates due to lack of adequate treatment. There is an immediate need for the development of effective medication for dengue fever. METHODS: Homology modeling of dengue virus (DENV) non-structural 4B (NS4B) protein was performed by SWISS-MODEL to predict the 3D structure of the protein. Structure validation was conducted using PROSA, PROCHECK, Ramachandran plot, and VERIFY-3D. MOE software was used to find out the in-Silico inhibitory potential of the five triterpenoids against the DENV-NS4B protein. RESULTS: The SWISS-MODEL was employed to predict the three-dimensional protein structure of the NS4B protein. Through molecular docking, it was found that the chosen triterpenoid NS4B protein had a high binding affinity interaction. It was observed that the NS4B protein binding energy for 15-oxoursolic acid, betulinic acid, ursolic acid, lupeol, and 3-o-acetylursolic acid were - 7.18, - 7.02, - 5.71, - 6.67 and - 8.00 kcal/mol, respectively. CONCLUSIONS: NS4B protein could be a promising target which showed good interaction with tested triterpenoids which can be developed as a potential antiviral drug for controlling dengue virus pathogenesis by inhibiting viral replication. However, further investigations are necessary to validate and confirm their efficacy.

Synthesis and evaluation of pentacyclic triterpenoids conjugates as novel HBV entry inhibitors targeting NTCP receptor.

Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157-165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.

Betulin: a novel triterpenoid anti-cancerous agent targeting cervical cancer through epigenetic proteins.

Worldwide, cervical cancer (CCa) is a major killer of women. As the conventional drugs used to treat cervical cancer are expensive and expose severe side effects, there is a growing demand to search for novel modifications. Therefore, in the current investigation employing a bioinformatic approach, we explored triterpenoids for their anti-cancer efficacy by targeting cervical cancer epigenetic proteins, namely DNMT3A, HDAC4, and KMT2C. The study utilized molecular docking, ADMET assay, Molecular Dynamic simulation, and DFT calculation to unveil Betulin (BE) as the potential lead compound. Comparative analysis with that standard drug indicated that BE has a better glide score with the target protein KM2TC (- 9.893 kcal/mol), HDAC4 (- 9.720 kcal/mol), and DNMT3A (- 7.811 kcal/mol), which depicts that BE could be a potent inhibitor of these three epigenetic proteins and exhibits favorable pharmacokinetic, pharmacodynamics and toxicity properties. Molecular Dynamics simulation revealed noteworthy structural stability and compactness. DFT analysis revealed higher molecular activity of BE and showed the most increased kinetic stability (δE = 0.254647 eV). Further, we employed In vitro analysis through MTT assay and found that BE has IC50 of 15 µg/ml. In conclusion, BE can potentially treat CCa upon further investigations using in vivo models for better understanding.

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry.

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant's leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations.

Discovery of new triterpenoids from Leptopus clarkei and their antiproliferative activity on cancer cells.

Two new triterpenoids (1-2), along with six known analogues (3-8) were obtained from the dried whole plant of Leptopus clarkei. Compound 1 is a 3,4-seco-lupane-type triterpenoid, and compound 2 is a phenylpropanoid-conjugated pentacyclic triterpenoid possessing trans-p-coumaroyl unit attached to oleanane-type skeleton. This is the first report on chemical investigation of the L. clarkei, and the triterpenoid derivatives were found in this plant for the first time. The structures of the new compounds were unequivocally elucidated by HRESIMS and 1D/2D NMR data. Additionally, the isolated compounds were evaluated for theircytotoxicities against four cancer cell lines including HepG2, MCF-7, A549 and HeLa. Notably, compound 2 exhibited the most significant antiproliferative activity with IC50 less than 20 µM for four cancer lines.

Two Undescribed Protostane Triterpenoids from the Rhizome of Alisma plantago-aquatica.

Two undescribed protostane triterpenoids, 11-deoxy-13(17),15-dehydro-alisol B 23-acetate (2) and alisol S (3), together with 21 known ones (1, 4-23), were isolated from the dried rhizome of Alisma plantago-aquatica. Of these compounds, 13(17),15-Dehydro-alisol B 23-acetate (1) and 11-deoxy-13(17),15-dehydro-alisol B 23-acetate (2) are two protostane triterpenoids containing conjugated double bonds in the five-membered ring D that are rarely found from nature resource, while alisol S (3) is a protostane triterpenoid with undescribed tetrahydrofuran moiety linked via C20 -O-C24 at the side chain. Additionally, compound 18 is a new natural product, and cycloartenol triterpenoid 23 is a non protostane triterpenoid firstly isolated from genus Alisma. Their structures were elucidated by extensive spectral analysis of the UV, IR, MS, 1D and 2D NMR, and comparison of the experimental and calculated CD curves.

Exploration of 1,2,3-triazolo fused triterpenoids as inhibitors of human coronavirus 229E targeting the viral nsp15 protein.

The coronavirus disease-19 (COVID-19) pandemic has raised major interest in innovative drug concepts to suppress human coronavirus (HCoV) infections. We previously reported on a class of 1,2,3-triazolo fused betulonic acid derivatives causing strong inhibition of HCoV-229E replication via the viral nsp15 protein, which is proposedly related to compound binding at an intermonomer interface in hexameric nsp15. In the present study, we further explored the structure-activity relationship (SAR), by varying the substituent at the 1,2,3-triazolo ring as well as the triterpenoid skeleton. The 1,2,3-triazolo fused triterpenoids were synthesized by a multicomponent triazolization reaction, which has been developed in-house. Several analogs possessing a betulin, oleanolic acid, or ursolic acid core displayed favorable activity and selectivity (EC50 values for HCoV-229E: 1.6-3.5 µM), but neither of them proved as effective as the lead compound containing betulonic acid. The 18ß-glycyrrhetinic acid-containing analogs had low selectivity. The antiviral findings were rationalized by in silico docking in the available structure of the HCoV-229E nsp15 protein. The new SAR insights will aid the further development of these 1,2,3-triazolo fused triterpenoid compounds as a unique type of coronavirus inhibitors.

Systematic qualitative analysis of terpenes in mastic (Pistacia lentiscus L.) extract and their fragmentations by UHPLC-Q-Orbitrap-HRMS.

INTRODUCTION: Mastic is a natural resin produced by Pistacia lentiscus L. (Anacardiaceae). The beneficial properties of this resin are attributed to its triterpenes and volatile compounds. OBJECTIVE: This study was conducted to screen and characterize the terpenes in mastic ethyl acetate extract (M-Ex). METHODS: An ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method was developed for the qualitative analysis of terpenes in M-Ex. We utilized in-house-isolated compounds as reference substance (Rs), including monoterpenes (A) with α-pinane structures, tetracyclic triterpene (B) containing tirucallane skeletons, and pentacyclic triterpene (C) belonging to olean, moronic, amyrone, and lupane types. Based on the mass spectrometric characteristics of the above compounds, and the difference in characteristic diagnostic fragment ions (DFIs) in isomeric compounds, the terpene compounds were further identified in M-Ex. RESULTS: Out of a total of 70 compounds, including monoterpenes and tetra-, and pentacyclic triterpenes, 20 were accurately determined by Rs, retention time (RT), and DFIs. Based on the cleavage patterns summarized from the above 20 compounds and with reference to the reported literature, another 50 compounds were putatively identified. Based on our discovery, six terpenic acids with A-seco-tirucallane types and one monoterpene dimer were identified for the first time in mastic. CONCLUSION: Our research serves not only as a foundation for the rapid identification and screening of terpene compounds in mastic but also as a supplementary basis for the identification of such compounds in other types of resins.

Centella asiatica (L.) Urb. and Hydrocotyle umbellata L. identification and quality assessment: A methodology comparison.

INTRODUCTION: Centella is an important genus in the Apiaceae family. It includes Centella asiatica, which has significant edible and medicinal values. However, this species is easily confused due to its similar morphological traits to Hydrocotyle umbellata, hindering its utilization in the consumer and pharmacological industries. OBJECTIVE: The study aims to differentiate these two closely related plant species using reliable methods of confirming the authenticity of natural herbal medicines. METHODS: Our work mainly focuses on the basic morphological characteristics, chemical markers, genetic fingerprints, and their biological responses. RESULTS: The plants can be clearly differentiated using their leaf shapes, stipules, petioles, inflorescences, and fruit structures. Although the phytochemical compositions of the C. asiatica extract were similar to that of H. umbellata which included flavonoids, tannins, and saponins important to the plant's ability to reduce inflammation and promote healing of wounds, the H. umbellata extract showed significantly higher toxicity than that of C. asiatica. High-performance liquid chromatography analysis was used to identify chemical fingerprints. The result revealed that C. asiatica had major triterpene glycoside constituents including asiaticoside, asiatic acid, madecassoside, and madecassic acid, which have a wide range of medicinal values. In contrast, triterpenoid saponins were not identified in H. umbellata. Furthermore, using SCoT1-6 primers was possible to effectively and sufficiently created a dendrogram which successfully identified the closeness of the plants and confirmed the differences between the two plant species. CONCLUSION: Therefore, differentiation can be achieved through the combination of morphometrics, molecular bioactivity, and chemical analysis.

New triterpenoids from the aerial parts of the Uygur medicine Salvia deserta.

Phytochemical investigation on the aerial parts of Salvia deserta led to the isolation of eight new pentacyclic triterpenoids including three oleanane- (1 - 3) and five ursane-type (4 - 8) triterpenoids, whose structures were elucidated based on extensive spectroscopic analysis and quantum chemical calculation. Weak immunosuppressive potency was observed for compounds 1, 2, and 4 - 8 via inhibiting the secretion of cytokines TNF-α and IL-6 in LPS-induced macrophages RAW264.7 at 20 µM. In addition, compounds 1, 2, and 4 - 6 exhibited moderate protective activity on t-BHP-induced oxidative injury in HepG2 cells.

Triterpenoids from Juglans mandshurica with anti-hyaluronidase activities.

Phytochemical study on 90% ethanol extract from the green walnut husks of Juglans mandshurica Maxim. resulted into the isolation of three undescribed triterpenoids, juglansmanoids A-C (1-3). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated components were evaluated in vitro for anti-hyaluronidase activities. As a result, triterpenoid 1 exhibited potent anti-hyaluronidase activity (IC50 = 9.78 µg/ml) three times more than the positive control drug oleanolic acid (IC50 = 40.12 µg/ml).

Triterpenoids from the hook-bearing stems of Uncaria rhynchophylla.

An unprescribed nortriterpenoid with an aromatic E ring, uncanortriterpenoid A (1), together with fourteen known triterpenoids (2-15), were isolated from the hook-bearing stems of Uncaria rhynchophylla Miq. Based on extensive spectroscopic analyses, the NMR data of 2, 5, and 10 in CD3OD were assigned for the first time, and the wrongly assigned δC of C-27 and C-29 of 2 were revised. Among the known compounds, 7, 13, and 15 were isolated from this species for the first time, and 15 represents the first lanostane triterpenoid bearing an extra methylidene at C-24 for the Rubiaceae family. Additionally, compounds 6 and 14 exhibited moderate ferroptosis inhibitory activity, with an EC50 value of 14.74 ± 0.20 µM for 6 and 23.11 ± 1.31 µM for 14.