Clustering Analysis of Natural D-borneol Resource Plants Based on Simple Sequence Repeat (SSR) Markers, Leaf Morphology, and Chemical Composition.

D-borneol is a double-loop monoterpene with a wide use in the pharmaceutical, food, and cosmetics industries. Natural D-borneol can be extracted from branches and leaves of D-borneol resource plants. With the widespread use of natural D-borneol, the identification of D-borneol resource plants and the protection of germplasm resources have become the focus of research. In this study, plant leaf morphology, chemical composition, and simple sequence repeat (SSR) molecular marker analysis were used to analyze and cluster 5 species of D-borneol resource plants and their closely related species. It was found that all three analysis methods could distinguish and cluster these D-borneol resource plants to some degree. The result of SSR analysis using capillary electrophoresis was the best, and it could distinguish Mei Pian tree from Yin Xiang as well as Longnao Zhang from An Zhang. The correlation analysis between SSR similarity matrix and leaf morphology analysis and between SSR similarity matrix and chemical composition similarity matrix revealed that they both had significant correlations (P < 0.0001) and the correlation (r = 0.588) between SSR and leaf morphology was a little higher than that (r = 0.519) between SSR and chemical composition. This indicated that the environment had a greater impact on the chemical composition than on leaf morphology. The research findings will offer efficient techniques to cluster natural D-borneol resource plants and establish a theoretical basis for their future development and utilization.

The history, stereochemistry, ethnopharmacology and quality assessment of borneol.

ETHNOPHARMACOLOGICAL RELEVANCE: Borneol (BO) represents a global trade-driven spreading of ethnic medicine traceable to the classical age, and won its name specific to its original habitat "Borneo". BO shows broad spectral pharmacological effects, such as anti-inflammatory, analgesic, antipyretic, inducing resuscitation, and widely applied in the protection and treatment of cardiovascular and cerebrovascular diseases, used singly or mostly in compound formulae. AIM OF THE STUDY: Three stereoscopic configuration forms of BO, l-borneol (LB), d-borneol (DB), and dl-borneol (synthetic, SB), are formulated in broad spectral application, yet their diverse pharmacodynamic and pharmacokinetic properties caused by configurations, and accurate assay and quality assessment are often overlooked. A systematic review and analysis of lumped studies and applications is necessary to clarify the relationship between configuration and its original plant, analysis method, activity and side effect BO in order to guarantee the efficacy and safety during their application. MATERIALS AND METHODS: The public databases including PubMed, Web of Science, Google Scholar, China National Knowledge Infrastructure were referenced to summarize a comprehensive research and application data of BO published up to date. RESULTS: This review includes following sections: History and current status, Stereochemistry, Ethnopharmacology, and Quality assessment. In the section of history, the changes of the plant origins of the two isomeric forms of natural BO were described respectively, and the methods for synthetic racemate SB were also included. The section of stereochemistry deals with the stereoscopic structures, physical/chemical property, optical rotation of the three forms of BO, as well as the main related substances like isoborneol, obtained in SB via chemical transformation of camphor and turpentine oil. In the section of Ethnopharmacology, pharmacological activities and pharmacokinetics of different forms of BO were discussed. BO is usually used as an "adjuvant", by enhancing the permeability of the blood-brain barrier and intervene the ADME/T pathways of the other ingredients in the same formulation. In the section of quality assessment, the analytical methods, including chromatography, especially GC, and spectroscopy were addressed on the chiral separation of the coexisting enantiomers. CONCLUSIONS: This overview systematically summarized three forms of BO in terms of history, stereochemistry, ethnopharmacology, and quality assessment, which, hopefully, can provide valuable information and strategy for more reasonable application and development of the globally reputed ethnic medicine borneol with characteristics in stereochemistry.

Full-Length Transcriptome Sequencing Combined with RNA-Seq to Analyze Genes Related to Terpenoid Biosynthesis in Cinnamomum burmannii.

Cinnamomum burmannii is a cinnamomum plant rich in natural D-borneol. Natural D-borneol is a bicycle monoterpenoid compound widely used in the food, pharmaceutical, and cosmetic industries. Therefore, analyzing the biosynthesis mechanism of natural D-borneol in C. burmannii at the molecular level is helpful for directional breeding in the future and further development and utilization of C. burmannii and its related gene resources. In our study, 76 genes related to terpene metabolism were analyzed through third-generation sequencing and second-generation sequencing. Of these genes, 57 were associated with the synthesis of the terpenoid skeleton, and 19 belonged to terpenoid synthase, including four monoterpenoid synthases, seven sesquiterpenoid synthases, and eight diterpenoid synthases. Two genes in diterpenoid synthase were differentially expressed in high D-borneol and low D-borneol plants. It was speculated that these two genes might be related to D-borneol synthesis. How these two genes participate in the synthesis of D-borneol needs further study.

d-Borneol enhances cisplatin sensitivity via autophagy dependent EMT signaling and NCOA4-mediated ferritinophagy.

BACKGROUND: d-Borneol has been widely used as a drug absorption enhancer, but there are few studies on the anti-resistance ability of d-borneol combined with cisplatin in cisplatin-resistant non-small cell lung cancer cells. Ferroptosis, autophagy and epithelial-mesenchymal transition (EMT) have been reported to be associated with drug resistance. PURPOSE: To investigate the molecular mechanisms and sensitizing effects of d-borneol combined with cisplatin to against drug cisplatin resistance from the perspective of ferroptosis, autophagy and EMT resistance. METHODS: H460/CDDP xenograft tumor model was established to verify the antitumor activity and safety in vivo. RNA sequencing was used to predict target molecules and signaling pathways. Reactive oxygen species (ROS) were used as marker of ferroptosis, and its level was determined by a dichlorodihydrofluorescein diacetate fluorescent probe and flow cytometry. Levels of glutathione (GSH), malondialdehyde (MDA), and antioxidants such as superoxide dismutase (SOD) and thioredoxin (Trx) involved in the balance of oxidative stress were measured by an assay kit or enzyme-linked immunosorbent assay. Western blotting and real-time polymerase chain reaction were used to assess the regulatory mechanism of EMT markers, autophagy, and ferroptosis signaling pathways. RESULTS: d-Borneol in combination with cisplatin reduced tumor volume and weight, enhanced tumor-inhibiting effects, and alleviated cisplatin-induced damage to the liver and kidney in vivo. RNA-sequencing showed that differentially expressed genes were enriched in ferroptosis. d-Borneol in combination with cisplatin promoted ROS accumulation, increased the content of MDA levels, and decreased GSH, SOD, Trx, and heme oxygenase-1 expression to induce oxidative damage. d-Borneol combination with cisplatin induced ferroptosis by promoting nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and regulating intracellular iron ion transport via upregulating PRNP and downregulating PCBP2. In addition, d-borneol combined with cisplatin promoted autophagy by upregulating expression of LC3II/ATG5/Beclin-1 and inhibited the EMT by increasing the expression of epithelial marker E-cadherin and decreasing mesenchymal markers (N-cadherin and vimentin) and transcription factors (Snail and ZEB1). CONCLUSION: For the first time, our study implies that d-borneol enhanced cisplatin sensitivity by inducing ferroptosis, promoting autophagy and inhibiting EMT progression, thereby enhancing antitumor activity. It suggests that d-borneol could be developed as a novel chemosensitizers.

d-Borneol enhances cisplatin sensitivity via p21/p27-mediated S-phase arrest and cell apoptosis in non-small cell lung cancer cells and a murine xenograft model.

BACKGROUND: Cisplatin (CDDP) is commonly used to treat non-small cell lung cancer (NSCLC), but the appearance of drug resistance greatly hinders its efficacy. Borneol may promote drug absorption; however, synergism between borneol and CDDP in suppressing NSCLC is not clearly understood. Hence, we investigated borneol as a novel chemosensitizer to support chemotherapeutic efficacy and reduce side effects. METHODS: We compared viability after exposure to d-borneol, l-borneol, and synthetic borneol in two NSCLC cell lines, A549 and H460, and selected the most sensitive cells. We then assessed synergy between borneol forms and CDDP in cisplatin-resistant NSCLC cells, H460/CDDP. Next, we identified effective concentrations and exposure times. Subsequently, we evaluated cell migration via wound healing and cell proliferation via clone formation assay. Then, we focused on P-glycoprotein (P-gp) function, cell cycle, apoptosis, and RNA sequencing to elucidate underlying molecular mechanisms for synergy. Finally, we used an H460/CDDP xenograft tumor model to verify antitumor activity and safety in vivo. Data were examined using one-way analysis of variance (ANOVA) for multiple datasets or t-test for comparisons between two variables. RESULTS: d-Borneol was more effective in H460 than A549 cells. d-Borneol combined with CDDP showed greater inhibition of cell proliferation, migration, and clone formation in H460/CDDP cells than CDDP alone. RNA sequencing (RNA-seq) analysis identified differentially expressed genes enriched in cell cycle pathways. The impact of d-borneol on CDDP chemosensitivity involved arrest of the cell cycle at S phase via p27/p21-mediated cyclinA2/D3-CDK2/6 signaling and activation of intrinsic apoptosis via p21-mediated Bax/Bcl-2/caspase3 signaling. Further, d-borneol ameliorated drug resistance by suppressing levels and activity of P-gp. Cotreatment with d-borneol and CDDP inhibited tumor growth in vivo and reduced CDDP-caused liver and kidney toxicity. CONCLUSIONS: d-Borneol increased the efficacy of cisplatin and reduced its toxicity. This compound has the potential to become a useful chemosensitizer for drug-resistance NSCLC.

Progress in Borneol Intervention for Ischemic Stroke: A Systematic Review.

Background: Borneol is a terpene and bicyclic organic compound that can be extracted from plants or chemically synthesized. As an important component of proprietary Chinese medicine for the treatment of stroke, its neuroprotective effects have been confirmed in many experiments. Unfortunately, there is no systematic review of these studies. This study aimed to systematically examine the neuroprotective effects of borneol in the cascade reaction of experimental ischemic stroke at different periods. Methods: Articles on animal experiments and cell-based research on the actions of borneol against ischemic stroke in the past 20°years were collected from Google Scholar, Web of Science, PubMed, ScienceDirect, China National Knowledge Infrastructure (CNKI), and other biomedical databases. Meta-analysis was performed on key indicators in vivo experiments. After sorting the articles, we focused on the neuroprotective effects and mechanism of action of borneol at different stages of cerebral ischemia. Results: Borneol is effective in the prevention and treatment of nerve injury in ischemic stroke. Its mechanisms of action include improvement of cerebral blood flow, inhibition of neuronal excitotoxicity, blocking of Ca2+ overload, and resistance to reactive oxygen species injury in the acute ischemic stage. In the subacute ischemic stage, borneol may antagonize blood-brain barrier injury, intervene in inflammatory reactions, and prevent neuron excessive death. In the late stage, borneol promotes neurogenesis and angiogenesis in the treatment of ischemic stroke. Conclusion: Borneol prevents neuronal injury after cerebral ischemia via multiple action mechanisms, and it can mobilize endogenous nutritional factors to hasten repair and regeneration of brain tissue. Because the neuroprotective effects of borneol are mediated by various therapeutic factors, deficiency caused by a single-target drug is avoided. Besides, borneol promotes other drugs to pass through the blood-brain barrier to exert synergistic therapeutic effects.

Enhanced extraction efficiency of natural D-borneol from Mei Pian tree leaves pretreated with deep eutectic solvents.

Stems and leaves of Mei Pian tree can be used to extract natural D-borneol. In this study, a variety of deep eutectic solvents (DESs) based on choline chloride were used to pretreat leaves of Mei Pian tree, from which natural D-borneol was extracted by Soxhlet extraction. The extraction efficiency of most pretreated leaves was higher than that of the unpretreated leaves, and that pretreated by DES based on choline chloride and sucrose or choline chloride and glycol was maximal. Moreover, The Box-Behnken design (BBD) of response surface method was used to optimize the water content, temperature, and pretreatment time. The optimal conditions included pretreatment of Mei Pian tree for 4.07 hr at 44.5°C by DES, which was synthesized using choline chloride, sucrose, and water at the molar ratio of 5:2:5.9, and the extraction efficiency (4.936 mg/g) was maximal, more than twice as much as that of unpretreatment. The results showed that the pretreatment process using DES based on choline chloride and sucrose could effectively improve the extraction efficiency of natural D-borneol from Mei Pian tree.

Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes.

BACKGROUND: Dextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol. RESULTS: Comparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis. CONCLUSIONS: We used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.