Discovery of potential antiauseterity agents from selected Southeast Asian medicinal plants

概要

Pancreatic cancer is the deadliest form of cancer having the lowest 5-year relative survival rate (˂ 5%). It ranks as the seventh leading cause of cancer-related deaths worldwide. Surgery, chemotherapy and radiotherapy are used to extend survival and/or relieve pancreatic cancer patients’ symptoms. However, for advanced stage pancreatic cancer cases, there is still no treatment. The aggressiveness of pancreatic cancer and the unavailability of definite treatment was further complicated due to the absence of appropriate screening tests and diagnostic markers for its early detection. Therefore, there is an urgent need for finding an effective treatment for this disease.

Cells in tumor microenvironment suffers from hypoxia and nutrition starvation due to the insufficient blood supply and excessive energy demand of intensively proliferating cells. For most cancer types, a process of angiogenesis takes place in order to sustain oxygen and nutrients supply. Despite the hypovascular nature of pancreatic tumors, pancreatic cancer cells have the remarkable ability to survive under metabolic stress conditions. Such phenomenon is referred to as “austerity”. The search of agents that inhibit cancer cells tolerance to nutrition starvation is a new approach for the anticancer drug discovery.

In the present study, extracts of Anneslea fragrans Wall., Betula alnoides, and Artemisia vulgaris showed preferential cytotoxicity against the PANC-1 human pancreatic cancer cells under nutrient-deprived conditions. Therefore, the phytochemical investigation of these active extracts was carried out in order to identify the active constituents responsible for the observed preferential cytotoxic activities.

Chemical constituents of Anneslea fragrans and their antiausterity activity against the PANC-1 human pancreatic cancer cell line
Anneslea fragrans Wall. (Theaceae) is a tree native to Southeast Asia. Traditionally, the leaves and bark of this plant are used to treat fever, diarrhea and liver inflammation. Leaves and twigs of this plant were collected from the Chiang Mai Province of Thailand, and showed preferential cytotoxicity against the PANC-1 cancer cells under a nutrient-deprived condition with a PC50 value of 9.6 and 16.4 µg/mL, respectively.

The phytochemical investigation of the leaves of A. fragrans resulted in the isolation of a new dihydrochalcone glucoside named fragranone A (1), and a new chalcone, fragranone B (2), together with fifteen previously reported compounds (Chart 1). Fragranone A (1) represents the first example of a rare natural product bearing an acetonide glucose moiety. Absolute configuration of the sugar unit in 1 was confirmed through acid catalyzed hydrolysis followed by extracting the sugar part and comparing its specific optical rotation with that reported for D-glucose. Both values are in very close agreement which confirmed the absolute configuration of the sugar part in 1 as D-glucose. Fragranone B (2) is representative of a rare class of natural products with a threonolactone unit linked to chalcone through an ether linkage. 1D NOE experiments and applying axial haloketone rule to the experimental ECD spectrum of 2, in addition to computational ECD calculations were used to unequivocally confirm the absolute configuration of the lactone unit in 2 as L-threono-1,4-lactone.1

The phytochemical investigation of the twigs of A. fragrans resulted in the isolation of an unprecedented flavanoid trimer named fragranol A (18), two new highly oxygenated spiro-biflavanoids named fragranols B and C (19 and 20), and a new dihydrochalcone glucoside, fragranone C (21), together with seven previously reported compounds (Chart 2). Fragranol A (18) represents a new class of structurally challenging and sterically hindered ""spiro-triflavanoid"" compounds possessing an unprecedented carbon skeleton. Compound 18 consists of a unique 45 carbon skeleton having six stereocenters, including two unprecedented spiro-chiral centers. Fragranols B and C (19 and 20) are the first spiro-biflavanoids incorporating a 2S,3R-flavan-3-ol unit. Extensive NMR spectroscopic analysis, taking consideration of the in-depth spatial anisotropic effects around spiro-centers, in addition to quantum computational ECD calculations have been employed for deciphering the absolute configuration of the chiral centers in fragranols A−C (18-20).2-4

All isolated compounds were tested for preferential cytotoxicity against PANC-1 human pancreatic cancer cell line utilizing the antiausterity strategy. Many compounds exhibited antiausterity activity against PANC-1 cells, and betulin (14) was found to be the most potent compound tested with a PC50 value of 8.4 μM. In addition, fragranone A (1) was found to suppress PANC-1 cancer cell migration in real time.1

Chemical constituents of Betula alnoides and their antiausterity activity against the PANC-1 human pancreatic cancer cell line
The 95% ethanolic extract of B. alnoides Ham. ex D. Don (Betulaceae) bark, collected from the Chiang Rai Province of Thailand, showed preferential cytotoxicity against the PANC-1 human pancreatic cancer cells under a nutrient-deprived condition with a PC50 value of 13.2 µg/mL. Phytochemical investigation of this active extract led to the isolation of six new benzophenones (27−32), together with five known compounds. The new compounds 27−31 incorporate open-sesquiterpene side chains. The new benzophenone 32 consists of a benzo[c]-2-oxadicyclo[3.3.1]nonane tricyclic system. The new compounds displayed potent antiausterity activity against PANC-1 cells in nutrient-deprived medium, without showing toxicity in normal, nutrient-rich conditions. Moreover, 27 and 32 induced alterations in PANC-1 cell morphology under nutrient-deprived conditions and also inhibited PANC-1 colony formation under nutrient-rich conditions.5

Chemical constituents of Artemisia vulgaris and their antiausterity activity against the PANC-1 human pancreatic cancer cell line
The genus Artemisia is one of the most diverse and widely distributed genera of Asteraceae family with over 500 species distributed in Europe, Asia, and North America. Ever since the discovery of the potent antimalarial agent artemisinin from Artemisia annua in 1972, a considerable focus has been made on finding new compounds with promising therapeutic activity from this genus. As such, several compounds with diverse biological activities have been reported.

In the present study, the 70% ethanolic extract of A. vulgaris showed preferential cytotoxicity against the PANC-1 cells under a nutrient-deprived medium (NDM) with a PC50 value of 12.5 µg/mL. The phytochemical investigation of this active extract resulted in the isolation of a new sesquiterpene named vulgaric acid (37), together with eight previously reported compounds (Chart 3). The structural elucidation of 37 was achieved by HRFABMS and NMR analysis. The absolute configuration of 37 was determined by computational calculations of ECD data. All isolated compounds were tested for preferential cytotoxicity against PANC-1 cells, and apigenin (39) showed the strongest activity with a PC50 value of 30.7 μM.6

Conclusion
Phytochemical investigation of the extracts A. fragrans, B. alnoides and A. vulgaris led to the isolation of 44 compounds including two new dihydrochalcones (1 and 21), a new chalcone (2), one unprecedented spiro- triflavanoid (18), two structurally challenging spiro-biflavanoids (19 and 20), six rare benzophenones (27−32) and a new sesquiterpene (37). Betulin (14) from A. fragrans and compounds 26−32 from B. alnoides showed potent preferential cytotoxicity against PANC-1 cells. Fragranone A (1) was found to suppress PANC-1 cancer cell migration in real time. Moreover, compounds 27 and 32 induced alterations in PANC-1 cell morphology under nutrient-deprived conditions and also inhibited PANC-1 colony formation under nutrient-rich conditions. In conclusion, in the present study, secondary metabolites isolated from the three selected Southeast Asian medicinal plants are interesting candidates for drug development against pancreatic cancer.

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