Bioassay-guided Isolation of Constituents of sarmentosum Using a Mitochondrial Transmembrane Potential Assay Li Pan,1 Susan Matthew,1,2 Daniel D. Lantvit,3 Xiaoli Zhang,4 Tran Ngoc Ninh,5 Heebyung Chai,1 Esperanza J. Carcache de Blanco,1,2 Djaja D. Soejarto,3,6 Steven M. Swanson,3 and A. Douglas Kinghorn*,1 1 Division of Medicinal Chemistry, 2 Division of Pharmacy Practice and Administration, College of Pharmacy, and 4 Center for Biostatistics, The Ohio State University, Columbus, Ohio 43210, USA, 3Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA, 5Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, , 6Department of Botany, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605, United States

INTRODUCTION BIOLOGICAL ACTIVITY EVALUATION Roxb. (), known as “wild ”, is a terrestrial herb with long creeping stems and a characteristic pungent odor. This is distributed mainly in Induction of apoptosis, or programmed cell death, has been considered as an important tropical and sub-tropical regions of Asia, from northeast to southern mainland mechanism of action for many antineoplastic agents, so targeting the apoptotic signaling and Malaysia, and the Andaman Islands.1-3 P. sarmentosum is used as a folk medicine in pathway is a promising approach for anticancer drug discovery. The mitochodria play an certain countries of for the treatment of various ailments including asthma, important role in regulation of the intrinsic apoptotic pathway, and the loss of mitochondrial cough, dyspepsia, fever, fungal dermatitis of the feet, headache, pleurisy, and toothache.1-4 transmembrane potential (MTP) is one of the key events that may occur during the Besides its medicinal uses, the of P. sarmentosum are also consumed as a popular apoptotic process. Thus, measurement of mitochondrial membrane potential (m) is a spice.5 suitable method to study signaling mechanisms involved in the initiation of the apoptotic

Several studies have been carried out to evaluate the biological activities of extracts of cascade. In a similar fashion, inhibition of the proteasome results in accumulation of many different parts of P. sarmentosum. An aqueous extract of the whole plant was reported to ubiquitinated proteins that are involved with multiple cellular functions and eventually show hypoglycemic effects.6 An aqueous extract of the leaves was found to possess triggers apoptosis Proteasome is the catalytic core of the complex ubiquitin-dependent antinociceptive and anti-inflammatory activities.7 Methanol extracts of the leaves exhibited protein degration pathway, and plays an important role in cell cycle regulation. Since tumor neuromuscular blocking action8 and antibacterial properties,9 and a crude methanol extract cells are more susceptible to proteasome inhibition than normal cells, targeting the of the roots exhibited antiamebic effects.10 A ethanol extract of the whole plant showed proteasome pathway has been demonstrated as a strategy to investigate potential new insecticidal activities against mosquitoes,11,12 while a chloroform extract of the leaves was cancer chemotherapeutic agents. Accordingly, the isolates obtained in this study were reported to have antimalarial activity.13 In addition, both the aqueous and organic phase evaluated for their effects in the MTP and proteasome assays. extracts of the fruits and leaves were found to exhibit antioxidant activity.14 Previous Table. 4. Bioactivity Evaluation of Compounds Isolated from P. sarmentosum.a,b phytochemical investigations on P. sarmentosum have resulted in the isolation of aromatic c c alkene, amide, lignan, phenylpropanoid, and sterol chemical constituents.15,16 Among compound MTP proteasome these compounds, certain amides from P. sarmentosum exhibit antiplasmodial, 1 3.7 >20 antimycobacterial, antifungal, and antituberculosis properties,15,16 and thus may be 2 6.8 >20 considered as a major class of active principles from this plant. 3 2.5 >20 4 3.9 >20 5 7.7 >20 6 13.6 >20 7 3.6 >20 8 1.6 3.45 ± 0.18 9 2.3 >20 10 >20 2.87 ± 0.26 11 4.6 >20 staurosporined 0.0026 - bortezomibe - 0.0025

a Compounds 1-11 and all other known compounds evaluated, were not cytotoxic against HT-29 cells (ED50 >10 M), using a standard protocol. b7-Methoxyisochamanetin (8) was not tested in any of the assays used due to the limited amount isolated. c d e Results are expressed as ED50 values (M). Used as a positive control in the MTP assay. Used as a positive control in the Field View of P. sarmentosum proteasome inhibition assay.

(D. D. Soejarto)) http://portal.ics.trieste.it/MAPs/MedicinalPlants_Plant.aspx?id=642 http://en.wikipedia.org/wiki/File:Piper_sarmentosum.jpg CONCLUSIONS

C-Benzylated dihydroflavones are rare plant-derived natural products, and have only been ISOLATION AND STRUCTURE ELUCIDATION OF COMPOUNDS found from several species of the family Annonaceae thus far. Accordingly, this is the first time that C-benzylated dihydroflavones have been isolated and characterized from a plant As part of an ongoing investigation on the discovery of natural anticancer agents from in the family Piperaceae. To date, evaluation of the bioactivity of C-benzylated tropical , a CHCl -soluble extract of the aerial parts of P. sarmentosum, including the 3 dihydroflavones has been limited. Several analogues have been reported to exhibit leaves, twigs, stems, and inflorescence, collected in Vietnam, exhibited mitochondrial moderate to marginal cytotoxicity against the KB and PS cell lines, and to possess transmembrane potential (MTP) inhibitory activity in HT-29 human colon cancer cells, antimicrobial activity. C-Benzylated dihydroflavones have not been evaluated using the using an initial screening procedure. This extract was not appreciably cytotoxic for HT-29 MTP and proteasome inhibition assays previously. Finally, as polyphenol derivatives, the cells, when evaluated using a standard protocol.17 In the present investigation, activity- C-benzylated dihydroflavones may be responsible for the previously reported antioxidant guided fractionation of this extract using this MTP assay led to the isolation of the four new effects of the water and organic solvent extracts of P. sarmentosum. C-benzylated dihydroflavones (1-4), together with 13 known compounds. The structures of the four new compounds 1-4 were established by spectroscopic data interpretation. The ACKNOWLEDGMENTS known compounds were identified as isochamanetin (5), 7-methoxychamanetin (6), This study was supported by grant P01 CA125066 (awarded to A.D. Kinghorn) from NCI, NIH. We thank Mr. dichamanetin (7), 7-methoxydichamanetin (8), 5-(2-hydroxybenzyl)uvarinol (2'''- John Fowble, College of Pharmacy, The Ohio State University, and Dr. Chun-Hua Yuan, OSU Campus hydroxy-5''-benzylisouvarinol-B) (9), pinocembrin (10), pipercallosine (11), pellitorine, 2,4- Chemical Instrument Center, for facilitating the acquisition of the 400 and 600 MHz NMR spectra. We dodecadienamide, pipercallosidine, 7-methoxyisochamanetin, sesamin benzoic acid, and acknowledge Ms. Nan Kleinholz, Mr. Mark Apsega and Dr. Kari Green-Church, Campus Chemical Instrument trans-cinnamic acid, by spectroscopic analysis and comparison of the data obtained with Center, The Ohio State University, for the mass spectrometric measurement. literature values. REFERENCES

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