Antitubercular constituents from Premna odorata Blanco

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Antitubercular constituents from Premna odorata Blanco Stephen B. Lirio, Allan Patrick G. Macabeo, Erickson M. Paragas, Matthias Knorn, Paul Kohls, Scott G. Franzblau, Yuehong Wang, Ma. Alicia M. Aguinaldo

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S0378-8741(14)00290-6 http://dx.doi.org/10.1016/j.jep.2014.04.015 JEP8742

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Journal of Ethnopharmacology

Received date: 13 November 2013 Revised date: 17 March 2014 Accepted date: 7 April 2014 Cite this article as: Stephen B. Lirio, Allan Patrick G. Macabeo, Erickson M. Paragas, Matthias Knorn, Paul Kohls, Scott G. Franzblau, Yuehong Wang, Ma. Alicia M. Aguinaldo, Antitubercular constituents from Premna odorata Blanco, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2014.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antitubercular constituents from Premna odorata Blanco  

Stephen B. Lirioa, Allan Patrick G. Macabeoa,b, Erickson M. Paragasb, Matthias Knornc, Paul Kohlsc, Scott G. Franzblaud, Yuehong Wangd, Ma. Alicia M. Aguinaldoa,b,* a

Graduate School, University of Santo Tomas, España, Manila 1015, Philippines

b

Phytochemistry Laboratory, Research Center for the Natural and Applied Sciences, Thomas

Aquinas Research Complex, University of Santo Tomas, España, Manila 1015, Philippines c

Institut fur Organische Chemie, Universitat Regensburg, Universitatsstrasse 31, 93053

Regensburg, Germany d

Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago

833 S. Wood St., Chicago, Illinois USA 60612-7231

*Corresponding author. Tel.: +63 02 4061611 local 4046; fax: +63 02 7314031. E-mail

addresses:

Aguinaldo).          

[email protected];

[email protected]

(A.M.

  Abstract Ethnopharmacological relevance: Premna odorata Blanco (Lamiaceae) is a medicinal plant traditionally used in Albay Province, in southeastern Luzon, Philippines to treat tuberculosis. This study aimed to determine the antitubercular property of the crude extract and sub-extracts of the leaves, and to isolate the bioactive principles from the active fractions. Materials and Methods: Through extraction, solvent polarity-based fractionation and silica gel chromatography purification of the DCM sub-extract, compound mixtures from the bioactive fractions were isolated and screened for their in vitro antimycobacterial activity against M. tuberculosis H37Rv using the colorimetric Microplate Alamar Blue assay (MABA). Results: The crude methanolic extract and sub-extracts showed poor inhibitory activity against M. tuberculosis H37Rv (MIC = >128 µg/mL). However, increased inhibitory potency was observed for fractions eluted from the DCM sub-extract (MIC = 54 to 120 µg/mL). Further purification of the most active fraction (MIC = 54 µg/mL) led to the isolation of a 1-heneicosyl formate (1), 4:1 mixture of β-sitosterol (2) and stigmasterol (3) and diosmetin (4), which were identified through GC-MS analysis (with dereplication) and NMR experiments. The MIC of compound 1 was 8 µg/ml. Conclusions: The results of this study provide scientific basis for the traditional use of P. odorata as treatment for tuberculosis.

Key words: Premna odorata, antitubercular, tuberculosis, 1-heneicosyl formate, diosmetin

1. Introduction As one of the global health concerns, tuberculosis (TB) was responsible for 1.4 million deaths of the world’s population in 2011, with an estimated 8.7 million new cases (Dye and Williams, 2010; WHO, 2012). The number of drugs currently in use against Mycobacterium tuberculosis is very limited and most of them were introduced more than 40 years ago (Pilcher, 2005; Lienhardt et al., 2012). Diverse factors, including limited drug efficacy, inadequate drug prescription or poor patient compliance, have resulted in the rise of TB cases worldwide, many of them being multidrug resistant (MDR) and extensively drug resistant (XDR). Drug resistant strains have acquired mutations in drug targets or enzymes activating pro-drugs (De Rossi et al. 2006). In spite of growing efforts to discover effective anti-tubercular agents of plant origin, the search for new bioactive compounds still represents a great challenge (Lienhardt et al., 2012). As part of our research endeavors in discovering potent antituberculosis compounds from Philippine medicinal plants (Aguinaldo et al., 2002; Macabeo et al., 2005; Aguinaldo et al., 2007; Mandap et al., 2007; Villaflores et al., 2010; Macabeo et al., 2011, Macabeo et al., 2012; Panlilio et al., 2012), we embarked our study on the Philippine medicinal plant, Premna odorata Blanco. The Lamiaceous plant, Premna odorata (“alagaw” in Filipino) is a small tree abundant in low-altitude thickets and secondary forests in the Philippines. The water decoction of the leaves is used to treat patients with tuberculosis problems. In addition, it is also utilized locally to manage phlegm, stomachache, headache, and cough (Quisumbing, 1978). The leaves of P. odorata are also used to treat wounds by direct application to the infected area. Chemical studies on this plant have elaborated a number of iridoid glycosides and some flavone from the butanol and dichloromethane extract, respectively (Otsuka et al., 1989a; Otsuka et al., 1989b; Otsuka et

al., 1992; Pinzon et al., 2011). So far, no report on the antimycobacterial activity of P. odorata has been indicated in the literature. In this paper, we report on the antitubercular evaluation of the crude-extract and sub-extracts of P. odorata in addition to the isolation of the bioactive principles from the active fractions of the DCM sub-extract. 2. Materials and methods 2.1 Plant material The leaves of Premna odorata were collected in Bacacay, Albay Province, Bicol Region, Philippines in May 2010, and were identified by Assoc. Prof. Rosie A. Madulid. A voucher specimen (USTH 5281) was deposited at the Herbarium of the Plant Sciences Laboratory of the Research Center for the Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines. 2.2 Extraction and isolation The air-dried powdered leaves of P. odorata (2.3 kg) were exhaustively extracted with methanol and the subsequent concentrated extract sequentially partitioned into n-hexane, dichloromethane, ethyl acetate and n-butanol sub-extracts. The dichloromethane sub-extract (26.3 g) was subjected to vacuum liquid chromatography using gradient elution (20% increments) dichloromethane in hexane and acetone in dichloromethane to give 20 pooled fractions. Fraction 4 (324.0 mg) was purified in silica gel using hexane-EtOAc mixtures (99:1 to 97:3) to afford five fractions from which the first fraction (26.0 mg) was further chromatographed on silica gel column (10% EtOAc in hexane) to finally give 1-heneicosyl formate (5.1 mg) (1) (Fig. 1). < Insert Fig. 1 >

Vacuum liquid silica gel column chromatography of fraction eight (2.4 g), using mixtures of hexane-DCM (20%), afforded seven fractions from which the fourth sub-fraction was further chromatographed on silica using hexane-EtOAc (5%) to give a mixture of 4:1 β-sitosterol (2) and stigmasterol (3) (6.0 mg) (Fig. 1) as white amorphous solid. Finally, vacuum liquid silica gel column chromatography of fraction twenty (1.7 g), using mixtures of chloroform-MeOH (20%), afforded eight fractions from which the seventh subfraction was further chromatographed using chloroform-MeOH (90%) to give five sub-fractions. The fourth sub-fraction was further chromatographed on silica using chloroform-MeOH (80%) to finally give pure diosmetin (4) (5,7,3’-trihydroxy-4’-methoxyflavone) (30 mgs) (Fig. 1) as yellow powder. 2.2.1 Compound (1) Obtained as a white powder; MS identical with Wiley and NIST library standards and mass spectral fragmentation patterns. 2.2.2 Compounds (2) and (3) Obtained as needle like crystals; MS, 1H and 13C NMR identical with literature data (Kongduang et al., 2008; Jamal et al., 2008). 2.2.3 Compound (4) Obtained as a yellow powder; MS, UV, 1H and 13C NMR identical with literature data (Park et al., 2007; Pinzon et al., 2011; Ahn et al., 2011). 2.3 Gas chromatography/mass spectrometry (GC/MS) analysis The GC/MS analysis of the isolated compounds was carried out using an Agilent 6890 N gas chromatograph with phenomenex zebron capillary column (15 m, 0.25 mm I.D., 0.25 µm FT, ZB – 5MS, 5 % Phenyl, 95% Methyl) equipped with an SSQ710A Finnigan MAT (San Jose)

selective detector with electron impact mode (Ionization energy: 70 eV). The carrier gas was ultra-pure helium at constant flow of 1.0 mL/min. The injector temperature was set at 300°C. The initial temperature of 80°C was held for 2 min; the temperature was increased by 10°C/min with 1 min incubation period; the temperature was increased at 300°C and held for 5 min. Injections of 1 µL were made in split less mode (split ratio: 20:1 to 100:1). The compounds were identified using Wiley and NIST library. 2.4 Ultraviolet-Visible (UV-Vis) analysis The UV-Vis experiment was recorded using Perkin Elmer Lambda 35 for compound 4. 2.5 Nuclear magnetic resonance spectroscopy (NMR) anlaysis The NMR experiments were recorded using Bruker AMX and Advance Bruker Kyro at 300 MHz field strengths. Samples were dissolved in deuterated chloroform (CDCl3) with tetramethylsilane (TMS) as internal standard. 2.6 Mycobacterial strain and growth conditions Mycobacterium tuberculosis H37Rv (ATCC 27294) was obtained from the American Type Culture Collection (Rockville, Md.). For the first three (of four) replicate experiments, H37Rv inocula were first passaged in radiometric 7H12 broth (BACTEC 12B; Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) until the growth index (GI) reached 800 to 999. For the fourth replicate experiment, H37Rv was grown in 100 mL of Middlebrook 7H9 broth (Difco, Detroit, Mich.) supplemented with 0.2% (vol/vol) glycerol (Sigma Chemical Co., Saint Louis, Mo.), 10% (vol/vol) OADC (oleic acid, albumin, dextrose, catalase; Difco), and 0.05% (vol/vol) Tween 80 (Sigma). The complete medium was referred to as 7H9GC-Tween. Bacterial cultures were subcultured in 500 mL nephelometer flasks on a rotary shaker (New Brunswick Scientific, Edison, N.J.) at 150 rpm and 37°C until they reached an optical density of 0.4 to 0.5 at 550 nm. Bacteria were washed and suspended in 20 ml of phosphate-buffered saline

and passed through an 8-mm-pore-size filter to eliminate clumps. The filtrates were aliquoted, stored at 280°C, and used within 30 days. 2.7 Minimum inhibitory concentration (MIC) determination All compounds were evaluated for MIC against M. tuberculosis H37Rv (ATCC 27294) using the microplate Alamar Blue assay (MABA) as previously described (Collins and Franzblau, 1997; Cho et al., 2007) except that we now use 7H12 media (3) (instead of 7H9 + glycerol + casitone + OADC). In the case of compounds exhibiting significant background fluorescence, luciferase reporter strains of M. tuberculosis H37Rv were utilized as well as measurement of intracellular adenosine triphosphate. Cultures were incubated in 200 ml medium in 96-well plates for 7 days at 37 oC. Alamar Blue and Tween 80 were added and incubation continued for 24 h at 37 oC. Fluorescence was determined at excitation/emission wavelengths of 530/590 nm, respectively. The MIC is defined as the lowest concentration effecting a reduction in fluorescence (or luminescence) of 90% relative to controls. Three control compounds were run in each experiment including isoniazid, rifampin and PA-824. The reported MIC values are an average of two individual experiments. 3. Results The crude methanolic extract, sub-extracts (hexane, DCM and n-butanol), DCM fractions and isolated compounds were subjected to the colorimetric Microplate Alamar Blue assay (MABA) (Collins and Franzblau, 1997) to assess their susceptibility against the virulent Mycobacterium tuberculosis H37Rv (Table 1). While low inhibitory potency was observed for < Insert Table 1 > the crude methanolic extract and sub-extracts (MIC >128 µg/mL), an improved antitubercular activity was observed in different fractions (MIC= 54 to 120 µg/mL) as the DCM sub-extract

was fractionated through silica gel chromatography.

Chromatographic purification of active

fraction four (MIC= 54 µg/mL) and fraction eight (MIC = 113 µg/mL) afforded compound 1 and the mixture (4:1) of 2 & 3, respectively (Fig. 1). With a sizeable amount for fraction twenty (1.7 g), further purification was conducted, affording compound 4. Due to a low yield and impurities present in fraction nine (MIC= 83 µg/mL), attempts to further purify were deemed impossible. Mass spectrometric identification by comparing with Wiley and NIST library standards and mass spectral fragmentation patterns, and NMR spectral data analysis obtained and comparison with literature showed the identity of the compounds known as 1-heneicosyl formate (1), mixture of ß-sitosterol (2) and stigmasterol (3) and diosmetin (4). Interestingly, compound 1 showed strong inhibitory activity against M. tuberculosis H37Rv (MIC= 8 µg/mL). This corroborates the aliphatic nature of some antimycobacterial compounds (Cantrell et al., 2001). It has been known that aldehydes play an important role in their antimicrobial activity, since they can behave as nonionic surfactants (Bisignano et al., 2001; Kubo et al., 2004). In addition, it is important to mention that M. tuberculosis is considered to be a gram-positive bacterium with the presence of mycolic acid in its cell wall (Esquivel-Ferriño et al., 2012). Thus, it is probable that the bactericidal activity of the aldehyde plus the heneicosyl group is due to a balance between the hydrophilic and hydrophobic portions of the molecule and the detergent-like effect on this organelle (Seidel et al., 2004). On the other hand, the mixture of compounds 2 and 3 exhibited 63% at >128 µg/mL while compound 4 showed no activity against M. tuberculosis H37Rv. It has been reported that steroids have antimycobacterial activity (Aguinaldo et al., 2002). The isolated ß-sitosterol and stigmasterol have MIC values of 128 and 32 µg/mL, respectively (Aguinaldo et al., 2002). With the result obtained, it shows that the mixture of compounds 2 and 3 is less active than the pure compounds.

4. Conclusions    In conclusion, our results demonstrate that the medicinal plant P. odorata possesses antimycobacterial compounds, and therefore, support the purported traditional use of this plant in the treatment of TB. This is the first report of the presence of compound 1 in P. odorata and its antimycobacterial activity. This study also reports on the absence of antimycobacterial activity of compound 4, and the weak activity of the sterol mixture of compounds 2 and 3 as compared to the pure compounds. Based on the results, it is strongly believed that the leaves of P. odorata represent a promising source of antimycobacterial agents that merits further investigation. Acknowledgement We thank the Department of Science and Technology - Philippine Council for Health Research and Development (DOST-PCHRD) for the scholarship grant to SL Lirio. References Aguinaldo, A.M., Saludes, J.P., Garson, M.J., Franzblau, S.G., 2002. Antitubercular constituents from the hexane fraction of Morinda citrifoilia Linn. (Rubiaceae). Phytotherapy Research 16, 683–685. Aguinaldo, A.M., Dalangin-Mallari, V.M., Macabeo, A.P.G, Byrne, L.T., Abe, F., Yamauchi, T., Franzblau, S.G., 2007. Quinoline alkaloids from Lunasia amara inhibit Mycobacterium tuberculosis H37Rv in vitro. International Journal of Antimicrobial Agents 29 (6), 744-746. Ahn, D., Lee, S.I., Yang, J.H., Cho, C.H., Hwang, Y.H., Park, J.H., Kim, D.K., 2011. Superoxide Radical Scavengers from the Whole Plant of Veronica peregrine. Natural Product Sciences 17 (2),142-146. Bisignano, G., Lagana, M., Trombetta, D., Arena, S., Nostro, A., Uccella, N., Mazzanti, G., Saija, A., 2001. In vitro antibacterial activity of some aliphatic aldehydes from Olea europea L. FEMS Microbiology Letters 198, 9–13. Cantrell, C., Franzblau, S.G., Fischer, N.H., 2001. Antimycobacterial Plant Terpenoids. Planta Medica 67, 685-694. Cho, S.H., Warit, S., Wan, B., Hwang, C.H., Pauli, G.F., Franzblau, S.G., 2007. Low-oxygenrecovery assay for high-throughput screening of compounds against non-replicating Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 51, 1380-1385.

Collins, L.A., Franzblau, S.G., 1997. Microplate Alamar blue assay versus BACTEC 460 System for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 41 (5), 1004-1009. De Rossi, E., Ainsa, J.A., Riccardi, G., 2006. Role of mycobacterial efflux transporters in drug resistance: an unresolved question. FEMS Microbiology Reviews 30 (1), 36–52. Dye, C., Williams, B.G., 2010. The population dynamics and control of tuberculosis. Science 328, 856–861. Esquivel-Ferriño, P.C., Favela-Hernández, J.M., Garza-Gonzalez, E., Waksman, N., Rios, M.Y., del Rayo Camacho-Corona, M., 2012. Antimycobacterial activity of constituents from Foeniculum vulgare var. dulce grown in Mexico. Molecules 17 (7), 8471-82. DOI: 10.3390/molecules17078471. Jamal, A.K., Yaacob, W.A., Din, L.B., 2008. A Chemical Study on Phyllanthus reticulatus. Journal of Physical Science 19 (2), 45–50. Kongduang, D., Wungsintaweekul, J., Eknamkul, W.D., 2008. Biosynthesis of β-sitosterol and stigmasterol proceeds exclusively via the mevalonate pathway in cell suspension cultures of Croton stellatopilosus. Tetrahedron Letters 49, 4067–4072. Kubo, I., Fujita, K., Kubo, A., Nihei, K., Ogura, T., 2004. Antibacterial activity of coriander volatile compounds against Salmonella choleraesuis. Journal of Agricultural and Food Chemistry 52, 3329–3332. Lienhardt, C., Raviglione, M., Spigelman, M., Hafner, R., Jaramillo, E., Hoelscher, M., Zumla, A., Gheuens J., 2012. New drugs for the treatment of tuberculosis: needs, challenges, promise, and prospects for the future. The Journal of Infectious Diseases 205 (suppl 2), S241-S249. doi: 10.1093/infdis/jis034. Macabeo, A.P.G., Krohn, K., Gehle, D., Read, R.W., Brophy, J.J., Cordell, G.A., Franzblau, S.G., Aguinaldo, A.M., 2005. Indole alkaloids from the leaves of Philippine Alstonia scholaris. Phytochemistry 66 (10), 1158-1162. Macabeo, A.P.G., Vidar, W.S., Chen, X., Decker, M., Heilmann, J., Wan, B., Franzblau, S.G., Galvez, E.V., Aguinaldo, A.M., Cordell, G.A., 2011. Mycobacterium tuberculosis H37Rv and cholinesterase inhibitors from Voacanga globosa. European Journal of Medicinal Chemistry 46 (7), 3118-3123. Macabeo, A.P.G., Tudla, F.A., Krohn, K., Franzblau, S.G., 2012. Antitubercular activity of the semi-polar extractives of Uvaria rufa. Asian Pacific Journal of Tropical Medicine. 5 (10), 777780.

Mandap, K., Marcelo, R., Macabeo, A.P.G., Yamauchi, T., Abe, F., Franzblau, S.G., Aguinaldo, A.M., 2007. Phenyldecanoids from the antitubercular fractions of the Philippine ginger (Zingiber officinale). ACGC Chemical Research Communications 21, 20-22. Otsuka, H., Kubo, N., Yamasaki, K., Padolina, W.G., 1989a. Two Iridoid Gylcoside Caffeoyl Esters from Premna odorata. Phytochemistry. 28(2), 513–515. Otsuka, H., Kubo, N., Yamasaki, K., Padolina, W.G., 1989b. Premnosides A-D: Diacyl 6-O-α-Lrhamnopyranosylcatapols from Premna odorata. Phytochemistry. 31(11), 3063-3067. Otsuka, H., Kashima, N., Hayashi, T., Kubo, N., Yamasaki, K., Padolina, W.G., 1992. “ Premnaodoroside A, B and C, Iridoid Glucoside Diester of an Acylic Monoterpenediol from Premna odorata”. Phytochemistry. 31(9), 3129–3133. Panlilio, B.G., Macabeo, A.P.G., Knorn, M., Kohls, P., Richomme, P., Kouam, S.F., Gehle, D., Krohn, K., Franzblau, S.G., Zhang, Q., Aguinaldo, M.A.M., 2012. A lanostane aldehyde from Momordica charantia. Phytochemistry Letters 5 (3), 682-684. Park, Y., Moon, B.H., Yang, H., Lee, H., Lee, E., Lim, Y., 2007. Spectral Assignments and Reference Data Complete assignments of NMR data of 13 hydroxymethoxyflavones. Magnetic Resonance Chemistry 45, 1072–1075. Pilcher, H., 2005. Drug giant to offer new, cut-price tuberculosis treatment. The Lancet Infectious Diseases 5 (12), 744. doi:10.1016/S1473-3099(05)70286-3. Pinzon, L.C., Uy, M.M, Sze, K.H., Wang, M., Chu, I.K., 2011. Isolation and characterization of antimicrobial, anti-inflammatory and chemopreventive flavones from Premna odorata Blanco. Journal of Medicinal Plants Research. 5(13), 2729-2735. Seidel, V., Taylor, P., 2004. In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria. International Journal of Antimicrobial Agents 23, 613–619. Quisumbing, E., 1978. Medicinal Plants of the Philippines., Bureau of Printing, Manila. Villaflores, O.B., Macabeo, A.P.G., Gehle, D., Krohn, K., Franzblau, S.G., Aguinaldo, M.A.M., 2010. Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis. Pharmacognosy Magazine 6(24), 339-344. World Health Organization, 2012. Global Tuberculosis Report 2012. Retrieved from http://www.who.int/tb/publications/global_report/en/index.html. Table 1. MIC values of P. odorata extracts, fractions and compounds tested against M. tuberculosis H37Rv. Sample Crude methanolic extract

% Inhibition (128 µg/mL) -108

MIC90 (µg/mL)a >128

Hexane sub-extract DCM sub-extract n-Butanol sub-extract DCM fractions Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Fraction 10 Fraction 11 Fraction 12 Fraction 13 Fraction 14 Fraction 15 Fraction 16 Fraction 17 Fraction 18 Fraction 19 Fraction 20 1 2&3 4 Rifampin Isoniazid PA-824 a Minimum inhibitory concentration.

-48 -44 -35

>128 >128 >128

4 20 25 99 97 75 97 97 94 21 29 29 21 28 94 53 25 33 54 40 96 63 -3 100 92 99

>128 >128 >128 54 120 >128 117 113 83 >128 >128 >128 >128 >128 109 >128 >128 >128 >128 >128 8 >128 >128 0.05 0.23 0.12

 

Fig. 1. Structures of compounds 1-4 isolated from Premna odorata.  

Graphical Abstract Highlights: Premna odorata Blanco, an endemic Philippine medicinal plant, is a known traditional herbal medicine used to manage phlegm, stomachache, headache, cough and tuberculosis patients. Bioassay guided isolation led to the identification of compounds from the dichloromethane subfractions of the leaves of Premna odorata Blanco.

   

Research Highlights

 

•

Premna odorata is a Philippine medicinal plant used to treat tuberculosis patients.

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The crude and semicrude extracts showed poor inhibitory activity against M.tb H37Rv.

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Increased activity was observed for fractions eluted from the DCM sub-extract.

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Bioassay guided isolation led to active compounds from the DCM sub-fractions.

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Results provide scientific basis for the traditional use of P. odorata for TB.