Pharmacognosy Research

RESEARCH ARTICLE
Year
: 2009  |  Volume : 1  |  Issue : 5  |  Page : 261--269

Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis


Oliver B Villaflores1, Allan Patrick G Macabeo1, Dietmar Gehle2, Karsten Krohn2, Scott G Franzblau3, Alicia M Aguinaldo1,  
1 Phytochemistry Laboratory, Research Center for the Natural Sciences, Thomas Aquinas Research Complex, University of Santo Tomas, Espaa, Manila 1015, Philippines
2 University of Paderborn, Department of Chemistry, Warburgerstrasse 100, 33098 Paderborn, Germany
3 Institute for TB Research, College of Pharmacy, MC 964 Rm 412, University of Illinois at Chicago, 833 S. Wood St., Chicago, Illinois 60612-7231, USA

Correspondence Address:
Alicia M Aguinaldo
Phytochemistry Laboratory, Research Center for the Natural Sciences, Thomas Aquinas Research Complex, University of Santo Tomas, Espaa, Manila 1015
Philippines

Abstract

Alpinia purpurata or red ginger was studied for its phytochemical constituents as part of our growing interest on Philippine Zingiberaceae plants that may exhibit antimycobacterial activity. The hexane and dichloromethane subextracts of the leaves were fractionated and purified using silica gel chromatography to afford a mixture of C 28 to C 32 fatty alcohols, a 3-methoxyflavone and two steroidal glycosides. The two latter metabolites were spectroscopically identified as kumatakenin (1), sitosteryl-3-O-6-palmitoyl-β-D-glucoside (2) and β-sitosteryl galactoside (3 ) using UV, IR, EIMS and NMR experiments, and by comparison with literature data. This study demonstrates for the first time the isolation of these constituents from A. purpurata. In addition to the purported anti-inflammatory activity, its phytomedicinal potential to treat tuberculosis is also described.



How to cite this article:
Villaflores OB, Macabeo AG, Gehle D, Krohn K, Franzblau SG, Aguinaldo AM. Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis.Phcog Res 2009;1:261-269


How to cite this URL:
Villaflores OB, Macabeo AG, Gehle D, Krohn K, Franzblau SG, Aguinaldo AM. Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis. Phcog Res [serial online] 2009 [cited 2019 Sep 15 ];1:261-269
Available from: http://www.phcogres.com/text.asp?2009/1/5/261/58052


Full Text

 Introduction



Several species of the genus Alpinia were reported to exhibit fungicidal, antioxidant and bactericidal properties [1],[2] . Alpinia purpurata (Vieill.) K. Schum (Family Zingiberaceae) is locally known in the Philippines as "luyang pula" or red ginger, and is a native to the Pacific [3]. Studies on its chemical constituents revealed the presence of α-pinene, β-pinene [4] , 1,8-cineole, (E)- methylcinnamate [5] , 6-shogaol, 8-gingerol, 6-gingerol, 10-gingerol, 10-shogaol and 4-shogaol [6] . A U.S. patent reported that its total anthocyanidin, shogaol and gingerol content shows promise in the treatment of inflammatory diseases such as arthritis [6],[7] .

With limited literature available as to the phytochemistry and biological activity of A. purpurata, and with the growing interest in Philippine Zingiberaceae species that inhibit the growth of Mycobacterium tuberculosis H37Rv [8],[9] , we embarked on further exploration on the isolation and identification of secondary metabolites from this Alpinia species. In addition to the fatty alcohol mixture, we report in this paper the chromatographic purification and spectroscopic identification of a flavone and two sitosteryl glycosides namely, kumatakenin 1, sitosteryl-3- O-6-palmitoyl-β-D-glucoside 2 and β-sitosteryl galactoside 3 from A. purpurata. The inhibitory activity against M. tuberculosis H37Rv of the extracts, fractions and the purified compounds is also presented.

 Results and Discussion



The MABA assay [10] result of the crude ethanolic extract of the various parts of Alpinia purpurata had shown the leaf extract to possess the highest activity followed by the rhizome and flower extracts. Among the sub-extracts, the DCM sub-extract exhibited the highest activity followed by hexane and n-butanol sub-extracts. All fractions obtained from the hexane and DCM sub-extracts showed low to moderate activity [Table 1].

Further chromatographic work-up was undertaken on fraction two of the hexane sub-extract, which afforded a white amorphous solid after crystallization. This compound was distinctly identified from the 1 H-NMR and 13 C-NMR spectra to be a fatty alcohol, but the LR-EIMS spectrum otherwise showed it to be a mixture of fatty alcohols. By careful analysis of the m/z values in the mass spectrum, it could be claimed that it is composed of montanyl alcohol (C28:0), melissyl alcohol (C30:0, major component) and domelissyl alcohol (C32:0) based on several characteristic peaks due to fragment ions of [M + -H 2 O] [11] .

The dichloromethane extract was likewise subjected for further investigation owing to its interesting phytochemical profile. Nine fractions after VLC were obtained from which fractions three and four yielded three solid compounds 1-3 .

Compound 1, a yellow crystalline substance (13.0 mg), was purified from fraction three after recrystallization. It was found to be a flavonoid after treating its TLC chromatograms with FeCl 3 -K 4 Fe(CN) 6 and 10% SbCl 3 in chloroform as shown by a blue-green spot and red-orange fluorescence under UV (365 nm), respectively [12] . In addition, major absorptions at 269 (Band I) and 352 nm (Band II) and a weak shoulder at 303 nm, which are typical for flavones, were observed in its UV spectrum [13],[14] . The structure, and degree and pattern of oxygenation in the flavonoid structure were examined by studying the effect of several wavelength shift reagents, NaOMe, NaOAc, NaOAc-1% aq. H 3 BO 3 , AlCl 3 and AlCl 3 -HCl in the UV spectral region of 1. With NaOMe, a 44 nm bathochromic shift and a significant increase in absorbance intensity were noted for Band II. This indicates the presence of a C-4' hydroxyl group. Treatment with NaOAc gave no observable change in the spectrum which shows that an alkoxy substituent is present in the C-7 position of the flavone nucleus. Addition of 1% boric acid produced no change in the spectrum which is symptomatic of the absence of ortho-hydroxyphenolic functionalities. This was also substantiated by the result of adding 0.1M HCl/ AlCl 3 in a separate experiment. The appearance of four absorption peaks (278, 304, 352 and 399 nm) after the complexation of AlCl 3 with 1 is a clear indication that a 5-hydroxyl moiety is present [14] .

The IR spectrum of 1 showed the presence of an enone (1665 cm -1 ) and phenolic OH's (3243 cm -1 ). The base peak at m/z 314 in the LR-EIMS mass spectrum was designated themolecular-ion peak. In the500 MHz 1 H NMR spectrum, two sets of AA'BB'-protons belonging to a para-substituted benzenoid moiety, two methoxy protons and meta-coupled protons were noted. In the proton-decoupled 13 C and DEPT-135 NMR spectrum, a total of 18 carbon atoms were accounted for 1 from which a conjugated ketone, six oxygenated olefinic/aromatic carbons, six aromatic methines and two methoxy carbons were deduced. The gross structure of the compound which is analogous to kumatakenin [15],[16] was elucidated via an HMBC experiment. Key 1 H- 13 C correlations are shown in [Figure 2].

It is noteworthy to report the isolation of kumatakenin (1) from A. purpurata. This rare ethyl ether flavonol was first isolated from the seeds of A. japonica [17] and A. kumatake [18] . Hence, the identification of 1 strengthens the chemical link of A. purpurata with the other species of Alpinia.

Compound 2 was afforded as a white amorphous solid (8.4 mg). TLC chromatograms of the isolate sprayed with Liebermann-Burchard and Molisch reagents [12] suggested a steroidal glycoside structure. The IR spectrum showed the presence of hydroxyl (3439 cm -1 ) and ester (1738 cm -1 ) functionalities. The molecular ion peak was not observed in the LR-EIMS spectrum. Instead, fragment ions corresponding to sitosterol (m/z 414, C 29 H 50 O) and a palmitoxy group (m/z 256, C 16 H 31 O 2 ) were noted. Signals characteristic of sitosterol resonances i.e. C-6 olefinic proton (δ 5.36), methyls associated to the cholestane skeleton(δ0.69-0.79), glucose(δ4.34 -4.38) and a palmitoyl group (δ 0.84, 1.26, 2.34) were evident in the 500 MHz 1 H NMR of 2. A total of 51 carbon atoms, of which 7 CH 3 , 25 aliphatic CH 2 , 7 aliphatic CH, two aliphatic quaternary C, one oxygenated C H 2 , 6 oxygenated CH and one each olefinic CH and C, were found in the 125 MHz 13 C NMR spectrum. HMBC correlations which were instrumental in finding the correct identity of 2 as sitosteryl-3-O-6-palmitoyl-β-D-glucoside are shown in [Figure 3]. The NMR values of 2 are in well agreement with those reported for sitosteryl-3-O-6-palmitoyl-β-D-glucoside by Pei-Wu et al. [19] , Gomes et al. [20] , and Shaiq Ali et al. [21] .

Metabolite 3 was obtained as white crystalline flakes (4.1 mg). The partial identity of 3 was revealed to be a OH steroidal glycoside as in 2 using the same phytochemical experiments. Only the occurrence of hydroxyl functionalities was inferred this time from the IRspectrum. The presence of a sitosterol fragment was ascertained in the LR-EIMS spectrum which was also verified by the 1 H and 13 C NMR spectra. The sugar moiety was deduced to be galactose upon comparison of the 1 H-NMR and 13 C-NMR values with those in literature [22],[23],[24] . Hence, the identity of 3 was established.

Sitosteryl glycosides 2-3 have been isolated from other several plant sources [19],[21] . These metabolites are also present in plant species belonging to the family Zingiberaceae [25] .

The fatty alcohols showed an MIC value of 64 μg/ mL and proved most active compared to the flavonoid kumatakenin and the steroidal glycosides previously reported to have MIC values > 128 μg/mL. Long chain alcohols show growth inhibitory activity to Gram-positive organisms including Staphylococcus aureus and Propionibacterium acnes [26] . Moreover, a study done by Togashi and co-workers in 2007 further supported the antibacterial activity of long chain aliphatic alcohols that had bactericidal activity and membrane-damaging activity on Staphylococcus aureus. Experimental results indicate that the antibacterial activity of long chain alcohols is mediated by damage to cell membranes that allows leakage of K + ions, with subsequent reactions that induce further leakage [27] . Thus, the ability of the isolated fatty alcohols to inhibit the growth of M. tuberculosis H 37 Rv gives further credence on the antibacterial activity of long chain alcohols particularly those bearing more than twenty carbon chains.

The activity of kumatakenin confirms the study by Murillo et al. [28] on its action against M. tuberculosis H 37 Rv. This compound has antiviral activity against HIV [29] , the virus that aggravates the problem on tuberculosis due to susceptibility to the lung pathogen. Plant sterols particularly β-sitosterol and its glucosides have been investigated as immune regulators of T-cell activity [30] and as agents in maintaining the CD4+ count in the absence of anti-retroviral therapy in HIV-infected patients [31] . Plant sterols were effective in patients treated for pulmonary TB, causing increase in their peripheral blood lymphocytes and eosinophil counts [32] .

To date, this is the first report of 2-3 from the genus Alpinia. More importantly, this paper cites for the first time the isolation of all compounds from A. purpurata. Furthermore, this study demonstrates the promise of this plant as a source of phytomedicinals that can fight TB.

 Methodology



General

The leaves of Alpinia purpurata (Vieill.) K. Schum. were collected in Los Baρos, Laguna (February 2004). Herbarium specimens (USTH 4717) were kept at the Botany Laboratory of the Research Center for the Natural Sciences, Thomas Aquinas Research Complex, University of Santo Tomas, Manila, Philippines.

Electron impact (EI) mass spectral analysis was carried out in JEOL D-300 FD mass spectrometer using m-nitrobenzyl alcohol/CHCl 3 as carrier at 60C. Proton ( 1 H) and 13 C NMR measurements were recorded in JEOL GX 400 MHz NMR spectrometer using CDCl 3 (δ 7.26 for 1H, δ 77.0 for 13 C) as internal reference.

Extraction and Isolation

The air-dried leaves of Alpinia purpurata (1.7 kg) were extracted with ethanol to give a crude extract (378 g) that was partitioned according to increasing polarity using n-hexane, dichloromethane and n-butanol.

The hexane extract (53.4 g) was subjected to VLC by gradient elution (20% increments) using hexane/ethyl acetate to give 11 fractions. Fraction 2 (8.4 g) was purified by GCC and gradient elution (5% increments) with hexane/chloroform and chloroform/acetone, to give four fractions. Sub-fraction 2 was further purified and gave 19 fractions. A solid in the eighth fraction was recrystallized in acetone to afford a white amorphous solid (10 mg) of a fatty alcohol mixture.

The dichloromethane extract (2.5 g) was subjected to vacuum liquid chromatography (VLC) (Si gel HF 254 Merck Art. 1.07739) by gradient elution (20% increments) using chloroform, chloroform-acetone, acetone and acetone-methanol to furnish nine fractions.

After evaporation of fraction 3, yellow needle-like crystals appeared and were recrystallized in acetone to give 1 (13 mg). UV Spectral analysis was also done on compound 1 using various shift reagents to determine the aromatic substitutions of the compound. Fraction four (458 mg) was gravity column chromatographed (GCC) (Si gel 60 Merck Art. 1.07734, 25 mm I.D.) by gradient elution with benzene-acetone (5:1); (5:2); (5:3), neat acetone, acetone-methanol (1:1) and neat methanol to give nine fractions. Sub-fraction eight gave an amorphous white powder that was recrystallized in acetone to give 2 (8.4 mg). Concentration and recrystallization of sub-fraction 6 in methanol afforded 3 (4.1 mg) as white flakes.

Fatty alcohols . 1 H NMR (500 MHz, CDCl 3 ) 3.57 (t, J 6.6Hz, H-1), 1.49 (m, H-2), 1.18 (br s), 0.81 (t, J 7Hz). 13 C NMR (DEPT-135, 125 MHz, CDCl 3 ) 63.1 (C-1), 32.8 (C-2), 31.9 (C-3), 29.7, 25.7(C-3), 22.6, 14.1. LR-EIMS m/z: 392.5 (C 28 H 57 OH-H 2 O) + , 420.5 (C 30 H 61 OH-H 2 O) + , 448 (C 32 H 65 OH-H 2 O) +

Kumatakenin (1) , yellow needles (13 mg), m.p. 248- 249C (uncorr., lit. 246-247C)(14). 1 H NMR (500 MHz, CDCl 3 /CD 3 OD) 3.69 (3H, s, 3-OMe), 3.78 (3H, s, 7-OMe), 6.24 (1H, d, J 2Hz, H-6), 6.37 (1H, d, J 2Hz, H-8), 6.82 (2H, d, J 9Hz, H-3', H-5'), 7.89 (2H, d, J 9Hz, H-2', H-6'. 13 C NMR (DEPT-135, 125 MHz, CDCl 3 /CD 3 OD) 156.7 (C-2), 138.4 (C-3), 178.7 (C-4), 161.3 (C-5), 97.8 (C-6), 165.4 (C-7), 92.2 (C-8), 156.8 (C-9), 105.8 (C-10), 121.3 (C-1'), 130.2 (C-2'), 115.5 (C-3', C-5'), 159.9 (C-4'), 130.2 (C-2', C-6'), 59.9 (3-OMe), 55.6 (7-OMe). LR-EIMS m/z: 314.1 (M + ), 271.1, 256.2, 167.0, 149.0, 97.1, 57.1, 43.0.

Sitosteryl-3-O-6-palmitoyl-β-D-glucoside (2), white amorphous powder (8.4 mg). 1 H NMR (500 MHz, CDCl 3 /CD 3 OD) Aglycone 3.58 (1H, m, H-3), 5.36 (1H, m, H-6), 0.70 (3H, s, 18-CH 3 ), 1.03 (3H, s, 19- CH 3 ), 0.94 (3H, d, J 6.5Hz, 21- CH 3 ), 0.85 (3H, d, J 6.8Hz, 26 CH 3 ), 0.84 (3H, d, J 6.8Hz, 27- CH 3 ), 0.90 (3H, t, J 6.9Hz, 29- CH 3 ) Sugar 4.38 (1H, d, J 7.7Hz, H-1'), 3.34 (1H, m, H-2'), 3.58 (1H, m, H-3'), 3.34 (1H, m, H-4'), 3.48 (1H, ddd, J 2Hz, 5Hz, 10Hz, H-5'), 4.29 (1H, dd, J 2Hz, 12Hz, H-6'a), 4.48 (1H, dd, J 5Hz, 12Hz, H-6'b) Fatty Acid 2.37 (2H, t, J 7.5Hz, H-2"), 1.72 (2H, m, H3"), 1.28 (24H, broad s, H-4"-15"), 0.87 (3H, t, J 7Hz, H-16"). 13 C NMR (DEPT-135, 125 MHz, CDCl 3 /CD 3 OD ) Aglycone 37.3 (C-1), 31.9 (C-2), 79.6 (C-3), 38.9 (C-4), 140.3 (C-5), 122.2 (C-6), 31.9 (C-7), 31.9 (C-8), 50.2 (C-9), 36.7 (C-10), 21.2 (C-11), 39.8 (C-12), 42.2 (C-13), 56.8 (C-14), 25.0 (C-15), 28.2 (C-16), 56.1 (C-17), 11.9 (C-18), 19.4 (C-19), 36.2 (C-20), 19.0 (C-21), 34.0 (C-22), 26.1 (C-23), 45.9 (C-24), 29.2 (C-25), 18.8 (C-26), 19.8 (C-27), 23.1 (C-28), 12.0 (C-29) Sugar 101.2 (C-1'), 73.6 (C-2'), 76.0 (C-3'), 70.1 (C-4'), 74.0 (C-5'), 63.2 (C-6') Fatty acid 174.7 (C=O), 34.2 (C-2"), 24.3 (C-3"), 28.9- 29.7 (C-4"-14"), 22.6 (C-15"), 14.1 (C-16"). LR-EIMS m/z: 414.7 (aglycone), 396.4, 381.3, 368.4, 329.3, 284.3,256.3, 241.2, 239.2, 227.0, 213.2.

β-sitosteryl galactoside (3) , white flakes (4.1 mg). 1 H NMR (500 MHz, CDCl 3 / CD 3 OD) Aglycone 3.47 (1H, m, H-3), 5.25 (1H, m, H-6), 0.57 (3H, s, 18- CH 3 ), 0.90 (3H, s, 19- CH 3 ), 0.73 (3H, d, J 7Hz, 21- CH 3 ), 0.70 (3H, d, J 7Hz, 26CH 3 ), 0.82 (3H, d, J 7.8Hz, 27- CH 3 ), 0.74 (3H, t, J 7.8Hz, 29- CH 3 ) Sugar 4.29 (1H, d, J 8Hz, H-1'), 3.12 (1H, dd, J 8Hz, 9Hz, H-2'), 3.32 (1H, dd, J 1Hz, 9Hz, H-3'), 3.32 (1H, dd, J 1Hz, 7Hz, H-4'), 3.18 (1H, m, H-5'), 3.63 (1H, dd, J 5Hz, 12Hz, H-6'a), 3.73 (1H, dd, J 3Hz, 12Hz, H-6'b). 13 C NMR (DEPT-135, 125 MHz, CDCl 3 /CD 3 OD) Aglycone 37.2 (C-1), 29.6 (C-2), 79.0 (C-3), 38.6 (C-4), 140.2 (C-5), 122.0 (C-6), 31.0 (C-7), 31.0 (C-8), 50.1 (C-9), 36.6 (C-10), 21.0 (C-11), 39.7 (C-12), 42.2 (C-13), 56.6 (C-14), 24.1 (C-15), 28.1 (C-16), 56.0 (C-17), 11.7 (C-18), 19.5 (C-19), 36.0 (C-20), 19.1 (C-21), 33.9 (C-22), 26.0 (C-23), 45.7 (C-24), 29.4 (C-25), 18.6 (C-26), 18.8 (C-27), 22.9 (C-28), 11.8 (C-29) Sugar 101.1 (C-1'), 73.4 (C-2'), 76.3 (C-3'), 70.2 (C-4'), 75.6 (C-5'), 61.7 (C-6'). LR-EIMS m/z 414.4 (aglycone), 397.4, 396.4, 381.4, 329.3, 288.3, 255.2, 213.2. Assignments were made by comparison with published data and confirmed by HMQC / COSY experiments.

Screening for antituberculosis activity

A Microplate Alamar Blue Assay (MABA) as described in the protocol of Collins and Franzblau [10] was used for anti-TB susceptibility of the extracts, fractions and purified compounds. Mycobacterium tuberculosis H 37 Rv (ATCC 27294; American Type Culture Collection, Rockville MD) was grown at 37C on a rotary shaker in Middlebrook 7H9 broth supplemented with 2% glycerol and 0.05% vv -1 Tween 80 until the culture density reached an optical density of 0.45-0.55 at 550 nm. Bacteria were pelleted, washed twice, resuspended in Dulbecco's phosphate-buffered saline, then filtered (8 μm) and aliquots frozen at -80C. After a night, the stocks were thawed, sonicated and successively diluted to get the CFU. Rifampin was obtained from Sigma and stock solutions were made in accordance to the manufacturer's instructions. The assay was performed in black, clear-bottomed, 96-well microplates (Black view plates: Packard Instrument company, Meriden, Conn.) in order to reduce background fluorescence. Initial drug dilutions were prepared in either dimethyl sulfoxide or distilled ionized water, and subsequent twofold dilutions were performed in 0.1 mL of 7H9GC (no Tween 80) in the microplates. BACTEC 12B-passaged inocula were initially diluted 1:2 in 7H9GC, and 0.1 mL was placed onto the wells. Frozen inocula were diluted 1:20 in BACTEC 12B medium followed by a 1:50 dilution in 7H9GC. Wells containing drug were used to monitor autofluorescence of compounds. Additional control wells consisted of bacteria only (B) and medium only (M). Plates were incubated at 37C. At day 4 of incubation were added 20 μL of 10x Alamar Blue solution (Alamar Biosciences/Accumed, Westlake, Ohio) and 12.5 μL of 20% Tween 80 to one B well and one M well, and plates were reincubated at 37 C. Detection of color change in wells was monitored at 12 and 24 h from blue to pink and for a measurement reading of > or equal to 50,000 fluorescence units (FU). Cytofluor II microplate flurometer (PerSeptive Biosystems, Framingham, Mass.) inbottom-readingsettingat530nmforexcitationand590 nm for emission was used in fluorescence measurement. In case that a pink color was observed with B wells after 24h, the colorimetric reagent was added to the entire plate. If a blue color persisted in the well or a reading of or equal to 90% was assigned as the MIC.

 Acknowledgments



This study was supported by the Philippine Council for Advanced Science and Technology Research and Development, Department of Science and Technology, Manila, and by the Department of Chemistry - College of Science, University of Santo Tomas, Manila. The authors thank Dr. Yuehong Wang for the MABA, Ms. Bernadette Macalino and Mr. Emerson Espadero for initial studies and Prof. Mary Garson for useful discussions.

Supplementary data for compounds 1-3

Compound 1 (kumatakenin)

Physical Properties of 1 :

Yellow needles, Rf: 0.5 in benzene acetone (6.4:3.6); 0.72 in diethyl ether-ethyl acetate (7:3), 0.37 in benzene-chloroform-methanol (5:4:1) and 0.38 in chloroform-acetone (9:1)

Melting point: 248-249 C (uncorrected, lit. 246-247 C) (Kimura et al., 1967)

UV: 269 nm (logε = 9.32) 352 nm (logε = 9.44)

IR: 3243.86 cm -1 , 1665.45 cm -1 , 1600.76 cm -1 , 1600.79 cm -1

LR-EIMS: m/z (rel. int.) m/z 314 (100)(M+ ), 271 (25), 167 (38), 149 (55), 97 (24), 57 (68), 43 (67).

Compound 2 and 3

Physical Properties of ( 3 )

White amorphous solid, Rf: 0.19 in benzene-acetone (6.4:3.6); 0.20 in diethyl ether-ethyl acetate (7:3), 0.15 in benzene-chloroform-methanol (5:4:1)

Melting point: not determined

UV: not determined

IR: 3419.45 cm -1 , 2559.3 cm -1 , 1073.94 cm -1

LR-EIMS: m/z 396, 381, 329, 255.2

Physical properties of ( 2 )

White amorphous solid, Rf: 0.13 in benzene- acetone (6.4:3.6); 0.26 in diethyl ether-ethyl acetate (7:3), 0.31 in benzene-chloroform-methanol (5:4:1) and 0.05 in chloroform-acetone (9:1)

Melting point: not determined

UV: 220 nm (logε = 6.43)

IR: 3439.20 cm -1 , 1738.78 cm -1 , 1600.76 cm -1 , 2923.87 cm -1 , 2852.06 cm -1 , 1467.34 cm -1

LR-EIMS: Fragment ion peaks at m/z 414, 396, 381, 368, 329.3, 256.3, 241.2 and 227. [Table 2], [Table 3], [Figure 1]

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