Analysis of homoisoflavonoids in Caesalpinia digyna by HPLC-ESI-MS, HPLC and method validation

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PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected]

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PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

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Analysis of Homoisoflavonoids in Caesalpinia digyna by HPLC-ESI-MS, HPLC and Method Validation Somendu K. Roy, Amit Srivastava and Sanjay M. Jachak* Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector- 67, SAS Nagar, Mohali- 160062 (Punjab), India [email protected], [email protected]

Received: May 17th, 2012; Accepted: July 18th, 2012

The roots of Caesalpinia digyna have been reported to contain gallic acid derivatives and minor homoisoflavonoids, but HPLC-ESI-MS and HPLC analyses of the homoisoflavonoids were challenging due to their low concentration in the roots. Separation and identification was accomplished by HPLC-ESI-MS and further elaborated for quantification using a C18 column with detection at 330 nm. A gradient mobile phase consisting of methanol and water (0.1% acetic acid) was used. The developed HPLC method showed good linearity (r2≥0.998), high precision (RSD<5%) and a good recovery (99.3-104.5%) of the compounds. The lowest detection limit was 0.75 ng and the method was found to be robust. All the validation parameters were found to be within the permissible limits and, therefore, the developed method is accurate and reliable for the quality control of C. digyna and other Caesalpinia species. This is the first report of sample preparation on Diaion HP-20 resin and characterization of homoisoflavonoids by HPLC-ESI-MS, extended by extensive quantitative HPLC analysis of homoisoflavonoids in C. digyna roots and method validation. Keywords: Caesalpinia digyna, HPLC, HPLC-ESI-MS, Homoisoflavonoids, Method validation.

Caesalpinia digyna Rottler (Leguminosae), commonly known as ‘Vakerimul’, is one of the ingredients of “Geriforte”, an ayurvedic formulation that has been used as a restorative tonic in old age and for curing senile pruritus, with excellent results [1]. The roots of C. digyna have been reported to possess antimicrobial, antioxidant, hypoglycaemic and hypolipidemic properties [2-5]. The detailed phytochemical investigation of C. digyna roots recently by us resulted in the isolation and characterization of fourteen polyphenolics that includes flavonoids, gallic acid derivatives and homoisoflavonoids [6]. Other Caesalpinia species, such as C. bonducella, C. pulcherrima and C. milletii, are also reported to contain homoisoflavonoids [7-10]. The content of homoisoflavonoids in cell suspension cultures of C. pulcherrima [11a], and inter-bulb surfaces of Scilla nervosa were analyzed by HPLC [11b]. On the basis of literature reports, we aimed to develop a simple, accurate and robust HPLC analytical method for the simultaneous determination of homoisoflavonoids (1-8) in C. digyna roots (Figure 1). As these homoisoflavonoids exist as isomers, HPLC-ESI-MS was used for their exact identification. The quantification of the homoisoflavonoids (1-8) in C. digyna has not been previously reported. Therefore, a reliable HPLC method was developed for the separation and determination of these compounds in homoisoflavonoid-rich fractions prepared from the methanol extracts of two different C. digyna root samples. After several attempts, a gradient mobile phase eluting with 0.1% acetic acid (A) and methanol (B) was optimized on a C18 reversed phase column to give a stable base line, and good peaks and resolution of all eight homoisoflavonoids in HRF-01 and HRF-01´ (Figure 2). The presence of homoisoflavonoids (1-8) in methanol extracts was confirmed by HPLC-ESI-MS prior to method validation; these were detected as protonated molecular ion [M+H]+ peaks in the MS (Table 1).

O

OR1

R2

R3OCH3

O

OR1

R2

R3

OCH3(2) R1= H, R2= R3= OH(4) R1= H, R2= OH, R3= OCH3(6) R1= R3= H, R2= OH(8) R1= R2= OH, R3= H

(1) R1= H, R2= R3= OH(3) R1= H, R2= OH, R3= OCH3(5) R1= R3= H, R2= OH(7) R1= R2= OH, R3= H

Figure 1: Homoisoflavonoids (1-8) detected in C. digyna roots. Table 1: Chromatographic and spectrometric characteristics of compounds 1-8 in the HRF-01 fraction of C. digyna.

Compound tR(min) λmax [M+H]+ (m/z) Isointricatinol (1) 20.35 347.3 298.99 Intricatinol (2) 21.59 355.6 299.00 Z-8-methoxybonducellin (3) 34.06 348.5 313.02 E-8-methoxybonducellin (4) 36.70 358.0 313.05 Isobonducellin (5) 37.47 355.6 282.97 Bonducellin (6) 38.65 363.9 283.03 Z-eucomine (7) 41.72 363.9 299.02 E-eucomine (8) 44.89 360.9 298.99

The validation of the developed method was accomplished using International Conference of Harmonization (ICH) guidelines [12]. Five point calibration curves were prepared for compounds 1-8 in the specified concentration range. The slopes, the intercepts and the correlation coefficients (r2) were calculated by regression analysis (Table 2). The correlation coefficient was found to be ≥0.998 for all the compounds studied, which indicated a high degree of correlation and good linearity for the method.The lowest value for LOD was observed for Z-eucomine (0.75 ng/mL), whereas the lowest LOQ value was found for Z-eucomine and Z-8-methoxybonducellin (2.00 ng/mL). For intra-day variations, the %RSD values of 1-8 were found in the range of 0.92-2.62%, while in the case of inter-day variations, the values were in the range 0.69-3.04%. These %RSD values are within the permissible limits and reflected the precision

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1190 Natural Product Communications Vol. 7 (9) 2012 Roy et al.

Table 2: Calibration curve, LOD, LOQ, precision, recovery and stability data of compounds 1-8 by HPLC.

Parameters 1 2 3 4 5 6 7 8

Concentration range (ng/mL) 31.2-4000 39.1-5000 39.1-5000 39.1-10000 31.2-8000 39.1-10000 31.2-2000 39.1-1250 Intercept 43943 -11078 24509 25274 80429 20940 -43531 78227 Slope 5306 3500 3163 3429 4255 2916 3508 2632 Correlation coefficient (r2) 0.999 0.999 0.999 0.999 0.999 0.998 0.998 0.999 LOD (ng/mL) 0.78 1.00 1.00 2.76 1.25 1.25 0.75 1.67 LOQ (ng/mL) 2.40 5.55 2.00 6.45 3.60 3.75 2.00 6.50 Intra-day (n=9) (%RSD) Day 1 0.92 2.11 1.68 2.62 2.43 2.17 1.01 2.17 Inter-day (n=9) (%RSD) Day 2 0.78 2.65 2.32 1.34 2.76 1.28 1.53 1.27 Inter-day (n=9) (%RSD) Day 3 0.69 2.42 1.94 1.57 3.04 1.35 2.25 1.35 Recovery [Mean±SD(%RSD)] (n=9) 101.27±1.22(1.20) 100.46±1.10(1.10) 101.83±2.29(2.25) 101.37±3.07(3.03) 104.53±2.30(2.21) 103.95±2.95(2.83) 99.27±2.98(2.99) 101.62±2.39(2.35)Stability (RSD%, n=6) 2.68 3.76 2.70 3.50 2.87 3.08 3.32 1.17

Figure 2: HPLC chromatograms of A) methanol extract (Sample 1); B) polar fraction of C. digyna methanol extract (Sample 1); C) HRF-01 and HRF-01´ of C. digyna methanol extracts detected at 330 nm (Samples 1 and 2).

of the method. The average recoveries for the determined compounds (1-8) were between 99.3-104.5% with a %RSD≤3.03%, which indicated good recovery of the respective samples, thus proving the effectiveness of the reported method (Table 2). The robustness of an analytical method is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For compounds 1-8 the %RSD values were found in the range of 0.080-0.897 for the mobile phase, 0.405-0.839 for the flow rate, 0.052-2.97 for the temperature, and 0.222-1.229 for the mobile phase gradient. As the observed values of %RSD were less than 5% for all the four studied parameters, the method developed could be considered as robust.

Table 3: Content (mg/g) of 1, 2, 5 and 6 in methanol extract and enriched fraction (HRF-1) of sample 1.

Compound HRF-01 [Mean±SD(%RSD)]

Methanol extract of sample 1 [Mean±SD (%RSD)]

Isointricatinol (1) 0.0265±0.0003(1.46) 0.0243±0.0008(3.40) Intricatinol (2) 0.0141±0.0002(1.10) 0.0147±0.0007(4.83) Isobonducellin (5) 0.0085±0.0001(1.31) 0.0084±0.0001(2.88) Bonducellin (6) 0.0049±0.0001(1.25) 0.0052±0.0002(3.88)

Table 4: Contents (mg/g) of 1-8 in two samples of C. digyna roots.

Compound HRF-01 [Mean±SD (%RSD)]

HRF-01´ [Mean±SD (%RSD)]

Isointricatinol (1) 0.0265±0.0003(1.46) 0.0179±0.0002(1.23) Intricatinol (2) 0.0141±0.0002(1.10) 0.1094±0.0030(2.76) Z-8-Methoxybonducellin (3) 0.0085±0.0001(1.31) 0.0563±0.0012(2.17) E-8-Methoxybonducellin (4) 0.0049±0.0001(1.25) 0.1817±0.0037(2.05) Isobonducellin (5) 0.0084±0.0001(2.88) 0.0389±0.0008(2.06) Bonducellin (6) 0.0052±0.0002(3.88) 0.2069±0.0042(2.04) Z-Eucomine (7) 0.0249±0.0003(0.13) 0.0201±0.0005(2.66) E-Eucomine (8) 0.0114±0.0001(1.48) 0.0112±0.0003(2.75)

The %RSD values for solution phase stability of compounds 1-8 were found to be in the range of 1.17-3.76, which demonstrated a good stability in methanol solution within the tested time period (Table 2). The proposed method was utilized for the determination of compounds 1-8 in two different samples of C. digyna roots. The results showed that homoisoflavonoids 1-8 were present in very low amounts in both samples (Figure 2A). Earlier, the bergenin content had been analyzed by LC-MS and found to be 39% in the methanolic extract of C. digyna [4].Therefore, prior to quantification of homoisoflavonoids by HPLC, methanolic extracts of the samples were loaded onto a Diaion HP-20 resin column to remove the major compounds, bergenin and 11-O-galloylbergenin. These fractions were considered to be polar and devoid of homoisoflavonoids (Figure 2B). Since there is ≥ 95.3% recovery obtained after loading the extracts on Diaion resin HP-20, the developed sample preparation method is reliable. The reliability of the sample preparation method was further confirmed by quantifying the homoisoflavonoids (1, 2, 5 and 6) in the methanol extract of sample 1 in which these were present at above the LOQ. The content of the homoisoflavonoids (1, 2, 5 and 6) in the methanol extract and enriched fraction HRF-01 of sample 1 were found almost similar, thus indicating the reliability of the sample preparation method using Diaion HP-20 resin (Table 3). A similarly enriched fraction of sample 2 was prepared and the homoisoflavonoids quantified (Figure 2C; Table 4). The sample preparation method was repeated three times for both samples on Diaion HP-20 resin to check reproducibility. The E-8-methoxybonducellin content in sample 2 showed significant variation when compared with sample 1. The homoisoflavonoids (1, 2, 4-8) in sample 2 showed less variation when compared with sample 1 (Table 4). These variations might be due to environmental and genetic factors.

Analysis of homoisoflavonoids in Caesalpinia digyna Natural Product Communications Vol. 7 (9) 2012 1191

In conclusion, the sample preparation using Diaion HP-20 resin allowed enrichment of homoisoflavonoids in C. digyna methanolic extracts. All the homoisoflavonoids were identified by LC-ESI-MS from their molecular ion peaks, and were further quantified in the enriched extracts of C. digyna roots. The developed HPLC analytical method was validated using the ICH guidelines for all the validation parameters. The %RSD values for all the validation parameters were found to be within the permissible limits and, therefore, the developed HPLC method is accurate, reliable and robust for the quality control of C. digyna. This validated method may also be used for the determination of E-and Z-isomers of homoisoflavonoids in other Caesalpinia species. Experimental

Materials: Two samples of C. digyna roots were purchased from Abirami Botanicals Corporation, Tuticorin, India (Sample 1) and SrijeeAyurved, Mumbai, India (Sample 2). The roots were authenticated by botanist Dr A. S. Sandhu, Department of Natural Products (DNP), NIPER and voucher specimens (NIPM-CD-013 and NIPM-CD-005) were deposited at the herbarium of DNP, NIPER, S.A.S. Nagar, India. Diaion resin (HP-20) was purchased from Supelco, PA, USA. HPLC grade methanol was obtained from J.T. Baker (Phillipsburg, USA) and ultrapure water (18 mΩ) was obtained from the Millipore system (Billerica, USA). Acetone was purchased from Merck (Mumbai, India), and acetic acid from CDH (Mumbai, India). Instrumentation: HPLC analysis was performed on a Waters 600 HPLC system equipped with a Delta 600 quaternary solvent pump, an in-line degasser, a 717 plus autosampler, a temperature control module, a 2996 photodiode array (PDA) detector and EmpowerTM2 software (Waters, Milford, MA, USA). The separation was carried out on C18 Symmetry column (4.6×250 mm, 5.0 µm) (Waters, Milford, MA, USA). HPLC-ESI-MS analysis was performed on a Thermo Scientific LTQ XL (Thermo Finnigan, San Jose, CA, USA) system equipped with an ACCELA autosampler, pump, PDA detector and linear ion trap mass analyzer. Sample preparation: Two samples of coarsely powdered roots (50 g) of C. digyna were refluxed separately with methanol for 2 h to give crude extracts. Each extract was further dissolved in 10 mL of methanol and loaded onto a Diaion HP-20 resin (50 g) column to remove gallic acid derivatives. The column was eluted with 60% methanol in water (1000 mL) to give polar fractions. The column was then eluted with methanol (250 mL) followed by acetone (200 mL). The methanolic and acetone solutions were pooled and concentrated on a rotary evaporator to give homoisoflavonoid rich fractions (HRF-01 and HRF-01´). The solutions (5 mg/mL) of HRF-01 and HRF-01´ were used for the analysis of compounds 1-8. The prepared sample solutions were sonicated and filtered through a 0.45 µm membrane prior to analysis. The sample solutions of HRF-01 and HRF-01´ were stored at -20C until analysis. Reference standards of homoisoflavonoids (1-8): Isointricatinol (1), intricatinol (2), Z-8-methoxybonducellin (3), E-8-methoxy-bonducellin (4), isobonducellin (5), bonducellin (6), Z-eucomine (7), and E-eucomine (8) previously isolated by us were used as reference standards [6]. Preparation of standard solutions: Stock solutions of homoisoflavonoids (1-8) (1 mg/mL) were prepared separately in methanol. All standard solutions were stored at -20C until analysis. The prepared standard solutions were sonicated and filtered through a 0.45 µm membrane filter prior to analysis.

HPLC and HPLC-ESI-MS analysis: The separation of HRF-01 and HRF-01´ was carried out on a C18 column using 0.1% acetic acid (solvent A) and methanol (solvent B) as mobile phase in gradient mode. The gradient elution profile used was: 0 min, 30% B; 3 min, 50% B; 8 min, 54% B; 28 min, 54% B; 38 min, 70% B; 45 min, 70% B; 45.01 min, 30%; 50 min, 30% B. The column was equilibrated with 30% B for 10 min before the next injection. The 20 µL of standards and sample were injected into the chromatographic system and the flow rate used was 1 mL/min. The analysis was carried out by maintaining the column oven at 30C. Homoisoflavonoids (1-8) were detected at 330 nm. The MS were obtained between m/z 0.00-500.00. The MS parameters were set as: ESI in positive ion mode; sheath gas flow rate: 40 L/min; aux. gas flow rate: 20 L/min; spray voltage: 5kV; capillary temperature: 250C; capillary voltage: 2 V; nebulizer: 40 psi. The HPLC peaks at tR 20.35, 21.59, 34.06, 36.70, 37.47, 38.65, 41.72 and 44.89 min, showed λmax of 347.3, 355.6, 348.5, 358.0, 355.6, 363.9, 363.9 and 360.9 nm, and exhibited [M+H]+ peaks at m/z 298.99, 299.00, 313.02, 313.05, 282.97, 283.03, 299.02 and 298.99, respectively. Validation of HPLC method: The HPLC method for the determination and quantification of homoisoflavonoids1-8 was validated in accordance with the ICH guidelines for linearity, precision, recovery, robustness and stability [12]. Linearity, LOD and LOQ: The linearity was performed for each compound separately and the concentration range was chosen on the basis of the expected values in the study. The calibration curves were generated from 5 concentrations of each compound, each in duplicate; calibration curves, regression coefficients, slopes and intercepts were calculated. The calibration curves thus obtained were used to quantify the respective compounds in HRF-01 and HRF-01´. LOD and LOQ of 1-8 were calculated based on the signal to noise (S/N) ratios of 3 and 10, respectively. Precision: For intra-day precision, the individual compound (1-8) was analyzed at 3 different concentrations in triplicate within one day. For inter-day precision, the standards were analyzed in triplicate on 3 consecutive days. The precision values were expressed by relative standard deviation (%RSD). Accuracy: Accuracy was determined using a recovery study performed by adding a known amount of individual homoisoflavonoids (1-8) at low, medium and high concentration into a 5 mg/mL solution of HRF-01. The 3 concentrations were analyzed in triplicate and the results expressed as the percentage recovery of the added analytes/compounds. Robustness: The robustness of the method for the analysis of compounds 1-8 was checked by making some deliberate changes in the chromatographic parameters. The changes made were: concentration of acetic acid in water (0.08 and 0.12%), flow rate (0.8 and 1.2 mL/min), and temperature (25 and 35 °C). The changes made in the mobile phase gradient were: 0 min, 29% B; 3 min, 49% B; 8 min, 53% B; 28 min, 53% B; 38 min, 69% B; 45 min, 69%B; 45.01 min, 29% B; 50 min, 29% B and 0 min, 32% B; 3 min, 52% B; 8 min, 56% B; 28 min, 56% B; 38 min, 72% B; 45 min, 72%, 45.01 min, 32% B; 50 min, 32% B. Each parameter was analyzed in triplicate and the variations in the retention time were noted. The variations were analyzed and expressed as the %RSD of the 3 determinations with respect to normal retention time.

1192 Natural Product Communications Vol. 7 (9) 2012 Roy et al.

Stability: The 0.1 mg/mL solutions of compounds 1-8 were stored at 25°C and 20 µL of each solution was injected into the HPLC system at intervals of 0, 2, 4, 8, 12 and 24 h, respectively, to evaluate stability of the compounds in the solution phase. The stability data are expressed as relative standard deviation (%RSD) of 3 determinations. Statistical analysis: The concentrations of compounds 1-8 in HRF-01 and HRF-01´ were determined from the respective areas

under the peaks obtained from the calibration curves and the data have been expressed as mean ± SD (%RSD). The intra- and inter-day variations for the homoisoflavonoids 1-8 have been expressed as %RSD, while LOD and LOQ of 1-8 were calculated using the HPLC software (EmpowerTM2 System Suitability, Waters) and expressed as ng/mL. Acknowledgments - Authors thank Director, NIPER for providing financial assistance and necessary facilities to carry out this work.

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[8] Ata A, Gale EM, Samarasekera R. (2009) Bioactive chemical constituents of Caesalpinia bonduc (Fabaceae). Phytochemistry Letters, 2, 106-109. [9] Srinivas K, Koteswara-Rao Y, Mahender I, Das B, Rama-Krishna KVS, Hara-Kishore K, Murty USN. (2003) Flavanoids from Caesalpinia

pulcherrima. Phytochemistry, 63, 789-793. [10] Chen P, Yang JS. (2007) Flavonolgalactosidecaffeate ester and homoisoflavones from Caesalpinia millettii HOOK. et ARN. Chemical and

Pharmaceutical Bulletin, 55, 655-657. [11] (a) Zhao P, Iwamoto Y, Kouno I, Egami Y, Yamamoto H. (2004) Stimulating the production of homoisoflavonoids in cell suspension cultures of

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Insecticidal Properties of Annonaceous Acetogenins and Their Analogues. Interaction with Lipid Membranes Lilian Di Toto Blessing, Juan Ramos, Sonia Diaz, Aída Ben Altabef, Alicia Bardón, Margarita Brovetto, Gustavo Seoane and Adriana Neske 1215

Activity of Alkanediol Alkanoates against Pathogenic Plant Fungi Rhizoctonia solani and Sclerotium rolfsii Paraj Shukla, Suresh Walia, Vivek Ahluwalia, Balraj S. Parmar and Muraleedharan G. Nair 1219

A Somaclonal Variant of Rose-Scented Geranium (Pelargonium spp.) with Moderately High Content of Isomenthone in its Essential Oil Swaroop S Kulkarni, Nagawara S Ravindra, Kalavagunta V N S Srinivas and Raghavendra N Kulkarni 1223

Comparative Study of the Chemical Composition of Essential Oils of Five Tagetes Species Collected in Venezuela Kaylin Armas, Janne Roja, Luis Rojas and Antonio Morales 1225

Chemical Composition and Antiphytoviral Activity of Essential Oil of Micromeria graeca Elma Vuko, Valerija Dunkić, Nada Bezić, Mirko Ruščić and Dario Kremer 1227

Chemical Composition and Biological Activity of the Essential Oil from Jamaican Cleome serrata Megil J. McNeil, Roy B. R. Porter and Lawrence A. D. Williams 1231

Chemical Compositions and Biological Activities of Amomum subulatum Essential Oils from Nepal Prabodh Satyal, Noura S. Dosoky, Brittany L. Kincer and William N. Setzer 1233

Chemical Composition and Antimicrobial Activity of Salvia x jamensis Essential Oil Daniele Fraternale, Guido Flamini, Angela Bisio, Maria Cristina Albertini and Donata Ricci 1237

Antimicrobial and Antioxidant Activities of Stachys lavandulifolia subsp. lavandulifolia Essential Oil and its Infusion Gökalp İşcan, Betül Demirci, Fatih Demirci, Fatih Göger, Neşe Kırımer, Yavuz B. Köse and Kemal Hüsnü Can Başer 1241

Composition, Anticancer, and Antimicrobial Activities in vitro of the Heartwood Essential Oil of Cunninghamia lanceolata var. konishii from Taiwan Yu-Chang Su, Kuan-Ping Hsu, Eugene I-Chen Wang and Chen-Lung Ho 1245

Review/Account Secondary Metabolites of Eichhornia crassipes (Waterhyacinth): A Review (1949 to 2011) Pottail Lalitha, Shubashini.K.Sripathi and Ponnusamy Jayanthi 1249

Natural Product Communications 2012

Volume 7, Number 9

Contents

Original Paper Page

Catalytic and Molecular Properties of Rabbit Liver Carboxylesterase Acting on 1,8-Cineole Derivatives María del H. Loandos, Ana C. Muro, Margarita B. Villecco, Marcelo F. Masman, Paul G.M. Luiten, Sebastian A. Andujar, Fernando D. Suvire and Ricardo D. Enriz 1117

Antiparasitic and Anticancer Carvotacetone Derivatives of Sphaeranthus bullatus Francis Machumi, Abiy Yenesew, Jacob O. Midiwo, Matthias Heydenreich, Erich Kleinpeter, Babu L. Tekwani, Shabana I. Khan, Larry A. Walker and Ilias Muhammad 1123

Naturally Occurring Limonene to Cinnamyl-type γ-Butyrolactone Substituted Aldol Condensation Derivatives as Antioxidant Compounds Pushpinder Kaur, Pralay Das, Abha Chaudhary and Bikram Singh 1127

Sesquiterpenes from Onopordum illyricum and their Antifeedant Activity Sergio Rosselli, Antonella Maria Maggio, Marisa Canzoneri, Monique S. J. Simmonds and Maurizio Bruno 1131

Three New Lactarane Sesquiterpenoids from the Mushroom Russula sanguinea Yasunori Yaoita, Moe Hirao, Masao Kikuchi and Koichi Machida 1133

Royleanumin, a New Phytotoxic neo-Clarodane Diterpenoid from Teucrium royleanum Shabir Ahmad, Muhammad Arfan, Naheed Riaz, Riaz Ullah, Ziarat Shah and Azhar Ul-Haq Ali Shah 1137

Bioactive-guided Identification of Labdane Diterpenoids from Aerial Parts of Aristeguietia glutinosa as anti-Trypanosoma cruzi agents Javier Varela, María L. Lavaggi, Mauricio Cabrera, Alejandra Rodríguez, Patricio Miño, Ximena Chiriboga, Hugo Cerecetto and Mercedes González 1139

Characterization of Yew Tree (Taxus) Varieties by Fingerprint and Principal Component Analyses Kalle Truus, Merike Vaher, Maria Borissova, Marju Robal, Tuuli Levandi, Rando Tuvikene, Peeter Toomik and Mihkel Kaljurand 1143

Identification of Triterpenes from Milkweed (Asclepias syriaca) Erzsébet Háznagy-Radnai, Edit Wéber, Szilvia Czigle and Imre Máthé 1147

Evaluation of the Anti-angiogenic Activity of Saponins from Maesa lanceolata by Different Assays Kenn Foubert, Annelies Breynaert, Mart Theunis, Rita Van Den Bossche, Guido R. Y De Meyer, André Van Daele, Ahmad Faizal, Alain Goossens, Danny Geelen, Edward M. Conway, Arnold Vlietinck, Luc Pieters and Sandra Apers 1149

Conversion of Protopanaxadiol Type Saponins to Ginsenoside Rg3 by Lemon Cheng-Peng Sun, Wei-Ping Gao, Bao-Zhong Zhao and Le-Qin Cheng 1155

Triterpene Glycosides from the Sea Cucumber Eupentacta fraudatrix. Structure and Biological Activity of Cucumariosides B1 and B2,Two New Minor Non-Sulfated Unprecedented Triosides Alexandra S. Silchenko, Anatoly I. Kalinovsky, Sergey A. Avilov, Pelageya V. Andryjaschenko, Pavel S. Dmitrenok, Ekaterina A. Martyyas and Vladimir I. Kalinin 1157

Osteoclastogenesis Inhibitory Effect of Ergosterol Peroxide Isolated from Pleurotus eryngii Satoru Yokoyama, Tran Hai Bang, Kuniyoshi Shimizu and Ryuichiro Kondo 1163

A Novel Sterol Sulfate and New Oligosaccharide Polyester from the Aerial Parts of Polygala sibirica Yue Lin Song, Si Xiang Zhou, He Lin Wei, Yong Jiang and Peng Fei Tu 1165

Angustinine – A New Benzopyridoquinolizine Alkaloid from Alangium lamarckii Mumu Chakraborty and Sibabrata Mukhopadhyay 1169

Antifungal Activity of Hydrochloride Salts of Tylophorinidine and Tylophorinine Mini Dhiman, Rajashri R. Parab, Sreedharannair L. Manju, Dattatraya C. Desai and Girish B. Mahajan 1171

Evaluation of Antibacterial and Anti-biofilm Activities of Cinchona Alkaloid Derivatives against Staphylococcus aureus Malena E. Skogman, Janni Kujala, Igor Busygin, Reko Leino, Pia M. Vuorela and Adyary Fallarero 1173

Simultaneous Determination of Alkaloids and Flavonoids from Aerial Parts of Passiflora Species and Dietary Supplements using UPLC-UV-MS and HPTLC Bharathi Avula, Yan-Hong Wang, Chidananda Swamy Rumalla, Troy J. Smillie and Ikhlas A. Khan 1177

Chemical Fingerprinting by RP-RRLC-DAD and Principal Component Analysis of Ziziphora clinopodioides from Different Locations Shuge Tian, Qian Yu, Lude Xin, Zhaohui Sunny Zhou and Halmuart·Upur 1181

Simultaneous Determination of Four Major Constituents of Semen Vaccariae Using HPLC-DAD Haijiang Zhang, Wei Yao, Yunyun Chen, Peipei He, Yao Chen, Peipei Chen, Jia Han and Xiaoyu Li 1185

Biological Activity of Isoflavonoids from Azorella madreporica Luisa Quesada, Carlos Areche, Luis Astudillo, Margarita Gutiérrez, Beatriz Sepúlveda and Aurelio San-Martín 1187

Analysis of Homoisoflavonoids in Caesalpinia digyna by HPLC-ESI-MS, HPLC and Method Validation Somendu K. Roy, Amit Srivastava and Sanjay M. Jachak 1189

Oligonol-induced Degradation of Perilipin 1 is Regulated through Lysosomal Degradation Machinery Junetsu Ogasawara, Kentaro Kitadate, Hiroshi Nishioka, Hajime Fujii, Takuya Sakurai, Takako Kizaki, Tetsuya Izawa, Hitoshi Ishida and Hideki Ohno 1193

Continued inside backcover