1. Introduction

AJPS

American Journal of Plant Sciences

2158-2742

Scientific Research Publishing

10.4236/ajps.2017.85073

AJPS-75666

Articles

Biomedical&Life Sciences

Profiling of Secondary Metabolites in Aerial Parts of <i>Phanera bracteata</i>

Phansuang

Udomputtimekakul

¹Wilart

Pompimon

¹Wipanoot

Baison

¹Punchavee

Sombutsiri

¹Nutthapol

Funnimid

¹Airada

Chanadee

¹Samneang

Apisantiyakom

Laboratory of Natural Products, Faculty of Science and Center for Innovation in Chemistry, Lampang Rajabhat University, Lampang, Thailand

Department of Chemistry, Faculty of Science and Technology, Valaya-Alongkorn Rajabhat University, Patumthani, Thailand

17042017

080511001134February 14, 2017Accepted: April 23, April 26, 2017

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

The flavonoid 4’,5,7-trihydroxy-6,8-dimethylflavanone (farresol) and 3’,4’,-4trihydroxy chalcone (isoliquiritigenin) together with triterpenoid (friedelin) were isolated from the aerial parts of P. bracteata. The structure of these compounds was determined on the basis of spectroscopic data including UV, IR, 1-D and 2-D, NMR and mass spectral analysis. This is the first report on the occurrence of these compounds in this plant.

Fabaceae <i>Phanera bracteata</i> Flavonoids

1. Introduction

One of the largest plant families is Fabaceae or Leguminosae dispersed in approximately 650 genera and 18,000 species [1] , which are found in several areas of the world especially the tropics. There are three sub families of Fabaceae: Mimosoideae, Faboideae and Caesalpinoideae [1] . Many of these species are applied as folk medicine and feed in remote areas all over the world [1] . Phytochemical investigation of the Fabaceae family has been widely accomplished by different groups and the presence of flavonoids [1] , alkaloids [2] , stilbene [3] , terpenoids [4] , phenolic compounds [5] , acylated triterpene glycosides [6] and nitro compounds [7] has been reported. Many of these constituents were found to show antimalarial [8] , antitumour [9] , anti-inflammatory [10] [11] , antihyperlipidemic [12] , anti-cancer [13] , antibacterial [14] , insecticidal [15] and antimicrobial activities [16] . Phanera bracteata Benth or Bauhinia bracteata (Graham ex Benth.) Baker is a woody climber tree native of the tropical area, located mainly in the Southeast Asian region [17] . The vine of P. bracteata is used in folk medicine for the treatment of irritated skin and as a rash ointment. There has been no previous report on the chemical investigation for this species. In the present study, we report herein the isolation and characterization of friedelin (1), farresol (2) and isoliquiritigenin (3) from the aerial parts of P. bracteata.

2. Materials and Methods2.1. General Procedures

Melting points were determined on a Büchi 322 micro melting point apparatus and remain uncorrected. Infrared spectra (IR) were recorded on KBr pellets with a Shimadzu 8900 FT-IR spectrophotometer. Silica gel 60 H (E. Merck. 70 - 230 mesh ASTM, cat. No. 7734) and Sephadex LH-20 (20 - 150 μm) were used for column chromatography. TLC analysis was performed on aluminium sheets of silica gel 60 PF₂₅₄ and the compounds were visualized under ultraviolet light. ¹H- and ¹³C-NMR spectra were recorded in CDCl₃ and CD₃OD solutions on a BrÜker AV-500 spectrometer. Chemical shifts are in δ (ppm) with tetramethylsilane (TMS) as an internal standard. Low resolution mass spectra were recorded on a Thermo Finnigan Polaris Q mass spectrometer at 70 eV (probe) and EIMS were measured with a BrÜker Esquire apparatus.

2.2. Plant Material

The aerial parts (fully grown trees) of P. bracteata were collected in May 2016 from tropical dry dipterocarp forest, Huai Yang Waterfall National Park, Prachuap Khiri Khan Province and authenticated by Mr. Narong Nuntasaen. The aerial parts were air-dried for one week and ground to a powder. A voucher specimen (BKF No. 147626) was deposited at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand.

2.3. Extraction and Isolation

The air-dried powdered aerial parts of P. bracteata (12 kg) were percolated with hexane (25 litters × 3 days × 5 times) and then extracted with ethyl acetate (25 litters × 3 days × 5 times) at room temperature, respectively, followed by filtration (cotton wool). The filtrates were combined and evaporated under reduced pressure to afford the hexane (31.21 g) and ethyl acetate extracts (314.40 g) respectively.

The hexane extract was chromatographed on a silica gel column with gradient mixtures of hexane: ethyl acetate (100:0 to 70:30). Fractions were collected and combined on the basis of TLC characteristic technique. The solvents were evaporated to dryness to afford four fractions (F₁-F₄). F₂ (2.88 g) was recrystallized from 95% ethanol to give compound (1) (470 mg) as white needles.

The ethyl acetate extract was subjected to column chromatography over silica gel, eluted with hexane, gradually enriched with ethyl acetate (100:0 to 0:100) to give fractions. Fraction (4.17 g) showed similar TLC characteristic to fraction (4.79 g), hence they were combined and subjected to column chromatography over silica gel eluted with a gradient hexane:ethyl acetate (95:5 to 80:20) to yield seven subfractions (B₁-B₇). Fraction B₂ was separated on a silica gel column eluted with a gradient of hexane:ethyl acetate (90:10 to 10:90) to obtain six subfractions (C₁-C₆). Fraction C₄ (1.71 g) was fractionated by Sephadex LH-20 (methanol) to afford six subfractions (An₁-An₆). Then, An₅ was recrystallized from 95% ethanol to provide compound (2) (107 mg) as yellow needles. Fraction (4.75 g) was purified by column chromatography over silica gel, eluted with various proportions of hexane: ethyl acetate (100:0 to 40:60) to give four subfractions (E₁-E₄). Fraction E₂ (0.24 g) was subjected to column chromatography over silica gel, eluted with hexane:ethyl acetate (100:0 to 50:50), followed by methanol in ethyl acetate (10:90) to afford three subfractions (G₁-G₃). Fraction G₂ (0.70 g) was subjected to repeated column chromatography by Sephadex LH-20 (methanol) to give three subfractions (Bn₁-Bn₃). Subfraction Bn₂ was recrystallized from 95% ethanol to yield compound (3) (89 mg) as pale yellow needles.

3. Results

Chromatographic separations of the hexane and ethyl acetate extracts from the aerial parts of P. bracteata afforded compound (1)-(3) respectively. The structure of these compounds was elucidated by comparison of ¹H and ¹³C NMR spectral data with those in previous reports [18] [19] [20] [21] [22] .

4. Discussion

Compound (1) was obtained from the crude hexane extract as white needles (470 mg) with m.p. 253˚C - 254˚C. The EI-MS gave the molecular ion peak at m/z 426, which indicated the molecular formula C₃₀H₅₀O. Comparison of the NMR spectral data with those in the literature [18] suggested that the compound (1) is a pentacyclic triterpene substituted by an oxo group at position 3 and by methyl groups at the 4, 5, 9, 13, 14, 17, and 20-positions. The ¹³C-NMR spectrum showed thirty carbons resonances. The DEPT spectrum indicated the presence of a ketone carbonyl at δ 213.2, eight methyl, eleven methylene, four methine and six quaternary carbons. By comparison of the ¹³C-NMR spectral data with previous reported spectral data [18] , the Compound (1) was identified as friedelin. Compound (2) was isolated from the crude ethyl acetate extract of P. bracteata as yellow needles (107 mg) with m.p. 210˚C - 211˚C. The EI-MS gave the molecular ion peak at m/z 300, which indicated the molecular formula C₁₇H₁₆O₅. The ¹H NMR spectral data of (2) showed resonances at δ 12.29 (OH), 7.35 (2H), 6.85 (2H), 5.31 (1H) 3.07 (1H), 2.72 (1H) 2.02 (3H), and 2.00 (3H) in agreement with the reported spectral data [18] . The ¹³C NMR spectral data of (2) showed seventeen carbons resonances, which were assigned to a ketone carbonyl at δ 197.0, four oxygenated carbon atoms at 157.4, 157.9, 158.9 and 162.7, four aromatic carbon atoms at δ 114.9 and 127.4, one methylene at δ 42.7, one oxymethine carbon atoms at δ 78.6, four quaternary aromatic carbon atoms at δ 101.9, 102.7, 103.4 and 130.1 and two methyl carbon atoms for the remaining signals at δ 6.0 and 6.7 respectively. The ¹H and ¹³C NMR spectral data of compound (2) were consistent with those of farresol [18] , hence the structure of compound (2) was determined to be farresol. Furthermore, compound (3) was obtained from the crude ethyl acetate as yellow needles (89 mg) with m.p. 192˚C - 193˚C and considered to be characteristic flavonoidal chalcone constituents of the Leguminosae family [23] . The EI-MS of (3) gave the M+1 peak at m/z 256, which indicated the molecular formula C₁₅H₁₁O₄. The ¹H NMR spectral date of (3) showed signals δ 8.00 (1H), 7.81 (1H), 7.65 (2H), 7.64 (1H), 6.87 (2H), 6.44 (1H), and 6.31 (1H). The ¹³C NMR spectral data of (3) showed fifteen carbon resonances, which were assigned to a ketone carbonyl at δ 192.2, three oxygenated carbon atoms at 160.1, 165.0 and 166.1, seven aromatic carbon atoms at δ 102.4, 107.8, 115.5, 130.4 and 132.0, two methine carbon atoms at δ 117.0 and 144.2 and two quaternary aromatic carbon for the remaining signals at δ 113.3 and 126.5 respectively. The ¹H and ¹³C NMR spectral data of compound (3) were consistent with those of isoloiquiritigenin [19] [20] [21] [22] , hence the structure of compound (3) was determined to be isoliquiritigenin.

Friedelin (1)

White needles (EtOH). C₃₀H₅₀O, m.p. = 253˚C - 254˚C [18] , EI-MS (m/z 426 [M]⁺ (20), 273 (48), 231 (83), 91 (100). ¹H NMR (CDCl₃, 500 MHz): was in agreement with [18] , ¹³C NMR (CDCl₃, 125 MHz): δ 213.2 (C-3), 59.6 (C-10), 58.3 (C-4), 53.3 (C-8), 43.0 (C-18), 42.3 (C-5), 41.7 (C-2), 41.4 (C-6), 39.8 (C-13), 39.4 (C-22), 38.4 (C-14), 37.6 (C-9), 36.2 (C-16), 35.8 (C-11), 35.5 (C-19), 35.2 (C-29), 32.9 (C-21), 32.6 (C-15), 32.2 (C-28), 31.9 (C-30), 30.6 (C-12), 30.2 (C-17), 28.3 (C-20), 22.4 (C-1), 20.4 (C-26), 18.8 (C-27), 18.3 (C-7), 18.1 (C-25), 14.8 (C-24), 6.9 (C-23).

4’,5,7-Trihydroxy-6,8-dimethylflavanone (Farresol) (2)

Yellow needles (MeOH). C₁₇H₁₆O₅, m.p. = 210˚C - 211˚C [19] , EI-MS (m/z) 300 [M]⁺ (17), 299 (100), 282 (12), 207 (27), 194 (60), 180 (75), 167 (28), 152 (76), 124 (51), 95 (33), 77 (21). UV (MeOH) λ_max (log ε): 416 (4.15), 317 (4.34), 214 (4.70), 210 (4.66) nm. IR (KBr) n_max 3794, 3448, 1637, 1600, 1521, 1323, 1191, 1176, 1124, 974, 945 cm⁻¹. ¹H NMR (CD₃OD, 500 MHz): δ 12.29 (br s, 5- OH), 7.35 (AA’BB’, J6’5’ = 8.6, H-6’), 7.35 (AA’BB’, J2’3’ = 8.6, H-2’), 6.85 (AA’BB’, J5’6’ = 8.6, H-5’), 6.85 (AA’BB’, J3’2’ = 8.6, H-3’), 5.31 (dd, J = 3.0, 12.9, H-2), 3.07 (dd, J = 12.9, 17.0, H-3ax), 2.72 (dd, J = 3.0, 17.0, H-3eq), 2.02 (s, 6- CH₃), 2.00 (s, 8-CH₃), ¹³C NMR (CD₃OD, 125 MHz): δ 197.0 (C-4), 162.7 (C-7), 158.9 (C-5), 157.9 (C-4’), 157.4 (C-9), 130.1 (C-1’), 127.4 (C-2’, 6’), 114.9 (C-3’, 5’), 103.4 (C-6), 102.7 (C-8), 101.9 (C-10), 78.6 (C-2), 42.7 (C-3), 6.7 (8-CH₃), 6.0 (6-CH₃), HMBC correlations, H/C: 7.35 (H-6’)/C-5’, 4’, 2’, 2; 7.35 (H-2’)/C- 6’, 4’, 3’, 2; 6.85 (H-5’)/4’, 3’, 1’; 6.85 (H-3’)/5’, 4’, 1’; 5.31 (H-2)/C-6’, 4, 2’, 1’; 3.07 (H-3ax)/C-4, 2, 1’; 2.72 (H-3eq)/C-4, 1’; 2.02 (6-CH₃)/C-7, 5); 2.00 (8- CH₃)/C-9. COSY correlations, H/H: 3’/2’; 5’/6’; 2ax/3ax, 3eq; 3ax/3eq.

3’,4’,4-Trihydroxychalcone (Isoliquiritigenin) (3)

Pale yellow needles (EtOH). C₁₅H₁₁O₄, m.p. = 192˚C - 193˚C [19] , EI-MS (m/z) 256 [M+1]⁺ (100), 241 (20), 164 (43), 152 (51), 138 (44), 121(31), 91 (34),

Figure 1 Chemical structure of Friedelin (1), Farresol (2) and Isoliquiritigenin (3)

65 (21), UV (MeOH) λ_max (log ε): 380 (1.86), 245 (1.15) nm. IR (KBr) n_max 3457, 1610, 1500, 1363, 1261, 1215, 1170, 1122, 520 cm⁻¹. ¹H NMR (CD₃OD, 500 MHz): δ 8.00 (d, J = 9.2, H-6’), 7.81 (d, J = 15.2, H-β), 7.65 (AA’BB’, J2,3 = 8.7, H-2), 7.65 (AA’BB’, J6,5 = 8.7, H-6), 7.64 (d, J = 15.2, H-α), 6.87 (AA’BB’, J3,2 = 8.7, H-3), 6.87 (AA’BB’, J5,6 = 8.7, H-5), 6.44 (dd, J = 2.7, 9.2, H-5’), 6.31 (d, J = 2.7, 3’), ¹³C NMR (CD₃OD, 125 MHz): δ 192.2 (C = O), 166.1 (C-2’), 165.0 (C-4’), 160.1 (C-4), 144.2 (C-β), 132.0 (C-6’), 130.4 (C-2, C-6), 126.5 (C-1), 117.0 (C-α), 115.5 (C-3, C-5), 113.3 (C-1’), 107.8 (C-5’), 102.4 (C-3’), HMBC correlations, H/C: 8.00 (H-6’)/C-1’, 2’, 4’, 5’, β’; 7.81 (H-β)/C-1, 2, 6, a, β’; 7.65 (H- 2)/C-1, 3, 4, 6, β; 7.65 (H-6)/C-1, 4, β; 7.64 (H-α)/C-1, β, β’; 6.87 (H-3)/C-1, 4, 5; 6.87 (H-5)/C-1, 3, 4; 6.44 (H-5’)/C-1’, 3’, 4’; 6.31 (H-3’)/C-1’, 2’, 4’, 5’. COSY correlaions, H/H: 5’/6’; β/α; 2/3; 6/5; 5’/3’ (Figure 1).

We have thus added two more flavonoids to the flavonoid constituents of P. bracteata. It is notable that flavonoids were found in the aerial part of this plant. The occurrence of flavonoids and chalcones may be a characteristic useful for further chemotaxonomic studies of the genus Phanera.

5. Conclusion

Our study of the aerial parts of P. bracteata (Fabaceae) led to the isolation and characterization of three compounds. These results reinforce the previous studies showing that the genus Phanera is considered a good source of flavonoids. We would like to note here that Friedelin (1), Farresol (2) and Isoliquiritigenin (3) were isolated for the first time from this genus. (Appendix)

Acknowledgements

This work was funded by a grant from Lampang Rajabhat University, through a Science Faculty Research Grant (04/2016). Our thanks are also due to Center for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Mrs. Suttiporn Pikulthong for measurement of NMR and MS.

Cite this paper

Udomputtimekakul, P., Pompimon, W., Baison, W., Sombutsiri, P., Funnimid, N., Chanadee, A. and Apisantiyakom, S. (2017) Profiling of Secon- dary Metabolites in Aerial Parts of Phanera bracteata. American Journal of Plant Sci- ences, 8, 1100-1134. https://doi.org/10.4236/ajps.2017.85073

Appendix FiguresFigure S1 Farresol-MS spectrum

Figure S2 Farresol-NMR spectrums.

Figure S3 Fridelin-NMR spectrums.

Figure S4 Friedelin-MS-spectrumFigure S5 Isoliquiritigenin-MS spectrum

Figure S6 Isoliquiritigenin-NMR spectrum.

References1

De Oliveira, J.C.S., David, J.P. and David, J.M. (2016) Biflavonoids from the Bark Roots of Poincianella pyramidalis (Fabaceae). Phytochemistry Letters, 16, 18-22. https://doi.org/10.1016/j.phytol.2016.02.017

Dade, J.M.E., Guessan, G.I.-N., Komlaga, G., Say, M., Okpekon, T.A., Boti, J.B., Kablan, B.J. and Bamba, E.H.S. (2016) Pyrrolidine Alkaloids and Their Glycosylated Derivatives from the Root Bark of Dichrostachys cinerea (L) Wight & Arn. (Fabaceae). Phytochemistry Letters, 16, 268-276. https://doi.org/10.1016/j.phytol.2016.05.002

Wanjala, C.C.W. and Majinda, R.R.T. (2001) A New Stilbene Glycoside from Elephantorrhiza Goetzei. Fitoterapia, 72, 649-655. https://doi.org/10.1016/S0367-326X(01)00295-7

Srinivasan, S., Wankhar, W., Rathinasamy, S. and Rajan, R. (2016) Free Radical Scavenging Potential and HPLC Analysis of Indigofera tinctoria Linn (Fabaceae). Journal of Pharmaceutical Analysis, 6, 125-131. https://doi.org/10.1016/j.jpha.2015.04.003

Kinfe, H.H., Long, H.S., Stander, M.A. and Wyk, B.-E.V. (2015) The Major Phenolic Compound of the Roots and Leaves of Rafnia amplexicaulis (Fabaceae). South African Journal of Botany, 100, 75-79. https://doi.org/10.1016/j.sajb.2015.05.014

Kozhamkulova, Z.A., Radwan, M.M. and Zhusupova, G.E. (2011) Halimodendrin I, a New Acylated Triterpene Glycoside from Halimodendron halodendron (Fabaceae). Phytochemistry Letters, 4, 323-327. https://doi.org/10.1016/j.phytol.2011.06.004

Na, C.S., Lee, Y.H., Kim, T.W. and Murai, Y. (2015) Aliphatic Nitro Compounds from Roots of Astragalus sikokianus (Fabaceae). Biochemical Systematics and Ecology, 60, 120-122. https://doi.org/10.1016/j.bse.2015.04.008

Kittakoop, P., Kirtikara, K., Tanticharoen, M. and Thebtaranonth, Y. (2000) Antimalarial Preracemosols A and B, Possible Biogenetic Precursors of Racemosol from Bauhinia malabarica Roxb. Phytochemistry, 55, 349-352. https://doi.org/10.1016/S0031-9422(00)00318-6

Rajkapoor, B., Jayakar, B. and Murugesh, N. (2003) Antitumour Activity of Bauhinia variegata on Dalton’s Ascetic Lymphoma. Journal of Ethnopharmacology, 89, 107-109. http://doi.org/10.1016/S0378-8741(03)00264-2

Gupta, M., Mazumder, U.K., Kumar, R.S., Gomathi, P., Rajeshwar, Y., Kakoti, B.B. and Selven, V.T. (2005) Anti-Inflammatory, Analgesic and Antipyretic Effects of Methanol Extract from Bauhinia racemosa Stem Bark in Animal Models. Journal of Ethnopharmacology, 98, 267-273. http://doi.org/10.1016/j.jep.2005.01.018

Campos, J.K.L., Araujo, C.S.F., Araujo, T.F.S., Santos, A.F.S., Teixeira, J.A., Lima, V.L.M. and Coelho, L.C.B.B. (2016) Anti-Inflammatory and Antinociceptive Activities of Bauhinia monandra Leaf Lectin. Biochimie Open, 2, 62-68. http://doi.org/10.1016/j.biopen.2016.03.001

Kumar, D., Parcha, V., Maithani, A. and Dhulia, I. (2012) Effect and Evaluation of Antihyperlipidemic Activity Guided Isolated Fraction from Total Methanol Extract of Bauhinia variegata (Linn). In Triton WR-1339 Induced Hyperlipidemic Rats. Asian Pacific Journal of Tropical Disease, 2, S909-S913.

Yuenyongsawad, S., Bunluepuech, K., Wattanapiromsakul, C. and Tewtrakul, S. (2013) Anti-Cancer Activity of Compounds from Bauhinia strychnifolia Stem. Journal of Ethnopharmacology, 150, 765-769. http://doi.org/10.1016/j.jep.2013.09.025

Pandey, S. (2015) Preliminary Phytochemical Screening and in Vitro Antibacterial Activity of Bauhinia variegata Linn. against Human Pathogens. Asian Pacific Journal of Tropical Disease, 5, 123-129. https://doi.org/10.1016/S2222-1808(14)60639-3

Poonsri, W., Plempanupat, W., Chitchirachan, P., Bullangpoti, V. and Koul, O. (2015) Insecticidal Alkanes from Bauhinia scandens Var. horsfieldii against Plutella xylostella L., (Leppidoptera: Plutellidae). Industrial Crops and Products, 65, 170-174. http://doi.org/10.1016/j.indcrop.2014.11.040

Sakthivel, M. and Palani, P. (2016) Isolation, Purification and Characterization of Antimicrobial Protein from Seedlings of Bauhinia purpurea L. International Journal of Biological Macromolecules, 86, 390-401. http://doi.org/10.1016/j.ijbiomac.2015.11.086

Larsen, K., Larsen, S.S. and Vidal, J.E. (1984). Leguminosae-Caesalpinioideae. Flora of Thailand, 4, 31.

Sousa, G.F., Duarte, L.P., Alcantara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M. and Takahashi, J.A. (2012) New Triterpenes from Maytenus ro-busta: Structural Elucidation Based on NMR Experimental Data and Theoretical Calculations. Molecules, 17, 13439-13456. https://doi.org/10.3390/molecules171113439

Youssef, D.T.A., Ramadan, M.A. and Khalifa, A.A. (1998) Acetophenones, a Chacone, a Chromone and Flavonoids from Pancratium maritimum. Phytochemistry, 49, 2579-2583. http://doi.org/10.1016/S0031-9422(98)00429-4

Ma, C.-J., Li, G.-S., Zhang, D.-L., Liu, K. and Fan, X. (2005) One Step Isolation and Purification of Liquiritigenin and Isoliquiritigenin from Glycyrrhiza uralensis Risch. Using High-Speed Counter-Current Chromatography. Journal of Chromatography A, 1078, 188-192. http://doi.org/10.1016/j.chroma.2005.01.053

Sano, S., Okubo, Y., Handa, A., Nakao, M., Kitaike, S., Nagao, Y. and Kakegawa, H. (2011) Reinvestigation of the Synthesis of Isoliquiritigenin: Application of Horner-Wadsworth-Emmons Reaction and Claisen-Schmidt Condensation. Chemical and Pharmaceutical Bulletin, 59, 885-888. https://doi.org/10.1248/cpb.59.885

Khamsan, S., Liawruangrath, S., Teerawutkulrag, A., Pyne, S.G., Garson, M.J. and Liawruangrath, B. (2012) The Isolation of Bioactive Flavonoids from Jacaranda obtusifolia H.B.K. ssp. rhombifolia (G.F.W. Meijer) Gentry. Acta Pharmaceutica, 62, 181-190.

Wollenweber, E. and Dietz, V.H. (1981) Occurrence and Distribution of Free Flavonoid Aglycones in Plants. Phytochemistry, 20, 869-932. https://doi.org/10.1016/0031-9422(81)83001-4