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Characterisation and some possible uses of Plukenetia conophora and Adenopus breviflorus seeds and seed oils
Bioresource Technology 85 (2002) 95–97
Short communication
Characterisation and some possible uses of Plukenetia conophora and Adenopus breviflorus seeds and seed oils E.T. Akintayo
*,1,
E. Bayer
Centre For Nucleic and Peptide Chemistry, Institute of Organic Chemistry, Universitat T€ubingen, Auf der Morgenstelle 18, 72076 T€ubingen, Germany Received 23 February 2002; received in revised form 7 March 2002; accepted 8 March 2002
Abstract Two non-conventional seeds, Plukenetia conophora (PKCP) and Adenopus breviflorus (ADB) were analysed for their proximate, fatty acids, sterols composition and physico-chemical characteristics. Crude protein was 25.65% for PKCP and 28.25% for ADB. ADB had lower moisture content (4.5%) than PKCP (8.0%) indicating that the former has better shelf life. Oil yields of the seeds were 49.58% for PKCP and 56.22% for ADB. The major sterols were stigmasterol and b-sitosterol in PKCP and ADB respectively. PKCP oil had 98.8% unsaturated fatty acids with linolenic acid predominating (70.1%) while ADB had 85.1% unsaturated fatty acids with linoleic acid being most abundant (65.3%). The very high saponification and iodine values of PKCP oil suggest its utilisation in alkyd resin, shoe polish, liquid soap and shampoo production. There is the possibility of using ADB oil in these regards as well as for edible purposes. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Plukenetia conophora; Adenopus breviflorus; Seed oil; Characteristics and uses
1. Introduction Plukenetia conophora (PKCP) (family Euphorbiacae) is a climbing shrub that is common in the South-western part of Nigeria. The seeds are eaten like wallnuts often along with maize. The leaves are edible and are often eaten with rice. The leaves are also used traditionally for curing headache and the fresh nuts are used for curing snakebites (Hutchinson and Dalziel, 1958). Much is known about the traditional use of this plant but very little on the characterisation and possible uses of its seed oil. The latter is part of the objectives of this study. Adenopus breviflorus (ADB) (family curcubitacae) is grown in the middle belt area of Nigeria. Like other members of the curcubitacae family, it possesses climbing stems. At maturity it produces fruits containing a varying number of seeds. The seeds are covered with a thin hull which when dried can easily be removed by hand. The seeds are mostly utilised as a soup ingredient in the Mid-western part of Nigeria. Oshodi (1992) has reported the proximate composition, nutritionally valuable minerals, and functional properties of the seeds flour and protein concentrate. The same author, Oshodi *
Corresponding author. Permanent address: Department of Chemistry, University of Ado-Ekiti, Ekiti-State, Nigeria. 1
(1996), also reported the amino and fatty acids composition of the plant seeds. In this work we report the characteristics and some possible uses of the ADB seeds and seed oil.
2. Methods Powdered seed samples were prepared by grinding in a C&N Junior laboratory mill size 5 (Christy and Norris limited, Engineers, Chelmsford England). Oil was extracted using chloroform/methanol (2:1) in a Soxhlet apparatus. Solvent was removed in a rotavapour at 35 °C. The extracted seed meals were thoroughly air dried to remove traces of solvent. The extracted seed oils were immediately analysed for iodine value, saponification value, acid value and unsaponifiable matter by methods described by the Association of Official Analytical Chemists (AOAC, 1984). Refractive indices of the oils (at room temperature) were determined with an Abbe refractometer and the specific gravity measurements were also done at room temperature using specific gravity bottles. The mean molecular mass was estimated from the relation ð56=SVÞ 1000 (Ajiwe et al., 1995). Fatty acid composition of the oils was determined as described by Akintayo (1997). The sterolic fractions obtained by TLC separation from the unsaponifiables of
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 7 3 - 1
96
E.T. Akintayo, E. Bayer / Bioresource Technology 85 (2002) 95–97
the oils were derivatised by refluxing with 20 ll of pyridine and bis(trimethylsilane)-trifluoroacetamide for 20 h at 80 °C. One ll of the derivatised sample was injected into a CP 9001 GC equipped with a flame ionisation detector and a SGE HTS capillary column (20 m, 0.22 m, 0.1 ll) coated with carborane polysiloxane. Injector and detector temperature were 280 and 340 °C. Temperature programming was 100 °C for 2 min, temperature increased at 5 °C/min up to 340 °C and held at 340 °C for 10 min. Proximate analysis of the seeds was carried out as described by the Association of Official Analytical Chemists (AOAC, 1995).
3. Results and discussion The proximate compositions of the seed flour of PKCP and ADB are presented in Table 1. Oil content of both is high, indicating that processing of their oils for industrial or edible purposes would be economical. They have low moisture content and this implies good shelf life characteristics. Going by their moisture content results, ADB will have a longer shelve life than PKCP. Their protein contents are comparable to those reported for some conventional oilseeds (Bailey’s Industrial Oil and Fat Products, 1964). They could, therefore, be recommended as protein supplements. It should, however, be borne in mind that the suitability of any plant material as food supplement depends on an interplay of factors like the digestibility of its nutrients and the presence of antinutritional factors. These concerns still need to be investigated with these oilseeds. The ash and crude fibre values are important in terms of the suitability of the seed cake for compounding of animal feeds. Although they both have favourable ash contents (<2.5%), that would have qualified them for compounding animal feeds, their low carbohydrate content renders them unsuitable for such purposes (Abighor et al., 1997). Table 2 presents the physico-chemical characteristics of the seeds oils. Both oils are yellow in colour. The iodine value of PKCP is very high (204.5 mg KOH/g) and places it in the drying group oil similar to linseed oil. Iodine value of ADB places it in the semi-drying group oil. These iodine values were confirmed by the Table 1 Proximate composition (%)a of PKCP and ADB seeds flours Assay
PKCP
ADB
Crude fat Crude protein Crude fibre Moisture Ash Carbohydrate by difference
49.58 1.14 23.65 0.62 6.00 0.22 8.00 0.12 2.10 0.24 10.67
56.22 0.86 28.25 0.24 3.40 0.14 4.50 0.11 1.30 0.10 6.33
a
Values are mean standard deviation of triplicate determinations.
Table 2 Physico-chemical characteristics of PKCP and ADB seeds oils Parameter
PKCP
ADB
Colour Free fatty acid (mg/g)a Acid value (mg KOH/g)a Saponification value (mg KOH/g)a Iodine value (mg iodine/g)a Mean molecular mass Unsaponifiable matter (%)a Refractive index (25 °C) Specific gravity (25 °C)
Light yellow 5.6 0.1 11.5 0.1 92.19 0.3
Light yellow 1.0 0.1 1.8 0.3 190.50 0.2
204.5 1.5 291.4 2.05 0.1 1.4835 0.9361
112.1 0.8 293.9 1.45 0.1 1.4721 0.9001
a
Values are mean standard deviation of triplicate determinations.
fatty acid composition of the oils presented in Table 3 which shows that PKCP oil consists of 98.8% unsaturated oil made up of mainly linolenic acid (70.1%) and ADB consists of 85.1% unsaturated oil with linoleic acid predominating (61.3%). Their iodine values suggest their use in alkyd resin production and other formulations like shoe polish. ADB oil may be of nutritional value with its 61.3% linoleic acid content, linoleic acid being an essential acid with cholesterol-lowering activity (Messink and Katan, 1992; Dery et al., 1993). Both oils show high saponification values suggesting their utilisation also in production of liquid soap and shampoos. The mean molecular masses of the oils are confirmed by the small amount of alkali required to saponify the oils. Free fatty acids can stimulate oxidative deterioration of oils by enzymatic and/or chemical oxidation to form off-flavour components. The free fatty acid and acid value of ADB are low which suggests its application as a good edible oil. Free fatty acid and acid value of PKCP are high, meaning that it would require refining to make it suitable for edible purposes, and may be better utilised for industrial purposes. Free fatty acid is also related to smoke point. ADB with low free fatty acid value will have a high smoke point, so it would be suitable for stirfry cooking. The low free fatty acid in ADB may be the reason why it is used for various edible purposes from frying to soup ingredient in the South-western part of Nigeria. Plant sterols have been suggested to have dietary significance and to protect vegetable oils from oxidative Table 3 Fatty acid composition (%)a of PKCP and ADB seeds oils Component
PKCP
ADB
Palmitic Stearic Oleic Linoleic Linolenic P Unsaturated acids
0.8 0.1 0.6 0.1 11.7 0.4 17.0 0.8 70.1 1.5 98.8
10.8 0.4 14.1 0.3 13.8 0.6 61.3 1.1 – 85.1
a
Values are mean standard deviation of duplicate determinations.
E.T. Akintayo, E. Bayer / Bioresource Technology 85 (2002) 95–97 Table 4 Sterol composition (%)a of PKCP and ADB seeds oils Component
PKCP
ADB
Cholesterol Campesterol Stigmasterol b-Sitosterol D5 -Avenasterol D7 -Stigmasterol Unknown
– 7.6 0.3 45.6 1.2 31.5 1.0 10.2 0.7 3.7 0.1 1.4
1.1 0.2 4.7 0.5 1.7 0.1 53.3 1.8 2.1 0.1 14.6 0.6 22.5
a
Values are mean standard deviation of duplicate determinations.
polymerisation during heating at frying temperatures (Gordon and Magos, 1983). Table 4 presents the sterol composition of the oils. Cholesterol, reported to be present in small amounts in vegetable oil (Kochar, 1983), was detected in ADB. b-Sitosterol was the major sterol in ADB while stigmasterol predominated in PKCP. D5Avenasterol was higher in PKCP than ADB. This sterol has an ethylidene group in the side chain. Such sterols are believed to improve the resistance of oils to darkening and polymerisation (Johansson and Appelquist, 1978). Two unknown peaks eluted after b-sitosterol in ADB and amounted to 22.5% of the sterol fraction in the oil. Further work is in progress to identify these peaks.
Acknowledgements Dr. E.T Akintayo is grateful to the Alexander von Humboldt Stifftung (AvH) of the Federal Republic of Germany.
97
References Abighor, R.D., Okpefa, E., Bafor, M.E., Udia, P.O., Osagie, A., 1997. The Physico-chemical properties of the seed and seed oil of Jatropha curcas L. Riv. Ital. Grasse. 74, 465–466. Ajiwe, V.I.E., Okeke, C.A., Agbo, H.N., 1995. Extraction and utilisation of Afzelia africana seed oil. Biores. Technol. 53, 89– 90. Akintayo, E.T., 1997. Chemical composition and Physico-chemical properties of Fluted pumpkin (Telfairia occidentalis) seed and seed oils. Riv. Ital. Grasse 74, 13–15. AOAC, 1984. Official Methods of Analysis, 14 ed., vol. 67. Association of Official Analytical Chemists, Arlington, VA, pp. 503–515. AOAC, 1995. Official Methods of Analysis, 16 ed., vol. 4. Association of Official Analytical Chemists, Arlington VA, pp. 1–45. Dery J. Kris., Etherton, P.M., Pearson, T.A., 1993. The role of fatty acid saturation on plasma lipids, lipoproteins and apolipoproteins II. The plasma total and low density lipoprotein cholesterol response of individual fatty acids. Metabolism 42, 130–134. Gordon, M.H., Magos, P., 1983. The effect of sterols on the oxidation of edible oils. Food chem. 10, 141–147. Hutchinson, J., Dalziel, J.M., 1958. Flora of West Tropical Africa, second ed., vol. 1, Part 2. Keay R.W.J Crown agents, London, pp. 365–367, 396–397. Johansson, A., Appelquist, L., 1978. The content and composition of sterols and sterol ester in low Erucic Rapeseed (Brassica napus). Lipids 13 (10), 658–665. Kochar, S.P., 1983. Influence of processing on sterols of edible vegetable oils. Prog. Lipid. Res. 22, 161–188. Messink, R.P., Katan, K.B., 1992. Effect of dietary fatty acids on serum lipids and lipoproteins. Arterioscler. Thromb. 12, 911– 912. Oshodi, A.A., 1992. Proximate composition, nutritionally valuable minerals and functional properties of Adenopus breviflorus benth seed flour and protein concentrate. Food Chem. 45 (2), 79–83. Oshodi, A.A., 1996. Amino acid and Fatty acid composition of Adenopus breviflorus benth seed. Int. J. Food Sci. Nutr. 47 (4), 295–298.
Short communication
Characterisation and some possible uses of Plukenetia conophora and Adenopus breviflorus seeds and seed oils E.T. Akintayo
*,1,
E. Bayer
Centre For Nucleic and Peptide Chemistry, Institute of Organic Chemistry, Universitat T€ubingen, Auf der Morgenstelle 18, 72076 T€ubingen, Germany Received 23 February 2002; received in revised form 7 March 2002; accepted 8 March 2002
Abstract Two non-conventional seeds, Plukenetia conophora (PKCP) and Adenopus breviflorus (ADB) were analysed for their proximate, fatty acids, sterols composition and physico-chemical characteristics. Crude protein was 25.65% for PKCP and 28.25% for ADB. ADB had lower moisture content (4.5%) than PKCP (8.0%) indicating that the former has better shelf life. Oil yields of the seeds were 49.58% for PKCP and 56.22% for ADB. The major sterols were stigmasterol and b-sitosterol in PKCP and ADB respectively. PKCP oil had 98.8% unsaturated fatty acids with linolenic acid predominating (70.1%) while ADB had 85.1% unsaturated fatty acids with linoleic acid being most abundant (65.3%). The very high saponification and iodine values of PKCP oil suggest its utilisation in alkyd resin, shoe polish, liquid soap and shampoo production. There is the possibility of using ADB oil in these regards as well as for edible purposes. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Plukenetia conophora; Adenopus breviflorus; Seed oil; Characteristics and uses
1. Introduction Plukenetia conophora (PKCP) (family Euphorbiacae) is a climbing shrub that is common in the South-western part of Nigeria. The seeds are eaten like wallnuts often along with maize. The leaves are edible and are often eaten with rice. The leaves are also used traditionally for curing headache and the fresh nuts are used for curing snakebites (Hutchinson and Dalziel, 1958). Much is known about the traditional use of this plant but very little on the characterisation and possible uses of its seed oil. The latter is part of the objectives of this study. Adenopus breviflorus (ADB) (family curcubitacae) is grown in the middle belt area of Nigeria. Like other members of the curcubitacae family, it possesses climbing stems. At maturity it produces fruits containing a varying number of seeds. The seeds are covered with a thin hull which when dried can easily be removed by hand. The seeds are mostly utilised as a soup ingredient in the Mid-western part of Nigeria. Oshodi (1992) has reported the proximate composition, nutritionally valuable minerals, and functional properties of the seeds flour and protein concentrate. The same author, Oshodi *
Corresponding author. Permanent address: Department of Chemistry, University of Ado-Ekiti, Ekiti-State, Nigeria. 1
(1996), also reported the amino and fatty acids composition of the plant seeds. In this work we report the characteristics and some possible uses of the ADB seeds and seed oil.
2. Methods Powdered seed samples were prepared by grinding in a C&N Junior laboratory mill size 5 (Christy and Norris limited, Engineers, Chelmsford England). Oil was extracted using chloroform/methanol (2:1) in a Soxhlet apparatus. Solvent was removed in a rotavapour at 35 °C. The extracted seed meals were thoroughly air dried to remove traces of solvent. The extracted seed oils were immediately analysed for iodine value, saponification value, acid value and unsaponifiable matter by methods described by the Association of Official Analytical Chemists (AOAC, 1984). Refractive indices of the oils (at room temperature) were determined with an Abbe refractometer and the specific gravity measurements were also done at room temperature using specific gravity bottles. The mean molecular mass was estimated from the relation ð56=SVÞ 1000 (Ajiwe et al., 1995). Fatty acid composition of the oils was determined as described by Akintayo (1997). The sterolic fractions obtained by TLC separation from the unsaponifiables of
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 7 3 - 1
96
E.T. Akintayo, E. Bayer / Bioresource Technology 85 (2002) 95–97
the oils were derivatised by refluxing with 20 ll of pyridine and bis(trimethylsilane)-trifluoroacetamide for 20 h at 80 °C. One ll of the derivatised sample was injected into a CP 9001 GC equipped with a flame ionisation detector and a SGE HTS capillary column (20 m, 0.22 m, 0.1 ll) coated with carborane polysiloxane. Injector and detector temperature were 280 and 340 °C. Temperature programming was 100 °C for 2 min, temperature increased at 5 °C/min up to 340 °C and held at 340 °C for 10 min. Proximate analysis of the seeds was carried out as described by the Association of Official Analytical Chemists (AOAC, 1995).
3. Results and discussion The proximate compositions of the seed flour of PKCP and ADB are presented in Table 1. Oil content of both is high, indicating that processing of their oils for industrial or edible purposes would be economical. They have low moisture content and this implies good shelf life characteristics. Going by their moisture content results, ADB will have a longer shelve life than PKCP. Their protein contents are comparable to those reported for some conventional oilseeds (Bailey’s Industrial Oil and Fat Products, 1964). They could, therefore, be recommended as protein supplements. It should, however, be borne in mind that the suitability of any plant material as food supplement depends on an interplay of factors like the digestibility of its nutrients and the presence of antinutritional factors. These concerns still need to be investigated with these oilseeds. The ash and crude fibre values are important in terms of the suitability of the seed cake for compounding of animal feeds. Although they both have favourable ash contents (<2.5%), that would have qualified them for compounding animal feeds, their low carbohydrate content renders them unsuitable for such purposes (Abighor et al., 1997). Table 2 presents the physico-chemical characteristics of the seeds oils. Both oils are yellow in colour. The iodine value of PKCP is very high (204.5 mg KOH/g) and places it in the drying group oil similar to linseed oil. Iodine value of ADB places it in the semi-drying group oil. These iodine values were confirmed by the Table 1 Proximate composition (%)a of PKCP and ADB seeds flours Assay
PKCP
ADB
Crude fat Crude protein Crude fibre Moisture Ash Carbohydrate by difference
49.58 1.14 23.65 0.62 6.00 0.22 8.00 0.12 2.10 0.24 10.67
56.22 0.86 28.25 0.24 3.40 0.14 4.50 0.11 1.30 0.10 6.33
a
Values are mean standard deviation of triplicate determinations.
Table 2 Physico-chemical characteristics of PKCP and ADB seeds oils Parameter
PKCP
ADB
Colour Free fatty acid (mg/g)a Acid value (mg KOH/g)a Saponification value (mg KOH/g)a Iodine value (mg iodine/g)a Mean molecular mass Unsaponifiable matter (%)a Refractive index (25 °C) Specific gravity (25 °C)
Light yellow 5.6 0.1 11.5 0.1 92.19 0.3
Light yellow 1.0 0.1 1.8 0.3 190.50 0.2
204.5 1.5 291.4 2.05 0.1 1.4835 0.9361
112.1 0.8 293.9 1.45 0.1 1.4721 0.9001
a
Values are mean standard deviation of triplicate determinations.
fatty acid composition of the oils presented in Table 3 which shows that PKCP oil consists of 98.8% unsaturated oil made up of mainly linolenic acid (70.1%) and ADB consists of 85.1% unsaturated oil with linoleic acid predominating (61.3%). Their iodine values suggest their use in alkyd resin production and other formulations like shoe polish. ADB oil may be of nutritional value with its 61.3% linoleic acid content, linoleic acid being an essential acid with cholesterol-lowering activity (Messink and Katan, 1992; Dery et al., 1993). Both oils show high saponification values suggesting their utilisation also in production of liquid soap and shampoos. The mean molecular masses of the oils are confirmed by the small amount of alkali required to saponify the oils. Free fatty acids can stimulate oxidative deterioration of oils by enzymatic and/or chemical oxidation to form off-flavour components. The free fatty acid and acid value of ADB are low which suggests its application as a good edible oil. Free fatty acid and acid value of PKCP are high, meaning that it would require refining to make it suitable for edible purposes, and may be better utilised for industrial purposes. Free fatty acid is also related to smoke point. ADB with low free fatty acid value will have a high smoke point, so it would be suitable for stirfry cooking. The low free fatty acid in ADB may be the reason why it is used for various edible purposes from frying to soup ingredient in the South-western part of Nigeria. Plant sterols have been suggested to have dietary significance and to protect vegetable oils from oxidative Table 3 Fatty acid composition (%)a of PKCP and ADB seeds oils Component
PKCP
ADB
Palmitic Stearic Oleic Linoleic Linolenic P Unsaturated acids
0.8 0.1 0.6 0.1 11.7 0.4 17.0 0.8 70.1 1.5 98.8
10.8 0.4 14.1 0.3 13.8 0.6 61.3 1.1 – 85.1
a
Values are mean standard deviation of duplicate determinations.
E.T. Akintayo, E. Bayer / Bioresource Technology 85 (2002) 95–97 Table 4 Sterol composition (%)a of PKCP and ADB seeds oils Component
PKCP
ADB
Cholesterol Campesterol Stigmasterol b-Sitosterol D5 -Avenasterol D7 -Stigmasterol Unknown
– 7.6 0.3 45.6 1.2 31.5 1.0 10.2 0.7 3.7 0.1 1.4
1.1 0.2 4.7 0.5 1.7 0.1 53.3 1.8 2.1 0.1 14.6 0.6 22.5
a
Values are mean standard deviation of duplicate determinations.
polymerisation during heating at frying temperatures (Gordon and Magos, 1983). Table 4 presents the sterol composition of the oils. Cholesterol, reported to be present in small amounts in vegetable oil (Kochar, 1983), was detected in ADB. b-Sitosterol was the major sterol in ADB while stigmasterol predominated in PKCP. D5Avenasterol was higher in PKCP than ADB. This sterol has an ethylidene group in the side chain. Such sterols are believed to improve the resistance of oils to darkening and polymerisation (Johansson and Appelquist, 1978). Two unknown peaks eluted after b-sitosterol in ADB and amounted to 22.5% of the sterol fraction in the oil. Further work is in progress to identify these peaks.
Acknowledgements Dr. E.T Akintayo is grateful to the Alexander von Humboldt Stifftung (AvH) of the Federal Republic of Germany.
97
References Abighor, R.D., Okpefa, E., Bafor, M.E., Udia, P.O., Osagie, A., 1997. The Physico-chemical properties of the seed and seed oil of Jatropha curcas L. Riv. Ital. Grasse. 74, 465–466. Ajiwe, V.I.E., Okeke, C.A., Agbo, H.N., 1995. Extraction and utilisation of Afzelia africana seed oil. Biores. Technol. 53, 89– 90. Akintayo, E.T., 1997. Chemical composition and Physico-chemical properties of Fluted pumpkin (Telfairia occidentalis) seed and seed oils. Riv. Ital. Grasse 74, 13–15. AOAC, 1984. Official Methods of Analysis, 14 ed., vol. 67. Association of Official Analytical Chemists, Arlington, VA, pp. 503–515. AOAC, 1995. Official Methods of Analysis, 16 ed., vol. 4. Association of Official Analytical Chemists, Arlington VA, pp. 1–45. Dery J. Kris., Etherton, P.M., Pearson, T.A., 1993. The role of fatty acid saturation on plasma lipids, lipoproteins and apolipoproteins II. The plasma total and low density lipoprotein cholesterol response of individual fatty acids. Metabolism 42, 130–134. Gordon, M.H., Magos, P., 1983. The effect of sterols on the oxidation of edible oils. Food chem. 10, 141–147. Hutchinson, J., Dalziel, J.M., 1958. Flora of West Tropical Africa, second ed., vol. 1, Part 2. Keay R.W.J Crown agents, London, pp. 365–367, 396–397. Johansson, A., Appelquist, L., 1978. The content and composition of sterols and sterol ester in low Erucic Rapeseed (Brassica napus). Lipids 13 (10), 658–665. Kochar, S.P., 1983. Influence of processing on sterols of edible vegetable oils. Prog. Lipid. Res. 22, 161–188. Messink, R.P., Katan, K.B., 1992. Effect of dietary fatty acids on serum lipids and lipoproteins. Arterioscler. Thromb. 12, 911– 912. Oshodi, A.A., 1992. Proximate composition, nutritionally valuable minerals and functional properties of Adenopus breviflorus benth seed flour and protein concentrate. Food Chem. 45 (2), 79–83. Oshodi, A.A., 1996. Amino acid and Fatty acid composition of Adenopus breviflorus benth seed. Int. J. Food Sci. Nutr. 47 (4), 295–298.