- Email: [email protected]
Screening of some medicinal plants for anti-lipase activity
Journal of Ethnopharmacology 97 (2005) 453–456
Screening of some medicinal plants for anti-lipase activity Niti Sharmaa , Vinay K. Sharmaa , Sung-Yum Seoa,b,∗ a
Applied Biotechnology Laboratory, Department of Biology, College of Natural Science, Kongju National University, Kongju 314-701, Korea b Korean Collection of Herbal Extracts, Inc., Kongju 314-701, Korea
Received 19 May 2004; received in revised form 13 October 2004; accepted 12 November 2004 Available online 1 February 2005
Abstract In order to find new pancreatic lipase (triacylglycerol lipase, EC 3.1.1.3) inhibitors from natural sources, 75 medicinal plants belonging to different families were screened for their anti-lipase activity, using a radioactive method. On evaluating the results, methanolic extracts of three plants namely, Eriochloa villosa (Thunb.) Kunth, Orixa japonica Thunb. and Setaria italica (L.) Palib., exhibited strong in vitro anti-lipase activity (above 80%). These plants will be studied further to elucidate the structure and chemical properties of the active compound responsible for anti-lipase action. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hyperlipidaemia; Pancreatic lipase; Anti-lipase activity; Medicinal plants
1. Introduction The effect of dietary fat on hyperlipidaemia is well known (Grundy and Denke, 1990) as it is directly or indirectly associated with various diseases like obesity, diabetes, hypertension and cardiovascular problems (Kopelman, 2000). The triglyceride absorption efficiency is one of the main factors contributing to the plasma triglyceride levels, however, the dietary triglycerides are not absorbed as such until hydrolyzed to free fatty acids by triacylglycerol lipases [EC 3.1.1.3] (Verger, 1997). A group of three important enzymes namely, gastric lipase, pancreatic lipase and colipase are involved in the triglyceride absorption from the small intestine to the enterocytes (Bernback et al., 1990; Borel et al., 1991), and if somehow this initial movement of triglycerides from the intestinal lumen is blocked, hyperlipidaemia can be prevented. Thus, an inhibitor of digestive lipase that helps to limit intestinal fat absorption at an initial stage could prove as a useful medication for the treatment of ∗
Corresponding author. Tel.: +82 41 850 8503; fax: +82 41 854 8503. E-mail address: [email protected] (S.-Y. Seo).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.009
hyperlipidaemia and holds great promise as an anti-obesity agent. Presently, the most common anti-obesity drug available in the market is Orlistat (Ro 18-0647), a hydrogenated derivative of lipstatin obtained from Streptomyces toxitricini. It is reported as a potent inhibitor of gastric, pancreatic and carboxylester lipase (Hadvay et al., 1988), and has proved to be effective for the treatment of human obesity (Hauptman et al., 1992; Drent et al., 1995; Sjostrom et al., 1998). Some hydrophobic proteins such as serum albumin and -lactoglobulin at concentrations more than micromolar are known to inhibit the activity of lipase (Gargouri et al., 1984) by interfering with the absorption of lipase at an oil–water interface (Brockerhoff, 1974). A wide variety of chemicals also exhibited anti-lipase activity (Gargouri et al., 1997). The presence of lipase inhibitors have also been reported in some natural sources like marine algae (Bitou et al., 1999), soybean (Gargouri et al., 1984; Satouchi et al., 1998), wheat (Borel et al., 1989; Tani et al., 1995), citrus (Kawaguchi et al., 1997), oolong tea (Han et al., 2001) and aqueous extract of some medicinal herbs (Shimura et al., 1992). However, there is a requirement
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N. Sharma et al. / Journal of Ethnopharmacology 97 (2005) 453–456
Table 1 Lipase inhibitory activity of various plants No.
Scientific name
Plant Part
Family
% Inhibition
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Acer pseudosieboldianum (Pax) Kom. Adenophora radiatifolia Nakai Agaricus bisporus (Lange) Imbach. Agastache rugosa (Fisch. & Mey.) O.Kuntze. Ailanthus altissima (Mill.) Swingle. Angelica dahurica (Hoffm.) Franch. & Sav. Aralia elata (Miq.) Seem. Artemisia capillaries Thunb. Artemisia scoparia Waldst. & Kit. Asparagus cochinchinesis (Lour.) Merr. Astragalus membranaceous (Fisch.) Bunge. Boehmeria longispica Steud. Bulbostylis barbata (Rottb.) C.B. Clarke Caltha palustris L. Carex kobomugi Ohwi. Carpesium abrotanoides L. Celosia cristate (L.) O. Kuntze Chrysosplenium grayanum Maxim. Cichorium endivia L. Citrus aurantifolium L. Corydalis heterocarpa Sieb. & Zucc. Cucurbita pepo L. Cuscuta japonica Choisy. Cyperus amuricus Maxim. Cyrtomium falcatum (L.f.) C.Presl. Elaeagnus macrophylla Thunb. Eriocaulon sieboldianum Sieb. & Zucc. Eriochloa villosa (Thunb.) Kunth Euonymus sachalinensis (Fr. Schmidt.) Maxim. Eupatorium chinense L. Gardenia jasminoides Ellis Gastrodia elata Bl. Geranium nepalense Sweet. Glycyrrhiza uralensis Fisch. Hemarthria sibirica (Gand.) Ohwi Inula Britannica L. Ixeris dentate (Thunb.) Nak. Juncus effuses L. Juncus gracillimus (Buchen.) V. Krecz. et Gontsch. Lespedeza cuneate G. Don Lindera glauca (Siebold. & Zucc.) Blume. Lonicera japonica Thunb. Lythrum salicaria L. Melandryum oldhamianum Rohrbach Meliotus suaveolens Ledeb. Melo pedicellus L. Momordica cochinchinesis (Lour.) Spreng. Narcissus tazetta L. Orixa japonica Thunb. Ostericum koreanum Maxim. Oxalis corniculate L. Panicum dichotomiflorum Michx. Plantago camtschatica Cham. Polygala tenuifolia Willd. Potamogeton distinctus Bennet. Prunella vulgaris L. Pueraria thunbergiana (Sieb. & Zucc.) Benth. Reynoutria elliptica (Koidz) Migo. Rhus sylvestris Sieb. & Zucc. Rubia akane Nak. Rubus croceanthus Lev.
WP WP WP WP WP WP WP WP WP R WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP F WP WP WP WP
Aeraceae Campandulaceae Agaricaceae Laminaceae Simaroubaceae Apiaceae Araliaceae Asteraceae Asteraceae Liliaceae Fabaceae Urticaceae Cyperaceae Ranunculaceae Cyperaceae Compositae Amaranthaceae Saxifragaceae Asteraceae Rutaceae Fumariaceae Cucurbitaceae Cuscutaceae Cyperaceae Aspidiaceaes Eleagnaceae Poaceae Poaceae Celastraceae Asteraceae Rubiacea Orchidaceae Geraniacea Fabaceae Poaceae Asteracese Asteracese Juncaceae Juncaceae Fabaceae Lauraceae Caprifoliaceae Lythraceae Caryophyllaceae Fabaceae Cucurbitaceae Cucurbitaceae Amaryllidaceae Rutaceae Apiceae Oxalidaceae Poaceae Plantaginaceae Polygalaceae Potamogetonaceae Laminaceae Fabaceae Polygonaceae Anacardiaceae Rubiaceae Rosaceae
62.2 6.4 61.6 40.5 51.0 50.8 35.7 69.3 59.5 58.9 36.9 67.7 40.5 20.5 48.6 20.9 10.8 73.2 26.0 44.6 61.1 66.3 22.9 57.3 76.7 60.0 41.7 83.0 51.8 22.5 60.2 63.0 72.4 57.6 66.6 18.7 50.1 63.5 39.0 71.7 60.3 40.9 43.9 70.6 35.6 75.4 58.9 11.2 81.3 42.6 55.6 66.6 35.7 65.0 72.5 68.0 40.2 58.2 70.4 58.7 35.9
N. Sharma et al. / Journal of Ethnopharmacology 97 (2005) 453–456
455
Table 1 (Continued ) No.
Scientific name
Plant Part
Family
% Inhibition
62 63 64 65 66 67 68 69 70 71 72 73 74 75
Sanicula chinensis Bunge. Sapium japonicum (Sieb. & Zucc.) Pax. & Hoff. Scilla scilloides (Lindl.) Druce. Securinega suffruticose (Pall.) Rehd. Selaginella tamariscina (Beauv.) Spring. Setaria italica (L.) Palib. Setaria viridis (L.) Palib. Solanum nigram L. Spirodela polyrhiza (L.) Schneid. Styrax obassia Sieb. & Zucc. Tilia taquetii Schneid Uncaria rhynchophylla (Miq.) Jacks. Viburnum dilalatum Thunb. Xanthium sibiricum Patrin ex Widder
WP WP WP WP WP WP WP WP WP WP WP WP WP WP
Apiacea Euphorbiaceae Liliaceae Euphorbiaceae Selaginellaceae Poaceae Poaceae Solanaceae Lemnaceae Styraceae Tiliaceae Rubiaceae Caprifoliacea Asteraceae
46.9 29.2 70.0 53.3 42.0 80.3 41.0 29.3 53.3 20.9 20.3 9.9 32.4 33.3
Plant Parts: WP: Whole Plant, R: Root, F: Flower. Five measurements were carried out per extract (n = 5).
of constant search for better lipase inhibitors from natural sources. Here, in the present article, we have screened methanolic extracts of various medicinal plants for their anti-lipase activity, using a radioactive method.
2. Materials and methods 2.1. Plant materials All the plants and their ethnobotanical information were obtained from “Korean Collection of Herbal Extracts”, a Biotech company in Korea. A collection of voucher specimen is available with the company (Korean Collection of Herbal Extracts, 2000). 2.2. Preparation of plant extracts The whole plant material was air dried and ground into fine powder. The powdered material (100 g) was extracted with 1000 ml of 80% methanolic solution (methanol:deionised water) and concentrated at 45 ◦ C in a rotatory vacuum evaporator (EYELA, Japan). The concentrated samples were stored at −20 ◦ C for further studies. 2.3. Chemicals Porcine pancreatic lipase (Type VI-S), colipase, triolein and sodium deoxycholic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Radioactive triolein (Carboxyl 14 C) was obtained from Perkin-Elmer (Boston, MA, USA) and the scintillation cocktail was purchased from Packard Bioscience Co. (Meriden, CT, USA).
with minor modifications. The enzymatic reaction was carried out in a final volume of 50 l assay buffer (27 mM Tris–HCl, pH 9.2, 0.1 mM CaCl2, 19.8 mM sodium deoxycholic acid) containing 62.5 ng porcine pancreatic lipase, 250 ng porcine colipase and 0.312 mM triolein including 1.0 × 105 dpm of radioactive triolein. The mixture was incubated at 23 ◦ C for 1 h. The reaction was terminated by adding 15 volumes of chloroform:methanol:heptane (12.5:14:10, by volume) followed by 5 volumes of 50 mM NaCO3 (pH 10.5). The mixture was vortexed and centrifuged in a micro centrifuge for 10 min. An aliquot of the upper phase was counted using Liquid Scintillation Analyzer (TRI-CARB 2100 TR, Packard, Canberra). 2.5. Assay for pancreatic lipase inhibitory activity For determining lipase inhibitory activity, the extracts (0.2 mg/ml) were pre-incubated with the enzyme for 1 h before assaying the enzyme activity. Negative controls were also applied to check the activity with and without inhibitor. The lipase inhibition (I) was calculated according the following formula: (A − a) − (B − b) I% = × 100 (A − a) where, A is the activity without inhibitor, a the negative control without inhibitor, B the activity with inhibitor and b is the negative control with inhibitor. In case of a, the reaction was immediately terminated after addition of the substrate to the assay buffer containing the enzyme while in case of b the reaction was stopped after incubating the inhibitor with the enzyme mixture and then the substrate was added.
3. Results and discussion 2.4. Assay for pancreatic lipase activity Pancreatic lipase activity was measured using a radioactive substrate, [14 C] triolein, as described (Lowe, 1992,1999)
The results of pancreatic lipase inhibition by the various plants have been summarized in Table 1, where lipase inhibition is expressed in percentage (%). Evaluating the results,
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36 extracts out of 75 can be regarded as poor lipase inhibitors (only nine plants with inhibition greater than 70%). Out of these nine plants only three plants namely, Eriochloa villosa (Thunb.) Kunth (83%), Orixa japonica Thunb. (81.3%) and Setaria italica (L.) Palib. (80.3%) exhibited anti-lipase activity greater than 80%. Interestingly, out of these three, Eriochloa villosa (Thunb.) Kunth and Setaria italica (L.) Palib. belong to same Poaceae family. To the best of our knowledge, the plants studied in this paper have not been screened earlier for their anti-lipase activity. The results suggest that these plants (Eriochloa villosa (Thunb.) Kunth, Orixa japonica Thunb. and Setaria italica (L.) Palib.) will prove a good source of effective crude drug for the treatment of obesity caused by a high fat diet. However, further biological investigations are needed, using animal models, to verify the inhibitory activities under in vivo conditions. These three plants will be examined in order to isolate, identify and characterize phytoactive compounds to further define the nature of their lipid-lowering activity.
Acknowledgements We are thankful to Prof. K. Wasan (Division of Pharmaceutics and Biopharmaceutics, faculty of Pharmaceutical Sciences, UBC, Canada) and Dr. M. Li (Pharma Division, Forbes Medi-Tech Inc., UBC, Canada) for their valuable suggestions. Financial help from the Ministry of Health and Welfare and RRC/NMR, Korea is gratefully acknowledged.
References Bernback, S., Blackberg, L., Hernell, O., 1990. The complete digestion of milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase dependent lipase, and bile salt-stimulated lipase. Journal of Clinical Investigation 85, 1221–1226. Bitou, N., Ninomiya, M., Tsujita, T., Okuda, H., 1999. Screening of lipase inhibitors from marine algae. Lipids 34, 441–445. Borel, P., Armand, M., Sentft, M., Lafont, H., Lairon, D., 1991. Gastric lipase. Gastroenterology 100, 1582–1589. Borel, P., Lairon, D., Termine, E., Grataroli, R., Lafont, H., 1989. Isolation and properties of lipolysis inhibitory proteins from wheat germ and wheat bran. Plant Foods and Human Nutrition 39, 339– 348.
Brockerhoff, H., 1974. Lipases. In: Brockerhoff, H., Jensen, R.G. (Eds.), Lipolytic Enzymes. Academic Press, New York, pp. 25–125. Drent, M.L., Larsson, I., William-Olsson, T., Quaade, F., Czubayko, F., Von Bergmann, K., Strobel, W., Sjostrom, L., Van der Veen, E.A., 1995. Orlistat (RO 18-0647), a lipase inhibitor, in the treatment of human obesity: a multiple dose study. International Journal of Obesity 19, 221–226. Gargouri, Y., Julien, R., Sugihara, A., Verger, R., Sarda, L., 1984. Inhibition of pancreatic and microbial lipases by proteins. Biochemical Biophysical Acta 795, 326–331. Gargouri, Y., Ransac, S., Verger, R., 1997. Covalent inhibition of digestive lipases: an in vitro study. Biochemical Biophysical Acta 1344, 6–37. Grundy, S.M., Denke, M.A., 1990. Dietary influences on serum lipids and lipoproteins. Journal of Lipid Research 31, 149–1172. Hadvay, P., Lengsfeld, H., Wolfer, H., 1988. Inhibition of pancreatic lipase in vitro by covalent inhibitor tetrahydrolipstatin. Biochemical Journal 256, 357–361. Han, L.K., Kimura, Y., Kawashima, M., Takaku, T., Taniyama, T., Hayashi, T., Zheng, Y.N., Okuda, H., 2001. Antiobesity effects in rodents of dietary teasaponin, a lipase inhibitor. International Journal of Obesity and Related Metabolic Disorders 25, 1459–1464. Hauptman, J.B., Jeunt, F.S., Hartmann, D., 1992. Initial studies in humans with the novel gastrointestinal lipase inhibitor Ro 18-0647 (tetrahydrolipstatin). American Journal of Clinical Nutrition 55, 309s–313s. Kawaguchi, K., Mizuno, T., Aida, K., Uchino, K., 1997. Hesperidin as an inhibitor of lipases from porcine pancrease and pseudomonas. Bioscience, Biotechnology and Biochemistry 61, 102–104. Kopelman, P.G., 2000. Obesity as a medical problem. Nature 404, 635–643. Korean Collection of Herbal Extracts, Kongju 314 701, South Korea. Lowe, M.E., 1992. The catalytic site residues and interfacial binding of human pancreatic lipase. Journal of Biological Chemistry 267, 17069–17073. Lowe, M.E., 1999. Assays for pancreatic TG lipase and colipase. Methods in Molecular Biology 109, 59–70. Satouchi, K., Hirano, K., Fujino, O., Ikoma, M., Tanaka, T., Kitamura, K., 1998. Lipoxygenase-1 from soybean seed inhibiting the activity of pancreatic lipase. Bioscience, Biotechnology and Biochemistry 62, 1498–1503. Shimura, S., Tsuzuki, W., Kobayashi, S., Suzuki, T., 1992. Inhibitory effect on lipase activity of extracts from medicinal herbs. Bioscience, Biotechnology and Biochemistry 56, 1478–1479. Sjostrom, L., Rissanen, A., Andersen, T., Boldrin, M., Golay, A., Koppeschaar, H.P.F., Krempf, M., 1998. Randomised placebocontrolled trial of orlistat for weight loss and prevention of weight regain in obese patients. Lancet 352, 167–172. Tani, H., Ohishi, H., Watanabe, K., 1995. Wheat flour lipase inhibitor decrease serum lipid levels in male rats. Journal of Nutrition and Vitaminology 41, 699–706. Verger, R., 1997. Interfacial activation of lipases: facts and antifacts. Trends in Bioscience and Technology 15, 32–37.
Screening of some medicinal plants for anti-lipase activity Niti Sharmaa , Vinay K. Sharmaa , Sung-Yum Seoa,b,∗ a
Applied Biotechnology Laboratory, Department of Biology, College of Natural Science, Kongju National University, Kongju 314-701, Korea b Korean Collection of Herbal Extracts, Inc., Kongju 314-701, Korea
Received 19 May 2004; received in revised form 13 October 2004; accepted 12 November 2004 Available online 1 February 2005
Abstract In order to find new pancreatic lipase (triacylglycerol lipase, EC 3.1.1.3) inhibitors from natural sources, 75 medicinal plants belonging to different families were screened for their anti-lipase activity, using a radioactive method. On evaluating the results, methanolic extracts of three plants namely, Eriochloa villosa (Thunb.) Kunth, Orixa japonica Thunb. and Setaria italica (L.) Palib., exhibited strong in vitro anti-lipase activity (above 80%). These plants will be studied further to elucidate the structure and chemical properties of the active compound responsible for anti-lipase action. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hyperlipidaemia; Pancreatic lipase; Anti-lipase activity; Medicinal plants
1. Introduction The effect of dietary fat on hyperlipidaemia is well known (Grundy and Denke, 1990) as it is directly or indirectly associated with various diseases like obesity, diabetes, hypertension and cardiovascular problems (Kopelman, 2000). The triglyceride absorption efficiency is one of the main factors contributing to the plasma triglyceride levels, however, the dietary triglycerides are not absorbed as such until hydrolyzed to free fatty acids by triacylglycerol lipases [EC 3.1.1.3] (Verger, 1997). A group of three important enzymes namely, gastric lipase, pancreatic lipase and colipase are involved in the triglyceride absorption from the small intestine to the enterocytes (Bernback et al., 1990; Borel et al., 1991), and if somehow this initial movement of triglycerides from the intestinal lumen is blocked, hyperlipidaemia can be prevented. Thus, an inhibitor of digestive lipase that helps to limit intestinal fat absorption at an initial stage could prove as a useful medication for the treatment of ∗
Corresponding author. Tel.: +82 41 850 8503; fax: +82 41 854 8503. E-mail address: [email protected] (S.-Y. Seo).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.009
hyperlipidaemia and holds great promise as an anti-obesity agent. Presently, the most common anti-obesity drug available in the market is Orlistat (Ro 18-0647), a hydrogenated derivative of lipstatin obtained from Streptomyces toxitricini. It is reported as a potent inhibitor of gastric, pancreatic and carboxylester lipase (Hadvay et al., 1988), and has proved to be effective for the treatment of human obesity (Hauptman et al., 1992; Drent et al., 1995; Sjostrom et al., 1998). Some hydrophobic proteins such as serum albumin and -lactoglobulin at concentrations more than micromolar are known to inhibit the activity of lipase (Gargouri et al., 1984) by interfering with the absorption of lipase at an oil–water interface (Brockerhoff, 1974). A wide variety of chemicals also exhibited anti-lipase activity (Gargouri et al., 1997). The presence of lipase inhibitors have also been reported in some natural sources like marine algae (Bitou et al., 1999), soybean (Gargouri et al., 1984; Satouchi et al., 1998), wheat (Borel et al., 1989; Tani et al., 1995), citrus (Kawaguchi et al., 1997), oolong tea (Han et al., 2001) and aqueous extract of some medicinal herbs (Shimura et al., 1992). However, there is a requirement
454
N. Sharma et al. / Journal of Ethnopharmacology 97 (2005) 453–456
Table 1 Lipase inhibitory activity of various plants No.
Scientific name
Plant Part
Family
% Inhibition
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Acer pseudosieboldianum (Pax) Kom. Adenophora radiatifolia Nakai Agaricus bisporus (Lange) Imbach. Agastache rugosa (Fisch. & Mey.) O.Kuntze. Ailanthus altissima (Mill.) Swingle. Angelica dahurica (Hoffm.) Franch. & Sav. Aralia elata (Miq.) Seem. Artemisia capillaries Thunb. Artemisia scoparia Waldst. & Kit. Asparagus cochinchinesis (Lour.) Merr. Astragalus membranaceous (Fisch.) Bunge. Boehmeria longispica Steud. Bulbostylis barbata (Rottb.) C.B. Clarke Caltha palustris L. Carex kobomugi Ohwi. Carpesium abrotanoides L. Celosia cristate (L.) O. Kuntze Chrysosplenium grayanum Maxim. Cichorium endivia L. Citrus aurantifolium L. Corydalis heterocarpa Sieb. & Zucc. Cucurbita pepo L. Cuscuta japonica Choisy. Cyperus amuricus Maxim. Cyrtomium falcatum (L.f.) C.Presl. Elaeagnus macrophylla Thunb. Eriocaulon sieboldianum Sieb. & Zucc. Eriochloa villosa (Thunb.) Kunth Euonymus sachalinensis (Fr. Schmidt.) Maxim. Eupatorium chinense L. Gardenia jasminoides Ellis Gastrodia elata Bl. Geranium nepalense Sweet. Glycyrrhiza uralensis Fisch. Hemarthria sibirica (Gand.) Ohwi Inula Britannica L. Ixeris dentate (Thunb.) Nak. Juncus effuses L. Juncus gracillimus (Buchen.) V. Krecz. et Gontsch. Lespedeza cuneate G. Don Lindera glauca (Siebold. & Zucc.) Blume. Lonicera japonica Thunb. Lythrum salicaria L. Melandryum oldhamianum Rohrbach Meliotus suaveolens Ledeb. Melo pedicellus L. Momordica cochinchinesis (Lour.) Spreng. Narcissus tazetta L. Orixa japonica Thunb. Ostericum koreanum Maxim. Oxalis corniculate L. Panicum dichotomiflorum Michx. Plantago camtschatica Cham. Polygala tenuifolia Willd. Potamogeton distinctus Bennet. Prunella vulgaris L. Pueraria thunbergiana (Sieb. & Zucc.) Benth. Reynoutria elliptica (Koidz) Migo. Rhus sylvestris Sieb. & Zucc. Rubia akane Nak. Rubus croceanthus Lev.
WP WP WP WP WP WP WP WP WP R WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP WP F WP WP WP WP
Aeraceae Campandulaceae Agaricaceae Laminaceae Simaroubaceae Apiaceae Araliaceae Asteraceae Asteraceae Liliaceae Fabaceae Urticaceae Cyperaceae Ranunculaceae Cyperaceae Compositae Amaranthaceae Saxifragaceae Asteraceae Rutaceae Fumariaceae Cucurbitaceae Cuscutaceae Cyperaceae Aspidiaceaes Eleagnaceae Poaceae Poaceae Celastraceae Asteraceae Rubiacea Orchidaceae Geraniacea Fabaceae Poaceae Asteracese Asteracese Juncaceae Juncaceae Fabaceae Lauraceae Caprifoliaceae Lythraceae Caryophyllaceae Fabaceae Cucurbitaceae Cucurbitaceae Amaryllidaceae Rutaceae Apiceae Oxalidaceae Poaceae Plantaginaceae Polygalaceae Potamogetonaceae Laminaceae Fabaceae Polygonaceae Anacardiaceae Rubiaceae Rosaceae
62.2 6.4 61.6 40.5 51.0 50.8 35.7 69.3 59.5 58.9 36.9 67.7 40.5 20.5 48.6 20.9 10.8 73.2 26.0 44.6 61.1 66.3 22.9 57.3 76.7 60.0 41.7 83.0 51.8 22.5 60.2 63.0 72.4 57.6 66.6 18.7 50.1 63.5 39.0 71.7 60.3 40.9 43.9 70.6 35.6 75.4 58.9 11.2 81.3 42.6 55.6 66.6 35.7 65.0 72.5 68.0 40.2 58.2 70.4 58.7 35.9
N. Sharma et al. / Journal of Ethnopharmacology 97 (2005) 453–456
455
Table 1 (Continued ) No.
Scientific name
Plant Part
Family
% Inhibition
62 63 64 65 66 67 68 69 70 71 72 73 74 75
Sanicula chinensis Bunge. Sapium japonicum (Sieb. & Zucc.) Pax. & Hoff. Scilla scilloides (Lindl.) Druce. Securinega suffruticose (Pall.) Rehd. Selaginella tamariscina (Beauv.) Spring. Setaria italica (L.) Palib. Setaria viridis (L.) Palib. Solanum nigram L. Spirodela polyrhiza (L.) Schneid. Styrax obassia Sieb. & Zucc. Tilia taquetii Schneid Uncaria rhynchophylla (Miq.) Jacks. Viburnum dilalatum Thunb. Xanthium sibiricum Patrin ex Widder
WP WP WP WP WP WP WP WP WP WP WP WP WP WP
Apiacea Euphorbiaceae Liliaceae Euphorbiaceae Selaginellaceae Poaceae Poaceae Solanaceae Lemnaceae Styraceae Tiliaceae Rubiaceae Caprifoliacea Asteraceae
46.9 29.2 70.0 53.3 42.0 80.3 41.0 29.3 53.3 20.9 20.3 9.9 32.4 33.3
Plant Parts: WP: Whole Plant, R: Root, F: Flower. Five measurements were carried out per extract (n = 5).
of constant search for better lipase inhibitors from natural sources. Here, in the present article, we have screened methanolic extracts of various medicinal plants for their anti-lipase activity, using a radioactive method.
2. Materials and methods 2.1. Plant materials All the plants and their ethnobotanical information were obtained from “Korean Collection of Herbal Extracts”, a Biotech company in Korea. A collection of voucher specimen is available with the company (Korean Collection of Herbal Extracts, 2000). 2.2. Preparation of plant extracts The whole plant material was air dried and ground into fine powder. The powdered material (100 g) was extracted with 1000 ml of 80% methanolic solution (methanol:deionised water) and concentrated at 45 ◦ C in a rotatory vacuum evaporator (EYELA, Japan). The concentrated samples were stored at −20 ◦ C for further studies. 2.3. Chemicals Porcine pancreatic lipase (Type VI-S), colipase, triolein and sodium deoxycholic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Radioactive triolein (Carboxyl 14 C) was obtained from Perkin-Elmer (Boston, MA, USA) and the scintillation cocktail was purchased from Packard Bioscience Co. (Meriden, CT, USA).
with minor modifications. The enzymatic reaction was carried out in a final volume of 50 l assay buffer (27 mM Tris–HCl, pH 9.2, 0.1 mM CaCl2, 19.8 mM sodium deoxycholic acid) containing 62.5 ng porcine pancreatic lipase, 250 ng porcine colipase and 0.312 mM triolein including 1.0 × 105 dpm of radioactive triolein. The mixture was incubated at 23 ◦ C for 1 h. The reaction was terminated by adding 15 volumes of chloroform:methanol:heptane (12.5:14:10, by volume) followed by 5 volumes of 50 mM NaCO3 (pH 10.5). The mixture was vortexed and centrifuged in a micro centrifuge for 10 min. An aliquot of the upper phase was counted using Liquid Scintillation Analyzer (TRI-CARB 2100 TR, Packard, Canberra). 2.5. Assay for pancreatic lipase inhibitory activity For determining lipase inhibitory activity, the extracts (0.2 mg/ml) were pre-incubated with the enzyme for 1 h before assaying the enzyme activity. Negative controls were also applied to check the activity with and without inhibitor. The lipase inhibition (I) was calculated according the following formula: (A − a) − (B − b) I% = × 100 (A − a) where, A is the activity without inhibitor, a the negative control without inhibitor, B the activity with inhibitor and b is the negative control with inhibitor. In case of a, the reaction was immediately terminated after addition of the substrate to the assay buffer containing the enzyme while in case of b the reaction was stopped after incubating the inhibitor with the enzyme mixture and then the substrate was added.
3. Results and discussion 2.4. Assay for pancreatic lipase activity Pancreatic lipase activity was measured using a radioactive substrate, [14 C] triolein, as described (Lowe, 1992,1999)
The results of pancreatic lipase inhibition by the various plants have been summarized in Table 1, where lipase inhibition is expressed in percentage (%). Evaluating the results,
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36 extracts out of 75 can be regarded as poor lipase inhibitors (only nine plants with inhibition greater than 70%). Out of these nine plants only three plants namely, Eriochloa villosa (Thunb.) Kunth (83%), Orixa japonica Thunb. (81.3%) and Setaria italica (L.) Palib. (80.3%) exhibited anti-lipase activity greater than 80%. Interestingly, out of these three, Eriochloa villosa (Thunb.) Kunth and Setaria italica (L.) Palib. belong to same Poaceae family. To the best of our knowledge, the plants studied in this paper have not been screened earlier for their anti-lipase activity. The results suggest that these plants (Eriochloa villosa (Thunb.) Kunth, Orixa japonica Thunb. and Setaria italica (L.) Palib.) will prove a good source of effective crude drug for the treatment of obesity caused by a high fat diet. However, further biological investigations are needed, using animal models, to verify the inhibitory activities under in vivo conditions. These three plants will be examined in order to isolate, identify and characterize phytoactive compounds to further define the nature of their lipid-lowering activity.
Acknowledgements We are thankful to Prof. K. Wasan (Division of Pharmaceutics and Biopharmaceutics, faculty of Pharmaceutical Sciences, UBC, Canada) and Dr. M. Li (Pharma Division, Forbes Medi-Tech Inc., UBC, Canada) for their valuable suggestions. Financial help from the Ministry of Health and Welfare and RRC/NMR, Korea is gratefully acknowledged.
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