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White poplar: Targeted isolation of pancreatic lipase inhibitors
Industrial Crops & Products 141 (2019) 111778
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
White poplar: Targeted isolation of pancreatic lipase inhibitors a
Marwa Elsbaey , Yhiya Amen
a,b
c
, Toshinori Nakagawa , Kuniyoshi Shimizu
b,⁎
T
a
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt Department of Agro-environmental Sciences, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 8190395, Japan c Department of Biological Resources Management, The University of Shiga Prefecture 2500, Hassaka-cho, Hikone-City, Shiga, 522-8533, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: poplar Lipase Obesity Bio-guided isolation Flavonoids Phenolic acids
In order to discover new anti-obesity agents, twenty two natural extracts from medicinal plants and agricultural wastes were screened by pancreatic lipase inhibition assay. The methanolic extract of poplar wood (Populus alba L.), an important raw material for paper industry, showed promising inhibitory activity, its IC50 value was 7.15 μg/mL. Through bio-guided fractionation, the ethyl acetate fraction (E) was found to be the major contributor to the activity, IC50 = 4.7 μg/mL. Hence, it was subjected to bio-guided isolation and nine compounds (1–9) were identified from the active sub-fractions. The isolates were identified by means of one- and twodimensional nuclear magnetic spectroscopy (1D, 2D-NMR), as well as high-performance liquid chromatography quadrupole time-of flight mass spectrometry (HPLC-QTOF-MS). (+)-Taxifolin (5) and (+)-ampelopsin (4) showed the highest activity, their IC50 values were 23.2 and 46.2 μM, respectively. The results indicated that flavonoids and phenolic acids in poplar wood could serve as potential candidates for development of new antiobesity agents.
1. Introduction Poplar wood is widely used for production of a variety of wood products, pulp, paper, and other fiber-based products. In the twentyfirst century, the worldwide demand for raw materials has grown extremely and hence poplar wood occupied an important position as a fiber resource in paper production (Przybysz and Przybysz, 2013). White poplar (Populus alba L.) has been particularly valued as a commercial wood source for its drought tolerant properties versus the changed climate and ecological conditions (Ištok et al., 2017). It is one of the most distinctive poplar species which is indigenous to Europe (Abdou and Ibrahim, 2015). Obesity is a significant public health problem that has become epidemic worldwide (Segula, 2014). Current evidence showed that obesity and overweight are globally the fifth leading risks for death (Buchholz and Melzig, 2015). Obesity is reported as a major contributor to diabetes mellitus, cardiovascular diseases and stroke, musculoskeletal disorders, dementia, and various types of cancer, including breast, endometrial, prostate, and colon cancer (Zhang et al., 2008). Pancreatic lipase is the key enzyme responsible for fat digestion, consequently its absorption. Inhibition of pancreatic lipase is an effective strategy for obesity management (de la Garza et al., 2011). Even
though physical activity and increased energy expenditure are the crucial cornerstones in body weight loss, drug treatment is indispensable for prevention of weight regain (de la Garza et al., 2011). Since 1999, orlistat® has been the only approved pancreatic lipase inhibitor as a pharmaceutical agent, according to the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) (Qi, 2018). Despite being an effective and a selective inhibitor, it is associated with many unpleasant gastrointestinal side effects. In this context, natural products can serve as a vast platform for development of safe and cost-effective drugs that can satisfy the need of the anti-obesity market (Birari and Bhutani, 2007; de la Garza et al., 2011). Particularly, exploring agricultural waste as an affordable and cheap source for natural products has many merits, including the environmental benefit. The aim of this research is to identify safe pancreatic lipase inhibitors of natural origin that could be of potential interest for the antiobesity market. In this study, the methanolic extracts of twelve medicinal plants and four agricultural wastes, were tested for pancreatic lipase inhibition activity. The young twigs of P. alba showed promising inhibitory activity. The extract was subjected to bio-guided fractionation and the active principles were identified.
⁎
Corresponding author at: Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail addresses: [email protected] (M. Elsbaey), [email protected] (Y. Amen), [email protected] (T. Nakagawa), [email protected] (K. Shimizu). https://doi.org/10.1016/j.indcrop.2019.111778 Received 8 May 2019; Received in revised form 29 July 2019; Accepted 9 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
Industrial Crops & Products 141 (2019) 111778
M. Elsbaey, et al.
2. Material and methods
of fraction 9E were applied onto the top of Sephadex LH-20 column (35 x 1.5 cm), eluted using MeOH, 10 mL fractions were collected to give 34 subfractions (9E.1–34). Subfractions (9E.6–8, 13 mg) were purified onto the top of RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (70:30) to give compound 7 (9.0 mg). Subfractions (9E.10–17, 42 mg) were chromatographed by a RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (50:50) to give compounds 8 (16.0 mg) and 9 (10.3 mg).
2.1. Chemistry Organic solvents, silica gel (75–120 mesh) and reversed phase (RPC18) silica (38–63 μm) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Preparative thin layer chromatography (PTLC) (RP-C18 F254 glass plates, 20 x 20 cm x 0.25 mm thick) and analytical TLC (silica gel 60 GF254 plates, 20 x 20 cm x 0.2 mm thick) or (RP-C18 F254 plates, 5 x 7.5 cm x 0.2 mm thick), both obtained from Merck (Darmstadt, Germany). 1D and 2D NMR were performed on a Bruker DRX 600 NMR spectrometer from Bruker Daltonics (MA, USA) using TMS as an internal standard. Liquid chromatography mass spectrometry (LC–MS) was carried out using Zorbax SB-C18 (2.1 × 150 mm, 1.8 μm) attached to Agilent 1260–6545 HPLC-QTOF-MS (Agilent, US). Detailed experimental procedure is available in supplementary material.
2.4. Lipase inhibition assay The pancreatic lipase inhibition assay was measured by an in vitro enzyme reaction, according to the method previously described (Tomohiro et al., 2009). Orlistat was used as a standard pancreatic lipase inhibitor. 3. Results 3.1. Biological screening of some medicinal plants and agricultural wastes for pancreatic lipase inhibition activity
2.2. Plant material The young twigs of P. alba were collected from El-Geneina, Menyet El-Nasr, Dakahlia Governorate, Egypt in August 2017. A voucher specimen has been deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University (08-17-PAMansoura). The agricultural wastes were collected from Gogar, Talkha, Dakahlia, while C. revoluta, I. carnea and C. arvensis were collected form the botanical garden of Mansoura university. The other plant materials were collected from Borg El Arab, Alexandria, Egypt. Authentication of the plant material has been done by Dr. Ibrahim Mashaly, Professor of Ecology, Faculty of Science, Mansoura University.
Twelve medicinal plants and four agricultural wastes were investigated for pancreatic lipase inhibition activity. The percentage of inhibition and the part used for each plant were recorded in (Table 1). Among the tested medicinal plants, P. alba (young twigs), Ononis vaginalis, and Asparagus stipularis demonstrated interestingly high percentage of inhibition, about 98, 94, and 92%, respectively; meanwhile Convolvulus arvensis and orlistat showed comparable activity about 90%. P. alba (leaves), Lotus polyphyllus, Euphorbia paralias, and Erygnium criticum showed moderate inhibition, about 80, 77, 72, and 70%, respectively. Other extracts showed percentage of inhibition ranging from about 66 to 46%. For the investigated agricultural wastes, Abelmoschus esculentus and Solanum melongena var. esculenta black demonstrated the highest percentage of inhibition, about 93 and 92%, respectively. Next in activity, Citrus limon (young twings) and Capsicum annuum, showing about 82 and 80%, respectively. These results are in accordance with literature, where the mucilage from Abelmoschus esculentus (Chukwuma et al.,
2.3. Extraction and isolation procedures About 2.5 kg of the powdered young twigs of P. alba were extracted exhaustively using 90% methanol (MeOH) to give 290.3 g of dried extract. The total extract was dissolved in the least amount of MeOH, diluted with 750 mL distilled water (H2O) in a separating funnel and then extracted successively with n-hexane, ethyl acetate (EtOAC) and finally with n-butanol. The solvents, in each case, were distilled to dryness under reduced to afford 31.8, 100.9 and 79.28 g, respectively of n-hexane (H), EtOAC (E) and n-butanol (B) fractions. The successive fractions (H, E and B) were subjected to lipase-inhibition assay and the results indicated that E was the most active, with IC50 4.73 μg/mL. Hence it was further subjected to fractionation on silica gel column followed by activity evaluation of the resulted fractions using lipaseinhibition assay. About 40 g of E were applied onto the top of a silica gel column (75 x 4.5 cm), previously packed in n-hexane, using a gradient elution of n-hexane–EtOAc (50:50→0:100), then EtOAc–MeOH (100:0→0:100). The effluents were collected in 250 mL fractions. Fractions with the same chromatographic pattern were pooled together and evaporated to dryness to give finally 11 fractions (1-11E). All of these fractions were subjected to lipase-inhibition assay. The results showed that 1E and 9E were the most active with IC50 of about 6.14 and 7.60 μg/mL, respectively. About 500 mg of fraction 1E were applied onto the top of sephadex LH-20 (35 x 1.5 cm), eluted with MeOH, 20 mL fractions were collected to give 36 subfractions (1E.1–36). Subfractions (1E.11–18, 149 mg) were applied onto the top of RP-C18 column (40 x 1.0 cm) using a gradient elution of H2O–MeOH (70:30→50:50) to give compounds 1 (6.2 mg) and 2 (12.3 mg) eluted with H2O–MeOH (60:40). Compound 3 (9.0 mg) was eluted with H2O–MeOH (55:45) and further purified using PTLC (on precoated RP-C18 F254 plates) using MeOH–H2O (50:50). Subfractions (1E.28–33, 56 mg) were applied on the top of RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (50:50) to give compounds 4 (5.5 mg), 5 (2.0 mg) and 6 (1.6 mg). About 200 mg
Table 1 Pancreatic lipase inhibitory activity of natural plant extracts. Methanol extracts
Part used
Lipase inhibition (%)
Asparagus stipularis Cakile martima Convolvulus althaeoides Convolvulus arvensis Cycas revoluta Erygnium criticum
Arial part Arial part Arial part Arial part Female cones Arial part Root Arial part Arial part Arial part Arial part Leaves Young twigs Arial part Leaves Leaves Leaves Young twigs Leaves Leaves Leaves Leaves
92.44 ± 1.52 63.26 ± 1.90 52.30 ± 4.11 89.99 ± 2.18 45.29 ± 1.25 70.27 ± 3.96 45.64 ± 2.98 72.02 ± 1.54 64.37 ± 2.21 76.87 ± 1.64 94.37 ± 0.78 80.10 ± 1.58 97.65 ± 0.23 65.53 ± 1.47 93.10 ± 0.91 79.65 ± 2.03 41.24 ± 3.66 81.93 ± 1.61 74.79 ± 4.44 54.73 ± 1.86 69.8 ± 1.38 91.60 ± 0.69 90.0 ± 2.18
Euphorbia paralias Ipomea cairica Lotus polyphyllus Ononis vaginalis Populus alba Zygophyllum album Abelmoschus esculentus Capsicum annuum Citrus limon Solanum melongena var. serpentinum purple Solanum melongena var. serpentinum black Solanum melongena var. serpentinum white Solanum melongena var.esculenta black Orlistat
The activity is expressed as percentage of inhibition (%) of extract at 800 μg/mL and orlistat at 1 μg/mL. Results are expressed as the mean value of triplicate data points. 2
Industrial Crops & Products 141 (2019) 111778
M. Elsbaey, et al.
Fig. 1. Structures of compounds (1–9), isolated from Populus alba.
2018), the fruit of Citrus limon, and the flower of Capsicum annuum, all were previously reported as inhibitors of pancreatic lipase (GironésVilaplana et al., 2014; Marrelli et al., 2016). Meanwhile, none of the remaining extracts has been previously investigated for pancreatic lipase inhibition activity.
Table 2 Pancreatic Lipase inhibitory activity of Populus alba.
3.2. Bio-guided isolation of the active constituents of the methanolic extract of white poplar Due to its interesting activity, the methanolic extract of white poplar was subjected to bio-guided fractionation in order to isolate the active principles. The extract was successively fractionated with n-hexane (H), ethyl acetate (E), and n-butanol (B). Through biological screening, it was found that (E) was accountable for the high activity of the extract, demonstrating IC50 value of 4.7 μg/mL (Table 2). Accordingly, E was subjected to column chromatography and active fractions 1E and 9 E afforded compounds 1–6 and 7–9, respectively (Fig. 1).
Extract/Compound
IC50
Total methanolic extract (PAS) n-Hexane fraction (H) Ethyl acetate fraction (E) n-Butanol fraction (B) p-Hydroxybenzoic acid (1) trans p-Coumaric acid (2) trans-Cinnamic acid (3) (+)-Ampelopsin (4) (+)-Taxifolin (5) (+)-Aromadendrin (6) Salicin (7) Quercetin-3-O-rutinoside (rutin) (8) Isorhamnetin-3-O-rutinoside (narcissin) (9) Orlistat
7.15 ± 0.44 73.32 ± 6.0 4.75 ± 0.10 6.65 ± 0.17 68.5 ± 2.3 112.2 ± 3.4 98.7 ± 1.0 46.2 ± 0.4 23.2 ± 0.8 173.1 ± 8.1 193.7 ± 6.2 152.3 ± 0.9 122.4 ± 1.5 1.35 ± 0.1
The activity was shown as IC50 value, (μg/mL for extract and fraction, μM for compounds). Results are expressed as the mean value of triplicate data points ± SD.
3.2.1. Characterization of the active compounds Phytochemical studies of the active fractions afforded 9 compounds (1–9). Based on NMR data and HPLC-QTOF-MS analysis (Fig. S1–S9), the isolates were identified as p-hydroxybenzoic acid (1) (Peungvicha et al., 1998), trans p-coumaric acid (2) (Liao et al., 2014), trans-cinnamic acid (3) (Liu et al., 2004), (+)-ampelopsin (4) (Sahidin et al., 2008), (+)-taxifolin (5) (Kuspradini et al., 2009), (+)-aromadendrin (6) (Jeon et al., 2011), salicin (7) (Otsuka et al., 1989), rutin (8) (Amen et al., 2015), and narcissin (9) (Sekii et al., 2015). Apart from compounds (2) (Kim et al., 2006), (6) and (7) (Tawfeek et al., 2019) these compounds are reported for the first time from P. alba in the present study.
by compound (1). The remaining compounds showed IC50 values ranging from 98.7 to 193.7 μM. The isolates were of two categories; flavonoids and phenolic acids. Both flavonoids and phenolic acids are the most commonly studied classes of natural products for lipase inhibition (Buchholz and Melzig, 2015). Generally flavonoids were the most active. The dihydro-quercetin derivatives, taxifolin 5 and ampelopsin 4 showed the lowest IC50 values of 23.2 μM and 46.2 μM, respectively, which is in accordance with previous literature regarding quercetin and taxifolin as a potent inhibitors of pancreatic lipase (Lee et al., 2011; Martinez-Gonzalez et al., 2017). Phenolic acids showed much lower activity, with p-hydroxybenzoic acid being the most active showing IC50 value of 68.5 μM, in accordance with previously reported by (De Pradhan et al., 2017). Meanwhile, the
3.2.2. Pancreatic lipase inhibition assay All the isolates were evaluated for pancreatic lipase inhibition potential (Table 2). Compounds (4) and (5) were the most active, followed 3
Industrial Crops & Products 141 (2019) 111778
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cinnamic acid derivatives (2) and (3) showed IC50 values of 112.2 and 98.7 μM, respectively, also compatible with literature (Karamać and Amarowicz, 1996). Previous reports in literature justified the superiority of flavonoids over phenolic acids in pancreatic lipase inhibition by their high molecular mass structure that can tolerate the increase in number of hydroxyl groups (Martinez-Gonzalez et al., 2017). Consequently, flavonoids and phenolic acids are accountable for the lipase inhibition potential of the extract or possibly a synergistic mechanism between them.
Jeon, Y.E., Yin, X.F., Choi, D.B., Lim, S.S., Kang, I.-J., Shim, J.-H., 2011. Inhibitory activity of aromadendrin from prickly pear (Opuntia ficus-indica) root on aldose reductase and the formation of advanced glycation end products. Food Sci. Biotechn. 20, 1283. https://doi.org/10.1007/s10068-011-0177-4. Karamać, M., Amarowicz, R., 1996. Inhibition of pancreatic lipase by phenolic acids examination in vitro. Z. Naturforsch. BC 51, 903–906. https://doi.org/10.1515/znc1996-11-1222. Kim, J.-K., Ham, Y.-H., Bae, Y.-S., 2006. Phenolic compounds from bark of Suwon poplar (Populus alba L. and Populus glandulosa Uyeki). Holzforschung. 60, 674–677. https:// doi.org/10.1515/HF.2006.113. Kuspradini, H., Mitsunaga, T., Ohashi, H., 2009. Antimicrobial activity against Streptococcus sobrinus and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (Koompassia malaccensis) extracts. J. Korean Wood Sci. Technol. 55, 308–313. https://doi.org/10.1007/s10086-009-1026-4. Lee, I.-C., Bae, J.-S., Kim, T., Kwon, O.J., Kim, T.H., 2011. Polyphenolic constituents from the aerial parts of Thymus quinquecostatus var. japonica collected on Ulleung Island. J. Korean Soc. Appl. Biol. Chem. 54, 811–816. https://doi.org/10.1007/BF03253166. Liao, C.-R., Kuo, Y.-H., Ho, Y.-L., Wang, C.-Y., Yang, C.-S., Lin, C.-W., Chang, Y.-S., 2014. Studies on cytotoxic constituents from the leaves of Elaeagnus oldhamii Maxim. in non-small cell lung cancer A549 cells. Molecules. 19, 9515–9534. https://doi.org/10. 3390/molecules19079515. Liu, R., Li, A., Sun, A., 2004. Preparative isolation and purification of hydroxyanthraquinones and cinnamic acid from the Chinese medicinal herb Rheum officinale Baill. by high-speed counter-current chromatography. J. Chromatogr. A 1052, 217–221. https://doi.org/10.1016/j.chroma.2004.08.101. Marrelli, M., Menichini, F., Conforti, F., 2016. Hypolipidemic and antioxidant properties of hot pepper flower (Capsicum annuum L.). Plant Foods Hum. Nutr. 71, 301–306. https://doi.org/10.1007/s11130-016-0560-7. Martinez-Gonzalez, A.I., Alvarez-Parrilla, E., Díaz-Sánchez, Á., de la Rosa, L.A., NúñezGastélum, J.A., Vazquez-Flores, A.A., Gonzalez-Aguilar, G.A., 2017. In vitro inhibition of pancreatic lipase by polyphenols: a kinetic, fluorescence spectroscopy and molecular docking study. Food Tech. Biotech. 55, 519–530. https://doi.org/10.17113/ftb. 55.04.17.5138. Otsuka, H., Yamasaki, K., Yamauchi, T., 1989. Alangifolioside, a diphenylmethylene derivative, and other phenolics from the leaves of Alangium platanifolium var. trilobum. Phytochemical 28, 3197–3200. https://doi.org/10.1016/0031-9422(89) 80306-1. Peungvicha, P., Temsiririrkkul, R., Prasain, J.K., Tezuka, Y., Kadota, S., Thirawarapan, S.S., Watanabe, H., 1998. 4-Hydroxybenzoic acid: a hypoglycemic constituent of aqueous extract of Pandanus odorus root. J. Ethnopharmacol. 62, 79–84. https://doi. org/10.1016/S0378-8741(98)00061-0. Przybysz, K., Przybysz, P., 2013. Poplar wood as raw material for the paper industry in the twenty-first century. Ann. WULS - SGGW, For. Wood Technol. 84, 56–59. Qi, X., 2018. Review of the clinical effect of orlistat. IOP Conf. Ser. Mater. Sci. Eng. 301. https://doi.org/10.1088/1757-899X/301/1/012063. Segula, D., 2014. Complications of obesity in adults: a short review of the literature. Malawi Med. J. 26, 20–24. Sekii, Y., Han, J., Isoda, H., Bouaziz, M., Dhouib, A., Sayadi, S., Shigemori, H., 2015. Two isorhamnetin glycosides from Arthrocnemum glaucum that inhibit adipogenesis in 3T3-L1 adipocytes. Chem. Nat. Compd. 51, 338–340. https://doi.org/10.1007/ s10600-015-1276-x. Sahidin, S., Hakim, E.S., Syah, Y.M., Juliawaty, L.D., Achmad, S.A., Din, L.B., Latip, J., 2008. Reservatrol dimers from stem bark of Hopea gergaria and their cytotoxic properites. Indo. J. Chem. 8 (2), 245–251. https://doi.org/10.22146/ijc.21629. Tawfeek, N., Sobeh, M., Hamdan, D.I., Farrag, N., Roxo, M., El-Shazly, A.M., Wink, M., 2019. Phenolic compounds from Populus alba L. and Salix subserrata willd. (Salicaceae) counteract oxidative stress in Caenorhabditis elegans. Molecules 24, 1999. https://doi.org/10.3390/molecules24101999. Tomohiro, M., Satoshi, I., Akihiro, I., Ei, K., 2009. Hypolipidemic effect of Pleurotus eryngii extract in fat-loaded mice. J. Nutr. Sci. Vitaminol. 56, 48–53. https://doi.org/10. 3177/jnsv.56.48. Zhang, J., Kang, M.-J., Kim, M.-J., Kim, M.-E., Song, J.-H., Lee, Y.-M., Kim, J.-I., 2008. Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo. Nutr. Res. Pract. 2, 200–203. https://doi.org/10.4162/nrp.2008.2.4.200.
4. Conclusion Upon biological screening of the twenty two plant extracts by pancreatic lipase inhibition assay, the methanolic extract of white poplar showed considerable activity. Through bio-guided fractionation and isolation, nine inhibitors of pancreatic lipase were identified. The flavonoids, (+) taxifolin (5) and (+)-ampelopsin (4) were the most active, followed by p-hydroxybenzoic acid (1). The results demonstrated that flavonoids and phenolic acids from white poplar could serve as basis for development of new anti-lipase drugs. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111778. References Abdou, M., Ibrahim, T., 2015. Response of Populus alba, L. Transplants to compost, biofertilizers and mineral NPK fertilization. Sci. J. Flowers Ornam. Plants. 2, 51–65. https://doi.org/10.21608/sjfop.2015.5099. Amen, Y.M., Marzouk, A.M., Zaghloul, M.G., Afifi, M.S., 2015. A new acylated flavonoid tetraglycoside with anti-inflammatory activity from Tipuana tipu leaves. Nat. Prod. Res. 29, 511–517. https://doi.org/10.1080/14786419.2014.952233. Birari, R.B., Bhutani, K.K., 2007. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Disco. Today. 12, 879–889. https://doi.org/10.1016/j. drudis.2007.07.024. Buchholz, T., Melzig, M.F., 2015. Polyphenolic compounds as pancreatic lipase inhibitors. Planta Med. 81, 771–783. https://doi.org/10.1055/s-0035-1546173. Chukwuma, C.I., Islam, M.S., Amonsou, E.O., 2018. A comparative study on the physicochemical, anti‐oxidative, anti‐hyperglycemic and anti‐lipidemic properties of amadumbe (Colocasia esculenta) and okra (Abelmoschus esculentus) mucilage. J. Food Biochem. 42, e12601. https://doi.org/10.1111/jfbc.12601. De la Garza, A.L., Milagro, F.I., Boque, N., Campión, J., Martínez, J.A., 2011. Natural inhibitors of pancreatic lipase as new players in obesity treatment. Planta Med. 77, 773–785. https://doi.org/10.1055/s-0030-1270924. De Pradhan, I., Dutta, M., Choudhury, K., De, B., 2017. Metabolic diversity and in vitro pancreatic lipase inhibition activity of some varieties of Mangifera indica L. fruits. Int. J. Food Prop. 20, S3212–S3223. https://doi.org/10.1080/10942912.2017.1357041. Gironés-Vilaplana, A., Moreno, D.A., García-Viguera, C., 2014. Phytochemistry and biological activity of Spanish Citrus fruits. Food Funct. 5, 764–772. https://doi.org/10. 1039/c3fo60700c. Ištok, I., Šefc, B., Hasan, M., Popović, G., Sedlar, T., 2017. Fiber characteristics of white poplar (Populus alba L.) juvenile wood along the Drava River. Drv. Ind. 68, 241–247. https://doi.org/10.5552/drind.2017.1729.
4
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
White poplar: Targeted isolation of pancreatic lipase inhibitors a
Marwa Elsbaey , Yhiya Amen
a,b
c
, Toshinori Nakagawa , Kuniyoshi Shimizu
b,⁎
T
a
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt Department of Agro-environmental Sciences, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 8190395, Japan c Department of Biological Resources Management, The University of Shiga Prefecture 2500, Hassaka-cho, Hikone-City, Shiga, 522-8533, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: poplar Lipase Obesity Bio-guided isolation Flavonoids Phenolic acids
In order to discover new anti-obesity agents, twenty two natural extracts from medicinal plants and agricultural wastes were screened by pancreatic lipase inhibition assay. The methanolic extract of poplar wood (Populus alba L.), an important raw material for paper industry, showed promising inhibitory activity, its IC50 value was 7.15 μg/mL. Through bio-guided fractionation, the ethyl acetate fraction (E) was found to be the major contributor to the activity, IC50 = 4.7 μg/mL. Hence, it was subjected to bio-guided isolation and nine compounds (1–9) were identified from the active sub-fractions. The isolates were identified by means of one- and twodimensional nuclear magnetic spectroscopy (1D, 2D-NMR), as well as high-performance liquid chromatography quadrupole time-of flight mass spectrometry (HPLC-QTOF-MS). (+)-Taxifolin (5) and (+)-ampelopsin (4) showed the highest activity, their IC50 values were 23.2 and 46.2 μM, respectively. The results indicated that flavonoids and phenolic acids in poplar wood could serve as potential candidates for development of new antiobesity agents.
1. Introduction Poplar wood is widely used for production of a variety of wood products, pulp, paper, and other fiber-based products. In the twentyfirst century, the worldwide demand for raw materials has grown extremely and hence poplar wood occupied an important position as a fiber resource in paper production (Przybysz and Przybysz, 2013). White poplar (Populus alba L.) has been particularly valued as a commercial wood source for its drought tolerant properties versus the changed climate and ecological conditions (Ištok et al., 2017). It is one of the most distinctive poplar species which is indigenous to Europe (Abdou and Ibrahim, 2015). Obesity is a significant public health problem that has become epidemic worldwide (Segula, 2014). Current evidence showed that obesity and overweight are globally the fifth leading risks for death (Buchholz and Melzig, 2015). Obesity is reported as a major contributor to diabetes mellitus, cardiovascular diseases and stroke, musculoskeletal disorders, dementia, and various types of cancer, including breast, endometrial, prostate, and colon cancer (Zhang et al., 2008). Pancreatic lipase is the key enzyme responsible for fat digestion, consequently its absorption. Inhibition of pancreatic lipase is an effective strategy for obesity management (de la Garza et al., 2011). Even
though physical activity and increased energy expenditure are the crucial cornerstones in body weight loss, drug treatment is indispensable for prevention of weight regain (de la Garza et al., 2011). Since 1999, orlistat® has been the only approved pancreatic lipase inhibitor as a pharmaceutical agent, according to the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) (Qi, 2018). Despite being an effective and a selective inhibitor, it is associated with many unpleasant gastrointestinal side effects. In this context, natural products can serve as a vast platform for development of safe and cost-effective drugs that can satisfy the need of the anti-obesity market (Birari and Bhutani, 2007; de la Garza et al., 2011). Particularly, exploring agricultural waste as an affordable and cheap source for natural products has many merits, including the environmental benefit. The aim of this research is to identify safe pancreatic lipase inhibitors of natural origin that could be of potential interest for the antiobesity market. In this study, the methanolic extracts of twelve medicinal plants and four agricultural wastes, were tested for pancreatic lipase inhibition activity. The young twigs of P. alba showed promising inhibitory activity. The extract was subjected to bio-guided fractionation and the active principles were identified.
⁎
Corresponding author at: Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail addresses: [email protected] (M. Elsbaey), [email protected] (Y. Amen), [email protected] (T. Nakagawa), [email protected] (K. Shimizu). https://doi.org/10.1016/j.indcrop.2019.111778 Received 8 May 2019; Received in revised form 29 July 2019; Accepted 9 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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2. Material and methods
of fraction 9E were applied onto the top of Sephadex LH-20 column (35 x 1.5 cm), eluted using MeOH, 10 mL fractions were collected to give 34 subfractions (9E.1–34). Subfractions (9E.6–8, 13 mg) were purified onto the top of RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (70:30) to give compound 7 (9.0 mg). Subfractions (9E.10–17, 42 mg) were chromatographed by a RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (50:50) to give compounds 8 (16.0 mg) and 9 (10.3 mg).
2.1. Chemistry Organic solvents, silica gel (75–120 mesh) and reversed phase (RPC18) silica (38–63 μm) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Preparative thin layer chromatography (PTLC) (RP-C18 F254 glass plates, 20 x 20 cm x 0.25 mm thick) and analytical TLC (silica gel 60 GF254 plates, 20 x 20 cm x 0.2 mm thick) or (RP-C18 F254 plates, 5 x 7.5 cm x 0.2 mm thick), both obtained from Merck (Darmstadt, Germany). 1D and 2D NMR were performed on a Bruker DRX 600 NMR spectrometer from Bruker Daltonics (MA, USA) using TMS as an internal standard. Liquid chromatography mass spectrometry (LC–MS) was carried out using Zorbax SB-C18 (2.1 × 150 mm, 1.8 μm) attached to Agilent 1260–6545 HPLC-QTOF-MS (Agilent, US). Detailed experimental procedure is available in supplementary material.
2.4. Lipase inhibition assay The pancreatic lipase inhibition assay was measured by an in vitro enzyme reaction, according to the method previously described (Tomohiro et al., 2009). Orlistat was used as a standard pancreatic lipase inhibitor. 3. Results 3.1. Biological screening of some medicinal plants and agricultural wastes for pancreatic lipase inhibition activity
2.2. Plant material The young twigs of P. alba were collected from El-Geneina, Menyet El-Nasr, Dakahlia Governorate, Egypt in August 2017. A voucher specimen has been deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University (08-17-PAMansoura). The agricultural wastes were collected from Gogar, Talkha, Dakahlia, while C. revoluta, I. carnea and C. arvensis were collected form the botanical garden of Mansoura university. The other plant materials were collected from Borg El Arab, Alexandria, Egypt. Authentication of the plant material has been done by Dr. Ibrahim Mashaly, Professor of Ecology, Faculty of Science, Mansoura University.
Twelve medicinal plants and four agricultural wastes were investigated for pancreatic lipase inhibition activity. The percentage of inhibition and the part used for each plant were recorded in (Table 1). Among the tested medicinal plants, P. alba (young twigs), Ononis vaginalis, and Asparagus stipularis demonstrated interestingly high percentage of inhibition, about 98, 94, and 92%, respectively; meanwhile Convolvulus arvensis and orlistat showed comparable activity about 90%. P. alba (leaves), Lotus polyphyllus, Euphorbia paralias, and Erygnium criticum showed moderate inhibition, about 80, 77, 72, and 70%, respectively. Other extracts showed percentage of inhibition ranging from about 66 to 46%. For the investigated agricultural wastes, Abelmoschus esculentus and Solanum melongena var. esculenta black demonstrated the highest percentage of inhibition, about 93 and 92%, respectively. Next in activity, Citrus limon (young twings) and Capsicum annuum, showing about 82 and 80%, respectively. These results are in accordance with literature, where the mucilage from Abelmoschus esculentus (Chukwuma et al.,
2.3. Extraction and isolation procedures About 2.5 kg of the powdered young twigs of P. alba were extracted exhaustively using 90% methanol (MeOH) to give 290.3 g of dried extract. The total extract was dissolved in the least amount of MeOH, diluted with 750 mL distilled water (H2O) in a separating funnel and then extracted successively with n-hexane, ethyl acetate (EtOAC) and finally with n-butanol. The solvents, in each case, were distilled to dryness under reduced to afford 31.8, 100.9 and 79.28 g, respectively of n-hexane (H), EtOAC (E) and n-butanol (B) fractions. The successive fractions (H, E and B) were subjected to lipase-inhibition assay and the results indicated that E was the most active, with IC50 4.73 μg/mL. Hence it was further subjected to fractionation on silica gel column followed by activity evaluation of the resulted fractions using lipaseinhibition assay. About 40 g of E were applied onto the top of a silica gel column (75 x 4.5 cm), previously packed in n-hexane, using a gradient elution of n-hexane–EtOAc (50:50→0:100), then EtOAc–MeOH (100:0→0:100). The effluents were collected in 250 mL fractions. Fractions with the same chromatographic pattern were pooled together and evaporated to dryness to give finally 11 fractions (1-11E). All of these fractions were subjected to lipase-inhibition assay. The results showed that 1E and 9E were the most active with IC50 of about 6.14 and 7.60 μg/mL, respectively. About 500 mg of fraction 1E were applied onto the top of sephadex LH-20 (35 x 1.5 cm), eluted with MeOH, 20 mL fractions were collected to give 36 subfractions (1E.1–36). Subfractions (1E.11–18, 149 mg) were applied onto the top of RP-C18 column (40 x 1.0 cm) using a gradient elution of H2O–MeOH (70:30→50:50) to give compounds 1 (6.2 mg) and 2 (12.3 mg) eluted with H2O–MeOH (60:40). Compound 3 (9.0 mg) was eluted with H2O–MeOH (55:45) and further purified using PTLC (on precoated RP-C18 F254 plates) using MeOH–H2O (50:50). Subfractions (1E.28–33, 56 mg) were applied on the top of RP-C18 column (40 x 1.0 cm) using an isocratic elution of H2O–MeOH (50:50) to give compounds 4 (5.5 mg), 5 (2.0 mg) and 6 (1.6 mg). About 200 mg
Table 1 Pancreatic lipase inhibitory activity of natural plant extracts. Methanol extracts
Part used
Lipase inhibition (%)
Asparagus stipularis Cakile martima Convolvulus althaeoides Convolvulus arvensis Cycas revoluta Erygnium criticum
Arial part Arial part Arial part Arial part Female cones Arial part Root Arial part Arial part Arial part Arial part Leaves Young twigs Arial part Leaves Leaves Leaves Young twigs Leaves Leaves Leaves Leaves
92.44 ± 1.52 63.26 ± 1.90 52.30 ± 4.11 89.99 ± 2.18 45.29 ± 1.25 70.27 ± 3.96 45.64 ± 2.98 72.02 ± 1.54 64.37 ± 2.21 76.87 ± 1.64 94.37 ± 0.78 80.10 ± 1.58 97.65 ± 0.23 65.53 ± 1.47 93.10 ± 0.91 79.65 ± 2.03 41.24 ± 3.66 81.93 ± 1.61 74.79 ± 4.44 54.73 ± 1.86 69.8 ± 1.38 91.60 ± 0.69 90.0 ± 2.18
Euphorbia paralias Ipomea cairica Lotus polyphyllus Ononis vaginalis Populus alba Zygophyllum album Abelmoschus esculentus Capsicum annuum Citrus limon Solanum melongena var. serpentinum purple Solanum melongena var. serpentinum black Solanum melongena var. serpentinum white Solanum melongena var.esculenta black Orlistat
The activity is expressed as percentage of inhibition (%) of extract at 800 μg/mL and orlistat at 1 μg/mL. Results are expressed as the mean value of triplicate data points. 2
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Fig. 1. Structures of compounds (1–9), isolated from Populus alba.
2018), the fruit of Citrus limon, and the flower of Capsicum annuum, all were previously reported as inhibitors of pancreatic lipase (GironésVilaplana et al., 2014; Marrelli et al., 2016). Meanwhile, none of the remaining extracts has been previously investigated for pancreatic lipase inhibition activity.
Table 2 Pancreatic Lipase inhibitory activity of Populus alba.
3.2. Bio-guided isolation of the active constituents of the methanolic extract of white poplar Due to its interesting activity, the methanolic extract of white poplar was subjected to bio-guided fractionation in order to isolate the active principles. The extract was successively fractionated with n-hexane (H), ethyl acetate (E), and n-butanol (B). Through biological screening, it was found that (E) was accountable for the high activity of the extract, demonstrating IC50 value of 4.7 μg/mL (Table 2). Accordingly, E was subjected to column chromatography and active fractions 1E and 9 E afforded compounds 1–6 and 7–9, respectively (Fig. 1).
Extract/Compound
IC50
Total methanolic extract (PAS) n-Hexane fraction (H) Ethyl acetate fraction (E) n-Butanol fraction (B) p-Hydroxybenzoic acid (1) trans p-Coumaric acid (2) trans-Cinnamic acid (3) (+)-Ampelopsin (4) (+)-Taxifolin (5) (+)-Aromadendrin (6) Salicin (7) Quercetin-3-O-rutinoside (rutin) (8) Isorhamnetin-3-O-rutinoside (narcissin) (9) Orlistat
7.15 ± 0.44 73.32 ± 6.0 4.75 ± 0.10 6.65 ± 0.17 68.5 ± 2.3 112.2 ± 3.4 98.7 ± 1.0 46.2 ± 0.4 23.2 ± 0.8 173.1 ± 8.1 193.7 ± 6.2 152.3 ± 0.9 122.4 ± 1.5 1.35 ± 0.1
The activity was shown as IC50 value, (μg/mL for extract and fraction, μM for compounds). Results are expressed as the mean value of triplicate data points ± SD.
3.2.1. Characterization of the active compounds Phytochemical studies of the active fractions afforded 9 compounds (1–9). Based on NMR data and HPLC-QTOF-MS analysis (Fig. S1–S9), the isolates were identified as p-hydroxybenzoic acid (1) (Peungvicha et al., 1998), trans p-coumaric acid (2) (Liao et al., 2014), trans-cinnamic acid (3) (Liu et al., 2004), (+)-ampelopsin (4) (Sahidin et al., 2008), (+)-taxifolin (5) (Kuspradini et al., 2009), (+)-aromadendrin (6) (Jeon et al., 2011), salicin (7) (Otsuka et al., 1989), rutin (8) (Amen et al., 2015), and narcissin (9) (Sekii et al., 2015). Apart from compounds (2) (Kim et al., 2006), (6) and (7) (Tawfeek et al., 2019) these compounds are reported for the first time from P. alba in the present study.
by compound (1). The remaining compounds showed IC50 values ranging from 98.7 to 193.7 μM. The isolates were of two categories; flavonoids and phenolic acids. Both flavonoids and phenolic acids are the most commonly studied classes of natural products for lipase inhibition (Buchholz and Melzig, 2015). Generally flavonoids were the most active. The dihydro-quercetin derivatives, taxifolin 5 and ampelopsin 4 showed the lowest IC50 values of 23.2 μM and 46.2 μM, respectively, which is in accordance with previous literature regarding quercetin and taxifolin as a potent inhibitors of pancreatic lipase (Lee et al., 2011; Martinez-Gonzalez et al., 2017). Phenolic acids showed much lower activity, with p-hydroxybenzoic acid being the most active showing IC50 value of 68.5 μM, in accordance with previously reported by (De Pradhan et al., 2017). Meanwhile, the
3.2.2. Pancreatic lipase inhibition assay All the isolates were evaluated for pancreatic lipase inhibition potential (Table 2). Compounds (4) and (5) were the most active, followed 3
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cinnamic acid derivatives (2) and (3) showed IC50 values of 112.2 and 98.7 μM, respectively, also compatible with literature (Karamać and Amarowicz, 1996). Previous reports in literature justified the superiority of flavonoids over phenolic acids in pancreatic lipase inhibition by their high molecular mass structure that can tolerate the increase in number of hydroxyl groups (Martinez-Gonzalez et al., 2017). Consequently, flavonoids and phenolic acids are accountable for the lipase inhibition potential of the extract or possibly a synergistic mechanism between them.
Jeon, Y.E., Yin, X.F., Choi, D.B., Lim, S.S., Kang, I.-J., Shim, J.-H., 2011. Inhibitory activity of aromadendrin from prickly pear (Opuntia ficus-indica) root on aldose reductase and the formation of advanced glycation end products. Food Sci. Biotechn. 20, 1283. https://doi.org/10.1007/s10068-011-0177-4. Karamać, M., Amarowicz, R., 1996. Inhibition of pancreatic lipase by phenolic acids examination in vitro. Z. Naturforsch. BC 51, 903–906. https://doi.org/10.1515/znc1996-11-1222. Kim, J.-K., Ham, Y.-H., Bae, Y.-S., 2006. Phenolic compounds from bark of Suwon poplar (Populus alba L. and Populus glandulosa Uyeki). Holzforschung. 60, 674–677. https:// doi.org/10.1515/HF.2006.113. Kuspradini, H., Mitsunaga, T., Ohashi, H., 2009. Antimicrobial activity against Streptococcus sobrinus and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (Koompassia malaccensis) extracts. J. Korean Wood Sci. Technol. 55, 308–313. https://doi.org/10.1007/s10086-009-1026-4. Lee, I.-C., Bae, J.-S., Kim, T., Kwon, O.J., Kim, T.H., 2011. Polyphenolic constituents from the aerial parts of Thymus quinquecostatus var. japonica collected on Ulleung Island. J. Korean Soc. Appl. Biol. Chem. 54, 811–816. https://doi.org/10.1007/BF03253166. Liao, C.-R., Kuo, Y.-H., Ho, Y.-L., Wang, C.-Y., Yang, C.-S., Lin, C.-W., Chang, Y.-S., 2014. Studies on cytotoxic constituents from the leaves of Elaeagnus oldhamii Maxim. in non-small cell lung cancer A549 cells. Molecules. 19, 9515–9534. https://doi.org/10. 3390/molecules19079515. Liu, R., Li, A., Sun, A., 2004. Preparative isolation and purification of hydroxyanthraquinones and cinnamic acid from the Chinese medicinal herb Rheum officinale Baill. by high-speed counter-current chromatography. J. Chromatogr. A 1052, 217–221. https://doi.org/10.1016/j.chroma.2004.08.101. Marrelli, M., Menichini, F., Conforti, F., 2016. Hypolipidemic and antioxidant properties of hot pepper flower (Capsicum annuum L.). Plant Foods Hum. Nutr. 71, 301–306. https://doi.org/10.1007/s11130-016-0560-7. Martinez-Gonzalez, A.I., Alvarez-Parrilla, E., Díaz-Sánchez, Á., de la Rosa, L.A., NúñezGastélum, J.A., Vazquez-Flores, A.A., Gonzalez-Aguilar, G.A., 2017. In vitro inhibition of pancreatic lipase by polyphenols: a kinetic, fluorescence spectroscopy and molecular docking study. Food Tech. Biotech. 55, 519–530. https://doi.org/10.17113/ftb. 55.04.17.5138. Otsuka, H., Yamasaki, K., Yamauchi, T., 1989. Alangifolioside, a diphenylmethylene derivative, and other phenolics from the leaves of Alangium platanifolium var. trilobum. Phytochemical 28, 3197–3200. https://doi.org/10.1016/0031-9422(89) 80306-1. Peungvicha, P., Temsiririrkkul, R., Prasain, J.K., Tezuka, Y., Kadota, S., Thirawarapan, S.S., Watanabe, H., 1998. 4-Hydroxybenzoic acid: a hypoglycemic constituent of aqueous extract of Pandanus odorus root. J. Ethnopharmacol. 62, 79–84. https://doi. org/10.1016/S0378-8741(98)00061-0. Przybysz, K., Przybysz, P., 2013. Poplar wood as raw material for the paper industry in the twenty-first century. Ann. WULS - SGGW, For. Wood Technol. 84, 56–59. Qi, X., 2018. Review of the clinical effect of orlistat. IOP Conf. Ser. Mater. Sci. Eng. 301. https://doi.org/10.1088/1757-899X/301/1/012063. Segula, D., 2014. Complications of obesity in adults: a short review of the literature. Malawi Med. J. 26, 20–24. Sekii, Y., Han, J., Isoda, H., Bouaziz, M., Dhouib, A., Sayadi, S., Shigemori, H., 2015. Two isorhamnetin glycosides from Arthrocnemum glaucum that inhibit adipogenesis in 3T3-L1 adipocytes. Chem. Nat. Compd. 51, 338–340. https://doi.org/10.1007/ s10600-015-1276-x. Sahidin, S., Hakim, E.S., Syah, Y.M., Juliawaty, L.D., Achmad, S.A., Din, L.B., Latip, J., 2008. Reservatrol dimers from stem bark of Hopea gergaria and their cytotoxic properites. Indo. J. Chem. 8 (2), 245–251. https://doi.org/10.22146/ijc.21629. Tawfeek, N., Sobeh, M., Hamdan, D.I., Farrag, N., Roxo, M., El-Shazly, A.M., Wink, M., 2019. Phenolic compounds from Populus alba L. and Salix subserrata willd. (Salicaceae) counteract oxidative stress in Caenorhabditis elegans. Molecules 24, 1999. https://doi.org/10.3390/molecules24101999. Tomohiro, M., Satoshi, I., Akihiro, I., Ei, K., 2009. Hypolipidemic effect of Pleurotus eryngii extract in fat-loaded mice. J. Nutr. Sci. Vitaminol. 56, 48–53. https://doi.org/10. 3177/jnsv.56.48. Zhang, J., Kang, M.-J., Kim, M.-J., Kim, M.-E., Song, J.-H., Lee, Y.-M., Kim, J.-I., 2008. Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo. Nutr. Res. Pract. 2, 200–203. https://doi.org/10.4162/nrp.2008.2.4.200.
4. Conclusion Upon biological screening of the twenty two plant extracts by pancreatic lipase inhibition assay, the methanolic extract of white poplar showed considerable activity. Through bio-guided fractionation and isolation, nine inhibitors of pancreatic lipase were identified. The flavonoids, (+) taxifolin (5) and (+)-ampelopsin (4) were the most active, followed by p-hydroxybenzoic acid (1). The results demonstrated that flavonoids and phenolic acids from white poplar could serve as basis for development of new anti-lipase drugs. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111778. References Abdou, M., Ibrahim, T., 2015. Response of Populus alba, L. Transplants to compost, biofertilizers and mineral NPK fertilization. Sci. J. Flowers Ornam. Plants. 2, 51–65. https://doi.org/10.21608/sjfop.2015.5099. Amen, Y.M., Marzouk, A.M., Zaghloul, M.G., Afifi, M.S., 2015. A new acylated flavonoid tetraglycoside with anti-inflammatory activity from Tipuana tipu leaves. Nat. Prod. Res. 29, 511–517. https://doi.org/10.1080/14786419.2014.952233. Birari, R.B., Bhutani, K.K., 2007. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Disco. Today. 12, 879–889. https://doi.org/10.1016/j. drudis.2007.07.024. Buchholz, T., Melzig, M.F., 2015. Polyphenolic compounds as pancreatic lipase inhibitors. Planta Med. 81, 771–783. https://doi.org/10.1055/s-0035-1546173. Chukwuma, C.I., Islam, M.S., Amonsou, E.O., 2018. A comparative study on the physicochemical, anti‐oxidative, anti‐hyperglycemic and anti‐lipidemic properties of amadumbe (Colocasia esculenta) and okra (Abelmoschus esculentus) mucilage. J. Food Biochem. 42, e12601. https://doi.org/10.1111/jfbc.12601. De la Garza, A.L., Milagro, F.I., Boque, N., Campión, J., Martínez, J.A., 2011. Natural inhibitors of pancreatic lipase as new players in obesity treatment. Planta Med. 77, 773–785. https://doi.org/10.1055/s-0030-1270924. De Pradhan, I., Dutta, M., Choudhury, K., De, B., 2017. Metabolic diversity and in vitro pancreatic lipase inhibition activity of some varieties of Mangifera indica L. fruits. Int. J. Food Prop. 20, S3212–S3223. https://doi.org/10.1080/10942912.2017.1357041. Gironés-Vilaplana, A., Moreno, D.A., García-Viguera, C., 2014. Phytochemistry and biological activity of Spanish Citrus fruits. Food Funct. 5, 764–772. https://doi.org/10. 1039/c3fo60700c. Ištok, I., Šefc, B., Hasan, M., Popović, G., Sedlar, T., 2017. Fiber characteristics of white poplar (Populus alba L.) juvenile wood along the Drava River. Drv. Ind. 68, 241–247. https://doi.org/10.5552/drind.2017.1729.
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