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Published in: BMC Complementary Medicine and Therapies 1/2014

Open Access 01-12-2014 | Research article

Phenolic contents, antioxidant and anticholinesterase potentials of crude extract, subsequent fractions and crude saponins from Polygonum hydropiper L

Authors: Muhammad Ayaz, Muhammad Junaid, Jawad Ahmed, Farhat Ullah, Abdul Sadiq, Sajjad Ahmad, Muhammad Imran

Published in: BMC Complementary Medicine and Therapies | Issue 1/2014

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Abstract

Background

We investigated Polygonum hydropiper L. (P. hydropiper) for phenolic contents, antioxidant, anticholinesterase activities, in an attempt to rationalize its use in neurological disorders.

Methods

Plant crude extract (Ph.Cr), its subsequent fractions: n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq) and saponins (Ph.Sp) were evaluated for 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) free radical scavenging potential. Further, acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities were performed using Ellman's assay. Moreover, total phenolic contents of plant extracts were determined and expressed in mg of gallic acid equivalent per gram of dry sample (mg GAE/g dry weight).

Results

Among different fractions, Ph.Cr (90.82), Ph.Chf (178.16), Ph.EtAc (203.44) and Ph.Bt (153.61) exhibited high phenolic contents. All fractions showed concentration dependent DPPH scavenging activity, with Ph.EtAc 71.33% (IC50 15 μg/ml), Ph.Bt 71.40% (IC50 3 μg/ml) and Ph.Sp 71.40% (IC50 35 μg/ml) were most potent. The plant extracts exhibited high ABTS scavenging ability i.e. Ph.Bt (91.03%), Ph.EtAc (90.56%), Ph.Sp (90.84%), Ph.Aq (90.56%) with IC50 < 0.01 μg/ml. All fractions showed moderate to high AChE inhibitory activity as; Ph.Cr, 86.87% (IC50 330 μg/ml), Ph.Hex, 87.49% (IC50 35 μg/ml), Ph.Chf, 84.76% (IC50 55 μg/ml), Ph.Sp, 87.58% (IC50 108 μg/ml) and Ph.EtAc 79.95% (IC50 310 μg/ml) at 1 mg/ml). Furthermore the BChE inhibitory activity was most prominent in Ph.Hex 90.30% (IC50 40 μg/ml), Ph.Chf 85.94% (IC50 215 μg/ml), Ph.Aq 87.62% (IC50 3 μg/ml) and Ph.EtAc 81.01% (IC50 395 μg/ml) fractions.

Conclusions

In this study, for the first time, we determined phenolic contents, isolated crude saponins, investigated antioxidant and anticholinestrase potential of P. hydropiper extracts. The results indicate that P. hydropiper is enriched with potent bioactive compounds and warrant further investigation by isolation and structural elucidation to find novel and affordable compounds for the treatment of various neurological disorders.
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Literature
1.
2.
go back to reference Sharma R: Medicinal plants of India-An encyclopedia. Daya Publishing House, 110035 Delhi, India. 2003, 46-47. Sharma R: Medicinal plants of India-An encyclopedia. Daya Publishing House, 110035 Delhi, India. 2003, 46-47.
3.
go back to reference Foster S, James AD: A field guide to medicinal plants: Eastern and central North America. Peterson Field Guide Series, 2nd edition, Houghton Mifflin Park Avenue, New York 10003. 1990, 241- Foster S, James AD: A field guide to medicinal plants: Eastern and central North America. Peterson Field Guide Series, 2nd edition, Houghton Mifflin Park Avenue, New York 10003. 1990, 241-
4.
go back to reference Chevallier A: The encyclopedia of medicinal plants. Dorling Kindersley London. 1996, London: Andrew DK Publishing, 185-ISBN 9-780751-303148 Chevallier A: The encyclopedia of medicinal plants. Dorling Kindersley London. 1996, London: Andrew DK Publishing, 185-ISBN 9-780751-303148
5.
go back to reference Peng ZF, Strack D, Baumert A, Subramaniam R, Goh NK, Chia TF, Ngin Tan S, Chia LS: Antioxidant flavonoids from leaves of Polygonum hydropiper L. Phytochemistry. 2003, 62 (2): 219-228. 10.1016/S0031-9422(02)00504-6.CrossRefPubMed Peng ZF, Strack D, Baumert A, Subramaniam R, Goh NK, Chia TF, Ngin Tan S, Chia LS: Antioxidant flavonoids from leaves of Polygonum hydropiper L. Phytochemistry. 2003, 62 (2): 219-228. 10.1016/S0031-9422(02)00504-6.CrossRefPubMed
6.
go back to reference Chan Y-C, Wang M-F, Chen Y-C, Yang D-Y, Lee M-S, Cheng F-C: Long-term administration of polygonum multiflorum thunb: reduces cerebral ischemia-induced infarct volume in gerbils. Am J Chin Med. 2003, 31 (1): 71-77. 10.1142/S0192415X03000734.CrossRefPubMed Chan Y-C, Wang M-F, Chen Y-C, Yang D-Y, Lee M-S, Cheng F-C: Long-term administration of polygonum multiflorum thunb: reduces cerebral ischemia-induced infarct volume in gerbils. Am J Chin Med. 2003, 31 (1): 71-77. 10.1142/S0192415X03000734.CrossRefPubMed
7.
go back to reference Chen LW, Wang YQ, Wei LC, Shi M, Chan YS: Chinese herbs and herbal extracts for neuroprotection of dopaminergic neurons and potential therapeutic treatment of parkinsons disease. CNS Neurol Disord Drug Targets. 2007, 6 (4): 273-281. 10.2174/187152707781387288.CrossRefPubMed Chen LW, Wang YQ, Wei LC, Shi M, Chan YS: Chinese herbs and herbal extracts for neuroprotection of dopaminergic neurons and potential therapeutic treatment of parkinsons disease. CNS Neurol Disord Drug Targets. 2007, 6 (4): 273-281. 10.2174/187152707781387288.CrossRefPubMed
8.
go back to reference Li X, Matsumoto K, Murakami Y, Tezuka Y, Wu Y, Kadota S: Neuroprotective effects of Polygonum multiflorum on nigrostriatal dopaminergic degeneration induced by paraquat and maneb in mice. Pharmacol Biochem Behav. 2005, 82 (2): 345-352. 10.1016/j.pbb.2005.09.004.CrossRefPubMed Li X, Matsumoto K, Murakami Y, Tezuka Y, Wu Y, Kadota S: Neuroprotective effects of Polygonum multiflorum on nigrostriatal dopaminergic degeneration induced by paraquat and maneb in mice. Pharmacol Biochem Behav. 2005, 82 (2): 345-352. 10.1016/j.pbb.2005.09.004.CrossRefPubMed
9.
go back to reference Kumpulainen J, Salonen J: Natural antioxidants and anticarcinogens in nutrition, health and disease. R Soc Chem UK. 1999, 240: 178-187. Kumpulainen J, Salonen J: Natural antioxidants and anticarcinogens in nutrition, health and disease. R Soc Chem UK. 1999, 240: 178-187.
10.
go back to reference Shah SM, Ayaz M, Khan A-u, Ullah F, Shah a-u-HA, Iqbal H, Hussain S: 1,1-Diphenyl,2-picrylhydrazyl free radical scavenging, bactericidal, fungicidal and leishmanicidal properties of Teucrium stocksianum. Toxicol Ind Health. 2013, doi:10.1177/0748233713487250 Shah SM, Ayaz M, Khan A-u, Ullah F, Shah a-u-HA, Iqbal H, Hussain S: 1,1-Diphenyl,2-picrylhydrazyl free radical scavenging, bactericidal, fungicidal and leishmanicidal properties of Teucrium stocksianum. Toxicol Ind Health. 2013, doi:10.1177/0748233713487250
11.
go back to reference Halliwell B: Free radicals, antioxidants, and human disease: curiosity, cause, or consequence?. Lancet. 1994, 344: 721-724. 10.1016/S0140-6736(94)92211-X.CrossRefPubMed Halliwell B: Free radicals, antioxidants, and human disease: curiosity, cause, or consequence?. Lancet. 1994, 344: 721-724. 10.1016/S0140-6736(94)92211-X.CrossRefPubMed
12.
go back to reference Kuhnan J: The flavonoids. A class of semi-essential food components; their role in human nutrition. World Rev Nutr Diet. 1976, 24: 117-191.CrossRef Kuhnan J: The flavonoids. A class of semi-essential food components; their role in human nutrition. World Rev Nutr Diet. 1976, 24: 117-191.CrossRef
13.
go back to reference Barlow A, Susan M: Toxicological aspects of antioxidants used as food additives. Food Antioxidants. 1990, Netherlands: Springer, 253-307.CrossRef Barlow A, Susan M: Toxicological aspects of antioxidants used as food additives. Food Antioxidants. 1990, Netherlands: Springer, 253-307.CrossRef
14.
go back to reference Kil HY, Seong ES, Ghimire BK, Chung IM, Kwon SS, Goh EJ, Hoe K, Kim MJ, Lim JD, Lee D, Yu CY: Antioxidant and antimicrobial activities of crude sorghum extract. Food Chem. 2009, 115: 1234-1239. 10.1016/j.foodchem.2009.01.032.CrossRef Kil HY, Seong ES, Ghimire BK, Chung IM, Kwon SS, Goh EJ, Hoe K, Kim MJ, Lim JD, Lee D, Yu CY: Antioxidant and antimicrobial activities of crude sorghum extract. Food Chem. 2009, 115: 1234-1239. 10.1016/j.foodchem.2009.01.032.CrossRef
15.
go back to reference Shahidi F, Wanasundara P: Phenolic antioxidants. Crit Rev Food Sci Nutr. 1992, 32: 67-103. 10.1080/10408399209527581.CrossRefPubMed Shahidi F, Wanasundara P: Phenolic antioxidants. Crit Rev Food Sci Nutr. 1992, 32: 67-103. 10.1080/10408399209527581.CrossRefPubMed
16.
go back to reference Atta-ur R, Wahab A, Nawas S, Choudhary M: New cholinesterase inhibiting bisbenzylisoquinoline alkaloids from Cocculus pendulus. Chem Pharm Bull. 2004, 52: 802-806. 10.1248/cpb.52.802.CrossRef Atta-ur R, Wahab A, Nawas S, Choudhary M: New cholinesterase inhibiting bisbenzylisoquinoline alkaloids from Cocculus pendulus. Chem Pharm Bull. 2004, 52: 802-806. 10.1248/cpb.52.802.CrossRef
17.
go back to reference Ahmad W, Ahmad B, Ahmad M, Iqbal Z, Nisar M, Ahmad M: In vitro inhibition of acetylcholinesterase, buty-rylcholinesterase and lipoxygenase by crude extract of Myricaria elegans Royle. J Biol Sci. 2003, 11: 1046-1049. Ahmad W, Ahmad B, Ahmad M, Iqbal Z, Nisar M, Ahmad M: In vitro inhibition of acetylcholinesterase, buty-rylcholinesterase and lipoxygenase by crude extract of Myricaria elegans Royle. J Biol Sci. 2003, 11: 1046-1049.
18.
go back to reference Jann MW: Preclinical pharmacology of metriphonate. Pharmacotherapy. 1998, 18: 55-67.PubMed Jann MW: Preclinical pharmacology of metriphonate. Pharmacotherapy. 1998, 18: 55-67.PubMed
19.
go back to reference Aisen PS, Davis KL: The search for disease-modifying treatment for Alzheimer’s Disease. Neurology. 1997, 48: 35-41. 10.1212/WNL.48.5_Suppl_6.35S.CrossRef Aisen PS, Davis KL: The search for disease-modifying treatment for Alzheimer’s Disease. Neurology. 1997, 48: 35-41. 10.1212/WNL.48.5_Suppl_6.35S.CrossRef
20.
go back to reference Bachman DL, Wolf PA, Linn RT: Prevalence of dementia and probable senile dementia of the Alzheimer's type in the Framingham study. Neurology. 1992, 42: 115-119. 10.1212/WNL.42.1.115.CrossRefPubMed Bachman DL, Wolf PA, Linn RT: Prevalence of dementia and probable senile dementia of the Alzheimer's type in the Framingham study. Neurology. 1992, 42: 115-119. 10.1212/WNL.42.1.115.CrossRefPubMed
21.
go back to reference Voet D, Voet JG: Serine proteases. Biochemistry. 1995, USA: John Wiley and Sons, 390-2 Voet D, Voet JG: Serine proteases. Biochemistry. 1995, USA: John Wiley and Sons, 390-2
22.
go back to reference Knapp MJ, Knopman DS, Solomon PR, Pendlebury WW, Davis CS, Gracon SI: A 30-week randomized controlled trial of high-dose tacrine in patients with Alzheimer’s disease.The Tacrine study group. J Am Med Assoc. 1994, 271: 985-991. 10.1001/jama.1994.03510370037029.CrossRef Knapp MJ, Knopman DS, Solomon PR, Pendlebury WW, Davis CS, Gracon SI: A 30-week randomized controlled trial of high-dose tacrine in patients with Alzheimer’s disease.The Tacrine study group. J Am Med Assoc. 1994, 271: 985-991. 10.1001/jama.1994.03510370037029.CrossRef
23.
go back to reference Schneider LJ: Treatment of Alzheimer’s disease with cholinesterase inhibitors. Clin Geriatr Med. 2001, 17 (2): 337-58. 10.1016/S0749-0690(05)70072-0.CrossRefPubMed Schneider LJ: Treatment of Alzheimer’s disease with cholinesterase inhibitors. Clin Geriatr Med. 2001, 17 (2): 337-58. 10.1016/S0749-0690(05)70072-0.CrossRefPubMed
24.
go back to reference Heinrich M, Teoh HL: Galanthamine from snowdrop-the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol. 2004, 92: 147-162. 10.1016/j.jep.2004.02.012.CrossRefPubMed Heinrich M, Teoh HL: Galanthamine from snowdrop-the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol. 2004, 92: 147-162. 10.1016/j.jep.2004.02.012.CrossRefPubMed
25.
go back to reference Perry NSL, Houghton PJ, Theobald A, Jenner P, Perry EK: In-vitro inhibition of human erythrocyte acetylcholinesterase by Salvia lavandulaefolia essential oil and constituent terpenes. J Pharm Pharmacol. 2000, 52: 895-902. 10.1211/0022357001774598.CrossRefPubMed Perry NSL, Houghton PJ, Theobald A, Jenner P, Perry EK: In-vitro inhibition of human erythrocyte acetylcholinesterase by Salvia lavandulaefolia essential oil and constituent terpenes. J Pharm Pharmacol. 2000, 52: 895-902. 10.1211/0022357001774598.CrossRefPubMed
26.
go back to reference Konan AB, Datte JY, Yapo P: Nitric oxide pathway-mediated relaxant effect of aqueous sesame leaves extract (Sesamum radiatum Schum. & Thonn.) in the guinea-pig isolated aorta smooth muscle. BMC Complement Altern Med. 2008, 8: 23-10.1186/1472-6882-8-23.CrossRefPubMedPubMedCentral Konan AB, Datte JY, Yapo P: Nitric oxide pathway-mediated relaxant effect of aqueous sesame leaves extract (Sesamum radiatum Schum. & Thonn.) in the guinea-pig isolated aorta smooth muscle. BMC Complement Altern Med. 2008, 8: 23-10.1186/1472-6882-8-23.CrossRefPubMedPubMedCentral
27.
go back to reference Khan FA, Ullah Z, Haider S: Phytochemicals screening and antimicrobial activities of selected medicinal plants of Khyber pakhtunkhwa Pakistan. Afr J Pharm Pharmacol. 2011, 5 (6): 746-750. 10.5897/AJPP11.175.CrossRef Khan FA, Ullah Z, Haider S: Phytochemicals screening and antimicrobial activities of selected medicinal plants of Khyber pakhtunkhwa Pakistan. Afr J Pharm Pharmacol. 2011, 5 (6): 746-750. 10.5897/AJPP11.175.CrossRef
28.
go back to reference Kim D, Jaeong S, Lee C: Antioxidant capicity of phenolic phytochemicals from various cultivars of plums. Food Chem. 2003, 81: 321-326. 10.1016/S0308-8146(02)00423-5.CrossRef Kim D, Jaeong S, Lee C: Antioxidant capicity of phenolic phytochemicals from various cultivars of plums. Food Chem. 2003, 81: 321-326. 10.1016/S0308-8146(02)00423-5.CrossRef
29.
go back to reference Braca A, Tommasi ND, Bari LD, Pizza C, Politi M, Morelli I: Antioxidant principles from Bauhinia terapotensis. J Nat Prod. 2001, 64 (7): 892-895. 10.1021/np0100845.CrossRefPubMed Braca A, Tommasi ND, Bari LD, Pizza C, Politi M, Morelli I: Antioxidant principles from Bauhinia terapotensis. J Nat Prod. 2001, 64 (7): 892-895. 10.1021/np0100845.CrossRefPubMed
30.
go back to reference Re R, Pellegrini N, Proteggente A, Pannala A, Yong M, Rice-Evas C: Antioxidant activity applying an improved FBTS radical cation decolorization assay. Free Rad Biol Med. 1999, 26 (9/10): 1231-1237.CrossRefPubMed Re R, Pellegrini N, Proteggente A, Pannala A, Yong M, Rice-Evas C: Antioxidant activity applying an improved FBTS radical cation decolorization assay. Free Rad Biol Med. 1999, 26 (9/10): 1231-1237.CrossRefPubMed
31.
go back to reference Trevisan MTS, Macedo FVV, Meent M, Rhee IK, Verpoorte R: Screening for acetylcholinesterase inhibitors from plants to treat Alzheimer's disease. QuÃmica Nova. 2003, 26 (3): 301-304.CrossRef Trevisan MTS, Macedo FVV, Meent M, Rhee IK, Verpoorte R: Screening for acetylcholinesterase inhibitors from plants to treat Alzheimer's disease. Química Nova. 2003, 26 (3): 301-304.CrossRef
32.
go back to reference Classics Ellman GL, Courtney KD, Andres V, Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961, 7: 88-95. 10.1016/0006-2952(61)90145-9.CrossRef Classics Ellman GL, Courtney KD, Andres V, Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961, 7: 88-95. 10.1016/0006-2952(61)90145-9.CrossRef
33.
go back to reference Aruoma O: Methodological considerations for characterizing potential antioxidant actions of bioactive components in food plants. Mutat Res. 2003, 9 (20): 523-524. Aruoma O: Methodological considerations for characterizing potential antioxidant actions of bioactive components in food plants. Mutat Res. 2003, 9 (20): 523-524.
34.
go back to reference Jadhav H, Bhutani K: Antioxidant properties of Indian medicinal plants. Phytother Res. 2002, 16 (8): 771-773. 10.1002/ptr.1063.CrossRefPubMed Jadhav H, Bhutani K: Antioxidant properties of Indian medicinal plants. Phytother Res. 2002, 16 (8): 771-773. 10.1002/ptr.1063.CrossRefPubMed
35.
go back to reference Yildirim A, Mavi A, Oktay M, Kara A, Algur O, Bilaloglu V: Comparison of antioxidant and antimicrobial activities of tilia (Tilia argenta Desf Ex DC), sage (Salvia triloba L.) and black tea (Camellia sinensis) extracts. J Agric Food Chem. 2000, 48 (10): 5030-5034. 10.1021/jf000590k.CrossRefPubMed Yildirim A, Mavi A, Oktay M, Kara A, Algur O, Bilaloglu V: Comparison of antioxidant and antimicrobial activities of tilia (Tilia argenta Desf Ex DC), sage (Salvia triloba L.) and black tea (Camellia sinensis) extracts. J Agric Food Chem. 2000, 48 (10): 5030-5034. 10.1021/jf000590k.CrossRefPubMed
36.
go back to reference Hasan S, Hossain M, Akter R, Jamila M, Mazumder M, Rahman S: DPPH free radical scavenging activity of some Bangladeshi medicinal plants. J Med Plants Res. 2009, 3 (11): 875-879. Hasan S, Hossain M, Akter R, Jamila M, Mazumder M, Rahman S: DPPH free radical scavenging activity of some Bangladeshi medicinal plants. J Med Plants Res. 2009, 3 (11): 875-879.
37.
go back to reference Saeed S, Urfi M, Ali T, Khimani F, Gilani A: Antioxidants: their role in health and disease. Int J Pharmacol. 2005, 1 (3): 210-217.CrossRef Saeed S, Urfi M, Ali T, Khimani F, Gilani A: Antioxidants: their role in health and disease. Int J Pharmacol. 2005, 1 (3): 210-217.CrossRef
38.
go back to reference Bashir S, Gilani A: Studies on the antioxidant and analgesic activities of Aztec marigold (Tagetes erecta) flowers. Phytother Res. 2008, 22 (12): 1692-1694. 10.1002/ptr.2550.CrossRefPubMed Bashir S, Gilani A: Studies on the antioxidant and analgesic activities of Aztec marigold (Tagetes erecta) flowers. Phytother Res. 2008, 22 (12): 1692-1694. 10.1002/ptr.2550.CrossRefPubMed
39.
go back to reference Soare J, Dinis T, Cunha A, Almeida L: Antioxidant activities of some extracts of Thymus zygis. Free Radic Res. 1997, 26 (5): 469-478. 10.3109/10715769709084484.CrossRef Soare J, Dinis T, Cunha A, Almeida L: Antioxidant activities of some extracts of Thymus zygis. Free Radic Res. 1997, 26 (5): 469-478. 10.3109/10715769709084484.CrossRef
40.
go back to reference Hagerman A, Riedl K, Jones G, Sovik K, Ritchard N, Hartzfeld P: High molecular weight plant polyphenolics (Tannins) asbiological antioxidants. J Agric Food Chem. 1998, 46: 1887-1892. 10.1021/jf970975b.CrossRef Hagerman A, Riedl K, Jones G, Sovik K, Ritchard N, Hartzfeld P: High molecular weight plant polyphenolics (Tannins) asbiological antioxidants. J Agric Food Chem. 1998, 46: 1887-1892. 10.1021/jf970975b.CrossRef
41.
go back to reference Ingkaninan K, Temkitthawon P, Chuenchon K, Yuyaem T, Thongnoi W: Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. J Ethnopharmacol. 2003, 89: 261-264. 10.1016/j.jep.2003.08.008.CrossRefPubMed Ingkaninan K, Temkitthawon P, Chuenchon K, Yuyaem T, Thongnoi W: Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. J Ethnopharmacol. 2003, 89: 261-264. 10.1016/j.jep.2003.08.008.CrossRefPubMed
42.
go back to reference Tildesley NTJ, Kennedy DO, Perry EK, Ballard CG, Savelev S, Sesnes KA, Scholey AB: Salvia lavandulaefolia (Spanish sage) enhances memory in healthy young volunteers. Pharmacol Biochem Behav. 2003, 75: 669-674. 10.1016/S0091-3057(03)00122-9.CrossRefPubMed Tildesley NTJ, Kennedy DO, Perry EK, Ballard CG, Savelev S, Sesnes KA, Scholey AB: Salvia lavandulaefolia (Spanish sage) enhances memory in healthy young volunteers. Pharmacol Biochem Behav. 2003, 75: 669-674. 10.1016/S0091-3057(03)00122-9.CrossRefPubMed
Metadata
Title
Phenolic contents, antioxidant and anticholinesterase potentials of crude extract, subsequent fractions and crude saponins from Polygonum hydropiper L
Authors
Muhammad Ayaz
Muhammad Junaid
Jawad Ahmed
Farhat Ullah
Abdul Sadiq
Sajjad Ahmad
Muhammad Imran
Publication date
01-12-2014
Publisher
BioMed Central
Published in
BMC Complementary Medicine and Therapies / Issue 1/2014
Electronic ISSN: 2662-7671
DOI
https://doi.org/10.1186/1472-6882-14-145

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