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F. U. Afifi , E. Hallaq, V. Kasabri and S. Litescu

Antidiabetic and antiobesity potential of Adiantum capillus-veneris used in the traditional medicine of Jordan. F. U. Afifi , E. Hallaq, V. Kasabri and S. Litescu School of Pharmacy, University of Jordan, Amman-Jordan Natural Products 2016, Melbourne, Australia - July 25-27 th , 2016.

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F. U. Afifi , E. Hallaq, V. Kasabri and S. Litescu

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  1. Antidiabetic and antiobesity potential of Adiantum capillus-veneris used in the traditional medicine of Jordan F. U. Afifi, E. Hallaq, V. Kasabri and S. Litescu School of Pharmacy, University of Jordan, Amman-Jordan Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  2. Overview • Project Background • Proposed research • Outline of methodology • Phytochemical evaluation • Acute toxicity determination • Biological evaluation • Anti-obesity activity • Anti-diabetic activity • Results and Conclusions Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  3. Objectives The herbalists recommend the infusion of the aerial parts of A. capillusveneris L. for the treatment of CVD, hypercholesterolemia and diabetes. This presentation aims to evidence the claimed antidiabetic and hypocholesteroemicactivities of A. capillusveneris L.using in vitro and in vivo experiments Phytochemical composition determination: CC, LC-MS Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  4. Objectives TM is part of the Jordanian culture Medicinal plants are the most common form of TM in Jordan. Main line treatment of a wide range of diseases, especially for Nomades and inhabitants of the remote areas Easy accessibility, low cost and broad acceptability of the TM in the local society as a safe and natural way to treat the diseases To explore and rejuvenate the ethnopharmacological wealth of Jordan To evaluate the efficacy of TM in treatment of chronic, non-communicable diseases 4 Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  5. Medicinal plants of Jordan • ~ 2500 vascular plants • 1/5 of them are classified as medicinal plants • There are more than 140 wild edible plants listed, many of them have medicinal value. • Only a small % of them has been phytochemically and biologically screened Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  6. Diabetes mellitus and obesity • 382 Mio people from across the world have Type 2 DM and by 2035 this will rise to 592 millions • Obesityis one of the greatest threats to global health (more than 1 billion overweight/300 Mio obese adults worldwide) • Diabetes mellitus is the 3rd killer of human (cancer – CVD and cerebrovascular diseases) • CVD [~20 Mio †] ← DM and HTN ← overweight and obesity Natural Products 2016, Melbourne, Australia - July 25-27th, 2016 8

  7. Why Diabetes mellitus? IDF, 2013 Melbourn Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  8. DM and obesity in Jordan • diabetes in Jordan is 10.1%; and ranks the 9th among the MENA countries • The incidence of type 2 DM and impaired fasting glycaemia in Jordan is increasing • Prevalence of related metabolic disorders (hypertension, obesity and the co-morbidities) are at alarming rates locally • obesity in Jordan varied greatly with age and sex but generally increasing, irrespective of the measurement used. • Men: 17.8%(20-29 years), 27.9%(30-39 years), 36.3%(40-49 years), 47.5%(50-59 years), 33.7%(>60 years), Total 32.7% • Female:25.9 %(20-29 years), 56.0%(30-39years), 76.1 %(40-49 years), 73.8%(50-59years),65.8%(>60 years), Total 59.8% 10 Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  9. Adiantumcapillus-venerisL.AdiantaceaeSouthern maidenhair fernBlack maidenhair fernCommon maidenhairVenus hair fern Adiantum: large genus of ferns, widely distributed throughout the world • Reported multiple biological activities Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  10. Adiantumcapillus-veneris Several biological activities have been reported: Antimicrobial activity Antioxidative activity Antiinflammatory activity Analgesic activity Antiurolithic activity Angiogenic activity Wound healing effect Hypoglycemic effect Protective effect against oxygen free radicals 13 Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  11. Phytochemical evaluation1-LC-MS 2-Isolation by CC Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  12. HPLC-MS analyses (1) gallic acid 3.44 mg/g; (2) caffeic acid 1.55 mg/g; (3) rutin 4.77 mg/g; (4) quercetin-3-O-glucoside 3.96 mg/g; (5) ferulic acid 3.88 mg/g; (6) ellagic acid 5.48 mg/g; (7) quercetin 0.43 mg/g Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  13. Phytochemical evaluation: Some LC-MS identified phenolic substances and plant acids Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  14. Phytochemical evaluation CC isolated substances R=H, Quercetin R=Rutinose, Rutin R =Glu, Quercetin-3-O-glucoside ß-sitosterol Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  15. Biological evaluation Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  16. Acute toxicity experiments with A. capillusveneris No LD50could be determined [mice and rats did not exhibit any abnormal behavior when p.o AE in doses up to 8.0 g/kg BW No lethality were observed within 24 hoursand afterwards during one weekobservation period No lethality were observed in rats after 10 weeks treatment with the highest utilized dose(500mg/kg bwt) [=35g/70kg BW] AE given animals exhibited normal values for RBC, WBC and Hb as the untreated control animals No abnormalities were observed in gross-anatomy Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  17. Antiobesity activity Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  18. 1-In vitro screening of the PL inhibitory activity of A. capillus-veneris extracts and of isolated major compounds 2-In vivo experiments: Simultanous administration of HCD and A. capillus-veneris extract and comparison to HCD given rats Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  19. In vitro screening of the PL inhibitory activity 22 The only clinically approved pharmacologic agent as PL - inhibitor is Orlistat (Xenical) One of the world’s best selling drugs The potential of developing successful NP with anti-pancreatic lipase activity poorly investigated Colorimetric method that measures the release of p-nitrophenol Extracts with an IC50higher than 1000μg were considered as inactive Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  20. Pancreatic Lipase (PL) Activity Assay • Colorimetrically , PL catalytic activity was determined by measuring the hydrolysis of p-nitrophenylbutyrate (p-NPB) into p-nitrophenol, and thus quantified by the increase of the rate of p-nitrophenol release at 410 nm estimated from the slope of the linear segment of absorbance vs. time profiles • PL inhibition (%) was calculated according to the formula:  Inhibition (%) = 100 – ((B/A) x100); where A is the PL activity without inhibitor or test extract/compound and; B is the PL activity with inhibitor or test extract/compound. 23 Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  21. In vitro inhibitory effects of A. capillus-venerisAE, chlorogenic acid, ellagic acid and ferulic acid, and orlistat concentrations on PL- activity. Results are mean ± SEM (n = 3 independent replicates). Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  22. In vitro PL IC50 values for increasing concentrations of A. capillus-veneris AEs and its bioactive consistuents versus Orlistat Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  23. Induction of hypercholesterolemia Animals were given standard pellet diet (ad libitum) and high cholesterol diet (HCD). HCD was prepared using : 37.5g cholesterol powder and 4.5g cholic acid dissolved in 300 ml 0.3% CMC concentration Daily 4 ml of this suspension were given orally to rats of 200g BW (2.2g/kg BW cholesterol and 0.26g/kg BW cholic acid) Pan et al., Eur J Pharmacol, 552, 170 (2006) Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  24. Treatment of hypercholesterolemic rats with plant extract or Atorvastatin • A. capillus-veneris AE (500mg/Kg b.w) or Atorvastatin (10mg/Kg b.w) was administered for 4 weeks (weeks 7-10) together with HCD to hypercholesterolemic animals and on weekly basis and the following parameters were determined: • Total cholesterol (TC) • Triglycerides • LDL • HDL Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  25. In vivo chronic effects of A. capillus veneris AE on the TC concentrations and the incremental AUC of 10 weeks treatment ***P<0.001 compared to control group, ∆∆∆ P<0.001 compared to HCD and P<0.05 compared to atorvastatin 10 mg/Kg b.wt; as determined by ANOVA followed by Dunnett post test Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  26. In vivo chronic effects of A. capillus veneris AE on the HDL concentrations and the incremental AUC of 10 weeks treatment Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  27. In vivo chronic effects of A. capillus veneris AE on the LDL concentrations and the incremental AUC of 10 weeks treatment ***P<0.001 compared to control group, ∆∆∆P<0.001 compared to HCD, as determined by ANOVA followed by Dunnett post test Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  28. In vivo chronic effects of A. capillus-venerisAE on the incremental AUC of 10 weeks treatment on triglycerides concentrations Results are mean ± SEM of AUC of TAG for 10 weeks intervention using ANOVA followed by Dunnett post test where *P<0.05 and **P<0.01 compared to negative control group, ∆P<0.05 and ∆∆∆P<0.001 compared to HCD group, and P<0.01 compared to atorvastatin group. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  29. In vivo chronic effects of A. capillus-veneris AE on the incremental AUC of 10 weeks treatment on the body weight Results are mean ± SEM of AUC of BW for 10 weeks intervention using ANOVA followed by Dunnett post test, where *P<0.05 and **P<0.01 compared to control group, ∆∆∆P<0.001 compared to HCD group, and P<0.01 compared to atorvastatin group. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  30. Antidiabetic activity Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  31. 1-In vitro screening of the antidiabetic activityEnzymatic starch digestion 24h Glucose dialysis 2-In vivoacute glucose /starch tolerance tests in normoglycemic rats (OGTT, OSTT) Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  32. Enzymatic starch digestion in vitro Gelatinised starch+-amylase Incubation (20 min at 80oC) with different A. capillus-veneris aqueous extracts concentrations Second incubation (30 min at 60oC) with -amyloglucosidase (pH 4.75) Liberated glucose concentrations –UV spectrophotometry 35 Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  33. In vitro inhibitory effects of acarbose concentrations on enzymatic starch digestion (IC50 of 0.2 ± 0.02g/mL) Results are means ± SEM (n=3). ***P<0.001 compared to control incubations Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  34. In vitro inhibitory effects of Adiantumcapillus-veneris (AE) concentrations in mg/mL on enzymatic starch digestion Results are mean ± SEM (n = 3 independent replicates). *P<0.05 and ***P<0.001 compared to control (drug-free or plant-free) incubations, as determined by ANOVA followed by Dunnett post test. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  35. In vitro inhibitory effects of chlorogenic acidconcentrations in μg/mL on enzymatic starch digestion Results are mean ± SEM (n = 3 independent replicates). ***P<0.001 compared to control (drug-free or plant-free) incubations, as determined by ANOVA followed by Dunnett post test. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  36. Effects of ascending concentrations of A. capillus-venerisAE (mg/mL) and chlorogenic acid (μg/mL) on %reduction of enzymatic starch digestion in vitro. Results expressed as % decrease in control values are mean ± SEM (n = 3 independent replicates). *P<0.05 and ***P<0.001 compared to control (drug-free or plant-free) incubations as determined by ANOVA followed by Dunnett post test Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  37. Lack of modulatory in vitro effects of A. capillus-veneris (AE) concentrations (mg/mL) on the incremental AUC of 24h glucose movement. ***P<0.001 compared to control (drug-free or plant-free) incubations, as determined by ANOVA followed by Dunnett post test. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  38. Lack of in vivo modulatory postprandial antihyperglycemic effects of A. capillus-venerisAE concentrations in mg/Kg b.wt on OGTT over 165 min and AUC in normoglycemic overnight fasting rats Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  39. In vivo modulatory postprandial antihyperglycemic effects of A. capillus-veneris(AE) concentrations in mg/Kg b.wt on OSTT over 165 min and AUC in normoglycemic overnight fasting rats *P<0.05 and ***P<0.001 compared to control untreated animals, as determined by ANOVA followed by Dunnett post test. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  40. SUMMARY • Comparable to acarbose, highly pronounced dose-dependent inhibitory effects on enzymatic starch digestion were exerted by the A. capillus-veneris AE Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  41. SUMMARY • Comparable to orlistat antilipase trend, a concentration dependent PL inhibition was obtained for the AE and chlorogenic acid Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  42. SUMMARY • A. capillus-venerisextracts (125, 250 and 500mg/kg b.wt) effected highly pronounced decreases in starch induced hyperglycaemia over 165 min in normal rats, parallel to acarbose acute efficacy Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  43. SUMMARY • Comparable to atorvastatin, A. capillus-veneris AE exhibited substantial hypocholesterolemic activity Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  44. CONCLUSION • A. capillus-venerisextracts demonstrated promising antiobesity/slimming activity in addition to hypocholesterolemic potential. Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  45. Ongoing experiments • In vivo experiments : Co-administarationof Adiantumcapillusvenerisextract at different doses with Atorvastatin to hypercholesterolemic rats where the concentration of the drug is used at descending doses Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  46. General Conclusions Plant derived drugs have an important place in both traditional and modern medicine Concerning therapy with phytomedicines, a clear distinction has to be made between its possibilities and limits The consumer has to be protected from inefficient and dangerous drugs or from those with fantastic claims of indications Both; users and practitioners should be enabled to make the best benefit – risk assessment before using any herbal medicine Despite tested for generations; safety and efficacy of herbal medicines should be confirmed scientifically Natural Products 2016, Melbourne, Australia - July 25-27th, 2016

  47. Acknowledgements Natural Products 2016, Melbourne, Australia - July 25-27th, 2016 This project was financially supported by Deanship of Academic Research of the University of Jordan The researchers would like to extend their thanks to Miss Lara Majdalawi and Mrs. Haneen Ramadan for their technical help

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