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Lithuanian University of Health Sciences Medical Faculty Institute Lithuanian University of Health Sciences Medical Faculty Institute of Physiology and Pharmacology Carl Ibe The effects of Elsholtzia ciliata extracts on isolated rat mesenteric arteries using wire myography Master Thesis Work Supervision: Doc. dr. Alė Laukevičienė KAUNAS, 2017/2018 Lithuanian University of Health Sciences Medical Faculty Institute of Physiology and Pharmacology I hereby certify: The effects of Elsholtzia ciliata extracts on isolated rat mesenteric arteries using wire myography Master Thesis Work supervisor Doc., Dr. Alė Laukevičienė Date Reviewer Work carried out by: Silvijus Abramavicius Data Carl Ibe 2018.05.11 KAUNAS, 2017/2018 2 Contents SUMMARY......................................................................................................................................4 ABBREVIATIONS AND DEFINITIONS.................................................................................................................................5 WORK OBJECTIVES...................................................................................................................................7 1.1 ELSHOLTZIA CILIATA .......................................................................................................... 8 1.2 CHEMICAL COMPOSITION OF ELSHOLTZIA CILIATA ESSENTIAL OIL AND 70% ETHANOL EXTRACT ................................................................................................................... 9 1.3 ANTICANCER ACTIVITY OF ELSHOLTZIA CILIATA ESSENTIAL OILS ........................ 9 1.4 ENDOTHELIUM-DEPENDENT AND -INDEPENDENT VASORELAXANT ACTIONS AND MECHANISMS INDUCED BY TOTAL FLAVONOIDS OF ELSHOLTZIA SPLENDENS IN RAT AORTAS. ........................................................................................................................................ 9 2.1. TEST MATERIALS ............................................................................................................... 12 2.2. REAGENTS USED ................................................................................................................ 14 2.4. MESENTERIC ARTERY FUNCTIONAL TESTS ............................................................... 14 2.5. STATISTICAL ANALYSIS .................................................................................................. 16 3.RESULTS....................................................................................................................................18 3.1 THE BIOLOGICAL EFFECTIVENESS OF TWO DIFFERENT PREPARATIONS OF ELSCHOLTZIA CILIATA: ETHANOLIC AND ESSENTIAL OIL EMULSION. 18 3.2 THE ROLE OF ENDOTHELIUM IN EOE INDUCED RELAXATION OF THE VESSELS 19 3.3 THE EFFECT OF EOE ON NO RELEASE……………………………………………………21 3.4 THE ROLE OF EXTRACELLULAR CA2+……………………………………………………22 4. DISCUSSION……………………………………………………………………………………23 5. CONCLUSION…………………………………………………………………………………..23 6. CONFLICTS OF INTEREST……………………………………………………………………24 7. ACKNOWLEDGEMENTS…………………………………………………………….………..24 8. REFERENCES…………………………………………………………………………….24,25,26 3 SUMMARY Master thesis by Carl Ibe. Supervisor doc. dr. Alė Laukevičienė. Lithuanian University of Health Sciences, Institute of Physiology and Pharmacology, Kaunas. Title: The effect of Elscholtzia ciliata extracts and essential oil on isolated rat mesenteric arteries using wire myography. The aim: To investigate the effect of Elscholtzia ciliata extracts on isolated rat mesenteric arteries’ contractility. Goals of the study: 1. To evaluate the biological effectiveness and effect character of two different preparations of Elscholtzia ciliata: ethanolic extract and essential oil emulsion.2. To investigate some pathways and mechanisms involved into relaxation of vessels by Elscholtzia ciliata essential oil emulsion (EOE).3. To evaluate effect of EOE on endothelium-induced relaxation by comparing relaxation of the vessels with and without endothelium, as well after inhibition of NO release by L- NAME.4. To compare the Ca2+ titration curve with and without Elscholtzia ciliata essential oil suspension in Ca2+ free PSS. Methods: Both vasorelaxing and vasoconstricting effects on rat isolated small mesenteric arteries were measured using DMT wire myography. Results and conclusions: E. ciliata ethanolic extract and essential oil emulsion reduce contractile force of small rat mesenteric arteries. The vasorelaxation is dose-dependent; higher doses caused more significant decreases in vasoconstriction. Essential oil emulsion proved to be more effective than ethanolic extracts in reducing contraction of vessels. E. ciliata ethanolic extract was slightly more effective than the ethanol control, while E. ciliata essential oil emulsion had a significant effect on inhibiting contraction of vessels. Emulsion relaxes intact vessels better, than vessels without endothelium; relaxation is slightly attenuated by NO inhibitor L-NAME. The findings indicate that relaxing Elscholtzia essential oil emulsion effect includes both smooth muscle and endothelial relaxing mechanisms. The Ca2+ titration curve results indicate slight inhibition of intracellular Ca2+ release induced by essential oil emulsion. 4 ABBREVIATIONS Ach- Acetylcholine ADE- Angelica decursiva extract CaCl2 – Calcium Chloride EDRF- endothelium derived relaxing factors EOE -E. ciliata essential oil emulsion g – grams KCl – Potassium chloride KH2PO4 - Potassium dihydro phosphate KPSS – potassium rich physiological salt solution l – litres L-NAME- N(ω)-nitro-L-arginine methyl ester MgSO4×7H20 – Magnesium Sulphate heptahydrate ml - millilitres M - moles NA – Noradrenalin NaCl - Sodium Chloride PSS – physiological salt solution ROCCS- receptor-operative Ca2+ channels VDCCC- voltage-dependent Ca2+ channels % – percent μl - microlitres 5 DEFINITIONS Wire myography- Wire myography is an in vitro technique to examine functional responses and vascular reactivity of isolated small resistance arteries. Elscholtzia ciliata- Elscholtzia ciliata is a flowering plant, commonly known as Vietnamese Balm or crested latesummer mint. INTRODUCTION Hypertension is a threat to global public health, itself being silent it is a major risk factor for heart disease, stroke, kidney failure, premature mortality and disability. The global prevalence of hypertension in adults above age 25 was 40% in 2008. [1] In 2012 it was reported to affect 1 billion people worldwide, with a higher incidence in developed countries. [2] Hypertension also is a major contributor to the global burden of disease.[3] This stresses the importance of effective blood pressure control in order to prevent its serious complications and improve global health. Although there is already a comprehensive set of antihypertensive drugs, the search for new drugs and supplements continues. Increasingly there is a focus on herbs and their potential blood pressure lowering effects. Hibiscus sabdariffa namely was shown to significantly decrease systolic and diastolic blood pressure in a 2015 meta-analysis to a degree comparable to common blood pressure reduction seen with Ramipril. [4] Such results are encouraging and warrant further research into the potential powers of many other plants, proving the particular relevance of this study. Elsholtzia ciliata, is a flowering plant of the Lamiaceae (Mint) family found mostly in Asia and Europe. It has been used both in traditional Chinese medicine for a variety of ailments such as diarrhea, fever, headache and edema, as well as for culinary purposes. [5] The scientific literature on E. ciliata is scarce, but there are studies that showed mild anti-leishmanial activity, attenuation of renal inflammation and interstitial fibrosis, inhibition of mast cell-mediated inflammation. [6,7] The health ministry of China has listed E.ciliata as an edible plant, which can be used as a powerful source of natural antioxidants. [8] There have not been any published studies or articles about the potential effects of E. ciliata extracts on blood vessels and blood pressure, making this a pilot study of this herb grown in Lithuania. Elscholtzia splendens on the other hand has already been successfully investigated for its endothelium- 6 dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids in rat aortas. [9] In the present study, the effects of ethanolic extract and an emulsion from E. ciliata essential oil on rat mesenteric arteries and potential mechanisms were investigated in order to differentiate between endothelial or vascular smooth muscle mechanisms of action. WORK OBJECTIVES Purpose: To investigate the effects of Elscholtzia ciliata extracts effects on small rat mesenteric blood vessel tone, possible pathways and mechanisms involved. Tasks: To evaluate the biological effectiveness of two different preparations of Elscholtzia ciliata: ethanolic and essential oil emulsion (EOE). To investigate whether the relaxing effect of EOE is conducted through smooth muscle- dependent or endothelium-dependent mechanisms by comparing relaxation of vessels with and without endothelium. To compare relaxation of the vessels with intact endothelium and after inhibition of NO release. To compare the Ca2+ titration curve with and without Elscholtzia ciliata essential oil emulsion. 7 1. LITERATURE REVIEW 1.1 Elsholtzia ciliata Elscholtzia ciliata is a flowering plant, commonly known as Vietnamese Balm or crested latesummer mint. Although native in Asia it can now be found in Europe and Northern America. [10,11] Elscholtzia ciliata grows up to 60cm in height with stalked, long and serrated leaves with up to 8.5cm
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