Lithuanian University of Health Sciences Medical Faculty Institute of Physiology and Pharmacology

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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

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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

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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.

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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

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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.

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endothelium-7 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-muscle-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.

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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 in length and 25 cm in width. Shape-wise they are lanceolate to ovate with a gland-dotted underside.

The flowers bloom in a purple color throughout September and October with seeds propagating within them. [10] It is commonly found on hills, waste areas, sunny terraces, riverbanks and forests from 0-3400m of elevation. [12]

The plants grow in sunny situations on dry to moderately moist soil with a sandy-loamy or gritty-loamy substrate. They tolerate temperatures down to -29°C. [13]

The plant is commonly used as medicinal and herbal plant containing essential oils. Its leaves are also commonly used in various cuisines.

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1.2 Chemical composition of Elsholtzia ciliata essential oil and 70% ethanol extract

To this date there has only been one study conducted to assess the chemical composition of fresh, frozen and dried E. ciliata herbal materials. The sample, grown in Lithuania, provided only a modest number of different compounds upon analysis. 16 volatile compounds could be identified. Dehydroelsholtzia and elsholtzia ketone, sesquiterpenes β-bourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene being the predominant compounds in fresh, dried and frozen herbal samples. [13]

1.3 Anticancer activity of Elsholtzia ciliata essential oils

Elsholtzia ciliata essential oil was shown to provide antiproliferative activity on a variety of

cell lines in a recent pilot study at the Lithuanian University of Health Sciences, which also provided inside to the chemical composition of E. ciliata essential oils and extracts.

It showed statistically significant inhibition of cancer cell growth for human glioblastoma (U87), Pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231) in vitro.

Ethanolic extracts on the other hand did not show such activity. They were not able to have any effect on U87 cells, and merely slightly able to decrease the viability of MDA-MB231 cells at maximum concentration, while having no negative effects on human fibroblasts. In conclusion, there is some promising data that encourages further research of E. ciliata and its potential anticancer activity. [13]

1.4 Endothelium-dependent and -independent vasorelaxant actions and

mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas.

A Chinese team of researchers from Zeihjang investigated Elsholtzia splendens, a close relative of E. ciliata, for its vasorelaxant properties in rat aortas. Rat aortic rings were put in an organ bath system to assess total flavonoids of E. splendens activity on vasorelaxation via endothelium-dependent and -inendothelium-dependent mechanisms.

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10 inhibitor and the guanylate cyclase inhibitor which both were able to significantly block endothelium-dependent vasorelaxation of TFES.

They were able to show that TFES concentration-dependently was able to elevate the activity of NOS.

The addition of indomethacin as a blocker of PGI2 showed no effect on TFES-induced vasorelaxation. The KATP channel mechanism was investigated using the KATP channel blocker glibenclamide, which was able to weaken endothelium-independent TFES-induced vasorelaxation.

TFES was also able to weaken calcium-induced contraction in aortic rings with endothelium removed that were stimulated with KCl or phenylephrine.

This led the researchers to believe that the NOS/NO/cGMP pathway is likely part of the endothelium-dependent vasorelaxation under addition of TFES by potential activation of KATP channels, inhibition of intracellular calcium release, blockade of calcium channels and reduction of calcium influx into vascular smooth muscle cells. [9]

1.5 Vasorelaxatory mechanisms of isopiole, an extract from lemonfragrant

Angelica root, in rat thoracic aortas.

Researchers from the Guangzouh University of Chinese Medicine investigated Isoapiole, being one of the main components of Lemonfragrant Angelica Root, and its potential vasorelaxant effects on rat thoracic aortas. This herb is known for its useful properties in treating angina pectoris, stomach pain and abdominal pain in tradition Chinese medicine, while the underlying mechanisms were poorly understood.

The rat thoracic aortas were assessed with a wire myograph suspended in an organ bath, similar to the one used for my experiment. Isoapiole was added to Norepinephrine or KCl-precontracted vessels and vessel relaxation was recorded. In order to assess isoapiole’s effect on NO release.

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11 This study is of particular interest, because similar equipment, methods and specimens were assessed. They were able to isolate a potentially responsible vasorelaxant mechanism and even were able to show an increase in NO production. They also made a most relevant comparison to HUVEC in order to show possibly similar effects in human vessels. [14]

1.6. Vasorelaxant effects of Angelica decursiva root on isolated rat aortic rings

A team of Korean researchers investigated possibly vasorelaxant properties of Angelica

decursiva extract (ADE) on rat aortic rings. Similar to the extract used in my extract dried herbs were

extracted using 70% ethanol. Angelica decursiva is a Korean herb that is known in Korean folk medicine, that until this study had not been evaluated for its vasorelaxant effects.

ADE was shown to cause vasorelaxation in endothelium-intact aortic rings in a concentration-dependent manner. Interestingly they found ADE to have the same vasorelaxant properties in endothelium-denuded aortic rings. The maximal relaxation was achieved at a concentration of 800 µg/ml. This is of particular interest and lets itself for further more detailed research in the future. Upon further investigation into the mechanism they were able to conclude that there might be a correlation between the vasorelaxant effect of ADE and vascular smooth muscle since glibenclamide, used for KATP channel blockade slightly altered its vasorelaxant effects.

Furthermore, they investigated the effects of ADE on extracellular calcium-induced contraction via receptor operative calcium channels and voltage-operated calcium channels. Preincubation of aortic rings with ADE was able to significantly inhibit extracellular CaCl2 induced contractions. ADE altered the permeation of ROCCS or VDCCC activated by Phenylephrine.This proves the correlation between ADE and vascular smooth muscle relaxation. Two compounds, decursin and nodakenin, also found in ADE have already been shown to be vasorelaxing in rabbit carotid arteries and rats. [15,16]

1.7 Mechanisms of blood vessel relaxation through NO/cGMP pathway

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12 The underlying mechanism is mediated by cGMP, which results in dephosphorylation of the myosin light chain, which then weakens the interaction between myosin and actin, which ultimately induces vasorelaxation. [17, 18]

Studies show that the vasorelaxant effect of some herbal preparations is induced by increased expression or activity of the endothelial NO synthase, and of soluble guanylatecyclase (sGS) in vascular smooth muscle. [17]

Active compounds have been identified, which can lead to increased eNOS expression. Namely these are pentacters, triterpenes, butyric acid, some flavonoids and alkaloids. Compounds, which actively acted on eNOS were also identified, e.g methyl eugenol, alpha terpineol, among others. [18, 19]

2. MATERIALS AND METHOD

2.1. Test materials

Materials

The E. ciliata preparations were received from the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Lithuania. Producers are referring, that E.

ciliata aerial parts were collected in Vilnius, Lithuania, in July 2016 and were purchased as fresh and

dried herbs from “Zolynu namai”, Vilnius, Lithuania. The herbs were identified by Dr. Prof. Nijole Savickiene, Medical Academy, Lithuanian University of Health Sciences. A voucher specimen (L 17710) was deposited at the Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences. Fresh and dried herb materials were mechanically ground in a laboratory mill.

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Tissue preparation

The male Wistar rats were obtained from the Lithuanian University of Health Sciences vivarium. Animals were killed by CO2 overdose and sacrificed for exsanguination. Abdominal cavity was opened and small intestine together with mesentery was isolated and immediately placed in PSS solutionof the following composition (mM) NaCl -118,4; KCl - 4,7; MgSO4 - 1,2; CaCl2 - 2,5; NaHCO3 - 25,0; KH2PO4 - 1,2 and glucose - 5,5, according to manufacturer recommendations. A part of the small intestine was fixed and small mesenterial blood vessels were dissected free from surrounding fat and connective tissue. A segment about 2 mm in lengths was transferred to organ bath of a dual isometric myograph (JP-Trading, Denmark). Two tungsten wires (40 μm diameter) were inserted into the lumen of the segment and mounted on a holder and on a force transducer. The solution was continuously bubbled with 95% O2 and 5% CO2 mixture. Vessels mounted were allowed to equilibrate for 30 minutes at physiological conditions (temperature 37°C, pH=7,4) and pre stretched to their optimal lumen diameter for active tension development, using semi-automated normalization system, developed by Danish Myo Technology (DMT, Denmark). The internal circumference-wall tension ratio of the vessel was adjusted by setting its internal circumference L0 to 90% of which the vessel would have had if it was exposed to a passive tension equivalent to that produced by a transmural pressure of 100 mm Hg. Segments were washed and left to equilibrate for 30 minutes. Before experiment the segments were pre-contracted several times with noradrenalin and PSS solution with 3 washes every time between the contractions, according to manufacturer recommendations. Endothelium function was evaluated using 10 μM of acetylcholine after pre-contraction with 5 μM of noradrenaline.

Contracting ability of E. ciliata was investigated by adding preparations (0.1-10% (v/v)) to the organ bath and after 15 min the result was registered. Relaxing ability was investigated on the similar way on pre-contracted blood vessels.

Organs used in the experiments:

Isolated rat small mesenteric arteries (250 – 350 μm in diameter).

Chemicals used

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2.2. Reagents used

Reagents used throughout the experiments: PSS

KPSS

Noradrenalin (NA). Concentration used throughout the experiments: 10 -2 M/l. Acetycholine (Ach). Concentration used throughout the experiments: 10 -2 M/l

Ethanolic Elscholtzia ciliata extract Ethanol 70%

Elscholtzia ciliata essential oil emulsion

2.4. Mesenteric artery functional tests

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2.4.1 Protocol 1: to evaluate the biological effectiveness of E. ciliata: ethanolic and

essential oil emulsion.

The effect of Elscholtzia ciliata ethanolic extract, the ethanol control and essential oil emulsion on vessels with intact endothelium pre-contracted with noradrenalin (NA, 5µM) was assessed by adding rising concentrations of the respective substance. (0.1-2.5% (v/v)). Subsequent effects on vessel tone were observed. The ethanol control was added in concentrations equivalent to the amounts of ethanol found in the E. ciliata ethanolic extract.

2.4.2 Protocol 2: to investigate some pathways and mechanisms involved into

relaxation of vessels by Elscholtzia ciliata essential oil suspension

For this experiment we contracted vessels with noradrenalin (NA, 5µM) and observed the maximum contraction. The vessels were either with intact endothelium or ones where the endothelium had been removed using a hair (effectiveness was noted by assessing Ach response). We then added e. ciliata essential oil emulsion cumulatively and observed the vasorelaxant activity at the maximum concentration. We then compared the effect EOE on endothelial-intact and removed vessels.

2.4.3 Protocol 3: to compare the Ca

2+

titration curve with and without Elscholtzia

ciliata essential oil suspension.

Vessels were pre-contracted using Ca2+ free PSS and NA 5µM. We then added EOE to one set of samples. This was followed by adding rising levels of Calcium (CaCl2) to the bath and observing the vessels reaction. We paid attention to whether the vessels with EOE added would show a step-wise contraction indicating that Ca channels were still intact and functioning normally.

2.4.4 Protocol 4: to compare relaxation of the vessels with intact endothelium and

after inhibition of no release.

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Fig. 2 The equipment used (scheme)[20,21,22,23]

2.5. Statistical analysis

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17 difference between the samples was detected using the Student’s test. The means of compared samples were considered significantly different when p<0.05

3. RESULTS

3.1 The biological effectiveness of two different preparations of Elscholtzia ciliata:

ethanolic and essential oil emulsion.

Fig.3. The effect of Elscholtzia ciliata ethanolic extract, the ethanol control and essential oil emulsion (EOE) on vessels with intact endothelium contracted with noradrenalin (NA, 5µM). NA induced contraction is 100% before respective addition of preparations. The data represents the Mean±SEM. The effect of the ethanolic extract is insignificant p>0,05. The effect of the essential oil significant p<0,05.

Elscholtzia ciliata ethanolic extract decreases NA induced contraction of the vessels by half

(50±7.74%) (Fig. 3). In comparison the ethanolic control with concentration equal to the E. ciliata ethanolic extract caused only slightly less of a decrease in contraction of the vessels.(41±7.17%).

Elscholtzia ciliata essential oil emulsion showed a drastic decrease in contraction of the vessels even at

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Fig.4 shows the effect of small concentration of EOE on vessels precontracted with NA. Na-induced contraction is at 100%. The data represents the Mean±SEM

3.2 The role of endothelium in eoe induced relaxation of the vessels

The relaxing effect may be dependent on inhibition of smooth muscle contraction mechanism or on stimulating of endothelial relaxing factors production. In order to reveal, which mechanisms are more important in EOE induced effect, we have compared contraction of the vessels with endothelium and without endothelium.

Fig 5 . Effect of E. ciliata essential oil emulsion (EOE; 0,4%( v/v) applied for 15 min.) To

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contraction before the application of EOE is =100%. Data are mean±SEM. * – the effect is significant (p<0,05).

Fig 6. Exposition to Elscholtzia EO (0,5% v/v; 15 min) decreases contraction of the vessels (intact endothelium) induced by noradrenaline (5µM).

E. ciliata essential oil (EOE) decreases noradrenaline induced contraction of the vessels

almost in half, both in endothelium intact (up to 66,62±9,96%; p<0,05 ) and endothelium denuded (up to 56,3±4,3%, p<0,05) blood vessels (Fig. 5). There is no essential difference between vessels with and without endothelium, the intact vessels investigated were contracting only slightly more, than these without endothelium. This finding indicates, that EOE probably affects smooth muscles contraction mechanisms by decreasing their effectiveness rather than impairing endothelium function. Also we may speculate that results may be slightly distorted by mechanical removing of endothelium, because this may affect contraction of the vessels.

EOE 0,5% v/v, 15min..

NA, 5µM

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20 After a washing the NA induced contraction remains only slightly decreased or returns to previous level (data not shown). This fact indicates that EOE affects contraction in competitive way.

3.3 The effect of EOE on no release

In order to evaluate, is acute application of EOE stimulating or inhibiting the release of one of most important endothelial factors – nitric oxide (NO) we have compared relaxation of blood vessels induced by EOE both in L-NAME pretreated and non-pretreated vessels.

Fig. 7. The relaxation of rat small mesenteric blood vessels, previously contracted by NA (5 µM) in vitro. Relaxations induced by Elscholtzia ciliata Essential Oil emulsion both in intact and l-NAME (30 µM) pretreated vessels. NA induced contraction before the EOE is =100%. Data are

mean±SEM. * – the relaxation in non- pretreated vessels is significantly higher (p<0,05).

E. ciliata essential oil (EOE) induces relaxation of vessels pre-contracted by NA (Fig.7). This

relaxation was only slightly weakened after pretreatment with L-NAME, being significantly lower at the EOE dose 0,25% (v/v), (60,34±1,36% of initial contraction remained before the treatment versus 71,31± 2,69% after the treatment with L-NAME). This data obtained reveals that EOE relaxes blood vessels by pathways, where endothelial NO release plays only some, but not essential role. Other endothelial factors (prostaglandins, EDRF or endothelial contracting factors) or smooth muscle mechanisms may be more important. Our data is comparable to others, they revealed NO-dependent and independent relaxation induced by total flavonoids of Elsholtzia splendens. [9]

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3.4 The role of extracellular Ca

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Fig.8 The effect of E. ciliata on the Ca2+_{titration curve. Vessels are pre-contracted in Ca}2+_{free PSS}

using NA 5µM with and without EOE. Data are mean±SEM. * -difference is statistically significant, p<0.05.

The initial contraction induced by NA in Ca2+ free solution was significantly higher without EOE than with its addition. (19.53±3.21 vs 8.855 ±2.669) and as well at lowest extracellular Ca2+ concentration added (1mM) (20.75±3.139 vs 9.95 ±2.69). At higher Ca2+ concentrations added (10-100mM) the contraction without EOE remains insignificantly higher. Our results suggest that EOE diminishes contractions to NA by inhibiting Ca2+ release from intracellular stores rather than influx of extracellular Ca2+ via the sarcolemmic Ca2+ channels.

Extracellular Calcium channels do not seem to be part of E. Ciliata essential oil emulsion’s underlying mechanism of decreasing contractility of the vessels. Vessels with EOE responded well to the addition of increasing levels of Ca2+ by causing an increase in contraction. Maximum levels of contraction with and without EOE were nearly identical.

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4. DISCUSSION

In the current study we were the first to assess E. ciliata and its vasoactive properties and some potential underlying mechanisms. We found that EOE induced endotheliumdependent and -independent vasorelaxation in small rat mesenteric artieries. Vasorelaxation in endothelium-intact vessels was slightly stronger than in endothelium-denuded vessels.

Almost 40 years ago endothelium derived relaxing factors (EDRFs) were described by Furchgott and Zawadzki. [25] PGI2 is a powerful vasoactive mediator produced and released by endothelium. [26] Furchgott was also able to show that NO is of major importance and that its release can be stimulated by Ach, histamine, and substance P. [24,25] We found that EOE showed significant vasorelaxation with and without endothelium, when removed mechanically. Under pretreatment with L-NAME the relaxation was partially, but significantly lower, than in vessels without pre-treatment. This indicates that potential underlying mechanisms are multiple and that NO does not appear to play a large role.

In the current study, EOE reduced the force of contraction in a concentration-dependent manner, similar to the finding of Hui-Ping Wang et al., who assessed ethanolic extract of E. splendens in rat aortic rings, this study is of great comparative value to E. ciliata, although this study assessed vessels of a different caliber. The underlying method to assess contraction and vasorelaxation is similar. It teases the potential that lies within the Elscholtzia species of plants and their potential vasorelaxant properties. [9]

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5. CONCLUSIONS

 Elscholtzia ciliata extracts investigated showed vasorelaxant properties. The essential oil emulsion proved to be significantly more effective than the ethanolic extract, which proved to be only marginally more effective than the ethanol control.

 It is shown that vasorelaxant properties of Elscholtzia ciliata extracts are dose-dependent.  E. ciliata essential oil emulsion relaxes blood vessels by both smooth muscle dependent and

endothelium dependent pathways, including slight attenuation of endothelial NO release.  EOE does not appear to affect Ca2+_{channels, because we were able to show that maximum}

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6. CONFLICTS OF INTEREST

The author declares that there are no conflicts of interest.

7. ACKNOWLEDGEMENTS

Prof. Jurga Bernatoniene and Lauryna Pudziuvelyte from the Department of Drug Technology and Social Pharmacy, and the Institute of Pharmaceutical Technologies from the Lithuanian University of Health Sciences produced and provided the extracts.

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26 20. Dual wire myograph system - 420A & 520A. Prieiga per internetą: https://www.dmt.dk/dual_wire_myograph_420a_520a.html

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Lithuanian University of Health Sciences Medical Faculty Institute of Physiology and Pharmacology