Investigation of Kelussia Odoratissima and

Angelica Sinensis Similarities in Zebrash-based

In-vivo Bioactivity Assays and Their Chemical

Composition

Mohammad Rezaei1, Parisa Fooladi1, 2, Mohamad Norani3, Alexander Crawford3,4, Shahram Eisa-Beygi5, Yaser

Tahamtani1, 6, Mahdi Ayyari3

1 Department of Stem Cells and Developmental Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and

Technology, ACECR, Tehran, Iran

2 Department of Developmental Biology, University of Science and Culture, Tehran, Iran

3 Department of Horticultural Science, Tarbiat Modares University, Tehran, Iran

4 Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway

5 Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

6 Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

GMJ.2023;12:e2793

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 Correspondence to:

Mahdi Ayyari, Department of Horticultural Science,

Faculty of Agriculture, Tarbiat Modares University,

Tehran, Iran.

Telephone Number: +98-21 48292093

Email Address: m.ayyari@modares.ac.ir

Received 2022-11-07

Revised 2023-01-04

Accepted 2023-04-17

Abstract

Background: Kelussia odoratissima (KO) and Angelica sinensis (AS) have been

used in their indigenous traditional medicine, for various diseases. This study

was conducted to evaluate the volatile oil composition of KO leaves (KVL) and

AS root (AVR) and biological activity of essential oils (EOs) and hydroalcohol-

ic extracts of both plants using two dierent transgenic zebrash (Danio rerio) models.

Materials and Methods: Both EOs were isolated by hydrodistillation and analysed by GC and

GC/MS. For viability tests, larvae were treated with dierent concentrations of extracts to deter-

mine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a

dose-dependent manner for their biological activity using tissue-specic transgenic zebrash Tg(-

i-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare

the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups.

Results: Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7%

of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that

both EOs blocked ISVs formation in the Tg (i-1: EGFP) embryos increased to 10% of the control

value, while both hydroalcoholic extracts did not show any anti-angiogenesis eects in these em-

bryos. In addition, AVR has been shown to signicantly induce PBC regeneration following abla-

tion in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5′-N-ethylcar-

boxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity

of both EOs could be attributed to the phthalide structures while for the PBC regenerative activi-

ty, other compounds including β-Thujaplicinol, exclusively existing in AVR, might be eective.

Conclusion: Although the genera, organs, and origin of these plants are dier-

ent, their similar chemical composition and biological activities make them valu-

able resources for further investigation in basic medical and pharmaceutical science.

[GMJ.2023;12:e2793] DOI:10.31661/gmj.v12i0.2793

Keywords: Angiogenesis Inhibitors; Pancreatic Beta Cell; Zebrash; Essential Oil

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Rezaei M, .Zebrash-based In-vivo Bioactivity Assays

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Introduction

Plants containing molecules with phthalide

structures have long been of interest in

traditional medicine around the world. Natu-

ral phthalides are a relatively small group of

compounds that exhibit a wide range of bio-

logical activities. They exist in several high-

er and lower plants and are used in many of

the traditional medicinal practices in Asia,

Europe, and North America [1]. Kelussia

odoratissima and Angelica sinensis are the

two most abundant sources of phthalides in

Iran and China, respectively. K. odoratissi-

ma known as “Kelows” or “Karafse kouhi”

grows exclusively in central Zagros Mountain

mainly in Chaharmahal & Bakhtiari prov-

inces. This plant is consumed traditionally

in yogurt-based products and pickles. It has

a pleasant aroma and is known for its warm

nature, according to Iranian traditional medi-

cine, especially for feminine disorders [2, 3].

Angelica sinensis, also known as “Don qui”,

is one of the most well-known medicinal

plants in traditional Chinese medicine for the

invigoration of blood and treatment of female

irregular menstruation cycles as well as toni-

fying and relieving pain [4].

It is also used in the formulation of other Chi-

nese medicinal plants for various treatments

[5, 6]. A long list of potential biological activ-

ities associated with phthalides was recently

published by León et al. [1], including anal-

gesic, antihyperglycemic, antithrombotic and

antiplatelet, neurological eects on Alzhei-

mer’s disease, Parkinson’s disease and other

cognitive impairments, GABAergic, sedative,

anticonvulsive and anti-stroke. Phthalides

have also been shown to display some proges-

togenic and cytotoxic eects.

Zebrash (Danio rerio) is a powerful verte-

brate model organism in genetics, drug dis-

covery, developmental biology, and regener-

ative studies. Zebrash have become an ideal

model for high-throughput screening systems

because of their high fecundity, transparency,

external development, and short generation

times [7-10]; additionally, ease of housing and

maintaining large numbers in captive [11-13].

For these reasons, zebrash have been recog-

nized as a unique model for various human

diseases, including Alzheimer’s disease [14,

15], diabetes [16], muscular dystrophy [17],

and cancer [18]. Zebrash have many con-

served disease proteins with humans. So, the

drugs used for humans often have the same

eects on these models [19, 20]. Even in some

of the diseases, zebrash are considered better

models than mice [21]; in this regard, there

is a list of small molecules that have been

screened in zebrash and are currently in clin-

ical trials [22].

In contrast to mammalian models, zebrash

can regenerate pancreatic beta cell (pBCs)

throughout their entire life [23, 24]. Tg(ins:FP-

NTR) zebrash line has been introduced as a

model to study pBC regeneration [25-27], and

the uorescent protein-nitroreductase (FP-

NTR) fusion protein is expressed in pBCs

around 24 hours post fertilization (hpf) under

the insulin promoter activity [28].

NTR converts metronidazole (MTZ) into

a toxin such that exposure of this model to

MTZ, results in ablation of NTR-expressing

PBCs [29]. Therefore, we generated and es-

tablished Tg(ins: GFP-NTR) transgenic mod-

el in our lab for conducting bioactivity tests of

natural products [30].

The promoter activity of i1, an endotheli-

al-specic transcription factor, was previous-

ly used to establish the endothelial-specic

transgenic zebrash lines including Tg(-

i1:EGFP) [31].

In this model, the enhanced green uorescent

protein (EGFP) signal is localized to the blood

vessels (arteries, veins, and capillaries). So,

this model is appropriate for vascular analy-

sis during zebrash embryonic development

[32].

The inhibition of the formation of interseg-

mental vessels (ISVs) and subintestinal ves-

sels (SIVs) is used as a measure of the po-

tential anti-angiogenesis eects of natural

compounds and small molecules; according-

ly, Tg(i1: EGFP) is also an ideal model for

cancer research [33-35].

The aim of this study was to compare the

chemical composition and biological activ-

ities/eects of two essential oils (EOs) and

hydroalcoholic extracts from K. odoratissi-

ma leaves and A. sinensis root. The potential

inhibition of angiogenesis and/or induction

of pBC regeneration was assessed by a ze-

brash-based bioassay system, which can be

Zebrash-based In-vivo Bioactivity Assays Rezaei M, et al.

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related to the anti-cancer and/or anti-diabet-

ic activity of the compounds, respectively.

To the best of our knowledge, this is the rst

report on the evaluation and comparison of

chemical composition and in vivo bioactivity

for K. odoratissima and A. sinensis.

Materials and Methods

Chemicals and Plant Materials

N-Phenylthiourea (PTU), MTZ, and methy-

lene blue was obtained from Sigma-Aldrich

(St. Louis, Missouri, United States). 5′-N-eth-

ylcarboxamidoadenosine (NECA) was pur-

chased from Torcis (Bristol, UK.) and SU5416

was obtained from Abcam (Cambridge, Unit-

ed Kingdom). Ethanol and other chemicals

were purchased from Merck (Darmstadt, Ger-

many).

The leaves of K.odoratissima was collected

in May 2018 from Bazoft (Chaharmahal &

Bakhtiari province, Iran), at an altitude of c.

2500 m. A voucher specimen was deposited

in the Herbarium of Medicinal Plants and

Drugs Research Institute in Shahid Beheshti

University, Tehran (Voucher No: MPH-1414).

A. sinensis roots were also collected in Gansu

province, China in the autumn of 2018.

Isolation and Analysis of the Essential Oil

One-hundred grams of air-dried leaves of K.

odoratissima were powdered and the EOs was

isolated by hydrodistillation in a Cleveng-

er-type apparatus for 3 h. The EOs was sepa-

rated and dried over anhydrous sodium sulfate

and kept in the freezer at -20°C until analysis.

The same procedure was performed to isolate

EOs from the root of A. sinensis.

Preparation of Hydroalcoholic Extracts

The ethanol/water (50%, 100ml) extracts of

10 grams of Kelussia and Angelica were sep-

arately prepared by sonication for 30 min.

Subsequently, the extract was ltered though

Wathman paper (no. 1).

The extracts were concentrated at 40 °C us-

ing a rotary evaporator and nally powdered

in a freeze drier to remove the residual water.

The hydroalcoholic extract of Kelussia leaves

(KEL) and Angelica root (AER) were stored

at -20 °C until analysis.

GC and GC/MS Analyses and Interpretation GC and GC/MS Analyses and Interpretation

of Volatile Oil Componentsof Volatile Oil Components

The isolated EOs were analysed using a GC

Agilent 7890B equipped with a ame ioniza-

tion detector (FID). The HP-5 column length,

inner diameter, and lm thickness were 30 m,

0.25 mm and 0.25 µm, respectively. The oven

temperature was programmed from 60 °C to

280 °C with the ramp of 5 °C/min and held at

280 °C for 2 minutes. Helium was used as the

carrier gas at a ow rate of 1.1 mL/min. The

detector and injector temperatures were kept

at 280 °C and 250 °C, respectively. A Thermo-

quest–Finnigan gas chromatograph coupled

with a trace mass spectrometer (GC/MS) with

the same parameter for fused silica column,

oven temperature, carrier gas, ow rate, and

injector temperature was also used to identify

individual peaks. The ionization voltage was

set at 70 eV and the interface temperature and

ion source were kept at 250 °C and 200 °C, re-

spectively. The identication of the individual

components of both EOs was performed by

comparing their mass spectra with those of the

Adams and Wiley 7.0 internal references mass

spectra library and was conrmed by compar-

ing their calculated retention indices (relative

to n-alkanes C8–C24) with those reported in

literature data [36]. The quantication of the

individual component in both samples was

performed by relative area percentages using

GC-FID.

Zebrash Models and Maintenance

Tg(ins: GFP-NTR) [28] and Tg(i1:EGFP)

[31] zebrash strains were used as transgen-

ic models to test the bioactivity of the com-

pounds. Adult zebrash, both male and fe-

male, were mixed and maintained at 28 °C

on a 14 h light/10 h dark cycle. Mating was

routinely carried out at 28 °C, male and fe-

male zebrash were placed in a breeder basket

the night before and embryos were collected

in the morning. The embryos were washed

and staged as described previously [37]. Ap-

proximately 300-400 embryos were generated

on average and cultured in E3 media (5 mM

NaCl, 0.33 mM MgSO4, 0.33 mM CaCl2,

0.17 mM KCl, and 0.1% methylene blue).

Some embryos were raised in the presence of

0.003% PTU, starting from 24 hpf, to prevent

pigmentation.

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Rezaei M, et al. Zebrash-based In-vivo Bioactivity Assays

Viability Tests

To test the viability of 12 hpf of Tg(i1: EGFP)

embryos and 4 dpf of Tg(ins: GFP-NTR) lar-

vae, we treated them with ve dierent con-

centrations of K. odoratissima leaves (KVL),

KEL, AER and A. sinensis root (AVR) (500,

125, 31.25, 7.81 and 1.95 µg/ml). At least ten

of 4 dpf of Tg(ins: GFP-NTR) larvae were

treated for 2 days. Next, the viability of em-

bryos/larvae at each concentration were then

reported as percentages and plotted as a via-

bility curve. A similar procedure was used to

evaluate the possible pBC regeneration ca-

pacities of Kelows extracts (Hexane (KHL),

EtOAc (KEtL), MeOH (KML)). The viability

test was used to determine the working con-

centration of each compound. The details are

provided in the supplementary data.

Testing Antiangiogenic Activity

Synchronized and healthy Tg (i1: EGFP)

embryos were harvested again at the 6-somite

stage (12 hpf). Embryos were placed into 24-

well plates, with 10 embryos per well, in 1.5

ml E3 medium and incubated at 28 °C with

desired concentrations of each compound that

were previously determined by the viabili-

ty test. Eighteen hours later, using an Olym-

pus SZX16 Fluorescence stereomicroscope

equipped with a Canon digital camera, embry-

os were observed and imaged for blood ves-

sel development, morphological changes, and

toxicity. We scored the anti-angiogenic activ-

ity of extracts by determining the extent of

the ISVs dorsal outgrowth in live transgenic

embryos. The mean extent that ISVs extended

dorsally from the dorsal aorta/posterior car-

dinal vein (100, 75, 50, 25, or 0%) was used

as a scoring value. The anti-angiogenic com-

pound, SU5416, previously described as the

inhibitor of vascular endothelial growth factor

(VEGF) receptor [38], was also used as a pos-

itive control. In all experiments DMSO treat-

ment (1%) was used as the vehicle, healthy

sh were denoted as an untreated group, and

NC as the negative control.

Testing PBC Regeneration Activity

Fertilized Tg (ins: GFP-NTR) transgenic

zebrash embryos were collected and incu-

bated at 28 °C for 24 hours. Dead embryos

were removed and the E3 medium was re-

placed with fresh embryo medium containing

0.003% PTU to prevent pigment formation.

GFP-positive larvae with green uorescent

pBCs were selected at 3 dpf, and subject-

ed to MTZ treatment at a concentration of 5

mM was performed to induce pBC ablation,

so heterozygous transgenic larvae were im-

mersed in MTZ solution for 24 hours. In the

following, 10 larvae were placed per well in

24-well plate containing 1.5 ml E3 medium

and optimum concentrations for each extract.

Forty-eight hours after treatment, live larvae

were imaged with a uorescent stereomicro-

scope.

PBC regeneration for each compound was

evaluated by measuring Pancreatic beta cell

arbitrary unit (pBC area AU) of each larva

by using ImageJ software (National Institutes

of Health (NIH), Bethesda, Maryland, USA).

NECA which increases pBC regeneration by

activating adenosine G protein-coupled recep-

tor (GPCR) signaling was used as a positive

control [39].

Statistical Analysis

Each experiment was carried out at least three

times, and all data are presented as mean ±

S.D. One-way ANOVA was used to analysis

the statistical signicance of the data pro-

duced from experiments using Graph Pad

Prism 6 software (GraphPad Software, Inc.,

San Diego, CA). P values less than 0.05 were

considered signicant.

Ethical Statement

All animal procedures were approved by the

Royan Institute Ethics Committee with the eth-

ical code IR.ACECR.ROYAN.REC.1398.211

and conducted in full compliance with the

guidelines of the Animal Care Committee.

Results

Chemical Composition of the EOs

Pale yellow colour EOs with especial aroma

in 0.03 and 0.04% yield (V/W % relative to

dry weight of plant materials) by hydrodistil-

lation process for volatiles of KVL and vola-

tiles of AVR, respectively.

A total of 52 compounds were identied in

both samples, representing 92.7 and 95.8% of

the EOs for Kelows and Don qui, respectively.

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Zebrash-based In-vivo Bioactivity Assays Rezaei M, et al.

GC chromatograms of AVR and KVL demon-

strated that the Z-ligustilide (24.5% in KVL

and 19.3 % in AVR) followed by E-ligustilide

(14.1% in KVL and 10.1% in AVR) are the

major compounds in both samples (Figure-1A

section i and ii, respectively).

More specically, eleven compounds were

found to be present in both oils, comprising

51.3% of Kelussia and 61.7% of Angelica

EOs, despite the dierences in genus, organs,

and origin bases.

For example, phthalide structures comprise

39.3% of Kelussia and 37.6% of Angelica, in-

cluding the (Z/E)-ligustilides and (3E/3Z)-bu-

tylidene phthalides. The structures of the main

components (with a relative percentage of

more than 5%) of both EOs included n-Prop-

ylbenzene, 2-Undecanone, α-Copaene, β-Thu-

japlicinol, (Z)-γ-Bisabolene, Sclarene as well

as other phthalide structures (Figure-1B).

Antiangiogenic Properties of the Compounds

The Tg (i1: EGFP) and Tg(ins: GFP-NTR)

transgenic zebrash embryos were used to

evaluate the possible anti-angiogenic and/or

pBC regeneration abilities of the compounds

derived from KEL and Angelica root (details

depicted in Figure-2A and -B). The viability

results showed that all compounds, including

KVL, KEL, AER, and AVR, were 100% via-

ble for 12 hpf Tg (i1: EGFP) embryos when

administered at nal concentrations lower

than 7.81 µg/ml (Figure-3A). Results showed

that KVL and AVR had signicantly (P<0.01)

decreased relative vascular outgrowth index

compared to the vehicle (1% DMSO) and

healthy groups (Figure-3B). However, other

compounds including KEL and AER showed

no signicant eect in this regard. As expect-

ed and consistent with previous reports, the

positive control, SU5416, decreased the rel-

Figure 1. A: GC chromatograms of essential oils of Kelussia odoratissima (i) and Angelica sinensis (ii);

B: Structures of some of the main compounds (more than 5%) 1: n-Propylbenzene, 2: 2-Undecanone,

3: α-Copaene, 4: β-Thujaplicinol, 5: (Z)-γ-Bisabolene, 6: (3E)-Butylidene phthalide, 7: (Z)-Ligustilide, 8:

(E)-Ligustilide, 9: Sclarene

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Rezaei M, et al. Zebrash-based In-vivo Bioactivity Assays

ative vascular outgrowth index signicantly

compared to the vehicle (1% DMSO) and

healthy groups (Figure-3B). To conrm the

above results, representative uorescence mi-

croscopy images were presented (Figure-3C).

PBC Regeneration Potentials

According to the viability test for Tg(ins:G-

FP-NTR) larvae at 4 dpf, KVL and AVR at a

concentration of 1.95 µg/ml, KEL and AER

at lower than 31.25 µg/ml showed 100% vi-

ability (Figure-4A). The average values of

pBC area (AU) for each compound is illus-

trated in the graph (Figure-4B). According to

the results, only the volatile oil from AVR was

eective in pBC regeneration (Figure-4C)

Figure 2. A: Schematics of zebrash anti-angiogenesis and pBC regeneration bioassay methods. B: (1)

Kelussia leave. (2) Angelica root. Bright-eld and GFP lter view of: (3) 12 hpf Tg (i: EGFP) embryo, (4) 3

dpf Tg(ins: GFP-NTR) larvae, (5) 4 dpf Tg(ins: GFP-NTR) larvae treated by MTZ, (6) 6 dpf Tg(ins: GFP-

NTR) larvae, (7) 30 hpf Tg(i: EGFP) embryo. Abbreviations: WT: Wild type; Tg: transgenic; dpf: days post

fertilization; hpf: hours post fertilization; MTZ: metronidazole; PS: primary stock; WS: working stock; NT:

Not treated; NC: Negative control; Ve: Vehicle; GFP: Green Fluorescent Protein NE: NECA.

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Zebrash-based In-vivo Bioactivity Assays Rezaei M, et al.

and pBC area (AU) signicantly (P<0.01) in-

creased in this sample compared to negative

and vehicle controls. Other samples, includ-

ing KVL, KEL and AER were not able to sig-

nicantly increase the pBC area (AU). NECA

as positive control showed the highest regen-

eration close to the healthy group.

To investigate the bioactivity of Kelussia in

more detail, we prepared a series of subse-

quent fraction extracts; obtained based on the

increasing polarities of solvents from KHL,

to KEtL and KML. The extraction method

for this step was also the same as what we

mentioned previously in the Materials and

Methods section. Whereas KHL and KEtL

induced 100% embryonic viability/survival at

Figure 3. The anti-angiogenesis bioassay in the Tg(i: EGFP) embryos A: Viability rate of zebrash em-

bryos exposed to di󰀨erent concentrations of extracts and essential oils. B: Relative vascular outgrowth in

30 hpf embryos after 18 h exposure to di󰀨erent samples in 7.81 µg/ml concentration (Larvae n:10). C: Vas-

cular system of controls and treated zebrash embryos. All the measurements were taken in triplicates and

the values were presented as mean ± SD of three independent experiments, **P<0.01, ***P<0.001. KVL:

Kelussia volatile oil leaves; KEL: Kelussia leaves; AER: Angelica root; AVR: Angelica volatile oil root.