Article, Journal of Ethnopharmacology 2023

Abstract

iziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is a subshrub that is widely distributed in China, Kazakhstan, Kyrgyzstan, Mongolia, Russia, Tajikistan, Turkmenistan, and Uzbekistan. The species is used in traditional medicine for the relief of symptoms connected to cardiovascular diseases like coronary heart disease or hypertension.

Full text

Journal of Ethnopharmacology 315 (2023) 116660
Available online 28 May 2023
0378-8741/© 2023 Elsevier B.V. All rights reserved.
Chemical composition and cardiotropic activity of Ziziphora clinopodioides
subsp. bungeana (Juz.) Rech.f.
A.O. Whaley
b
, D.Y. Ivkin
a
,
1
, K.A. Zhaparkulova
e
, I.N. Olusheva
b
, E.B. Serebryakov
d
,
S.N. Smirnov
d
, E.D. Semivelichenko
a
, A. Yu. Grishina
a
, A.A. Karpov
a
, E.I. Eletckaya
a
,
K.K. Kozhanova
e
, L.N. Ibragimova
e
, K.T. Tastambek
f
,
g
, A.M. Seitaliyeva
h
, I.I. Terninko
c
,
Z.B. Sakipova
e
, A.N. Shikov
b
, M.N. Povydysh
b
,
*
, A.K. Whaley
b
,
1
a
Saint Petersburg State Chemical Pharmaceutical University, Saint Petersburg, Department of Pharmacology and Clinical Pharmacology, Russia
b
Saint Petersburg State Chemical Pharmaceutical University, Saint Petersburg, Department of Pharmacognosy, Russia
c
Saint Petersburg State Chemical Pharmaceutical University, Saint Petersburg, Center for Quality Control of Medicines, Russia
d
Saint Petersburg State University, Universitetskii pr. 26, St. Petersburg, 198504, Russia
e
School of Pharmacy, S.D. Asfendiyarov Kazakh National Medical University, Tole-bi 94, 050012, Almaty, Kazakhstan
f
Ecology Research Institute, Khoja Akhmet Yassawi International Kazakh-Turkish University, Turkistan, 161200, Kazakhstan
g
Department of Biotechnology, M. Auezov South Kazakhstan University, Shymkent, 160012, Kazakhstan
h
Higher School of Medicine, Al-Farabi Kazakh National University, Tole-bi 96, 050012, Almaty, Kazakhstan
ARTICLE INFO
Keywords:
Z. clinopodioides subsp. bungeana
Extracts
Cardioprotective activity
Flavonoids
Polyphenols
Monoterpenoids
ABSTRACT
Ethnopharmacological relevance: Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is a subshrub that is widely
distributed in China, Kazakhstan, Kyrgyzstan, Mongolia, Russia, Tajikistan, Turkmenistan, and Uzbekistan. The
species is used in traditional medicine for the relief of symptoms connected to cardiovascular diseases like
coronary heart disease or hypertension.
Aim of the study: was to validate traditional use of Z. clinopodioides subsp. bungeana for the treatment of coronary
hearth diseases using in vivo models and to nd active compounds responsible for the activity.
Materials and methods: Multiple extracts were obtained from the aerial parts of Z. clinopodioides subsp. bungeana
using maceration, liquid-liquid extraction, CO2 extraction and ultrasound-assisted extraction. Preliminary
screening studies for the evaluation of the efcacy of Z. clinopodioides subsp. bungeana extracts on the model of
hemic hypoxia were performed. The most effective samples were selected and included in the main study. Stage 2
of the study evaluated the cardiotropic activity of the selected extracts on a model of chronic heart failure.
Preparations were administered to animals intragastrically once a day for 28 days. For the isolation of individual
compounds plant material was extracted with 96% ethanol. The obtained crude extract was sequentially
extracted with n-hexane and dichloromethane and separated by chromatography on a Diaion HP-20 column. The
obtained fractions were further subjected to Sephadex LH-20 column chromatography and eluted isocratically
with 96% ethanol (EtOH) to yield subfractions, which were further separated by preparative HPLC to obtain 13
individual compounds.
Results: Extracts obtained from Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. herb were subjected to
pharmacological screening for the evaluation of their efcacy on hemic hypoxia. Based on the obtained results,
out of the sixteen tested extracts two (AR and US 60%) were selected for further evaluation of their cardiotropic
activity. Modeling of chronic heart failure was carried out in accordance with the following stages: 1) anesthesia
with chloral hydrate at a dose of 450 mg/kg, intraperitoneally, 2) articial ventilation of the lungs, 3) thora-
cotomy, 4) modeling of permanent ischemic or ischemic-reperfusion damage. Both extracts effected the in-
dicators of contraction and output, comparable to the reference drug - Monopril. Based on the extraction
methods used to obtain RAF and US60 and data from the literature, it can be assumed that they contain com-
pounds with medium polarity, including polyphenols and terpenoids. At the next stage three previously unde-
scribed monoterpenoid derivatives – Ziziphoric acid (1), Ziziphoroside D (2) and 6′-malonylziziphoroside A (3),
* Corresponding author. Department of Pharmacognosy, Saint Petersburg state Chemical Pharmaceutical University, Saint Petersburg, Prof. Popov str. 14, Russia.
E-mail address: maria.povydysh@pharminnotech.com (M.N. Povydysh).
1
The main authors have the same role in the article.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
https://doi.org/10.1016/j.jep.2023.116660
Received 18 April 2023; Received in revised form 13 May 2023; Accepted 17 May 2023

Journal of Ethnopharmacology 315 (2023) 116660
2
along with two previously described megastigmane glucosides – blumenol C glucoside (4), blumenol C 9-O-(6′-O-
malonyl-beta-D-glucopyranoside (5) and two previously described monoterpenoids 7a-hydroxymintlactone (6),
7-hydroxypiperitone (7) together with six polyphenols – pinocembrine-7-O-rutinoside (8), chrysine-7-O-
rutinoside (9), acacetin-7-O-rutinoside (10), luteolin-7-O-rutinoside (11), rutin (12) and rosmarinic acid (13)
were isolated from Z. clinopodioides subsp. bungeana extracts.
Conclusion: Our results support the traditional use of Z. clinopodioides subsp. bungeana for the treatment of
coronary diseases. As a result of Z. clinopodioides subsp. bungeana extracts screening in vivo, two extracts were
selected as potential cardiotropic agents. Phytochemical analysis of the plant material led to the isolation of ve
terpenoid derivatives, two megastigmane glycosides, ve avonoids and one cinnamic acid derivative, which
could be responsible for the reported biological activity. Future experiments are required to understand the
mechanisms of action for the isolated compounds.
1. Introduction
Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is a subshrub
from the Lamiaceae family with a characteristic aroma that grows in
open xeric habitats in China, Kazakhstan, Kyrgyzstan, Mongolia, Russia,
Tajikistan, Turkmenistan, and Uzbekistan (Karlygash et al., 2016a; Sri-
vedavyasasri et al., 2018). The genus Ziziphora L. (Lamiaceae) includes
about 30 species, widely distributed in Asia, Africa and Europe. Zizi-
phora species are used in folk medicine in the countries of Southeast and
Central Asia. The plant is used in Turkey as a wound healing and anti-
septic agent (Senejoux et al., 2012) and as a sedative and expectorant in
traditional Iranian medicine (Naghibi and O’Malley, 2005).
Z. clinopodioides subsp. bungeana is used in the folk medicine of
Kazakhstan in the form of infusions, alcohol tinctures and decoctions as
sedative, expectorant, carminative agents, along with as a remedy for
cold, inammation, cough, migraine, fever, diarrhea, and depression.
Aqueous tinctures are used as a sedative for the heart, for scrofula and
colds, externally for rheumatism and toothache (Loseva, 2008; Karly-
gash et al., 2016a, b; Zhaparkulova et al., 2016). The antimicrobial ac-
tivity of essential oils from a number of Ziziphora species has been
reported: Z. clinopodioides, Z. taurica (Sonboli et al., 2006), Z. hispanica
(Rabah et al., 2013), Z. tenuior (Dakah et al., 2019), etc. Combined drug
preparations, containing dried EtOH extracts of Z. clinopodioides subsp.
bungeana herb are present on the pharmaceutical market in the form of
granules and capsules, which are prescribed as a remedy for coronary
heart disease (ˇ
Smejkal et al., 2016). Additionally, an oral health spray
was created with the use of Ziziphora clinopodioides essential oil (Beik-
mohammadi, 2011).
Especially interesting is the use of Z. clinopodioides subsp. bungeana
tincture in traditional medicine for the relief of symptoms connected to
cardiovascular diseases like coronary heart disease or hypertension.
Other species from the Ziziphora genus are also used in traditional
medicine for the treatment of cardiovascular conditions, including -
Z. clinopodioides used for the treatment of heart disease, high blood
pressure (ˇ
Smejkal et al., 2016).
The pharmacological effects observed for Z. clinopodioides subsp.
bungeana are mediated by the rich diversity of bioactive compounds
found in the plant, which include oleanolic and ursolic acids, phenolic
compounds, avonoids (pigenin, chrysin, linarin and acacetin) and
essential oil components (menthone (6.7%), isomenthone (28%), pule-
gone (55.2%) and thymol (2.5%)) which have all been previously iso-
lated from aerial parts of Z. clinopodioides subsp. bungeana (Aliakbarlu
and Shameli, 2013; He et al., 2020).
It has been found that Z. clinopodioides avonoids protect myocardial
tissue from ischemia-reperfusion injury by reducing reactive oxygen
species related damage (Li et al., 2018). Likewise, acacetin, the major
avonoid of Z. clinopodioides subsp. bungeana, was demonstrated to
protect against myocardial ischemia reperfusion injury (Yang et al.,
2014). Later it was also shown that acacetin protected H9c2 car-
diomyocytes from H/R damage by enhancing autophagy. Application of
acacetin increased activation of the PI3K/Akt signaling pathway,
whereas co-treatment with the PI3K inhibitor LY294002 reversed the
inhibition of apoptosis and autophagy induced by acacetin (Liu et al.,
2021). Apigenin and chrysin additionally proved to be effective vaso-
relaxant compounds (Senejoux et al., 2012).
The aim of this study was to validate traditional use of Z. clinopo-
dioides subsp. bungeana for the treatment of coronary heart diseases
using in vivo models and to nd the active compounds responsible for the
activity.
2. Materials and methods
2.1. Material
2.1.1. Plant material
The aerial parts ofZ. clinopodioides subsp. bungeana were collected in
the summer of 2021 in the owering stage in the Turkestan region of the
Republic of Kazakhstan and identied by the Institute of Botany and
Phytointroduction, Science Committee, Ministry of Education and Sci-
ence of the Republic of Kazakhstan. A voucher sample (N◦01–05/337
from October 5, 2021) has been deposited in the herbarium of the
Institute of Botany and Phytointroduction, Almaty, Republic of
Kazakhstan. The fresh plants were air-dried, and stored in a cold, dark,
dry place until the analyses were carried out.
2.1.2. Preparation of the extracts
2.1.2.1. Maceration. Dried and ground aerial parts of Z. clinopodioides
subsp. bungeana (3000 g) were macerated with ethanol 95% (30 L) at
room temperature for 7 days. The extraction was repeated twice more
until the raw material was depleted. The resulting EtOH extracts (EE)
were combined and evaporated at 50 ◦C using rotary evaporator EYELA
N-1300. The resulting crude aqueous residue (300g) (AR) was subjected
to fractionation in a separatory funnel with solvents according to the
order of increasing polarity (Abubakar and Haque, 2020).
2.1.2.2. Liquid-liquid extraction. 100 g of AR was dissolved in puried
water in a 1:1 ratio, the resulting solution was ltered. The solution was
extracted successively in a 1:1 ratio by volume in a separating funnel in
triplicate with petroleum ether, dichloromethane, ethyl acetate, and n-
butanol. The obtained fractions were then concentrated on a rotary
evaporator at 50 ◦C. As a result, 6.3 g of petroleum ether extract (PE),
4.3 g of dichloromethane extract (DC), 1.3 g of ethyl acetate extract (EA)
and 13.14 g of butanol extract (B) were obtained. The residual aqueous
fraction (RAF) after liquid extraction was concentrated on a rotary
evaporator at a temperature of 50 ◦C.
2.1.2.3. CO2 extraction. 1 kg of dried and ground aerial parts of
Z. clinopodioides subsp. bungeana was subjected to extraction under
subcritical conditions, at a working pressure of 62 atm and a tempera-
ture of 22 ◦C for 10 h, using liquid CO2 as an extractant (GOST
8050–85). 500 ml of liquid CO2 extract (CO2) was obtained.
2.1.2.4. Ultrasound-assisted extraction. 600 g of dried and ground aerial
parts of Z. clinopodioides subsp. bungeana were placed in an ultrasonic
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
3
extractor together with 4.8 L of EtOH for a duration of 3 h. After 3 h,
ultrasound was turned on at 40 Hz for 60 min. The process was repeated
two times until the complete depletion of the raw material. The resulting
corresponding extracts were then united and ltered. The ltered so-
lution was dried on an EYELA N-1300 rotary evaporator at 50 ◦C. Using
this method, 60 g of 40% EtOH extract (US40), 60 g of 50% EtOH extract
(US50), 60 g of 60% EtOH extract (US60), and 60 g of 70% EtOH extract
(US70) were obtained.
2.1.2.5. Heat-assisted extraction. 50 g of dried and ground aerial parts of
Z. clinopodioides subsp. bungeana were placed in a ask with 400 ml of
EtOH of various concentrations and macerated for 3 h. Extraction was
then further carried out on a boiling water bath under reux for 60 min
and the process was repeated two times until the raw materials were
completely depleted. The resulting corresponding extracts were united
and ltered. The ltered solutions were concentrated on a rotary
evaporator at 50 ◦C, as a result of which 40% (E40), 50% (E50), and 60%
(E60) EtOH extracts were obtained.
2.2. Animals
The hemic hypoxia model was performed on male mice weighing
20.2 g, while the cardiotropic activity model was performed on outbred
laboratory male rats weighing 180 ±10 g. All the laboratory animals
were obtained from the Federal State Unitary Enterprise PLZh “Rappo-
lovo” (Leningrad region).
Before the study, the animals underwent quarantine in a separate
room for fourteen days. The animals were kept under standard condi-
tions in accordance with «OECD. Principles of good laboratory prac-
tice». The animals were provided with standard feed (“Complete feed for
laboratory animals”, LLC “Laboratorkorm”, Moscow, Russia) and water
(GOST 2874–82 “Drinking water”) ad libitum. Access to food was
restricted prior to blood collection. Each animal was assigned an indi-
vidual number (a mark on the tail area with a permanent indelible
marker, periodically updated). All studies were carried out in accor-
dance with the International Rules for Working with Laboratory Animals
and approved by the Bioethical Commission of the Federal State
Budgetary Educational Institution of Higher Education SPCPU of the
Russian Ministry of Health.
The animals were kept under standard conditions in accordance with
the sanitary and epidemiological requirements for the design, equip-
ment and maintenance of experimental biological clinics (vivariums),
guidelines for the care and use of laboratory animals.The research was
conducted in accordance with the internationally accepted principles for
laboratory animal use and care - (Guide for the care and use of labora-
tory animals. National Academy press. - Washington, DC, 1996) and
Directive 2010/63/EU of the European Parliament and of the Council of
the European Union of September 22, 2010 on the protection of animals
were used during scientic research (Protocol of the Bioethical Com-
mission Mice/Rats-01-21).
2.3. Experimental procedures
2.3.1. Antihypoxic effects of Z. clinopodioides subsp. bungeana extracts
Screening evaluations of the efcacy of Z. clinopodioides subsp. bun-
geana extracts on the model of hemic hypoxia was preliminarily carried
out. The studies were performed on male mice weighing 20.2 g. Tested
Z. clinopodioides subsp. bungeana extracts were administered in a dose of
10 mg/kg orally through a probe half an hour before the injection of the
prohypoxant. To induce acute hemic hypoxia, animals were injected
intraperitoneally with sodium nitrite (300 mg/kg) in the form of a 10%
solution, and then the lifespan of the mice was recorded. Based on the
obtained results, the most effective samples were selected and included
in the main study (Stage 2).
2.3.2. Cardiotropic effects of Z. clinopodioides subsp. bungeana extracts
The efcacy of Ziziphora extracts has been known for a long time,
there is evidence of cardiotropic activity in the model of myocardial
infarction in rabbits in a wide range of doses, so in this study the optimal
dose was selected as a result of the screening performed through a
hypoxia model. Cardiotropic activity of Ziziphora was shown through
the ability of the plant extracts to decrease the consequences of induced
myocarditis in rabbits. In rabbits treated with Ziziphora infusion, the
heart rate slowed down by 40%, the R–R interval decreased by
0.08–0.03 s, the ventricular complex increased by 0.02 s, and the sys-
tolic index was restored by 11% (Dzhumagalieva, 1963).
During Stage 2 of the study, we evaluated the cardiotropic activity of
the selected extracts on a model of post heart attack chronic heart fail-
ure. Outbred male laboratory rats weighing 180 ±10 g were used as a
test system for the study. Four groups with 10 animals in each group
were formed (n =40): Group One - pathology without treatment (left
coronary artery ligation +puried water); Group Two - pathology +
administration of Z. clinopodioides subsp. bungeana aqueous extract;
Group Three - pathology +administration of Z. clinopodioides subsp.
bungeana ultrasound extract; Group Four - pathology +administration
of iAPP (Monopril). Tested Z. clinopodioides subsp. bungeana extracts and
the control substance were administered to animals intragastrically once
a day for 28 days. Simulation of post heart attack chronic heart failure
was performed according to the following steps: 1) anesthesia with
chloral hydrate at a dose of 450 mg/kg, intraperitoneally, 2) articial
ventilation, 3) thoracotomy, and 4) simulation of permanent ischemic or
ischemia-reperfusion injury. Formation of pathology and percentage of
necrotic tissue was assessed postoperatively using ECG, EchoCG and
pathomorphological/histological examination. Statistical processing of
the obtained data was performed using GraphPad Prism 8.0.2 software
package (GraphPad Software; USA). Normal distribution of quantitative
characteristics was checked using the Shapiro-Wilk’s W test. Median
(Me) and quartile range (Q1; Q3) were calculated if the data did not
conform to the law of normal distribution. Intergroup differences were
analyzed using the Mann-Whitney test. For data from several groups
(more than two) not subject to the law of normal distribution, we used
Kruskal-Wallis test, with further use of nonparametric mean ranks
method for multiple comparisons in case of signicant inuence of the
investigated factor. Differences were determined at 0.05 level of
signicance.
2.3.3. Phytochemical analysis
Analytical high-performance liquid chromatograms along with UV-
spectra were obtained using a Prominence LC-20 with a SPD-M20A
diode-array detector (Shimadzu corp., Japan) on a Supelcosil LC18
column (250 х 4.6, 5 mm). Preparative HPLC was carried out using a
Smartline system (Knaur, Germany) with a spectrophotometric detector
equipped with a Kromasil C18 preparative HPLC column (250 ×30 mm,
5 mm). HPLC grade acetonitrile used for HPLC and preparative HPLC
analysis was J.T. Baker HPLC gradient grade. TLC analysis was per-
formed using TLC Silica gel 60 (Merk, Germany). HR-ESI-MS spectra
were acquired on a Bruker Micromass Q-TOF spectrometer (Bruker,
Germany). NMR spectra were recorded on a Bruker Avance III 400 NMR
Spectrometer (Bruker, Germany). Chemical shifts (δ) are expressed in
ppm with reference to the solvent signal (DMSO‑d
6
).
2.3.4. Isolation of individual compounds
For the isolation of semipolar compounds from Z. clinopodioides
subsp. bungeana, air-dried and powdered aerial parts of Z. clinopodioides
subsp. bungeana (844 g) were repeatedly extracted (x11) through
maceration with 9 L of 96% EtOH at room temperature. The obtained
extracts were combined and concentrated under reduced pressure to a
volume of 450 mL giving a crude extract. The crude extract was
sequentially extracted with n-hexane and dichloromethane (DCm). The
obtained DCm fraction was evaporated to dryness, and the residue was
dissolved in 350 mL 96% EtOH that was further separated by
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
4
chromatography on a Diaion HP-20 column into 10 sub-fractions (SFr.)
using gradient elution of EtOH: Water (from 0:100 to 100:0, v/v, with
gradient steps of 10%). Eluting sub-fractions were combined based on
TLC analysis in a n-buthanol-acetic acid-water (BAW) (4:1:2) system.
The resulting SFr. 8 and 9 were combined. The combined fraction was
then subjected to Sephadex LH-20 column chromatography and eluted
isocratically with 96% EtOH to yield 5 fractions (Frs
1
1–5). Frs
1
2 was
further separated by preparative HPLC, using a gradient solvent system:
0.01–5.0 min 5% B; 5.0–45.75 min 5–70% B; (eluent A – 0.1% v/v tri-
uoroacetic acid (TFA) in water, and eluent B – 0.1% v/v TFA in
acetonitrile) to obtain compounds 1 (17.43 mg, t
R
=24.675), 3 (23.13
mg, t
R
=23.080), 6 (12.65 mg, t
R
=23.847) and 8 (14.35 mg, t
R
=
23.722).
SFr. 3 and 4 were combined and subjected to Sephadex LH-20 col-
umn chromatography using isocratic elution with 96% EtOH to 10
fractions (Frs
2
1–10). Frs
2
3 was further separated by preparative HPLC,
using a gradient solvent system: 0.01–5.0 min 5% B; 5.0–45.75 min
5–60% B; (eluent A – 0.1% v/v triuoroacetic acid (TFA) in water, and
eluent B – 0.1% v/v TFA in acetonitrile) to obtain compounds 4 (9.32
mg, t
R
=22.129), 5 (11.87 mg, t
R
=22.588) and 7 (13.98 mg, t
R
=
21.392). The SFr 2 was subjected to column chromatography with
Sephadex LH-20 and eluted isocratically with 96% EtOH to yield 5
fractions (Frs
3
1–5). Frs
3
2 was puried by preparative HPLC using a
gradient solvent system: 0.01–5.0 min 5% B; 5.0–45.75 min 5–60% B;
(eluent A – 0.1% v/v triuoroacetic acid (TFA) in water, and eluent B –
0.1% v/v TFA in acetonitrile) to yield compound 2 (8.93 mg, t
R
=
21.257).
For the isolation of polar compounds from Z. clinopodioides subsp.
bungeana, 300 ml of US60 extract was evaporated under reduced pres-
sure to a volume of 40 ml and then subjected to column chromatography
using LH-20 as the stationary phase and 96% EtOH as the mobile phase.
The eluate from the column was collected in 20 ml glass test tubes,
which were combined based on TLC analysis in a n-buthanol-acetic acid-
water (BAW) (4:1:2) system. Test tubes found to contain solutions with a
similar composition were further united and evaporated under reduced
pressure to a volume of 10 ml – affording fractions 1–8 (Frs
4
1–8). Frs
4
2–6 were puried by preparative HPLC using a gradient solvent system:
0.01–5.0 min 5% B; 5.0–45.75 min 5–60% B; (eluent A – 0.1% v/v tri-
uoroacetic acid (TFA) in water, and eluent B – 0.1% v/v TFA in
acetonitrile) to yield compounds 8 (31.6 mg, t
R
=22.129), 9 (6.8 mg, t
R
=22.918), 10 (2.8 mg, t
R
=23.417), 11 (2.2 mg, t
R
=19.275), 12 (2.2
mg, t
R
=19.156) and 13 (3.6 mg, t
R
=21.257) were isolated in indi-
vidual form.
2.4. Statistical analysis
The obtained data were statistically processed using the GraphPad
Prism 8.0.2 software package (GraphPad Software; USA). The normality
of the distribution of quantitative traits was checked using the Shapiro-
Wilk’s W test. In case of data discrepancy to the normal distribution law,
the median (Me) and quartile range (Q1; Q3) were calculated. Inter-
group differences were analyzed using the Mann-Whitney test. For data
of several groups (more than two) that do not obey the law of normal
distribution, the Kruskal-Wallis test was used, with further use of the
non-parametric method of average ranks for multiple comparisons if a
signicant inuence of the studied factor was found. Differences were
determined at a 0.05 signicance level.
3. Results
3.1. Acute hemic hypoxia
Initial screening assessments were conducted to establish the effec-
tiveness of the studied extracts. The model of acute hemic hypoxia was
chosen as the most appropriate for Z. clinopodioides subsp. bungeana
extract screening.
Due to the large number of extracts the use of a large range of doses is
not a viable option, therefore one concentration was chosen, on which
the antihypoxic activity was evaluated for every extract. The tested
extracts of Z. clinopodioides subsp. bungeana were administered at a dose
of 10 mg/kg orally through a probe half an hour before the formation of
the pathology.
For the creation of the acute hemic hypoxia model, the animals were
intraperitoneally injected with sodium nitrite (300 mg/kg) in the form
of a 10% solution, after which the life span of the mice was recorded.
The data is presented in Table 1. The results of the study to identify the
optimal dose of extracts are presented in Table 2.
In the course of the results obtained, the two most effective samples
(AR and US 60%) were selected and included in the main study.
3.2. Cardiotropic activity
At the second stage of the study, the cardiotropic activity of the ex-
tracts selected at the rst stage was evaluated. After arrival from the
nursery, the animals were acclimatized for 5 days at standard daylight
hours (12/12) with open access to water and food. Four groups of ani-
mals were formed with 10 animals in each group (n =40): the rst group
- pathology without treatment (ligation of the left coronary artery +
puried water); the second group - pathology +the introduction of the
RAF extract of Ziziphora; the third group - pathology +the introduction
Table 1
Life expectancy of animals against the background of exposure to hemic hypoxia
with the introduction of the studied extracts (M ±m).
Extract
number
Name of the extract/control substance
(abbreviation)
Survivability, sec
1 Crude aqueous residue (AR) 926,6 ±70,4
2 Ethyl acetate (EA) 745,5 ±90,0
3 Dichloromethane (DC) 866,6 ±133,1
4 Petrolium ether (РЕ) 973,9 ±255,7
5 Buthanol (B) 861,0 ±281,2
6 Residual aqueous fraction (RAF) 1234,2 ±249,9*
7 Carbon dioxide (CO2) 826,0 ±136,2
8 96% Ethanol (EE) 871,3 ±136,7
9 Ultrasound extract 50% (US50) 824,4 ±76,5
10 Ultrasound extract 60% (US60) 1204,1 ±
351,1**
11 Ultrasound extract 40% (US40) 911,3 ±127,5
12 Ultrasound extract 70% (US70) 967,7 ±236,6
13 Reux 40% (E40) 908,4 ±162,9
14 Reux 50% (E50) 904,4 ±238,8
15 Reux 60% (E60) 907,1 ±150,5
16 Lipophilic 834,9 ±192,8
* - p-value compared to control group (p <0.01).
**- p-value compared to control group (p <0.05).
* - p-value compared to control group (p <0.01).
**- p-value compared to control group (p <0.05).
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
5
of US60 extract of Ziziphora; the fourth group - pathology +adminis-
tration of an ACE inhibitor (Monopril).
The test extracts of Z. clinopodioides subsp. bungeana and the control
substance were administered to animals intragastrically one time per
day for twenty-eight days.
At the second stage of the study, modeling of post heart attack
chronic heart failure was carried out in accordance with the following
stages:
1) anesthesia with chloral hydrate at a dose of 450 mg/kg,
intraperitoneally,
2) articial ventilation of the lungs,
3) thoracotomy,
4) modeling of permanent ischemic or ischemic-reperfusion damage.
Animals were anesthetized with chloral hydrate, which was admin-
istered intraperitoneally at a dose of 450 mg/kg during the operation.
The duration of anesthesia was, on average, 1–1.5 h. The animals were
placed on a thermostat table.
Articial lung ventilation was carried out using the SAR-830/AP
device (USA). Respiratory rate: 60/min, tidal volume: 3 ml/100 g
body weight. The connection of the ventilator system to the respiratory
system of the animal was carried out using tracheal intubation. To do
this, the larynx was treated with 2% lidocaine solution to suppress vagal
reactions, then the larynx was visualized by hyperextension of the cer-
vical spine (the longitudinal axis of the head coincides with the longi-
tudinal axis of the body of the rat), after which a conductor was inserted
into the trachea (during the opening of the glottis in inspiratory phase),
an endotracheal tube was placed into the trachea along the guidewire,
after which the guidewire was removed.
Thoracotomy was performed through the fourth intercostal space
with an L-shaped skin incision from the upper edge of the body of the
sternum to the xiphoid process along the midline and further along the
VII rib to the middle axillary line and with dilution of the pectoral
muscles. After thoracotomy, the heart was visualized through the fourth
intercostal space. Further, in a blunt way, using the branches of
anatomical tweezers, the pericardium was removed. At the border of the
free edge of the left atrial appendage, the left coronary artery (LCA) was
visualized, under which a ligature (prolene 6/0, Ethicon, Germany) was
applied, directly at the edge of the left atrial appendage.
The formation of pathology and the percentage of necrotic tissue
were assessed after surgery using ECG and echocardiography, as well as
pathomorphological examination with histological analysis.
ECG were recorded in standard intervals before the surgery and 10
min after ligation of the left coronary artery. The onset of ischemia was
veried visually by electrocardiographic criteria: ST segment elevation,
the onset of ischemic arrhythmias.
Next, layer-by-layer suturing of the surgical wound was performed.
The seam on the skin was treated with an alcohol 5% solution of iodine.
After the operation, ceftriaxone (50 mg/kg) was injected intraperito-
neally to prevent postoperative infectious complications.
The parameters of the shortening fraction (FU) and ejection fraction
(EF) are comparable with the used ACE inhibitor (Monopril), which
differ from the control group and indicate the cardiotropic effect of the
extracts (Figs. 1–2).
Additionally, a macroscopic examination of the heart, liver and
kidneys was carried out. As a result of the examination, no visible pa-
thology was observed. During the histological examination, the heart,
liver and kidneys were analyzed. In all cases, the myocardium of the left
ventricle contained foci of transmural macrofocal cardiosclerosis with
the formation of an aneurysm (Fig. 3). In some cases, mild focal
myocarditis was observed.
The kidneys were found to have a normal structure. Glomeruli with
moderately plethoric capillaries, without pathology. The epithelium of
the tubules of the cortical and medulla had small-focal vacuolar dys-
trophy. Interstitium with extremely poor inltration of lymphocytes.
The mucosa of the calyces and pelvis were without pathology. No
Table 2
Life expectancy of animals against the background of exposure to hemic hypoxia
when choosing the optimal dose with an antihypoxic effect (M ±m).
Extract
number
Name of the extract/control substance
(abbreviation)
Survivability, sec
1 Residual aqueous fraction (RAF) 1 mg/kg 772,0 ±23,6*
2 Residual aqueous fraction (RAF) 10 mg/kg 1237,2 ±
157,0**
3 Residual aqueous fraction (RAF) 50 mg/kg 940,5 ±54,5
4 Ultrasound extract 60% (US60) 1 mg/kg 780,3 ±8,9*
5 Ultrasound extract 60% (US60) 10 mg/kg 1205,4 ±190**
6 Ultrasound extract 60% (US60) 50 mg/kg 932,3 ±49,6
7 Trimetazidine (TMZ) 5 mg/kg 1139 ±86,6**
* - p-value compared to control group (p <0.05).
** - p-value compared to control group (p <0.0001).
* - p-value compared to control group (p <0.001).
** - p-value compared to control group (p <0.0001).
Blue highlighting - measurement area of the control group (AR).
Fig. 1. Shortening fraction in the experiment
* - p-value compared to control group (p <0.05).
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
6
changes were found in the liver tissue.
3.3. Phytochemical analysis
For the identication of the main compounds responsible for the
observed pharmacological effects phytochemical analysis of
Z. clinopodioides subsp. bungeana was performed, which resulted in the
isolation of compounds 1–13.
Compound 1 was obtained as a white crystalline solid. The UV-
absorption spectrum displayed one maximum at 208 nm. Its positive
HR-ESIMS data m/z 207.0952 [M+Na]
+
(calcd. for C
10
H
14
O
3
Na
+
207.0991) suggested that the molecular formula was C
10
H
16
O
3
. NMR
data implied that compound 1 was a linear monoterpenoid (Table 3).
The
1
H NMR and COSY spectra showed the presence of two spin systems
starting at opposite sides of the molecule. The rst spin system started
from the carboxy group and consisted of three methylene groups, two of
which showed diastereotopicity - δ
H
2.02 (1H, dd, J =15.5, 8.0 Hz, H-
2a), 2.24 (1H, dd, J =15.5, 6.0 Hz, 2b), 1.39 (1H, m, H-4a), 1.53 (1H, m,
H-4b) and 2.73 (2H, m, H-5), one methine group at δ
H
1.83 (1H, m, H-3)
and one methyl group at δ
H
0.9 (3H, d, J =6.6 Hz, Me-3). The second
spin system started at the opposite side of the molecule from the car-
boxylic acid group and consisted of two geminal olenic protons at δ
H
5.85 (1H, d, J =0.7 Hz, H-8a) and 6.09 (1H, brs, H-8b), along with one
methyl group at 1.79 (3H, brs, Me-7) (Fig. 4). The orientation of both
gemilar olenic protons was established through NOESY correlations
with the adjacent methyl group. The
13
C NMR data revealed ten signals
that were assigned to two methyl groups, four methylene, one methine
and three quaternary carbon atoms (including one carboxylic, keto and
olenic carbons) with the help of a DEPT experiment (Table 3). The
above spectroscopic data suggested that compound 1 was a linear
monoterpenoid derivative. The structure of compound 1 was conrmed
through HMBC data (Fig. 4). HMBC correlations from Me-3 → C-2, 3, 4,
from Me-8 → 6, 7, 8, from H-3 → C-2, 4, 5 and Me-3 along with corre-
lations from H-5 → 3, 4, 6 established the structure of compound 1 as
3,7-dimethyl-6-ketooct-7-enoic acid and was given the name Ziziphoric
acid.
Compound 2 was obtained as a white crystalline solid. The UV-
absorption spectrum displayed one maximum at 241 nm. Its positive
HR-ESIMS data m/z 429.1730 [M+Na]
+
(calcd. for C
20
H
34
O
10
Na
+
429.1731) suggested that the molecular formula was C
18
H
30
O
10
. The
1
H
NMR and COSY spectra implied that compound 2 consists of a central
glycoside core with two modied aliphatic substituents - one of which is
connected through the glycosidic bond, and the other at position 6
through an ester group (Table 4). The hydrocarbon substituent con-
nected through the glycoside bond displayed one continuous spin system
consisting of three methylene groups at δ
H
3.43 (1H, m, H-1a′), 3.67 (1H,
m, H-1b′), 2.27 (2H, q, J =7.0 Hz, H-2′) and 2.02 (2H, q, J =7.4 Hz, H-
5′), two methine groups at δ
H
5.34 (1H, m, H-3′) and 5.42 (1H, m, H-4′)
and one methyl group at 0.93 (3H, t, J =7.4 Hz, H-6′). The methylene
group closest to the glycosidic bond is diastereotopic. The substituent at
position 6 of the glucose residue contained two spin systems each con-
sisting of two diastereotopic geminal protons at δ
H
2.57 (1H, d, J =14.2
Hz, H-2a’’), 2.66 (1H, d, J =14.2 Hz, H-2b’’) and 2.49 (1H, m, H-4a’’),
2.55 (1H, d, J =14.8 Hz, H-4b’’), respectively. The central glycoside
core consisted of seven proton signals characteristic of a β-D-glucopyr-
anoside residue at δ
H
4.17 (1H, d, J =7.7 Hz, H-1), 2.96 (1H, m, H-2),
3.16 (1H, m, H-3), 3.06 (1H, m, H-4), 3.34 (1H, m, H-5), 4.01 (1H, dd, J
=11.6, 6.9 Hz, H-6a) and 4.32 (1H, dd, J =11.6, 1.3 Hz, H-6b) (Fig. 4).
The
13
C NMR data revealed the presence of twenty signals that included
six methylene groups, seven methine groups, two methyl groups and
two quaternary (one ester and one carboxylic acid) carbons with the
Fig. 2. Ejection fraction in experiment
* - p-value compared to control group (p <0.05).
Fig. 3. Transmural large focal cardiosclerosis with aneurysm formation.
Staining with hematoxylin and eosin, pv. 50.
Table 3
1
H NMR and
13
C NMR data for compound 1.
1
Position δ
H
δ
C
1-COOH 12.00 (1H, s) 174.3
2a 2.02 (1H, dd, J =15.5, 8.0 Hz) 41.5
2b 2.24 (1H, dd, J =15.5, 6.0 Hz)
3 1.83 (1H, m) 29.8
4a 1.53 (1H, m) 31.2
4b 1.39 (1H, m)
5 2.73 (2H, m) 34.9
6 – 202.1
7 – 143.9
8a 6.09 (1H, brs) 125.7
8b 5.85 (1H, d, J =0.7 Hz)
3-Me 0.9 (3H, d, J =6.6 Hz) 19.9
7-Me 1.79 (3H, brs) 17.9
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
7
help of a DEPT experiment (Table 4). The structure of compound 2 was
conrmed through HMBC data (Fig. 4). HMBC correlations from H-1’ →
C-1, 2′, 3′, from H-2’ → C-1′, 3′, 4′, from H-3’ → C-5′, from H-4’ → C-5′, 6′
and from H-5’ → C-3′, 4′, 6′established the structure and connectivity of
the terpenoid substituent in position 1-O position of the glucoside core.
HMBC correlations from H-2’’ → C-1′′, 3′′, 4′′, 6′′, from H-4’’ → C-2′′, 3′′,
6′′, 7′′ and from H-6’’ → C-2′′, 3′′, 4′′ along with HMBC correlations from
H-6’ → C-1′′ established the structure and connectivity of the carboxylic
acid substituent in the 6-O position of the glucoside core and found it to
be a dicrotalic acid residue. As a result, the structure of compound 2 was
established as 1-O-(8′-methylhept-3′-en-1′-yl)-6-O-(3′′,5′′ -dihydoxy-5′′-
carbonyl-3′′-methylpentanoyl)-β-D-glucopyranoside, and named Zizi-
phoroside D.
Compound 3 was obtained as a white crystalline solid. The UV-
absorption spectrum displayed one maximum at 233 nm. Its positive
HR-ESIMS data m/z 439.1614 [M+Na]
+
(calcd. for C
19
H
28
O
10
Na
+
439.1574) suggested that the molecular formula was C
19
H
28
O
10
. The
1
H
NMR and COSY spectra implied that compound 3 consists of a cyclic
monoterpenoid aglycone and glycoside part (Table 4). The mono-
terpenoid aglycone showed one spin system consisting of two diaster-
eotopic methylene groups at δ
H
2.10 (1H, m, H-2a), 2.33 (1H, m, H-2b),
2.04 (1H, m, H-6a) and 2.44 (1H, m, H-6b), two methine groups at 2.06
(1H, m, H-1) and 7.34 (1H, m, H-5) and one methyl group at 0.97 (3H, d,
J =5.5 Hz, H-7). Additionally present in the monoterpenoid aglycone
were two methyl groups at 1.35 (3H, s, H-9) and 1.38 (3H, s, H-10). The
glycoside consisted of seven proton signals characteristic of a 6′-
substituted β-D-glucopyranoside residue at δ
H
4.34 (1H, d, J =7.6 Hz, H-
1′), 3.03 (1H, m, H-2′), 3.15 (1H, m, H-3′), 3.04 (1H, m, H-4′), 3.32 (1H,
m, H-5′), 4.07 (1H, m, H-6a′) and 4.31 (1H, m, H-6b′) (Fig. 5). The
substituent in the 6′-O position of the glucoside residue was found to be
a malonyl group with diastereotopic methylene group signals at δ
H
3.33
(1H, m, H-2a’’) and 3.38 (1H, m, H-2b’’). The
13
C NMR data revealed
the presence of nineteen signals that included four methylene groups,
seven methine groups, three methyl groups and ve quaternary (one
ester, one carboxylic acid, one keto, one olenic and one ether) carbons
with the help of a DEPT experiment (Table 4). The structure of com-
pound 3 was conrmed through HMBC data (Fig. 4). HMBC correlations
from H-7 → C-1, 2, 6, from H-2 → C-1, 3, 4, 6, from H-5 → C-1, 3, 4, 8 and
from H-9 → C-4, 8, 10 established the structure and connectivity of the
monoterpenoid aglycone. HMBC correlations from H-1’ → C-8, from H-6
→ C-1′′ and from H-2’’ → C-1′′ , 3′′ established the connectivity of the
glucoside to the monoterpenoid and malonic acid residues. Thus, the
structure of compound 3 was established as 8-O-(6′′-O-malonyl-β-D-
glucopyranosyl)-menth-4(5)-en-3-one, and named 6′-malonylzizi-
phoroside A.
4. Discussion
The choice of optimal methods for sample preparation is key to
obtaining correct data about the bioactive components of medicinal
plants. Therefore, the choice of extraction method is of great importance
and is the main parameter when planning research of the biological
activity of herbal products and herbal remedies. However, nding a
universal phase or extraction method is nearly impossible due to the
complexity of plant systems and the metabolites chemical diversity. To
identify the active substances of the herb Z. clinopodioides subsp. bun-
geana, various approaches to extraction were applied: sequential
exhaustive extraction with solvents of different polarity, ultrasonic
extraction and maceration. This made it possible to obtain fractions with
different chemical composition and, presumably, with different bio-
logical activity. The model of acute hemic hypoxia was chosen as the
primary screening method. Based on the obtained results, the most
active extracts were identied as – RAF and US60. Both extracts effected
the indicators of contraction and output, comparable to the reference
drug - Monopril. Based on the extraction methods used to obtain RAF
and US60 and data from the literature, it can be assumed that they
contain compounds with medium polarity, including polyphenols and
terpenoids (Karpov et al., 2019).
Phytochemical analysis of Z. clinopodioides subsp. bungeana extracts
was performed to nd the active components responsible for the
Fig. 4. Key HMBC and COSY correlations for compounds 1–3.
Table 4
1
H NMR and
13
C NMR data for compounds 2–3.
2 3
Position δ
H
δ
C
Position δ
H
δ
C
1a′3.43 (1H, m) 68.8 1 2.06 (1H,
m)
30.3
1b′3.67 (1H, m) 2a 2.10 (1H,
m)
48.1
2′2.27 (2H, q, J
=7.0 Hz)
28.0 2b 2.33 (1H,
m)
3′5.34 (1H, m) 125.5 3 – 198.7
4′5.42 (1H, m) 133.4 4 – 142.4
5′2.02 (2H, q, J
=7.4 Hz)
20.6 5 7.34 (1H,
m)
145.9
6′0.93 (3H, t, J
=7.4 Hz)
14.6 6a 2.04 (1H,
m)
34.0
1 4.17 (1H, d, J
=7.7 Hz)
103.2 6b 2.44 (1H,
m)
1
4.17 (1H, d, J
=7.7 Hz)
103.2
7
0.97 (3H, d,
J =5.5 Hz)
21.2
2 2.96 (1H, m) 73.7 8 – 77.5
3 3.16 (1H, m) 76.9 9 1.35 (3H, s) 25.5
4 3.06 (1H, m) 70.6 10 1.38 (3H, s) 27.8
5 3.34 (1H, m) 74.1 1′4.34 (1H, d,
J =7.6 Hz)
97.4
6a 4.01 (1H, dd,
J =11.6, 6.9
Hz)
65.0 2′3.03 (1H,
m)
74.1
6b 4.32 (1H, dd,
J =11.6, 1.3
Hz)
3′3.15 (1H,
m)
77.1
1′′ – 170.9 4′3.04 (1H,
m)
70.7
2a’’ 2.57 (1H, d, J
=14.2 Hz)
46.0 5′3.32 (1H,
m)
73.8
2b’’ 2.66 (1H, d, J
=14.2 Hz)
6a′4.07 (1H,
m)
65.1
3′′ – 69.4 6b′4.31 (1H,
m)
4a’’ 2.49 (1H, m) 45.7 1′′ – 167.2
4b’’ 2.55 (1H, d, J
=14.8 Hz)
2′′ 3.33 (2H, s) 41.9
5′′ – 172.82b’’
3.38 (1H,
m)
6′′ 1.28 (3H, s) 27.9 3′′ – 168.2
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
8
characterized cardiotropic activity, which led to the isolation of 13
compounds (Fig. 5). Blumenol C glucoside (4) (Miyase et al., 1988),
blumenol C 9-O-(6′-O-malonyl-beta-D-glucopyranoside (5) (Schliemann
et al., 2008), 7a-hydroxymintlactone (6) (Woodward and Eastman,
1950; N¨
af and Velluz, 1998), 7′-hydroxypiperitone (7) (Guillermo,
1991; Grudniewska et al., 2010), pinocembrin-7-O-rutinoside (8) (Xu
et al., 2011), chrysin-7-O-rutinoside (9) (Sankara Subramanian et al.,
1972), acacetin-7-O-rutinoside (10) (Rios et al., 2018),
luteolin-7-O-rutinoside (11) (Yasuaki et al., 1984; Kim et al., 2000) and
rutin (12) were isolated from the arial parts of Z. clinopodioides subsp.
bungeana for the rst time, whereas rosmarinic acid (13) was previously
characterized through HPLC-ESI-QTOF-MS/MS analysis of
Z. clinopodioides subsp. bungeana extracts (Zhaparkulova K. et al., 2022).
The three previously unreported compounds were identied as 3,
7-dimethyl-6-ketooct-7-enoic acid (Ziziphoric acid) (1),
1-O-(8′-methylhept-3′-en-1′-yl)-6-O-(3′′,5′′ -dihydox-
y-5′′-carbonyl-3′′ -methylpentanoyl)-β-D-glucopyranoside (Ziziphoro-
side D) (2) and 8-O-(6′′-O-malonyl-β-D-glucopyranosyl)-menth-4
(5)-en-3-one (6′-malonylziziphoroside A) (3).
Phytochemical analysis of Z. clinopodioides subsp. bungeana extracts
led to the isolation of ve terpenoid, two megastigmage glycosides, ve
avonoid and one cinnamic acid derivative. Part of the isolated terpe-
noid derivatives most likely underwent interesting structural modica-
tions during their biosynthesis. Ziziphoric acid could be possibly formed
through the oxidative ring opening of isopulegone between positions 3
and 4. The structure of Ziziphoroside D is extremely interesting, due to it
containing two different substituents – a terpenoid derived pentenyl
group and a mevalonic acid derived dicrotalic acid residue. Ziziphoro-
side D could also show interesting bioactivities due to alkyl glucosides
being well known as non-ionic surfactants and dicrotalic acid (also
known as Meglutol) being a hypolipidemic agent. 6′-malonylzizi-
phoroside A is the 6′-malonylated glucoside of the monocyclic mono-
terpene shizonol, which is a known component of the herbal drug
“keigai” (Oshima et al., 1989).
The avonoid components isolated from Z. clinopodioides subsp.
bungeana pertain to three different avonoid subclasses – avanones,
avones and avonols all of which are known to possess different types
of bioactivities. For example, pinocembrin-7-O-rutinoside was shown to
have signicant liver-protecting effects, though the aglycone pinocem-
brin was found to be the active component (Guo et al., 2016). Pino-
cembrin is also one of the most abundant avonoids in propolis, which is
known for a wide scope of useful properties (Shen et al., 2019). Both
chrysin and acacetin are also known for displaying a large range of
bioactivities (Naz et al., 2019; Singh, S. et al., 2020). Rosmarinic acid is
considered a wide occurring component of many herbal remedies (Guan
et al., 2022). Of special interest is that all ve isolated avonoids are
rutinosides, in addition to the fact that pinocembrine, chrysine, acacetin
and luteolin all share the rutinoside residue in the 7-O position of ring B.
The shared 7-O position of the rutinoside residue along with similar
aglycone structures hints at potentially similar pharmacokinetic proles
of the isolated pinocembrine, chrysine, acacetin and luteolin glycosides
along with potentially related pharmacodinamic characteristics.
The use in traditional medicine of Z. clinopodioides subsp. bungeana,
the observed experimental cardiotropic properties for the extracts along
with the established phytochemical composition of the plant are in good
accordance with each other. The cardiotropic activity of the main
avonoid components – pinocembrin (Zheng et al., 2020), chrysin
(Farkhondeh et al., 2019), acacetin (Wu et al., 2022) and luteolin (Luo
et al., 2017) are well known and denitely play a crucial role in the
pharmacological activity of Z. clinopodioides subsp. bungeana extracts
and raw materials, especially considering their high content in the plant.
The monoterpenoids and their glycosides found in Z. clinopodioides
subsp. bungeana most likely also play a role in the observed cardiotropic
activities based on data present in the literature (Silva et al., 2021).
The achievements of modern phytopharmacology open up new op-
portunities in the development of new, more effective herbal remedies
with targeted medicinal properties based on traditional herbal remedies.
Analytical screening of plant extracts of different polarity for their bio-
logical activity has shown to be an effective approach, bringing us closer
to understanding which of the secondary metabolites contained in
traditional herbal remedies are responsible for the displayed pharma-
cological effects. The pharmacological effects of Z. clinopodioides subsp.
bungeana extracts are most likely associated with their rich content of
terpenoid and avonoid derivatives. The applied approach also allows a
more reasonable strategy in the choice of extraction technology for the
development of new targeted herbal preparations based on
Fig. 5. Structures of compounds 1–13.
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
9
Z. clinopodioides subsp. bungeana raw materials.
5. Conclusions
Our results support the traditional use of Z. clinopodioides subsp.
bungeana for the treatment of coronary diseases. In the result of in vivo
pharmacological screening of extracts of Z. clinopodioides subsp. bun-
geana, two extracts were selected as potential cardiotropic agents.
Phytochemical analysis of the most active extracts led to the isolation of
ve terpenoid derivatives, two megastigmane glucosides and ve a-
vonoids and one cinnamic acid derivative, which due to their presence
in the raw plant material likely mediate the observed experimental
pharmacological activity.
Funding
This work was supported by a grant IRN N◦AP09259196 from the
Science Committee of the Ministry of Science and Higher Education of
the Republic of Kazakhstan.
The analyses were performed out on the equipment in the Core
Shared Research Facilities “Analytical Center” of the Saint Petersburg
State Chemical and Pharmaceutical University, with nancial support
from the Ministry of Science and Higher Education of the Russian
Federation (Agreement No. 075-15-2021-685; dated July 26, 2021 on
the provision of the Federal budget grants).
CRediT authorship contribution statement
A.O. Whaley: Investigation. D.Y. Ivkin: Conceptualization, Inves-
tigation. K.A. Zhaparkulova: Conceptualization. I.N. Olusheva:
Investigation. E.B. Serebryakov: Investigation. S.N. Smirnov: Investi-
gation. E.D. Semivelichenko: Formal analysis. A. Yu. Grishina:
Investigation. A.A. Karpov: Investigation. E.I. Eletckaya: Investiga-
tion. K.K. Kozhanova: Resources. L.N. Ibragimova: Formal analysis.
K.T. Tastambek: Writing – original draft. A.M. Seitaliyeva: Resources.
I.I. Terninko: Writing – original draft. Z.B. Sakipova: Conceptualiza-
tion. A.N. Shikov: Conceptualization. M.N. Povydysh: Writing – orig-
inal draft, Writing – review & editing. A.K. Whaley: Conceptualization,
Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
HR-ESI-MS and NMR data was obtained using equipment from the
Center for Chemical Analysis and Materials Research, Magnetic Reso-
nance Research Center of the Research Park of the Saint Petersburg State
University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2023.116660.
References
Abubakar, A.R., Haque, M., 2020. Preparation of medicinal plants: basic extraction and
fractionation procedures for experimental purposes. J. Pharm. BioAllied Sci. 12 (1),
1.
Aliakbarlu, J., Shameli, F., 2013. In vitro antioxidant and antibacterial properties and
total phenolic contents of essential oils from Thymus vulgaris, T. kotschyanus,
Ziziphora tenuior and Z. clinopodioides. Turk. J. Biochem. 38 (4).
Beikmohammadi, M., 2011. The evaluation of medicinal properties of Ziziphora
clinopodioides. World Appl. Sci. J. 12 (9), 1635–1638.
Dakah, A., Zaid, S., Suleiman, M., Dakka, M., 2019. Chemical components and
antibacterial activities of essential oil of wild, in vitro and acclimatised plants of
Ziziphora tenuior L. Int. Food Res. J. 26 (2).
Dzhumagalieva, F.D., 1963. Prevention and Treatment of Experimental Myocarditis and
Myocardial Infarction. Academy of Sciences of the Kazakh SSR, Alma-Ata, p. 153 (in
Russ).
Farkhondeh, T., Samarghandian, S., Bafandeh, F., 2019. The cardiovascular protective
effects of chrysin: a narrative review on experimental researches. Cardiovasc.
Hematol. Agents Med. Chem. 17 (1), 17–27.
Grudniewska, A., Gniłka, R., Wawrze´
nczyk, C., 2010. Enantioselectivity of hydroxylation
of racemic piperitone by fungi. Chirality 22 (10), 929–935.
Guan, H., Luo, W., Bao, B., Cao, Y., Cheng, F., Yu, S., Fan, Q., Zhang, L., Wu, Q., Shan, M.,
2022. A comprehensive review of rosmarinic acid: from phytochemistry to
pharmacology and its new insight. Molecules 27 (10), 3292.
Guo, W.W., Qiu, F., Chen, X.Q., et al., 2016. In-vivo absorption of pinocembrin-7-O-β-D-
glucoside in rats and its in-vitro biotransformation. Sci. Rep. 6 (1), 1–8.
He, J., Yang, W., Cheng, B., Ma, L., Tursunjiang, D., Ding, Z., Li, G., 2020. Integrated
metabolomic and transcriptomic proling reveals the tissue-specic avonoid
compositions and their biosynthesis pathways in Ziziphora bungeana. Chin. Med. 15,
1–15.
Karlygash, Z.A., Zuriydda, S.B., Inna, T.I., Vijayasankar, R., Natalya, K.V., Samir, R.A.,
Ikhlas, K., 2016. Macroscopic and morpho-anatomical diagnostic features of
Ziziphora bungeana Juz. from Kazakhstan. Int. J. Pharmacogn. Phytochem. Res. 8,
812–819.
Karpov, A.A., Puzanov, M.V., Ivkin, D.Y., Krasnova, M.V., Anikin, N.A., Docshin, P.M.,
Moiseeva, O.M., Galagudza, M.M., 2019. Non-inferiority of microencapsulated
mesenchymal stem cells to free cells in cardiac repair after myocardial infarction: a
rationale for using paracrine factor(s) instead of cells. Int. J. Exp. Pathol. 100 (2),
102–113.
Kim, N.M., Kim, J., Chung, H.Y., Choi, J.S., 2000. Isolation of luteolin 7-O-rutinoside and
esculetin with potential antioxidant activity from the aerial parts of Artemisia
montana. Arch Pharm. Res. (Seoul) 23 (3), 237–239.
Li, Q., Tursun, D., Shi, C., Heyrulla, M., Zhang, X., Yang, W., 2018. Ziziphora
clinopodioides avonoids protect myocardial cell damage from myocardial ischemia-
reperfusion injury. Evid. Based Complementary Altern. Med. 2018.
Liu, C., Zhang, M., Ye, S., Hong, C., Chen, J., Lu, R., Gu, Z., 2021. Acacetin protects
myocardial cells against hypoxia-reoxygenation injury through activation of
autophagy. J. Immunol. Res. 2021.
Luo, Y., Shang, P., Li, D., 2017. Luteolin: a avonoid that has multiple cardio-protective
effects and its molecular mechanisms. Front. Pharmacol. 8, 692.
Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., Oguchi, H., 1988. Studies on the
Glycosides of Epimedium grandiorum MORR. var. thunbergianum. Chem. Pharm. Bull.
36 (7), 2475–2484.
N¨
af, R., Velluz, A., 1998. Phenols and lactones in Italo-Mitcham peppermint oil Mentha
×piperita L. Flavour Fragrance J. 13, 203–208.
Naghibi, N., O’Malley, A., 2005. Estranging the familiar:" east" and" west" in Satrapi’s
persepolis. ESC Engl. Stud. Can. 31 (2), 223–247.
Naz, S., Imran, M., Rauf, A., Orhan, I.E., Shariati, M.A., Ul-Haq, Iahtisham, Iqra, Y.,
Shahbaz, M., Qaisrani, T.B., Shah, Z.A., Plygun, S., Heydari, M., 2019. Chrysin:
pharmacological and therapeutic properties. Life Sci. 235, 116797.
Oshima, Y., Takata, S., Hikino, H., 1989. Schizonodiol, schizonol, and schizonepetosides
D and E, monoterpenoids of Schizonepeta tenuifolia Spikes. Planta Med. 55 (2),
179–180.
Rabah, B., Lograda, T., Ramdani, M., Chalard, P., Feguiredo, G., 2013. Chemical
composition and antibacterial activity of essential oil of Ziziphora hispanica L. Glob.
J. Res. Med. Plants Indig. Med. 2 (2), 73.
Rios, M.Y., C´
ordova-Albores, L.C., Ramírez-Cisneros, M.´
A., King-Díaz, B., Lotina-
Hennsen, B., Le´
on Rivera, I., Miranda-S´
anchez, D., 2018. Phytotoxic potential of
zanthoxylum afne and its major compound linarin as a possible natural herbicide.
ACS Omega 3 (11), 14779–14787.
Sankara Subramanian, S., Nagarajan, S., Sulochana, N., 1972. Chrysin-7-rutinoside from
the leaves of Dolichandrone falcate. Phytochemistry 11 (1), 438–439.
Schliemann, W., Kolbe, B., Schmidt, J., Nimtz, M., Wray, V., 2008. Accumulation of
apocarotenoids in mycorrhizal roots of leek (Allium porrum). Phytochemistry 69 (8),
1680–1688.
Senejoux, F., Demougeot, C., Kerram, P., Aisa, H.A., Berthelot, A., B´
evalot, F., Girard-
Thernier, C., 2012. Bioassay-guided isolation of vasorelaxant compounds from
Ziziphora clinopodioides Lam.(Lamiaceae). Fitoterapia 83 (2), 377–382.
Shen, X., Liu, Y., Luo, X., Yang, Z., 2019. Advances in biosynthesis, pharmacology, and
pharmacokinetics of pinocembrin, a promising natural small-molecule drug.
Molecules 24 (12), 2323.
Silva, E.A.P., Santos, D.M., de Carvalho, F.O., Menezes, I.A.C., Barreto, A.S., Souza, D.S.,
Quintans-Júnior, L.J., Santos, M.R.V., 2021. Monoterpenes and their derivatives as
agents for cardiovascular disease management: a systematic review and meta-
analysis. Phytomedicine 88, 153451.
A.O. Whaley et al.

Journal of Ethnopharmacology 315 (2023) 116660
10
Singh, S., Gupta, P., Meena, A., Luqman, S., 2020. Acacetin, a avone with diverse
therapeutic potential in cancer, inammation, infections and other metabolic
disorders. Food Chem. Toxicol. 145, 111708.
ˇ
Smejkal, K., Malaník, M., Zhaparkulova, K., Sakipova, Z., Ibragimova, L., Ibadullaeva, G.,
ˇ
Zemliˇ
cka, M., 2016. Kazakh Ziziphora species as sources of bioactive substances.
Molecules 21 (7), 826.
Sonboli, A., Mirjalili, M.H., Hadian, J., Ebrahimi, S.N., Yousefzadi, M., 2006.
Antibacterial activity and composition of the essential oil of Ziziphora clinopodioides
subsp. bungeana (Juz.) Rech. f. from Iran. Z. Naturforsch. 61 (9–10), 677–680.
Srivedavyasasri, R., Zhaparkulova, K.A., Sakipova, Z.B., Ibragimova, L., Ross, S.A., 2018.
Phytochemical and biological studies on Ziziphora bungeana. Chem. Nat. Compd. 54,
195–197.
Woodward, R., Eastman, R., 1950. The autoxidation of menthofuran. J. Am. Chem. Soc.
72 (1), 399–403.
Wu, C., Yan, J., Li, W., 2022. Acacetin as a potential protective compound against
cardiovascular diseases. Evid. Based Compl. Alternat. Med. 2022, 6265198.
Xu, X., Xie, H., Hao, J., Jiang, Y., Wei, X., 2011. Flavonoid glycosides from the seeds of
Litchi chinensis. J. Agric. Food Chem. 59 (4), 1205–1209.
Yang, W.J., Liu, C., Gu, Z.Y., Zhang, X.Y., Cheng, B., Mao, Y., Xue, G.P., 2014. Protective
effects of acacetin isolated from Ziziphora clinopodioides Lam.(Xintahua) on neonatal
rat cardiomyocytes. Chin. Med. 9, 1–6.
Yasuaki, H., Schuichi, S., Yoeriteru, I., Junzo, S., 1984. Studies on the constituents of
palmae plants. I. The constituents of Trachycarpus fortunei (Hook.) H. Wendl. Chem.
Pharm. Bull. 32 (1), 295–301.
Zhaparkulova, K.A., Srivedavyasasri, R., Raman, V., Sakipova, Z.B., Ibragimova, L.,
Khan, I.A., Ross, S.A., 2016. Standardization of medicinal herb Ziziphora bungeana
Juz.(Lamiaceae) and quantitative determination of pulegone in the essential oil.
Planta Med. 82 (5), PC76.
Zhaparkulova, K., Karaubayeva, A., Sakipova, Z., Biernasiuk, A., Gaweł-Bęben, K.,
Laskowski, T., Kusniyeva, A., Omargali, A., Bekezhanova, T., Ibragimova, L.,
Ibadullayeva, G., 2022. Multidirectional characterization of phytochemical prole
and health-promoting effects of Ziziphora bungeana Juz. extracts. Molecules 27 (24),
8994.
Zheng, Y., Wan, G., Yang, B., Gu, X., Lin, J., 2020. Cardioprotective natural compound
pinocembrin attenuates acute ischemic myocardial injury via enhancing glycolysis.
Oxid. Med. Cell. Longev. 2020, 4850328.
Glossary
ACE: angiotensin-converting enzyme
AR: aqueous residue
B: butanol extract
BAW: n-buthanol-acetic acid-water
COSY: correlation spectroscopy
DC: dichloromethane extract
DCm: dichloromethane
DEPT: distortionless enhancement by polarization transfer
DMSO: dimethyl sulfoxide
E40: 40% ethanol extract
E50: 50% ethanol extract
E60: 60% ethanol extract
EA: ethyl acetate extract
ECG: electrocardiography
EchoCG: echocardiography
EE: ethanol extracts
EF: ejection fraction
EtOH: ethanol
Frs: fractions
FU: shortening fraction
GOST: governmental standard
HMBC: eteronuclear multiple bond correlation
HPLC: high performance liquid chromatography
HR-ESI-MS: high-resolution electrospray ionization mass spectrometry
LCA: left coronary artery
NMR: nuclear magnetic resonance
NOESY: nuclear overhauser effect correlation spectroscopy
PE: petroleum ether extract
PLZh Rappolovo: pitomnik laboratornyh zhivotnyh Rappolovo (Rus)
Q-TOF: quadrupole time-of-ight
RAF: residual aqueous fraction
SFr: sub-fractions
SPCPU: Saint Petersburg State Chemical Pharmaceutical University
TFA: triuoroacetic acid
TLC: thin layer chromatography
t
R
: retention time
US40: 40% ultrasound ethanol extract
US50: 50% ultrasound ethanol extract
US60: 60% ultrasound ethanol extract
US70: 70% ultrasound ethanol extract
UV-spectra: ultraviolet–visible spectra
A.O. Whaley et al.