Chemical Papers (2024) 78:5191–5207

https://doi.org/10.1007/s11696-024-03458-7

ORIGINAL PAPER

Novel findings on the bioactivities of black ginger of Vietnam

and optimization of its extraction using response surface
methodology
Thi Van Anh Nguyen1 · Thanh Hang Nguyen1 · Thi Kieu Oanh Nguyen1,2 · Phuong Nhung Nguyen3 ·
Hong Luyen Le1

Received: 1 March 2023 / Accepted: 9 April 2024 / Published online: 23 April 2024
© The Author(s), under exclusive licence to the Institute of Chemistry, Slovak Academy of Sciences 2024

Abstract
The present study aimed to evaluate the antioxidant activity and antithrombotic effect of the n-hexane extract from rhi-
zomes Distichochlamys citrea (DC-R), an endemic ginger species of Vietnam. Results showed that DC-R expressed a more
potent antioxidative effect against ABTS than DPPH radical (p < 0.05). DC-R contained a total phenolic content of 3.41 g
GAE/100 g and a total flavonoid content of 18.33 g QE/100 g of the dried extract. At concentrations of 1, 2 and 4 mg/mL,
DC-R reduced the percentage of platelet aggregation triggered by ADP, collagen and ristocetin in a dose-dependent manner
(Pearson’s correlation, r > 0.95, p < 0.05). DC-R also exhibited anticoagulant activity by significantly prolonging the activated
prothrombin time (APTT) at all concentrations tested (p < 0.05 compared to DMSO 0.1%). GC–MS revealed 41 volatile
components in DC-R, in which several compounds were firmly bound to receptors COX-1 and P2Y12 of the platelet aggrega-
tion and factors X and II of the blood coagulation cascades. Using response surface methodology with central composition
design, the extraction yield of DC-R reached the highest value (1.011 ± 0.008%) at optimized conditions: liquid/ solid ratio
13.5 mL/g, ultrasonic power level 80%, extraction time 30 min, and extraction temperature 34 °C.

Keywords Distichochlamys citrea · Antioxidative effect · Antiaggregatory activity · Anticoagulant activity · Optimization
of extraction · Response surface methodology

Introduction to prevent platelet aggregation (antiplatelet drugs) or blood

In 2019, cardiovascular diseases (CVDs) killed 17.9 million
people, representing approximately one-third of all deaths
globally (WHO 2021). Heart attacks and strokes, which
account for more than 80% of all cardiovascular-related
deaths, are most commonly caused by thrombosis, the devel-
opment of thrombi in blood arteries (Mackman 2008). The
main antithrombotic medications on the market now work
* Hong Luyen Le
le-hong.luyen@usth.edu.vn
1 citrea, D. orlowii, D. benenica, and D. rubrostriata (Larsen
Department of Life Sciences, University of Science
and Technology of Hanoi, Vietnam Academy of Science
and Technology, Hanoi, Vietnam
2
Laboratory Mixte International of Drug Resistance
of Southeast Asia (LMI DRISA), Hanoi, Vietnam
3
Department of Basic Science, Hanoi University of Pharmacy,
Hanoi, Vietnam
clot formation (anticoagulant medicines). Those agents are
relatively effective but responsible for several side effects,
such as bleeding and drug resistance, limiting their use
(Mackman et al. 2020). This fact leads to a growing need
for safer and more potent antithrombotic agents for CDVs
patients. Moreover, consuming natural antioxidants is also
believed to contribute considerably to reducing the risk of
atherosclerosis and other heart-related diseases (Allouche
et al. 2010; Halliwell 1994; Mary et al. 2003).
Distichochlamys, belonging to the Zingiberaceae family,
is an endemic ginger genus in Vietnam. Up to date, four
Distichochlamys species have been revealed, including D.
and Newman 2001; Nguyen and Leong-Škorničková 2012).
D. citrea, also called black ginger, has been used by local
people to treat many health issues, such as stimulating diges-
tion, immunity enhancement, diabetes disease, and heart
diseases. Only a few reports on the chemical profile of this
plant have been documented. Earlier authors showed that the

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principal compounds contained in D. citrea’s essential oil
included α-pinene, β-pinene, β-linalool, α-terpineol, 1,8-cin-
eole, cis-geraniol, β-citral, and α-citral (Le et al. 2017; Pham
Viet Ty et al. 2017). Similarly, limited scientific evidence
on the pharmacological benefits of D. citrea has previously
been published, such as antioxidant and alpha-glucosidase
inhibitory effects (Van Chen et al. 2022), and antimicro-
bial activity (Thinh et al. 2022; Van Hue et al. 2022). But
as far as we know, there has not been any research on the
antithrombotic properties of this plant.
The present study investigated the antioxidative activity
and the inhibitory effect of the n-hexane extract of D. cit-
rea (DC-R) on platelet aggregation and blood coagulation
for the first time. Volatile components of this fraction were
identified using GC–MS. The main compounds in DC-R
were then docked with several platelet receptors and coagu-
lant factors to predict their potential interactions that might
induce the antithrombotic effect of this plant. Finally, the
ultrasonic-assisted extraction of DC-R was optimized using
response surface methodology (RSM) with central compos-
ite design (CCD).
Experimental
Chemicals and reagents
Aspirin, ticagrelor, ADP, collagen, ristocetin, heparin,
and dimethyl sulfoxide (DMSO) were provided by Sigma-
Aldrich. Reagents for blood clotting assay were obtained
from Dade Behring Marburg GmbH (Marburg, Germany).
Reagents for antioxidant activity and hexane (LC–MS grade)
were purchased from Thermo Fisher. Ultrapure water was
taken from a Millipore System (Merck).
Plant materials
D. citrea rhizomes were collected in Thua Thien Hue prov-
ince, Vietnam (16.4674° N, 107.5905° E), in June 2021.
Dr. Nguyen Quoc Binh of the Vietnam National Museum
of Nature, Vietnam Academy of Science and Technology
(VAST) identified it. The Department of Life Sciences at the
University of Science and Technology of Ha Noi received a
voucher specimen with the identification number SH 1195.
10 g of dried rhizomes were macerated under sonication
with 100 mL of hexane for 2 h. The crude extract (DC-R:
430.2 mg) was obtained after evaporation under reduced
pressure and then stored at 4 °C until analysis.
DPPH assay
This experiment was performed on 96-well plates using
the previous method with slight modifications (Zhao et al.
Chemical Papers (2024) 78:5191–5207
2010). In each well, 10 µL of the plant extract was incubated
with 190 µL of DPPH (0.1 mM in methanol) at 37 °C for
20 min. The absorbance of the mixture was measured at
517 nm using a Microplate spectrophotometer (xMark, Bio-
Rad). Ascorbic acid was used as the positive control. The
scavenging percentage was calculated as follows:
As
% Inhibition = 100 − ( x100%)
Ac
where As and Ac are the absorbance of the sample and the
control, respectively.
IC50 values are the concentration of samples that scav-
enged 50% of radicals.
ABTS assay
This study was performed according to the method of Bel-
lik (2014). The ABTS.+ radical cation was generated by the
reaction between potassium persulfate solution at 2.45 mM
in water and ABTS+ solution at 7 mM in water (ratio 1:1,
v/v) at 25 °C for 14 h in the dark. 10 μL of the plant extract
was mixed with 190 μL of the diluted solution of ABTS.+ (in
ethanol) at room temperature for exactly 10 min in the dark.
The absorbance was measured at a wavelength of 734 nm
with a microplate reader. Trolox was used as the positive
control.
Total phenolic content
The Folin–Ciocalteu assay was applied in this experiment
according to the previous method (Ali et al. 2018). 10 μL of
DC-R was incubated with 95 μL of Folin–Ciocalteu reagent
and 95 μL of ­Na2CO3 6% at 40 °C for 15 min in a 96-well
plate. The absorbance was measured at 765 nm.
A standard curve of gallic acid was established at con-
centrations from 31.25 to 500 μg/mL. The total phenolic
content (TPC) of DC-R was calculated as grams of gallic
acid equivalents per 100 g of dried sample (g GAE/100 g
sample): TPC = (CGAE × 100)/C0.
where CGAE is the concentration of gallic acid equivalent
(g/mL) and C0 is the concentration of DC-R (g/mL).
Total flavonoid content
This study was done according to the previous method with
slight modifications (Ali et al. 2018). 40 μL of DC-R was
mixed with 10 μL of ­NaNO2 5% and incubated for 6 min at
room temperature. Then, 10 μL ­AlCl3 10% was added to the
mixture and the solution was set for 6 min. Finally, 80 μL
of NaOH and 60 μL of EtOH were consecutively added
and incubated for another 15 min at room temperature. The
absorbance was measured at 510 nm.
Chemical Papers (2024) 78:5191–5207
A standard curve of quercetin was made at concentrations
ranging from 15.625 to 1000 μg/mL. The total flavonoid con-
tent (TFC) of DC-R was determined as grams of quercetin
equivalents per 100 g of dried sample (g QE/100 g sample)
according to the following equation: TPC = (CQE × 100)/C0.
Where CQE is the concentration of quercetin equivalent
(g/mL) and C0 is the concentration of DC-R (g/mL).
Blood samples preparation
Healthy volunteers were free of antiplatelet or anticoagulant
agents at least three weeks before the test. Briefly, blood
samples were anticoagulated with 3.2% sodium citrate
before being centrifuged at 500 rpm and then 3000 rpm for
10 min to obtain platelet-rich plasma (PRP) and platelet-
poor plasma (PPP). After being collected from volunteers,
all blood samples were used within three hours.
Antiplatelet aggregation assay
The method described by Lordan et al. (2020) was used with
slight modifications. 50 µL of DC-R (at final concentrations
of 1, 2 and 4 mg/mL in DMSO 0.1%) were gently mixed
with 450 µL of PRP and incubated for 3 min at 37 °C. Then,
the platelet aggregation was triggered by several agonists
such as ADP (5 µL, 10 µM), collagen (1 µL, 2 µg/mL), or
ristocetin (5 µL, 1.25 µg/mL). The negative control was
DMSO 0.1%; while, the positive controls were ticagrelor at
0.002 mg/mL (for ADP) and aspirin at 0.1 mg/mL (for col-
lagen and ristocetin). The maximum percentage of aggrega-
tion (%) was recorded by a Chrono-Log 530 VS aggregator
(USA). All samples were tested with three blood samples.
Anticoagulant assay
This study used classical coagulant assays (Dhahri et al.
2020). Briefly, 50 μL of DC-R and 450 μL of PPP were incu-
bated at 37 °C for 5 min. Then clotting reagents were added
to the mixture according to the machine’s instruction (ACL
TOP500, USA). The APTT (activated partial thromboplas-
tin time), PT (prothrombin time), and TT (thrombin time)
values were measured after a 6 min curve was recorded. The
negative control was DMSO 0.1%, and the positive control
was heparin 0.2 IU/mL (for APTT and TT parameters) or
2 IU/mL (for PT parameter). The experiment was done in
triplicate.
GC–MS analysis
The analysis of volatile components in DC-R was investi-
gated with some modifications to the method of Madhumita
5193
and colleagues (Madhumita et al. 2019). 1 μL of DC-R
was analyzed by a GC–MS system (a Thermo Scientific
GC-TRACE™ 1300 combined with an MS- DSQ II). All
runs were performed using a capillary column TG-5MS
(30 m × 0.25 mm × 0.25 μm) (Thermo Fischer Scientific). A
gradient oven temperature was carried out with the program
as follows: the initial temperature of 50 °C was kept for 2 min
before being progressively raised to 180 °C at the rate of 5 °C
/min and remained at 180 °C for 3 min. The temperature was
then increased to 280 °C at 4 °C/min and kept at 280 °C for
5 min. 1.2 mL/min of helium was used as the gas carrier. The
mass scan was set for acquisition from 40 to 800 m/z. A mix-
ture of n-alkanes (­ C8–C20) was used as a reference in calcu-
lating retention indices (KRI). The raw files were converted
into cdf files by Xcalibur software (Thermo Scientific); then
processed by Automated Mass Spectral Deconvolution and
Identification System (AMDIS) software. Volatile compounds
were identified and quantified using the NIST mass spectral
library (NIST27, WILEY7) and essential oil components by
GC/MS Version 4 by Robert Adams (Adams 2007).
Molecular docking
The elucidated compounds of DC-R were docked against sev-
eral targets to study ligand protein interactions by Autodock
Vina 4 software. The binding affinity of all 41 compounds
and the positive control (aspirin and ticagrelor) to COX-1 and
P2Y12 (PDB-IDs of 3N8Z and 4NTJ, respectively) was evalu-
ated for antiplatelet aggregation. In the case of anticoagulant
activity, 41 components in DC-R were docked to factor II
and factor X (PDB-IDs of 5JZY and 1KSN), and the positive
control was heparin. The structure of receptors was obtained
from Protein Data Bank (https://​www.​rcsb.​org/) and after
ligands and solvent removal, the proteins were prepared by
Autodock Tools version 1.5.7 by adding hydrogen and assign-
ing Gasteiger charges. The structure of small molecules was
extracted from the PubChem database (https://​pubch​em.​ncbi.​
nlm.​nih.​gov/) in 2D sdf format, converted to pdb format by
MarvinSketch version 22.3, and prepared to Autodock format
(pdbqt) by Raccoon plugin (Forli et al. 2016) with hydrogen
addition, Gasteiger charge calculation, and torsion assignment.
The docking protocols were validated by redocking the co-
crystallized ligands through their ability to recover the docking
pose and essential interactions of the co-crystallized ligands.
Through docking procedures, the binding affinity of mol-
ecules in DC-R and positive controls with specific receptors
was evaluated by the best pose’s binding energy (BE) (kcal/
mol). Ligand protein interaction was visualized by Discovery
Studio Visualizer.
5194
Optimization of DC‑R extraction yield
Ultrasound‑assisted extraction
2 g of D. citrea rhizomes powder was soaked with 100 mL
of n-hexane in an ultrasonic bath (WUC-D22H, 528 W,
40 kHz, Daihan, Korea) with the ultrasonic power levels
ranging from 30 to 100%. Several extraction conditions
were varied using preliminary single factor experiments.
Then, the extract was obtained after being centrifuged at
10,000 rpm for 10 min and filtered to remove the solid resi-
dues. Next, a rotary evaporator was employed to remove the
solvent at 50 °C. Each experiment was performed twice, and
the extraction yield was calculated as follows:
me
Yield(%) = × 100
ms
where me is the mass of DC-R extract, and ms is the mass of
the dried sample.
Single factor experiments
The impact of four factors, including liquid/ solid ratio,
level of ultrasonic power, extraction time, and extraction
temperature was evaluated by varying one factor while
holding the other three constants. For instance, the effect
of ultrasonic power was examined at levels 30, 40, 60,
80, and 100%; while, the extraction time, extraction tem-
perature, and liquid/ solid ratio were, respectively, set at
15 min, 40 °C, and 10:1 mL/g. The impact of extraction
time on the yield of DC-R was studied at 5, 15, 30, 60,
and 90 min with the ultrasonic power of 30%, the extrac-
tion temperature of 40 °C, and the liquid/ solid ratio of
10:1 mL/g. The effect of the liquid/ solid ratio was exam-
ined from 5:1 to 30:1 mL/g; while, the ultrasonic power,
extraction temperature, and extraction time were kept at
30%, 40 °C, and 15 min, respectively. Five temperature
points were chosen to examine the impact of temperature,
Table 1  Fractional factorial Run Ratio (mL/g)
design with the mean of
extraction yield for each run
1 5
2 15
3 5
4 15
5 5
6 15
7 5
8 15
Chemical Papers (2024) 78:5191–5207
30, 40, 50, 60, and 70 °C; while, the other parameters were
kept constant at 30%, 10:1 mL/g, and 15 min, respectively.
Screening of factors
The four factors mentioned above were simultaneously
investigated to determine their effect on the response of
the experiment. Two levels (high and low) for each factor
were chosen from the preliminary studies, as shown in
Table 1. A total of 8 experiments were carried out. Minitab
statistical software (Release 18, Minitab Inc.) was used to
analyze the data of this study statistically.
Optimization of extraction yield
Using RSM, CCD with axial points was used to assess
the effective combinations of several variables needed to
optimize the yield of DC-R extraction. The range of each
significant extraction parameter was chosen based on the
result of the screening step: the ratio between solvent and
dried material from 5:1 to 15:1 mL/g, and extraction tem-
perature from 30 °C to 60 °C. A total of 14 experiments
generated with six replications at central points for the
complete design matrix of CCD was presented in Table 2.
A second-order polynomial model, represented in the
equation below, was used to fit all the experimental results:
Y = X0 + XA A + XB B + XAA A2 + XBB B2 + XAB AB
where Y is the predicted extraction yield, X0 is the offset
term, XA , XB are the regression coefficients for factors A
and B, XAA , XBB are quadratic effects, and XAB is interaction
effects. A and B are liquid/ solid ratio, and extraction tem-
perature, respectively. The matrix of experimental design,
regression coefficients, and graphical optimization was ana-
lyzed by using Design expert version 12 software.
Temperature Power (%) Time (min) Extraction yield (%)
(°C)
30 30 5 0.253 ± 0.004
60 30 5 1.020 ± 0.038
60 80 5 0.390 ± 0.010
30 80 5 0.707 ± 0.018
60 30 30 0.328 ± 0.002
30 30 30 0.722 ± 0.008
30 80 30 0.395 ± 0.010
60 80 30 0.953 ± 0.010
Chemical Papers (2024) 78:5191–5207 5195

Table 2  Experiment design Run Ratio (mL/g) Temperature Power (%) Time (min) Extraction yield (%)
matrix of the central composite (oC)
design
1 6.5 55.6 80 30 0.750 ± 0.014
2 10 45 80 30 0.850 ± 0.011
3 10 45 80 30 0.848 ± 0.006
4 13.5 55.6 80 30 0.915 ± 0.013
5 10 45 80 30 0.854 ± 0.020
6 6.5 34.4 80 30 0.545 ± 0.002
7 13.5 34.4 80 30 0.992 ± 0.006
8 10 45 80 30 0.862 ± 0.004
9 5 45 80 30 0.506 ± 0.018
10 10 60 80 30 0.906 ± 0.018
11 10 45 80 30 0.860 ± 0.017
12 10 30 80 30 0.823 ± 0.023
13 15 45 80 30 1.028 ± 0.007
14 10 45 80 30 0.851 ± 0.009

Statistical analysis essential oil of Z. officinale rhizomes also had a significantly

GraphPad Prism 9.1 software was used to analyze the data of
this study statistically. Data were determined as mean ± SD.
Group means were compared using an independent t test
and one-way ANOVA. Differences were significant when
p < 0.05.
Results and discussion
Antioxidant activity
DC-R exerted an antioxidative effect in a concentration-
dependent manner (Pearson’s correlation, r > 0.995,
p < 0.01) against both DPPH and ABTS radicals (Table 3).
In addition, the ­IC50 obtained for ABTS was significantly
lower than DPPH (1.50 vs. 2.26 mg/mL, respectively). How-
ever, the extract exhibited much higher I­ C50 values than the
positive controls (p < 0.001). The TFC value (18.33%) of
DC-R was remarkably more elevated than the TPC value
(3.41%) (p < 0.01).
In the literature, the DPPH scavenging capacity of ginger
Zingiber officinale fractions (­ IC50: 2.81 to 5.57 mg/mL) (Yeh
et al. 2014) seemed to be weaker than the effect of black gin-
ger in the present study ­(IC50: 2.26 mg/mL). Similarly, the
higher ­IC50 value (110.14 mg/mL) (Bellik 2014) than DC-R
­(IC50 = 1.50 mg/mL) when scavenging ABTS radical. In this
study, the TPC value of DC-R (3.41 g GAE/100 g extract)
seemed to be smaller than the one of Z. officinale rhizomes
(60.34 mg GAE/g extract), but the TFC of DC-R (18.33 g
QE/100 g extract) was found to be much higher than the
content of flavonoid in Z. officinale (40.25 mg QE/g extract)
(Ali et al. 2018).
Earlier studies led to the suggestion that the antioxidant
activity of DC-R may be possibly associated with the pres-
ence of several major volatile components such as citral (Shi
et al. 2016), β-selinene (Chandra et al. 2017) or eucalyptol
(Lee and Shibamoto 2001). Moreover, the ability to scav-
enge free radicals of DC-R might come from many minor
constituents with strong antioxidative effects, such as n-hex-
adecanoic acid Kalpana Devi et al. (2012), β-bisabolene
(Kazemi and Rostami 2015) or β-sesquiphellandrene (Zhao
et al. 2010).
Antiplatelet aggregation activity
Three agonists ADP, collagen and ristocetin were used to
induce platelet aggregation. Figure 1 showed that DC-R
reduced the percentage of platelet aggregation triggered
by all agonists in a dose-dependent manner (Pearson’s

Table 3  Antioxidant activity of Sample DPPH ­(IC50, mg/mL) ABTS ­(IC50, mg/mL) TPC (g TFC (g QE/100 g DW)
DC-R GAE/100 g
DW)

Positive control 0.008 ± 0.002 0.009 ± 0.005

DC-R 2.26 ± 0.06 1.50 ± 0.04 3.41 ± 0.16 18.33 ± 1.08
5196
Fig. 1  Effect of DC-R on the maximum percentage of the plate-
let aggregation. (−) The negative control, ( +) The positive control,
*p < 0.05, **p < 0.01, ***p < 0.001 compared to the negative control
correlation, r > 0.95, p < 0.05). DC-R significantly inhibited
the platelet aggregation at 2 and 4 mg/mL compared to the
negative control (p < 0.05). Moreover, the highest percent-
age of inhibition (%I = 61.8 ± 3.5) was obtained by DC-R at
4 mg/mL in the case of ADP. However, DC-R at all doses
showed a considerably less potent inhibitory impact than the
positive control, regardless of the agonists used (p < 0.01).
Platelet aggregation is primarily attributed to thrombo-
sis; thus, antiplatelet agents would be crucial in decreasing
the risk of thrombosis and cardiovascular-related diseases
(Mackman et al. 2020). Several in vitro and in vivo stud-
ies on the antiplatelet aggregation of ginger Z. officinale
were reported a long time ago. In the 1980s, Srivastava and
colleagues demonstrated the inhibitory effect of this gin-
ger species on various agonists-induced platelet aggrega-
tion (Srivastava 1984; Srivastava and Mustafa 1989). The
n-hexane extract of ginger also reduced platelet thrombox-
ane formation and platelet aggregation (Srivastava 1986).
Primarily, gingerol isolated from Z. officinale remarkably
inhibited aggregating of platelets triggered by collagen,
arachidonic acid, or thrombin (Guh et al. 1995). In 1997,
a three months study performed on those with coronary
artery disease indicated a considerable restraint of ADP and
epinephrine-induced platelet aggregation by a 10 g-dose of
ginger powder each day (Bordia et al. 1997). More recently,
ginger was considered a potential antithrombotic agent by
reducing platelet thromboxane-B2 ­(TXB2) production in rats
with daily oral administration of 500 mg/kg for four weeks
(Thomson et al. 2002). In the current study, DC-R exerted
the inhibitory effect for all three agonists. This finding added
scientific evidence of the anti-aggregatory effect of D. citrea
for the first time.
Anticoagulant activity
In this experiment, DC-R was investigated for its anti-
coagulant activity through three parameters: the blood
Chemical Papers (2024) 78:5191–5207
Fig. 2  Effect of DC-R on the coagulation time (s). (−) The negative
control, ( +) The positive control, *p < 0.05, **p < 0.01, ***p < 0.001
compared to the negative control
clots following the intrinsic pathway were represented by
the APTT parameter; the PT parameter demonstrated the
extrinsic coagulation pathway, and the common coagula-
tion pathway where fibrin is generated from fibrinogen was
represented by TT value. Results showed that DC-R sig-
nificantly increased the APTT value at all concentrations
tested (p < 0.05), but it possessed no prolongation effect on
two other parameters (p > 0.05 compared to DMSO 0.1%)
(Fig. 2). The extract exhibited remarkably lower APTT, PT,
and TT compared to heparin (p < 0.001).
Like the antiplatelet aggregation activity, Z. officinale was
also documented to have an inhibitory effect on blood coag-
ulation. An aqueous extract of rhizomes Z. officinale was
shown to significantly inhibit the in vitro blood coagulation
by increasing prothrombin time (Taj Eldin et al. 2016). In
2017, Ajala and colleagues showed that the ginger’s metha-
nol extract remarkably exhibited an in vivo prolongation
of three coagulation pathways in rats (Ajala et al. 2017).
Recently, sulfated ginger extracts were shown to effectively
prolong clotting times through different pathways (Abd
El-Hameid et al. 2020; Wang et al. 2020). In the current
study, the non-polar fraction of Vietnamese black ginger was
able to inhibit blood coagulation only through the intrinsic
pathway by extending the APTT parameter. The possible
interaction between components in DC-R and receptors of
the intrinsic coagulation pathway was predicted in further
studies.
Volatile components analysis
A total of 41 compounds accounted for 98.95% of the
extract were annotated by matching their retention times
and the similarity in the mass spectra with those that were
dedicated in the libraries (Table 4). In detail, the oxygen-
ated hydrocarbons represented the highest abundance with
Chemical Papers (2024) 78:5191–5207 5197

Table 4  Volatile components in DC-R

ID KRI RI Chemical name Formula %

1 1027 1032 Eucalyptol C10H18O 6.67

2 1162 1176 Rosefuran epoxide C10H14O2 0.83
3 1167 1177 Terpinen-4-ol C10H18O 1.62
4 1169 1184 E-Isocitral C10H16O 0.76
5 1181 1189 α-Terpineol C10H18O 2.88
6 1234 1240 Neral C10H16O 14.69
7 1269 1270 Geranial C10H16O 17.24
8 1298 1300 Geranyl formate C11H18O2 0.25
9 1388 1382 Geranyl acetate C12H20O2 12.18
10 1397 1391 β-Elemene C15H24 1.19
11 1404 1404 Vanillin C8H8O3 0.52
12 1410 1391 Sesquithujene < 7-epi- > C15H24 0.42
13 1421 1415 cis-α-Bergamotene C15H24 0.35
14 1452 1447 cis-Eudesma-6,11-diene C15H24 0.49
15 1463 1454 Humulene C15H24 0.48
16 1463 1464 Sesquisabinene C15H24 0.47
17 1485 1492 4a,8-Dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene C15H24 0.44
18 1498 1486 β-Selinene C15H24 14.04
19 1506 1494 α-Selinene C15H24 1.55
20 1517 1509 β-Bisabolene C15H24 1.53
21 1521 1514 β-Curcumene C15H24 0.4
22 1533 1524 β-Sesquiphellandrene C15H24 1.58
23 1567 1581 trans-Sesquisabinene hydrate C15H26O 0.61
24 1595 1581 Caryophyllene oxide C15H24O 0.93
25 1610 1596 Guaiol C15H26O 0.26
26 1621 1606 Humulene epoxide II C15H24O 0.51
27 1670 1660 Intermedeol C15H26O 0.87
28 1677 1678 Aromadendrene oxide-(2) C15H24O 1.66
29 1695 1698 4-(1,5-Dimethylhex-4-enyl)cyclohex-2-enone C14H22O 1.52
30 1803 1809 Ambrial C16H26O 0.65
31 1904 1909 3a,9-Dimethyldodecahydrocyclohepta[d]inden-3-one C16H26O 1.2
32 1942 1960 m-Camphorene C20H32 0.5
33 1966 1962 geranyl-α-terpinene C20H32 0.48
34 1981 1968 n-Hexadecanoic acid C16H32O2 2.5
35 2063 2150 Pallensin C15H20O4 0.32
36 2133 2113 6-Methyl-4,6-bis(4-methylpent-3-en-1-yl)cyclohexa-1,3-dienecarbaldehyde C20H30O 2.2
37 2161 2141 Oleic acid C18H34O2 1.13
38 2184 2172 Octadecanoic acid C18H36O2 0.42
39 2248 2251 Androstan-17-one, 3-ethyl-3-hydroxy-, (5α)- C21H34O2 0.4
40 2366 2383 (E)-Labda-8(17),12-diene-15,16-dial C20H30O2 0.82
41 2682 2777 Geranyl palmitate C26H48O2 1.41
Diterpenes (2 compounds: 32, 33) 0.98
Sesquiterpenes (12 compounds: 10, 12, 13, 14–22) 22.94
Oxygenated hydrocarbons (27 compounds: 1–9, 11, 23–41) 75.03
Total 98.95

RT Retention time, KRI Calculated Retention Index, RI Theory Retention Index reported by library
5198 Chemical Papers (2024) 78:5191–5207

Table 5  BE of top compounds with platelet receptors and coagulation factors
For platelet receptors
No Chemical name % in DC-R COX-1 (kcal/mol) P2Y12
(kcal/
mol)

1 Sesquithujene < 7-epi- > 0.42 − 7.7 − 7.3

2 Eudesma-6,11-diene < cis- > 0.49 − 7.8 − 6.7
3 Curcumene < beta- > 0.40 − 8.3 − 7.6
4 Sesquiphellandrene < beta- > 1.58 − 8.0 − 7.7
5 Guaiol 0.26 − 6.9 − 8.7
6 4-(1,5-Dimethylhex-4-enyl) cyclohex-2-enone 1.52 − 7.7 − 7.2
7 m-Camphorene 0.50 − 7.6 − 8.5
8 Geranyl-α-terpinene 0.48 − 7.2 − 8.8
9 Androstan-17-one, 3-ethyl-3-hydroxy-, (5α)- 0.40 − 4.3 − 7.9
10 Aspirin − 6.9
11 Ticagrelor − 8.9
For coagulation factors
No Chemical name % in DC-R Factor X (kcal/mol) Factor
II (kcal/
mol)

1 Geranyl-α-terpinene 0.48 − 7.7 − 7.2

2 m-Camphorene 0.50 − 7.5 − 8.0
3 Pallensin 0.32 − 7.5 − 7.8
4 Selinene < beta- > 14.04 − 7.2 − 6.8
5 6-Methyl-4,6-bis(4-methylpent-3-en-1-yl) cyclohexa-1,3-di- 2.2 − 7.2 − 7.2
enecarbaldehyde
6 α-Selinene 1.55 − 7.0 − 6.8
7 Heparin − 4.9 − 2.6
8 Apixaban − 9.2
9 Pegnivacogin − 6.0

75.03%, followed by sesquiterpenes (22.94%) and diterpenes
(0.98%). The main volatile constituents in DC-R included
geranial (17.24%), neral (14.69%), β-selinene (14.04%),
geranyl acetate (12.18%), and eucalyptol (6.67%).
In the literature, 1,8-cineole (eucalyptol, 23.0%), (E)-cit-
ral (geranial, 18.9%), (Z)-citral (neral, 15.0%), and geraniol
(9.3%) were reported by earlier workers as the key compo-
nents of D. citrea rhizome oils (Le et al. 2017). The variation
of chemical composition and each component’s yield of the
same species might be due to the location of the sample,
cultivars, the extraction method, or other environmental fac-
tors. Among volatile components elucidated from DC-R,
several compounds were demonstrated to have an inhibition
action on platelet aggregation, such as citral (Huang et al.
2017), eucalyptol (Alatawi et al. 2021), oleic acid (Nunez
et al. 1990) or caryophyllene oxide (Lin et al. 2003). These
compounds’ presence might contribute to the anti-aggre-
gatory effect of DC-R. However, the main compounds in
DC-R, such as geranial, neral, β-selinene, or eucalyptol were
not previously documented to prolong coagulation time.
Therefore, the anticoagulant action of DC-R might come
from minor constituents or the synergic effect of compounds
in this extract.
Besides the antiplatelet effect, geranial and eucalyptol in
DC-R were shown to display several pharmacological effects
such as anti-inflammatory activity (Juergens et al. 2003;
Liao et al. 2015), immunomodulatory capacity (Farhath
et al. 2013), and the inhibition of human 5-HT3 receptors
(Jarvis et al. 2016) or the treatment of chronic diseases (Seol
and Kim 2016). Moreover, other key components in DC-R,
such as neral and geranyl acetate also showed potential in
treating different types of cancer (Qi et al. 2018; Quintans-
Júnior et al. 2013; Silva et al. 2022), making them com-
pounds of great therapeutic value.
Molecular docking
According to the above result, DC-R exhibited a good
inhibitory effect on platelet aggregation and an inhibition
Chemical Papers (2024) 78:5191–5207
Fig. 3  Interactions of top ligands to COX-1
action on blood coagulation through the intrinsic pathway by
prolonging the APTT parameter. Therefore, 41 compounds
elucidated by GC–MS from this extract were docked against
two typical receptors in the aggregation process, including
COX-1 and P2Y12, and two factors involved in the intrinsic
pathway, including FX and FII.
Antiplatelet targets
Docked compounds exhibited a wide range of binding affin-
ity, and the binding energy (BE) of top molecules and the
positive controls were shown in Table 5.
For COX-1, several constituents of DC-R had better
BE than aspirin used as the positive control. Compounds
with the strongest binding affinity to the receptor, such as
β-curcumene (BE = − 8.3 kcal/mol), β-sesquiphellandrene
(BE = − 8.0 kcal/mol), or cis-eudesma-6,11-diene
(BE = − 7.8 kcal/mol) were found as minor compounds
in DC-R. The interaction of aspirin with the protein was
stabilized by hydrogen bonding between the oxygen of the
5199
carboxylic group and hydrogen of a residue in the protein
(SerA:530), as well as the alkyl—alkyl or alkyl—π inter-
action. Meanwhile, compounds in DC-R were bound to
the receptor mainly by the later interactions and, in some
cases, Van der Waals force (Fig. 3). This might explain the
weaker inhibitory effect of DC-R than the positive controls
on platelet aggregation.
For P2Y12, geranyl-α-terpinene (BE = − 8.8 kcal/
mol), guaiol (BE = − 8.7 kcal/mol), and m-camphorene
(BE = − 8.5 kcal/mol) from DC-R showed the highest
affinity to the receptor, which is close to the affinity of
ticagrelor. Apart from hydrogen bonds, π—π stacking,
especially the stacking between TyrA:105 and an aromatic
ring of ligand, plays a vital role in strengthening the bind-
ing of ligands and P2Y12 (Fig. 4).
Anticoagulant targets
Many compounds of DC-R had a stronger affinity with FX
and FII than heparin (− 4.9 and − 2.6 kcal/mol for factors
5200
Fig. 4  Interactions of top ligands to P2Y12
X and II) but were weaker than other standard compounds
(Table 5).
For factor X, apixaban has a high affinity to the recep-
tor with BE of − 9.2 kcal/mol. This fact can be explained
by the contribution of many π—π stacking interac-
tions between aromatic amino acid residues (TrpA:215,
TyrA:99, PheA:174) and phenyl group as well as amide—π
stacking of CysA:191 and SerA:214 with six-member aro-
matic ring (Fig. 5). Heparin had low BE of − 4.9 kcal/mol
although this ligand can form multiple hydrogen bonds
with the receptor. Geranyl-α-terpinene displayed the best
BE (− 7.7 kcal/mol), but the binding affinity with factor X
remained weaker than apixaban. The weaker interactions
of tested compounds to the receptor may be due to the
absence of stacking interactions.
Similarly, the ligand with π—π and amide—π stack-
ing interactions, such as m-camphorene, show good BE
(− 8.0 kcal/mol) to factor II (Fig. 6). Heparin has a high
polarity, so it mainly forms hydrogen bonds with the
receptor and the interaction with factor II is even weaker
than that with factor X. Like Heparin, pegnivacogin did
not create stacking interaction; hence, its binding energy
is also low (− 6 kcal/mol).
Chemical Papers (2024) 78:5191–5207
Optimization of DC‑R extraction
Single factor experiments
The impact of each independent variable on the DC-R’s
extraction yield was shown in Fig. 7. The result given in
Fig. 7a indicated that the extraction efficiency was improved
with the increase in the ultrasound power from 30 to 80%.
This was in agreement with the basis of mass transfer since
the higher ultrasound power caused extensive cavitation,
quickly disrupting the plant tissue, and thereby enhancing
the extraction of intracellular compounds into the solvent
(Samaram et al. 2015). However, the yield sharply dropped
when the ultrasound power exceeded 80%. This could be due
to the loss or degradation of volatile compounds because
of the thermal reaction at a high ultrasound power value
(Mohammadpour et al. 2019). Therefore, 30–80% range
power was chosen for the subsequent experiments.
Figure 7b showed that the liquid/ solid ratio increas-
ing from 5:1 to 15:1 mL/g resulted in significant growth
in extraction yield, reaching a peak at a ratio of 15:1 mL/g.
Because of the possibility that the high rate of solvent-to-
sample increased the solvent’s diffusivity into the cell, lead-
ing to a higher extraction efficiency. However, the extraction
Chemical Papers (2024) 78:5191–5207
Fig. 5  Interactions of top ligands to factor X
yield significantly decreased as the liquid/ solid ratio rose.
The presence of excess solvent had a detrimental effect on
the intense cavitation due to the viscosity of the medium.
In the wave function’s rarefaction zone, negative pressure
was necessary for cavitation to emerge to resist the forces
of nature that act cohesively (Xu et al. 2014). Hence, the
higher viscosity of the solvent, the higher the cohesive forces
would be, which might result in lowering cavitation phenom-
ena (Gogate and Pandit 2004). Consequently, the suitable
range of liquid/ solid ratio was determined to be from 5:1
to 15:1 mL/g.
The extraction process by UAE method contains two
steps: washing process and slow diffusion. Washing is the
first stage to extract bioactive compounds on the sample’s
surface. Then, the compounds inside the cell plant are
transferred into solvent through slow diffusion. Therefore,
increasing the extraction time resulted in increasing the
5201
extraction yield. From Fig. 7c, increasing extraction time
resulted in a rise in the extraction yield of DC-R until get-
ting a maximum value of 0.872% at 30 min. However, pro-
longed extraction time over 30 min led to a slight decline
in extraction efficiency, possibly because some components
were degraded or oxidized by prolonged exposure to ultra-
sonic settings (Tiwari et al. 2009). As a result, the extraction
time ranging from 5 to 30 min was selected for the future
experimental design.
As illustrated in Fig. 7d, the extraction yield of DC-R
increased by about 52.6% when the temperature was raised
from 30 °C to 60 °C. An increase in extraction tempera-
ture referred to a rise in cavitation bubbles, enhancing the
extraction efficiency (Febriana et al. 2016). Nevertheless,
it is witnessed a detrimental effect on extraction efficiency
at a temperature higher than 60 °C. The breakdown of
numerous chemicals may have produced this observation
5202
Fig. 6  Interactions of top ligands to factor II
during extraction at an unusually high temperature or by
the vaporization of solvent, which caused the solvent loss
(Khemakhem et al. 2017). Again and colleagues (Again
et al. 2022) also obtained the maximum extraction yield
of antioxidative compounds from Bentong ginger at 60 °C.
Hence, the chosen temperature applied for extraction
ranged between 30 °C and 60 °C.
Determinant factors affecting the extraction yield
Table 6 displays the results of the analysis of variance
(ANOVA), which included the regression coefficients for
the intercept, linear, and terms that interacted with one
another in the model. The model was statistically signifi-
cant (p value = 0.001, F = 55.30).
The regression model's coefficient (R2) is 0.9765, indi-
cating that it accurately fits the data and may be used to
forecast future results. Table 6 showed that the liquid/
solid ratio, extraction temperature, and their interaction
Chemical Papers (2024) 78:5191–5207
significantly impacted the response (p < 0.05). On the con-
trary, other factors’ effects and interactions were insignifi-
cant (p > 0.05), not presented in Table 6.
Response surface optimization
The Model F-value of 179.08 with p < 0.05 indicated the
model’s validity (Table 7). As observed, A, B, AB, and A2
were significant model terms. It is seen that the liquid/ solid
ratio and extraction temperature were the main determi-
nant parameters of the response. The model’s coefficient of
determination (R2) was 0.9922, and the predicted (R2) was
0.9200, which means the regression model could be consid-
ered to navigate future outcomes. The response of the model
was expected using the following equation:
Y = −0.9535 + 0.2081 × A + 0.0205 × B − 0.0018
(1)
× AB − 0.0037 × A2 + 0.00001 × B2
Chemical Papers (2024) 78:5191–5207 5203

Fig. 7  Effect of ultrasonic power level (a), liquid/solid ratio (b), extraction time (c), and extraction temperature (d) on the DC-R’s extraction
yield (%)

variables but also to attain the best value of each factor for
Table 6  ANOVA for screening factor experiments
the outcome of the experiments (Dang et al. 2017).
Source DF Adj SS Adj MS F-Value p value The obtained result identified variable levels that favor
the desired extraction efficiency. Figure 8 illustrates the
Model 3 0.59331 0.197769 55.30 0.001
impact of two significant factors on the DC-R’s extraction
Linear 2 0.56518 0.282591 79.02 0.001
yield at a fixed extraction time (30 min), and ultrasonic
A-Ratio 1 0.51832 0.218320 144.94 0.000
power (80%). Increasing the extraction temperature to a
B-Temperature 1 0.04686 0.046862 13.10 0.022
specific value (optimum point) with increasing the liq-
2-Way Interactions 1 0.02812 0.028124 7.86 0.049
uid/ solid ratio enhanced extraction efficiency. Based on
Ratio*Temperature 1 0.02812 0.028124 7.86 0.049
Eq. (1), the best parameters for the highest extraction yield
Error 4 0.01430 0.003576
of DC-R were the liquid/ solid ratio of 13.5 mL/g, and
Total 7 0.60761
R2 0.9765
extraction temperature of 34.4°C. The predicted outcome
Adj.R2 0.9588
with these conditions was 1.017%.
Pred.R2 0.9058
For operational convenience, the optimal conditions
were slightly modified: liquid/ solid ratio of 13.5 mL/g,
DF Degrees of freedom, Adj SS Adjusted sum of squares, Adj MS extraction temperature of 34 °C, ultrasonic power of 80%
Adjusted mean square, Adj R2 Adjusted R2, Pred.R2 Predicted R2 and extraction time of 30 min. Three recovery experi-
ments were conducted under the adjusted extraction con-
ditions to validate the predicted model. A mean extrac-
where A is the liquid/ solid ratio, and B is the extraction tion yield obtained from verification experiments was
temperature. 1.011 ± 0.008% compared to 1.017% predicted by the
3D surface plots and 2D contour plots were constructed model (coefficient of variation % = 0.42%), demonstrat-
not only to visualize the relationship between two different ing that the anticipated model can precisely forecast the
optimum parameters of DC-R extraction.
5204 Chemical Papers (2024) 78:5191–5207

Table 7  ANOVA for the Source Sum of squares df Mean square F-value p-value Note
experimental results in the
optimization process Block 0.0005 1 0.0005
Model 0.2723 5 0.0545 179.08 < 0.0001 Significant
A-Ratio 0.2281 1 0.2281 750.15 < 0.0001 Significant
B-Temperature 0.0075 1 0.0075 24.80 0.0016 Significant
AB 0.0199 1 0.0199 65.49 < 0.0001 Significant
A2 0.0165 1 0.0165 54.33 0.0002 Significant
B2 0.0000 1 0.0000 0.0488 0.8315 Not significant
Residual 0.0021 7 0.0003
Lack of fit 0.0020 3 0.0007 28.50 0.0037 Significant
Pure error 0.0001 4 0.0000
Cor total 0.2749 13
R2 0.9922
Adjusted R2 0.9867
Predicted R2 0.9200

Fig. 8  Effect of liquid/ solid ratio and extraction temperature on DC-R extraction’s yield demonstrated by 2D-contour plot (a) and 3D-surface
plot (b)

Conclusions DC-R had a strong binding affinity to COX1 and P2Y12

The current work is the first study on DC-R’s antioxi-
dant components and antithrombotic action. This extract
showed moderate antioxidative activity but promising anti-
aggregatory and anticoagulant effects. GC–MS revealed
that the oxygenated hydrocarbons (75.03%) dominated the
composition profile of DC-R, followed by sesquiterpenes
(22.94%) and diterpenes (0.98%). Many constituents of
receptors of the platelet aggregation and FII and FX fac-
tors of the intrinsic coagulation pathway. The optimal
parameters for DC-R’s extraction were determined using
response surface methodology to reach the highest extrac-
tion yield of 1.011 ± 0.008%. In conclusion, DC-R might
be a potential source of active phytoconstituents for treat-
ing cardiovascular-associated diseases.
Chemical Papers (2024) 78:5191–5207
Acknowledgments The authors thank the Vietnam Academy of Sci-
ence and Technology (VAST) for financial support with the grant num-
ber: THTETN.09/21-23.
Declarations
Conflict of interest The authors declare no conflict of interest in writ-
ing upon manuscript submission.
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