Chemical compositions and biological activities of Serevenia buxifolia essential oil leaves cultivated in Vietnam (Thua Thien Hue)

Abstract Serevenia buxifolia is an evergreen citrus plant and has attracted considerable attention due to its bioactive components and biological activities. In the present study, the essential oil (EO) from S. buxifolia cultivated in Vietnam was demonstrated to exhibit the in vitro antioxidant, thrombolytic, anti‐hemolysis, anti‐inflammatory, and antidiabetic activities. Briefly, the gas chromatography coupled to mass spectrometry analysis revealed that the leaf EO of S. buxifolia was composed of 33 components, with the main constituents being β‐carypphyllene (32.5%), and elixene (9.8%). The extracted oil possessed a fairly high free radical scavenging activity against 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH), with an IC50 value of 190.7 μg/mL compared with positive control, α‐tocopherol, IC50 value of 42.6 μg/mL. The EO also exhibited thrombolytic activity: the percentage of inhibition was found to be 70.75% at 100 μL, in comparison with 87.2% for the positive control, streptokinase. For hemolytic activity, the percentage of inhibition of the EO was from 27.4% to 59.6% at concentrations from 10 to 100 μg/mL, respectively. The results of in vitro anti‐inflammatory activity indicated that the EO of S. buxifolia leaves effectively protects the heat‐induced denaturation, with an IC50 value of 40.25 μg/mL. The EO also exhibited antidiabetic potential, with IC50 values of 87.8 and 134.9 μg/mL against α‐amylase and α‐glucosidase, respectively. It is noteworthy that the potent biological activities of the obtained S. buxifolia oil increased in a dose‐dependent manner. The results achieved show that the EO of S. buxifolia leaves can be a potential source for oxidative stress, inflammatory, and diabetic management.

Among the 20 species of genus Serevenia (Rutaceae family),
In Vietnam, although S. buxifolia is widely used as a traditional medicine for cough, snakebites, malaria, chronic rheumatism, influenza, sore throat, and pain (Satyal et al., 2019;Truong et al., 2019Truong et al., , 2021, studies on the chemical compositions and pharmaceutical effects of this plant are still limited. In previous studies, we have detected the presence of phenolics, flavonoids, alkaloids, and terpenoids in the crude and fraction extracts of S. buxifolia branches and stems. The potent in vitro antioxidant and anti-inflammatory action of such extracts have also been investigated. Considering the EO of S. buxifolia, Satyal et al. (2019) have recently identified the oil compositions of S. buxifolia leaves cultivated in Da Nang city (in the middle of Vietnam), with sabinene, β-caryophyllene, bicyclogermacrene, germacrene D, (E)-nerolidol, globulol, and linalool being the main components. The EO also showed larvicidal activity and repellent activity against Triatoma rubrofasciata, Aedes aegypti, and A. albopictus.
In order to exploit local species and to continue the previously successful studies on S. buxifolia extracts, we aimed in this work on the investigation of biological activities of EO from S. buxifolia leaves.
No scientific studies have been conducted regarding the in vitro antioxidant, thrombolytic, anti-hemolytic, anti-inflammatory, and antidiabetic activities of S. buxifolia leaf oil. Therefore, the present study was undertaken to evaluate the in vitro antioxidant, thrombolytic, anti-hemolytic, anti-inflammatory, and antidiabetic activities of the essential oil of S. buxifolia leaves collected from Thua Thien Hue, Vietnam. Besides, the phytochemical composition of the leaf oil was also identified by using gas chromatography coupled to mass spectrometry (GC/MS).

| Plant materials and extraction procedure
The fresh leaves were collected from S. buxifolia in the Phu Loc district, Thua Thien Hue province, Vietnam in the summer season (from August to September, 2020). The plant was taxonomically identified by identified by Dr. Son V. Dang, Curator of the VNM Herbarium, Institute of Tropical Biology, Vietnam. The leaves were lightly washed with tap water to remove debris and then ground into a powder using a mill (Jehmlich) and kept at 4-6°C until further use.
The leaf EO of S. buxifolia was extracted by steam distillation for 5 h, as described by Božović et al. (2017), with some minor modifications. The mixture of EO and water was then dried over anhydrous sodium sulfate and stored at 4°C until required in the bioassay. The extract yields (EY) were calculated as the ratio of the mass of EO to the mass of the starting plant materials and were expressed as a percentage (w/w).

| Chemical analysis of essential oil
The EO of Serevenia buxifolia leaves was analyzed by GC/MS, as described by Liu et al. (2014).
The components of the EO were separated by GC and identified by MS using an Agilent GC 6890 equipped with an HP-5MS (5%-Phenylmethylpolysiloxane) fused silica column (30 m × 0.25 mm × 0.25 μm) interfaced with a quadrupole detector (Model 5973). The GC settings were as follows: the initial oven temperature was held at 50°C for 2 min and ramped at 2°C/min to 80°C, then ramped at 5°C/min to 150°C, where it was held for 5 min, before being ramped at 10°C/min to 200°C and then at 20°C/min to 300°C, where it was held for 10 min. The sample (1 μL diluted 1:100 in hexane) was injected, with a split ratio of 1:50. The carrier gas was helium, at a flow rate of 1 mL/min. Spectra were obtained over the scan range from 20 to 500 m/z at 2 scans/s.

| Qualitative analysis
Most components were identified based on the retention indices (RI) determined with reference to a homologous series of n-alkanes, under identical experimental conditions, co-injection with authentic samples or known EO compositions, MA library search (NIST 08 and Wiley 9th Version), and by comparing with MS literature data of Adams (2007).

| Quantitative analysis
The percentage compositions of the oil depending on whether there was computed by the normalization method from the GC peak areas, assuming an identical mass response for all components. In addition, the content of the main oil component was also calculated according to the standard curve of the commercial compound. where A blank is the absorbance of the control (containing all reagents except the test sample) and A sample is the absorbance of the tested EO solution.

| Assessment of biological activities
The antioxidant potential of the EO or standard was expressed as an IC 50 value (μg/mL), defined as the concentration of the tested sample required to cause a 50% decrease in initial DPPH concentration.

| Thrombolytic activity
The in vitro thrombolytic activity of the EO was established according to the method of Prasad et al. (2006), with some minor modifications. A 5 mL sample of venous blood was drawn from healthy human volunteers (n = 5), without recent history of anticoagulant and contraceptive therapy (at least 7-10 days duration).
Then, the blood samples were immediately transferred in different pre-weighed sterile Eppendorf tubes (500 μL/tube; m 0 ) and incubated for 45 min at 37°C. Next, the serum was completely removed after clot formation (working carefully to avoid disturbing the formed clot). Later, each tube including clot was weighed again (m 1 ) to determine the clot weight according to the following equation: Each Eppendorf tube with clot was properly labeled, and 100 μL of different dilutions of EO in dimethyl sulfoxide (DMSO; 10, 20, 50, and 100 μg/mL) was added to the tubes. DMSO was considered as a negative thrombolytic control, while the thrombolytic drug streptokinase (30,000 IU) was used as a positive standard stock (10, 20, 50, and 100 μg/mL). After incubation for 90 min at 37°C, the fluids obtained in all the tubes were removed, and tubes were again weighed to observe the variation in weight after clot disruption. The difference obtained from the weight taken before and after clot lysis was expressed as a percentage of clot lysis:

| Anti-hemolytic activity against H 2 O 2− induced hemolysis
The anti-hemolytic potential of S. buxifolia leaf oil was measured according to the method of Vinodhini and Kalaiselvi (2021), with slight modifications. First of all, 5 mL samples of venous blood from healthy human volunteers were collected in EDTA tubes (10%) and centrifuged at 1008 g (EPA 200) for 10 min. After centrifugation, the plasma was discarded from the tubes, and the settled red blood cells (RBCs) were washed three times with an equal volume of normal saline (0.9% NaCl). The RBCs were then diluted with PBS (pH 7.4) to give a 4% (v/v) suspension, and 0.4 mL of EO diluted in PBS to different concentrations (10, 20, 50, and 100 μg/ mL) was then added to 2.0 mL of the RBC suspension, and the volume was made up to 5.0 mL with saline. This mixture was later incubated for 5 min at room temperature before adding 0.5 mL of weight of clot = weight of clot filled tube − weight of empty tube % of clot lysis = (Weight of the clot before lysis − Weight of the clot after lysis) ∕ Weight of the clot before lysis x 100 H 2 O 2 (PBS) solution in buffered saline to induce oxidative degradation of the membrane lipids. All tubes were incubated for 1 h at room temperature, and the reaction mixture was then centrifuged at 252 g (Hematokrit 210) for 10 min. Finally, the hemolysis inhibitory ability was measured spectrophotometrically at 540 nm. The relative hemolysis of the EO was assessed compared to the hemolysis in the H 2 O 2 -treated sample (negative control), which was set as 100%. PBS was used as a positive control. The percent inhibition of hemolysis was calculated as follows: The concentrations of extracts causing 50% inhibition of enzyme activity (IC 50 ) were determined graphically. All products were evaluated with the comparison of their IC 50 values estimated from the dose-response curves.

| In vitro anti-inflammatory activity
The in vitro anti-inflammatory ability of EO from S. buxifolia leaves was determined by the inhibition of albumin denaturation, as described by (Vinodhini & Kalaiselvi, 2021), with a few modifications.
The reaction mixture consisted of 1 mL of EO at different concentrations (10, 20, 50, and 100 μg/mL diluted in DMSO) and 1 mL of 1% aqueous solution of BSA. The pH of the mixture was adjusted to 6.3 using 1 N HCl. Next, the samples were incubated at 37°C for 20 min before being heated to 57°C for 20 min. The sample was cooled and the turbidity was measured spectrophotometrically at 660 nm. The inhibitory percentage of protein denaturation was calculated as follows:

| Determination of in vitro antidiabetic activity
The potential antidiabetic activity of the EO of S. buxifolia leaves was evaluated based on the percentage of α-amylase and α-glucosidase inhibition.

| α-Amylase inhibitory method
The α-amylase inhibition assay of the EO of S. buxifolia leaves was adapted from Jaradat et al. (2020), with slight modifications. A working solution (1 mg/mL) of EO was prepared by dissolving 25 mg in a small amount of DMSO; later, a buffer solution was added up to 25 mL. This solution was then diluted with DMSO to obtain various dilutions (50, 100, 150, and 200 μg/mL). The α-amylase enzyme working solution (2 U/mL) was produced by dissolving 12.5 mg of αamylase enzyme powder in a small amount of DMSO, and the buffer solution was made up to 100 mL. Corn starch solution was obtained by dissolving 1 g of starch in 100 mL of distilled water. The reaction mixture containing 200 μL of each EO stock solution and 200 μL of α-amylase stock solution was incubated at 30°C for 10 min. Next, 200 μL of corn starch solution was added, before continuing the incubation at 30°C for 3 min. The enzyme reaction was terminated by adding 200 μL of DNS and heating at 85-90°C for 10 min. The solution was then cooled and 5 mL of distilled water was added. Acarbose was used as the reference standard (positive control), while DMSO (200 μL) was used as the blank solution. The α-amylase inhibitory potential was measured spectrophotometrically at 540 nm and estimated using the following formula: where %I is the percentage of α-amylase inhibition Ali et al. (2006). The results were expressed in terms of IC 50 values, indicating the concentration of EO required to cause 50% enzyme inhibition.

| α-Glucosidase inhibitory method
The α-glucosidase assay was performed according to Siahbalaei et al. (2020), with some modifications. The reaction mixture containing 300 μL of each concentration of EO (50, 100, 150, and 200 μg/ mL diluted in DMSO from stock solution (1 mg/mL)) and 200 μL of α-glucosidase (0.3 U/mL) was incubated at 37°C for 15 min in the dark. Next, 100 μL of pNPG (10 mM) was added before incubating at 37°C for 30 min. Later, 300 μL of Na 2 CO 3 (100 mM) was added, and the absorbance was recorded at 405 nm. Acarbose was used as the reference standard, while DMSO was used as the blank control.
The percentage of α-glucosidase inhibitory potential of the EO was calculated in the same manner as in the α-amylase assay. Acarbose, a prescribed drug for α-glucosidase inhibition, was also used as a control. The blank solution was prepared by replacing the EO with 200 μL of DMSO.

| Statistical analysis
All experiments were performed in triplicates, and values were represented as the mean ± standard deviation (SD). Analysis of variance (ANOVA; Minitab 16 software) and Tukey's multiple comparison test were used to compare the variation between assays; p values <.05 were considered significant.

| Essential oil analysis
Essential oil obtained by steam distillation of the S. buxifolia leaves was extracted with a yield of 0.31 ± 0.01 (w/w). The density of the concentrated oil was 0.87 g/mL. The isolated EO was a yellowish clear The results of GC-MS analysis showed that the EO of S. buxifolia leaves exhibited 33 identified components by comparison of their retention times and the mass spectra of each GC composition with those of the standards, accounting for 97.5% of the total oil (Table 1 & Figure 1). The main components of the EO were β-caryophyllene (32.5%), elixene (9.8%), germacrene D (6.9%), β-farnesene (6.6%), b Retention indices according to literature (Adams, 2007).
TA B L E 1 Chemical constituents of essential oil derived from Serevenia buxifolia leaves cultivated in Thua Thien Hue, Vietnam.

| Thrombolytic activity
Thrombolytic agents, like tissue plasminogen activator (t-PA), urokinase (UK), and streptokinase (SK) drugs, are used to dissolve blood clots that can obstruct the flow of blood through the circulatory system (Azad et al., 2015;Furie & Furie, 2008). SK can unite and activate the inactive precursor of the enzyme plasmin (plasminogen). It is noted that plasmin is an important proteolytic enzyme that leads to the degradation of fibrin clots (Vinodhini & Kalaiselvi, 2021).
However, evidence has indicated that these drugs could cause some side effects. For example, treatment with SK is restricted because of immunogenicity (Jennings, 1996). Thus, the development of recombinant variants of these drugs is necessary (Azad et al., 2015;Farnsworth, 1993;Marder, 1993). Evidence has indicated that herbal plants have an antithrombotic effect preventing thus coronary heart disease and stroke (Azad et al., 2015;Prasad et al., 2007;Ramjan et al., 2014). In the present study, the in vitro thrombolytic potential of the EO of S. buxifolia leaves was investigated (Figure 2

| Anti-hemolytic activity
Increased hemolysis occurs when RBCs are exposed to some toxic factors like hydrogen peroxide (Arfan et al., 2016). Therefore, we assessed whether S. buxifolia leaf oil could prevent oxidative damage to the erythrocyte membrane. The results showed that the EO exhibited potent anti-hemolytic action in a concentrationdependent way ( TA B L E 2 DPPH radical scavenging activity of essential oil of Serevenia buxifolia leaves and α-tocopherol.
F I G U R E 2 Thrombolytic activity of essential oils from Serevenia buxifolia leaves.
mL. Lysis of erythrocytes was shown to increase with an increase in concentration of EO, that is, %I of hemolytic activity from 27.4% to 59.6% at concentrations from 10 to 100 μg/mL (Table 3)

| In vitro anti-inflammatory activity: inhibition of albumin denaturation
It is known that proteins, namely albumin and trypsin, can activate and release inflammatory mediators from humans and may be involved in inflammation and immunity (Foudah et al., 2021;Ullah et al., 2014). In the present experiment, anti-denaturation was established using the BSA method to estimate the in vitro anti-inflammatory property of the EO of S. buxifolia leaves. The results are summarized in Table 4. The reference drug, aspirin, also exhibited a concentration-dependent inhibition of albumin denaturation over the same concentration range. Indeed, the effect of aspirin against BSA heat denaturation was found to be more than 1.1-fold higher than that of S.buxifolia EO. The percentage of protected BSA increased with increasing dose of EO and showed a significant difference (p < .001). In particular, we showed a dose-dependent inhibition of protein (albumin) by S. buxifolia leaf oil in the range from 10 to 100 μg/mL. The effect of the reference drug, aspirin, against heat denaturation of the BSA was found to be lower than that of the S. buxifolia oil at the same concentration range. These observations were confirmed through IC 50 values, with 60.2 and 40.3 μg/mL for aspirin and EO, respectively (Table 4) Brito et al., 2019;Dahham et al., 2015;Francomano et al., 2019). This could be explained by the fact that the inflammatory action of S. buxifolia oil may be due to its main component, β-caryophyllene, besides the other compounds.

| In vitro antidiabetic activity
Diabetes is a well-known serious metabolic disorder which occurs because of the deficiency of insulin secretion or the resistance against insulin. Up to now, significant developments, such as diet therapy, insulin therapy, and pharmacotherapy, are used in the treatment of diabetic patients (Kooti et al., 2016;Mrabti et al., 2018).
However, evidence indicates that these treatments still have some side effects, drug resistance (reduction of efficiency), or even toxicity (Dey et al., 2002;Kooti et al., 2015;Mrabti et al., 2018). It is worth noting that, in traditional medicines, a medical plant can be used to treat diabetes without side effects (Kooti et al., 2016).
Diabetic patients always need adequate food management to prevent hyperglycemia. It has been documented that inhibition of αamylase and α-glucosidase can lead to slow and prolonged release of glucose into the circulation, thereby retarding glucose release and absorption after food consumption, reducing thus postprandial hyperglycemia (Kumar et al., 2011). Thus, these enzymes play an important role in the management of hyperglycemia-linked diabetic disease (Aazza et al., 2016;Inthongkaew et al., 2017).
Our previously shown data on S. buxifolia verified its antioxidant and anti-inflammatory potential (Truong et al., 2019(Truong et al., , 2021. In the present study, the EO from S. buxifolia leaves was found to be rich in terpenoids, and the most active compounds, such as limonene, linalool, caryophyllene, elixene, and cadinene, which are responsible for the observed in vitro biological activity (i.e., antioxidant, thrombolytic, anti-hemolytic, anti-inflammation, and antidiabetic) were nominated based on documentation in the literature (Brito et al., 2019;Elgendy & Semeih, 2019;Francomano et al., 2019;Xu et al., 2021).

TA B L E 5
The α-amylase and αglucosidase inhibitory activity by acarbose drug and Serevenia buxifolia leave oil in addition to their IC 50 values (μg/mL).

| CON CLUS ION
In the present study, the chemical composition of the EO extracted from S. buxifolia leaves was determined. Thirty-three components were identified using GC/MS analysis. As a valuable source for traditional medicine, the S. buxifolia EO demonstrated in vitro antioxidant, thrombolytic, anti-hemolysis, anti-inflammatory, and antidiabetic activities. Thus, the EO of this plant could be considered as an alternative food supplement to treat oxidative stress and stress-related diseases.
Nevertheless, this study was limited to an in vitro evaluation of the EO biological activities; therefore, the observed potential and bioavailability aspects should be addressed in animal models in the future. Conceptualization (equal); data curation (equal); investigation (equal); methodology (equal); project administration (equal); resources (equal); software (equal); supervision (equal); validation (equal); writing -original draft (equal); writing -review and editing (equal).

ACK N OWLED G M ENTS
The authors thank Ton Duc Thang University, Ho Chi Minh City, for its support.

CO N FLI C T O F I NTE R E S T S TATE M E NT
We, the authors, declare that we have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available by contacting the corresponding author by email.

E TH I C S S TATEM ENT
Ethical approval is not applicable to this study.