Chemical Constituents, Antibacterial and Coagulation Activity of the Essential Oil from the Stem of Artemisia argyle

Corresponding Author: Hongli Zhou School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin 132022, PR China E-mail: zhouhongli@jlict.edu.cn Abstract: Artemisia argyle H. Lév. (A. argyle) is a plant of historical importance known as the "mother of herbs" in the Middle Ages. As the leaves of A. argyle have been studied more, but the stems has not been reported. This paper explores the chemical constituents, antibacterial and coagulation activities of the Essential Oil from the Stems of A. argyle (EOSAA) for better exploitation and utilization of resources. EOSAA was extracted by hydrodistillation and its chemical constituents were determined by GC-MS. Escherichia coli, Staphylococcus aureus and Bacillus pumilus were used as test microbial strains to evaluate the antimicrobial potential of EOSAA. Three indicators of Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) were used to evaluate the coagulation activity. The extraction rate was 0.20% and 36 compounds were identified, accounting for 93.75% of the total content. Among the identified compounds, 41.72% belong to oxiterpenes, 21.31% belong to sesquiterpenes and terpenoids are extremely abundant. The main constituents were as follows: 3Biphenylmethanol (16.01%), 2,6-Dimethyl-8-(tetrahydropyran-2yloxy)-octa-2,6-dien-1-ol (9.22%), Germacrene D (7.22%), α-Bisabolol (5.85%), L(-)-Borneol (5.75%), Eucalyptol (5.71%) and so on. EOSAA exhibited certain inhibitory effects against Escherichia coli, Staphylococcus aureus and Bacillus pumilus, the minimum inhibitory concentrations were 40, 42 and 48 mg/mL, respectively. Compared with Yunnan Baiyao, APTT, PT and TT time of high concentration (60 mg/mL) EOSAA were shortened by 28.83, 42.86 and 68.99%, respectively. The experimental results show that EOSAA is a natural coagulant with antibacterial effect and has a broad application prospect.


Introduction
Artemisia argyle H. Lév. (A. argyle) is a plant of the genus Artemisia in the compositae family which appears perennial herbs or slightly subshrub forms with strong fragrance. It widely distributes around the world . A. argyi has been received considerable attention due to its abundant bioactive substances. The leaves of A. argyle smoke have obvious antibacterial effect on the affected area, reducing the number of bacterial colonies in the air and completely inhibiting the growth of pyogenic bacteria (Zhang et al., 2014). The leaves of A. argyle are one of the common gynecological drugs, which were recorded as "hemostatic drugs" in medical records of past dynasties (Tan et al., 1992;Zheng et al., 2004), regulating the meridians and protecting the fetus, etc. (Adams et al., 2012). Pharmacological studies show that A. argyle has the effect of anti-fibrinolysis hemostasis by reducing capillary permeability (Yu et al., 2012). A. argyle is a traditional Chinese medicine and the whole grass can be used as medicine (Li et al., 2008).
The leaves of A. argyle contain essential oil, flavonoids, glycosides, terpenoids and other active compounds, among which the essential oil is the most important chemical component. For example, the Essential Oil of A. argyi Leaves (EOLAA) showed antihistaminic effects and antifungal activity (Huang et al., 2012). Moreover, EOLAA had strong antibacterial effects against Staphylococcus aureus, Escherichia coli and Salmonella enteritidis (Smith-Palmer et al., 2001). The chemical constituents of the EOLAA are extensive and thorough, mainly including eucalyptol, thujone, alcanfor and borneol (Xiang et al., 2018;Junjie et al., 2016). According to traditional knowledge and medical records, the roots, stems, leaves, buds and flowers of fresh or dried plants all contain some active ingredients that can cure some diseases (Lae et al., 2019). For A. argyle, in addition to the leaves, the stems are also rich in chemical constituents. In the current study, usually only the leaves are fully utilized, while the stems are discarded as waste, which not only causes environmental pollution but also wastes resources.
At present, there are numerous reports about the leaves of A. argyle, but few on stems. Herewith, the chemical constituents and its antibacterial and coagulation activity of Essential Oil from the Stems of A. argyle (EOSAA) obtained by hydrodistillation were done in this study, which has a broad application prospect as a kind of natural coagulant with antibacterial effect.

Extraction of Essential Oil
Fresh stems of A. argyle (60 g) were placed into a 1000 mL two-necked flask equipped with 600 mL of distilled water and connected with a Clevenger-type device, then heated together for 3 h. The essential oil was collected, dried with anhydrous Na2SO4 and hermetically stored at 0℃ for GC-MS analysis (Sparkman, 2005).

GC-MS Analysis
The chemical composition of EOSAA was analyzed by GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan) and the column was Rxi-5sil columnn (30 m, 0.25 mm, film thickness 0.25 μm). The carrier gas was nitrogen and flow rate was 1 ml/min. The collision energy for Mass Spectrometry (MS) detection was 70 eV and data were recorded within 40-450 amu. The vaporizer temperature and ion-source temperature were respectively adjusted to 280 and 230℃. Each chemical composition was identified by comparing the retention index obtained from a database (NIST05) with the retention indexes calculated on the basis of n-alkanes (C9-C46) (Rehman et al., 2021;Chzhu et al., 2020).
An essential oil sample dissolved in diethyl ether (60 mg/ml) was injected automatically into a vaporizer at 250℃ with a split ratio of 1:30, the conditions of the column temperature were as follows: Starting at 60℃ and maintaining it for 6min; then increasing 60 to 300℃ at 3℃/min; finally keeping 300℃ for 10 min (Padalia et al., 2016).

Antibacterial Activity
Escherichia coli ATCC 33456, Staphylococcus aureus ATCC 49775 and Bacillus pumilus ATCC 700814 were used as test microbial strains to evaluate the antimicrobial potential of EOSAA. Using 50% Dimethyl Sulfoxide (DMSO) as solvent, then the EOSAA solution was successively diluted to 100~10 mg/ml by 2 folds dilution method. Adding 50 microliters solution to 96-well plate and 150 µL microorganism liquid which was prepared by fresh nutrient medium containing 10 7~1 0 8 CFU/ml microbial strains were added into each hole, then it was placed in an incubator at 37℃ for 24 h, after that the absorbance was measured at 600 nm. Chloramphenicol solution (100~1 mg/ml) was used as the positive control and 50% DMSO as the negative control (Zheng et al., 2019;Toledo et al., 2020).

Coagulation Activity
Taking 0.50% Yunnan Baiyao (a well-known hemostatic on the market, positive control) and 0.90% NaCl (blank control) as reference, the coagulation test was carried out using CL-2000BV coagulometer (Jiangsu Xenova medical Technology Co., Ltd, China) to determine the coagulation activity of EOSAA. Three indicators of Activated Partial Thromboplastin Time (APTT), Thrombin Time (TT) and Prothrombin Time (PT) were used to evaluate the coagulation activity. Plasma pretreatment: 0.109 mol/L sodium citrate was accurately mixed with fresh plasma kits (Dade Behring Marburg Gmbh) at a volume ratio of 1:9 and centrifuged at 3000 r/min for 20 min. The supernatant of platelet-poor plasma was collected, sealed with plastic tubes and took the refrigerated preservation. Before the experiment, the plasma preheating was carried out at 37℃. Note that all experiments in this process were carried out at 37℃ and should be completed within 2 h (Dore et al., 2013).
PT Assay 20 µL of five different concentrations of EOSAA (20, 30, 40, 50 and 60 mg/mL) and same plasma were incubated for 30 s, then 200 µL of PT reagent was added to the above mixture (Sun et al., 2018).
TT Assay 20 µL of five different concentrations of EOSAA (20, 30, 40, 50 and 60 mg/mL) were incubated with same plasma for 30 s, then 0.1 mL of preheated TT reagent was added to the above mixture (Wang et al., 2013).
In a word, the flow chart of the experiment methods is shown in Fig. 1.

Antibacterial Activity
The Minimum Inhibitory Concentration (MIC) values of EOSAA against Escherichia coli, Staphylococcus aureus and Bacillus pumilus were shown in Table 2. EOSAA has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus and Bacillus pumilus, which may be related to its a large amount of terpenoids containing oxygen, such as, α-Bisabolol, L(-)-Borneol, Eucalyptol and so on (Popović et al., 2010;de Morais et al., 2016). Borneol is a valuable medicinal ingredient, advanced flavor and chemical used in food and folk medicine in China and India for its anti-inflammatory and neuroprotective properties (Asadollahi et al., 2019). Eucalyptol has antibacterial, anti-inflammatory and antioxidation effects (Jiang et al., 2019). So antibacterial activity of EOSAA may be related to terpenoids, terpenoids have antimicrobial and bactericidal activities, which can destroy the permeability of cell membranes (Yang et al., 2019), lead to the leakage of nucleic acid and other macromolecular substances and interfere with the synthesis and accumulation of cell bacterial proteins. In addition, terpene interactions can induce changes in cellular respiration, leading to the subsequent decoupling of microbial oxidative phosphorylation (Zengin and Baysal, 2014), which has synergistic inhibitory effects on drug-resistant bacteria. (Zacchino et al., 2017).  .37 RI a Retention indices relative to C8-C46 n-alkanes on a HP-5MS column RI b is based and calculated on retention time relative to C9-C46

Coagulation Activity
Coagulation is a series of enzymatic reactions, activated by Pre correlation factors, ultimately produces thrombin and fibrin. APTT is a screening test to test whether the endogenous blood coagulation system is normal. PT is a screening test that reflects whether the exogenous coagulation pathway is normal. TT refers to the time required for blood clotting after thrombin is added to plasma. It is commonly used to test the function of blood coagulation, anticoagulation and fibrinolytic system (Jastrzebski et al., 2014). As shown in Table 3, the coagulation activity was positively correlated with the sample concentration. Moreover, compared with Yunnan Baiyao, APTT, PT and TT time of high concentration EOSAA were shortened by 28.83, 42.86 and 68.99%, respectively. Since EOSAA affects APTT, PT and TT, it is indicated that it affects the coagulation function through the way of endogenous and exogenous coagulation or/and common. This is consistent with the application of A. argyle leaves to the treatment of menorrhagia, leakage, hemostasis and other blood syndromes (Dhanapal et al., 2016). Since the coagulation activity of the EOSAA was reported for the first time and the pharmacological study of the essential oil from A. Argyi was mainly conducted in animal experiments, the coagulation mechanism of the specific components remains to be further studied.

Conclusion
EOSAA was extracted by hydrodistillation and the extraction rate was 0.20%. A total of 36 compounds were identified by GC-MS, accounting for 93.75% of the total coment, which its large amount of constituents is terpenoids such as eucalyptol, borneol, camphor and so on. The EOSAA has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus and Bacillus pumilus. The time of APTT, PT and TT were shortened, indicating that EOSAA had a certain coagulation effect. Therefore, EOSAA is a kind of coagulant with antibacterial effect, which has potential application prospects in cosmetics and medicine. This research provides value for the further development and utilization of the stem of A. argyle.