茶树精油主要成分松油烯-4-醇抑制金黄色葡萄球菌作用机理分析
Analysis of the mechanism of terpinene-4-ol, the main component of tea tree essential oil, in inhibiting Staphylococcus aureus

摘要：分析茶树精油（Tea Tree Oil，TTO）的化学组成及其主要成分松油烯-4-醇（Turpentene-4-ol，T-4-O）的抗抑菌作用机理，通过GC-MS测定了TTO的组成成分，再分别从T-4-O体外抑菌性能，对金黄色葡萄球菌菌株生长、菌体微观形态、细胞膜和细胞壁影响再到对菌株细胞溶出物、蛋白质泄露、细胞周期和基因转录表达的影响分析了T-4-O对金黄色葡萄球菌抑菌机理。结果表明，TTO包括醇类、烯类、烷烃类和酯类共15个化合物，其中T-4-O占31.09%，为主要的物质。T-4-O对金黄色葡萄球菌具有良好的抑菌效果，MIC和MBC分别为2.12 mg/mL和8.47 mg/mL。2MIC T-4-O能基本抑制金黄色葡萄球菌的生长，在该浓度下，T-4-O干扰了细菌生物膜形成的代谢途径，对金黄色葡萄球菌生物膜形成抑制率可达50%左右；能使得细菌细胞膜可能发生局部位移，并影响了细胞壁的形成，细胞溶出物、蛋白质等物质发生泄露，菌体无法正常分裂、生长，细胞生长周期一直停留在R期，最终使菌体细胞膜发生破裂、死亡，从而起到了抑菌效果。
Abstract:The chemical composition of Tea Tree Oil (TTO) and the antibacterial mechanism of its main component Turpentene-4-ol (T-4-O) were analyzed, and the composition of TTO was determined by GC-MS. The effects of cell cycle and gene transcriptional expression were used to analyze the antibacterial mechanism of T-4-O on Staphylococcus aureus. The results showed that TTO included 15 compounds, including alcohols, alkenes, alkanes and esters, of which T-4-O accounted for 31.09%, which was the main substance. T-4-O had a good antibacterial effect on Staphylococcus aureus, with MIC and MBC of 2.12 mg/mL and 8.47 mg/mL, respectively. 2MIC T-4-O can basically inhibit the growth of Staphylococcus aureus, and at this concentration, T-4-O interferes with the metabolic pathway of bacterial biofilm formation, and the inhibition rate of Staphylococcus aureus biofilm formation can reach about 50%. It can make the bacterial cell membrane shift locally, and affect the formation of the cell wall, the leakage of cell extractables, proteins and other substances, the bacteria can not divide and grow normally, and the cell growth cycle has been stuck in the R phase, and finally the cell membrane of the bacteria is ruptured and died, thus playing a bacteriostatic effect.

关键词：茶树精油；GC-MS；松油烯-4-醇；抑菌；金黄色葡萄球菌
Keywords: tea tree essential oil; GC-MS； terpinene-4-ol; bacteriostatic; Staphylococcus aureus

植物精油是植物次级代谢产物，其组成非常复杂，主要是萜烯及其衍生物^[1]。植物精油功能因组分复杂而多样化，具有抑菌、抗炎、抗氧化、促进动物采食、提高生产性能和增强机体免疫等生物学功能，以及无毒副作用和无药残留等优点，有望成为促生长类抗生素替代品，成为近年来国内外的研究热点^[2-3]。互叶白千层（Melaleuca ahemifolia）属于桃金娘科白千层属植物，约有230个品种，在中国福建、四川、云南、广东、广西等省份广泛分布^[4]。茶树精油（Tea Tree Oil，TTO）又称互叶白千层精油，是一种无色至浅黄色的液体，它是从澳大利亚桃金娘科互叶白千层植物的嫩枝和叶片中通过水蒸气萃取而得，含有香樟和薄荷的香气^[5]。TTO被广泛运用于制作香料、食品工业、农业和化妆品等领域，具有极高的经济价值，其独特功效及安全性获得了欧美多个国家的认可，是第7版欧洲药典中收录的少数几种精油之一^[6]。
Plant essential oils are secondary metabolites of plants with a very complex composition, mainly terpenes and their derivatives ^[1] . The functions of plant essential oils are diverse due to their complex compositions, and they have the advantages of antibacterial, anti-inflammatory, antioxidant, promoting animal feeding, improving production performance and enhancing immunity, as well as no toxic side effects and no drug residues, which are expected to become a growth-promoting antibiotic substitute and become a research hotspot at home and abroad in recent years ^[2-3] . Melaleuca ahemifolia belongs to the genus Melaleuca in the Myrtaceae family, with about 230 varieties and is widely distributed in Fujian, Sichuan, Yunnan, Guangdong, Guangxi and other provinces in China ^[4] . Tea Tree Oil (TTO), also known as Melaleuca alternifolia essential oil, is a colorless to light yellow liquid, which is extracted from the shoots and leaves of the Australian Myrtaceae alterniflora plant through water vapor, containing the aroma of camphor and ^[5] peppermint. TTO is widely used in the production of spices, food industry, agriculture and cosmetics and other fields, has high economic value, its unique efficacy and safety has been recognized by many countries in Europe and the United States, is one of the few essential oils included in the 7th edition of the European Pharmacopoeia ^[6] .

TTO是一种含有一百多种化学成分的混合物，其中主要以单萜、倍半萜烯和醇类化合物为主^[7]。TTO主要成分中具有六种化学类型，分别为萜品油烯化学型、松油烯-4-醇化学型和四种1,8-桉叶素化学型^[8]，其中松油烯-4-醇（Turpentene-4-ol，T-4-O）、1,8-桉叶素（1,8- Cineole）占总含油量的50%以上，是TTO具有强大抗菌活性的物质基础^[9]。TTO作为一种植物提取物，其抗菌等作用已得到大量的临床证据支持，如。Groppo^[10]等采用随机对照单盲法研究了6 茶树油凝胶治疗复发性唇疱疹临床疗效，表现出明显的治疗效果。Arweiler^[11]用0.34%茶树油漱口水研究了其对口腔内细菌的杀灭效果，得出了茶树油可明显减少口腔细菌数。但由于种植地域的差异性，不同提取来源的TTO化学成分差异较大，且其抑菌作用也有所区别^[12]。因此，本研究采用GC-MS（Gas chromatography-mass spectrometry）对市售茶树精油组成成分分析进行测定分析，研究其主要成分T-4-O体外抑菌活性，并进一步分析其对金黄色葡萄球菌的抑菌机理，以期为茶树精油及其单体的进一步开发利用提供理论基础依据。
TTO is a mixture of more than 100 chemical components, mainly monoterpenes, sesquiterpenes and alcohols ^[7] . There are six chemical types in the main components of TTO, namely terpinolene, terpinene-4-ol and four 1,8-cineol, ^[8] among which Turpentene-4-ol (T-4-O) and 1,8-cineole account for more than 50% of the total oil content, which is the material basis for TTO with strong antibacterial activity ^[9] . As a plant extract, TTO's antimicrobial and other effects have been supported by a large number of clinical evidence, such as: Groppo ¹⁰ et al. used a randomized controlled single-blind method to study the clinical efficacy of 6 tea tree oil gel in the treatment of recurrent cold sores, and showed a significant therapeutic effect. Arweiler ¹¹ studied the killing effect of 0.34% tea tree oil mouthwash on oral bacteria and concluded that tea tree oil significantly reduced the number of oral bacteria. However, due to the differences in planting regions, the chemical composition of TTO from different extraction sources is quite different, and its antibacterial effect is also different ^{[1 2} . Therefore, in this study, GC-MS (Gas chromatography-mass spectrometry) was used to determine and analyze the composition of commercially available tea essential oil, to study the antibacterial activity of its main component T-4-O in vitro, and to further analyze its antibacterial mechanism against Staphylococcus aureus, in order to provide a theoretical basis for the further development and utilization of tea essential oil and its monomers.

材料和方法
Materials and methods

1.3试验方法
1.3 Test methods

1.3.1 TTO、T-4-O的配制
1.3.1 Preparation of TTO and T-4-O

本研究前期预试验证实将TTO、T-4-O与无水乙醇按照1:19体积比混合时，无水乙醇对菌液的生长状况无影响。取TTO、T-4-O的溶于无水乙醇，质量浓度分别为880mg/mL、846.5 mg/mL。
The preliminary test of this study confirmed that when TTO, T-4-O and absolute ethanol were mixed at a volume ratio of 1:19, absolute ethanol had no effect on the growth of bacterial solution. TTO and T-4-O were dissolved in absolute ethanol at concentrations of 880 mg/mL and 846.5 mg/mL, respectively.

1.3.2 TTOGC-MS成分分析
1.3.2 TTOGC-MS composition analysis

色谱分析条件：毛细管柱（30m × 0.32mm × 0.25μm），以高纯氦气（纯度不小于 99.999%）为载体，恒流流速 1.2 mL/min；进样口温度250 °C，为不分流进样；柱温60°C保持1min，15°C/min升温至230°C。质谱分析条件：电子轰击离子源（EI），离子源温度230 °C，四级杆温度150 °C，质谱接口温度280 °C，电子能量 70 eV，扫描模式：全扫描；扫描范围：50～650m/z。
Chromatographic analysis conditions: capillary column (30m × 0.32mm × 0.25μm), with high-purity helium (purity not less than 99.999%) as the carrier, constant flow rate 1.2 mL/min; The inlet temperature is 250 °C, which is a splitless injection; The column temperature was kept at 60°C for 1min, and the temperature was raised to 230°C at 15°C/min. Mass spectrometry analysis conditions: electron bombardment ion source (EI), ion source temperature 230 °C, quadrupole temperature 150 °C, mass spectrometry interface temperature 280 °C, electron energy 70 eV, scanning mode: full scan; Scanning range: 50~650m/z.

1.3.3 T-4-O对不同菌株的体外抑菌性能测定
1.3.3 Determination of the in vitro bacteriostatic performance of T-4-O on different strains

（1）牛津杯法抑菌圈的测定
(1) Determination of the Oxford Cup inhibition zone

将待测各菌株（大肠杆菌、金黄色葡萄球菌和白色念珠菌）在对应培养基中划线、活化、培养，待用，采用牛津杯法测定T-4-O对不同菌株的抑菌性能。将各菌株转接至无菌生理盐水试管中制成菌悬液，浓度108cfu/mL。取0.1mL菌悬液到各菌对应的平板培养基中，涂布均匀。将牛津杯放置平板（牛津杯D=6mm），加入150μL配置的T-4-O药液，在生化培养箱中（细菌37℃、真菌30℃）培养24～48h，测定各药液抑菌圈大小，同时以无水乙醇为对照。
The strains to be tested (Escherichia coli, Staphylococcus aureus and Candida albicans) were marked, activated and cultured in the corresponding medium, and the antibacterial performance of T-4-O against different strains was determined by the Oxford cup method. Each strain was transferred to a sterile saline test tube to make a bacterial suspension at a concentration of 108 cfu/mL. 0.1mL of bacterial suspension was added to the plate medium corresponding to each bacterium and evenly coated. The Oxford cup was placed on a plate (Oxford cup D=6mm), 150μL of T-4-O liquid was added, and incubated in a biochemical incubator (37 °C for bacteria and 30 °C for fungi) for 24~48 h, and the size of the inhibition zone of each solution was determined, and absolute ethanol was used as the control.

（2）MIC和MBC的测定
(2) Determination of MIC and MBC

将T-4-O药液接种至LB肉汤进行对倍稀释法测定各样品MIC（Minimum Inhibitory Concentration）及MBC（MinimumBactericidal Concentration）。取10支无菌试管，依次编号。首先所有试管中均加入1mLLB培养基，然后将稀释后的T-4-O药液1mL加入到第1根试管中，混合均匀；再取出1mL加入到第2根试管中，再混合均匀，依次再倍比稀释至第7根试管；第7根试管混合均匀后，取去多余的1mL混合液丢弃。再将10根试管中分别加入3.8mL的LB培养基，然后1～7根试管加入0.2mL的实验菌液（浓度10⁸cfu/mL），第8根试管加入0.2mL T-4-O药液，第9根试管加入0.2mL培养基，第10根试管加入0.2mL上述待测各菌株的实验菌液。以9根试管进行调O，测定各试管OD₆₀₀值，抑菌率（%）=（1-OD每管测定值/第10管测定值）×100%，其中抑菌率≧50%的最大稀释倍数为最小抑菌浓度（MIC），抑菌率≧90%的最大稀释倍数为最小杀菌浓度（MBC）。
T-4-O solution was inoculated into LB broth for dilution to determine the MIC (Minimum Inhibitory Concentration) and MBC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) of each sample. Take 10 sterile test tubes and number them sequentially. First, 1 mLLB medium was added to all test tubes, and then 1mL of diluted T-4-O solution was added to the first test tube, and mixed evenly. Take out 1mL and add it to the second test tube, mix it evenly, and dilute it to the 7th test tube in turn. After the 7th tube is mixed well, remove the excess 1 mL of the mixture and discard. Then 3.8mL of LB medium was added to 10 test tubes, then 0.2mL of experimental bacterial solution (concentration of 10 ⁸ cfu/mL) was added to 1~7 test tubes, 0.2mL of T-4-O liquid was added to the 8th test tube, 0.2mL of medium was added to the 9th test tube, and 0.2mL of the experimental bacterial solution of the above strains to be tested was added to the 10th test tube. 9 test tubes were used to adjust O, and the OD ₆₀₀ value of each test tube was measured, and the inhibition rate (%) = (1-OD measured value per tube / 10th tube measured value) × 100%, in which the maximum dilution factor of the inhibition rate ≧50% was the minimum bactericidal concentration (MIC), and the maximum dilution factor of the bacteriostatic rate ≧90% was the minimum bactericidal concentration (MBC).

1.3.4 T-4-O对金黄色葡萄球菌抑菌机理研究
1.3.4 Study on the antibacterial mechanism of T-4-O against Staphylococcus aureus

（1）对菌株生长和菌体结构的影响
(1) Effect on strain growth and cell structure

对菌株生长影响的测定：将金黄色葡萄球菌的菌悬液培养至生长对数期，调节其浓度为1×10⁸CFU/mL。将T-4-O以3种不同浓度（1MIC、2MIC和3MIC）加入到调节完浓度的菌悬液中，同时以蒸馏水最为对照。混合后的样品在摇床中37℃培养，分别在0、0.5、1、2、3、4h分别取出1mL样品通过分光光度计测定菌体浓度OD₆₀₀值，分析不同浓度的精油对菌株生长的影响。
Determination of the effect on the growth of strains: The bacterial suspension of Staphylococcus aureus was cultured to the logarithmic phase of growth, and its concentration was adjusted to 1×10 ⁸ CFU/mL. T-4-O was added to the regulated bacterial suspension at three different concentrations (1MIC, 2MIC and 3MIC), and distilled water was used as the control. The mixed samples were incubated at 37°C in a shaker, and 1mL of samples were taken out at 0, 0.5, 1, 2, 3 and 4 h, respectively, and the OD ₆₀₀ value of cell concentration was determined by spectrophotometer, and the effects of different concentrations of essential oils on the growth of the strain were analyzed.

扫描电镜（SEM）观察菌体整体结构：采用扫描电子显微镜（SEM）观察T-4-O对金黄色葡萄球菌细胞整体形态的影响，菌种活化及菌悬液制备的操作同上。将加完不同浓度T-4-O（1MIC、2MIC和3MIC）的菌悬液置于摇床180r/min中培养，同时以蒸馏水为对照，37℃培养1h后，分别从每份试样中取出2mL细菌悬浮液，以4000r/min 的转速离心10min。然后将收集的细胞用50%乙醇/水溶液洗涤2次。然后将20μL悬浮液涂布在盖玻片上，并将100μL含有体积分数为2.5%的戊二醛溶液（用磷酸盐缓冲液稀释）加入到盖玻片上固定2～3h，然后进行喷金和SEM观察。
Scanning electron microscopy (SEM) was used to observe the overall structure of the cell: The effect of T-4-O on the overall morphology of Staphylococcus aureus cells was observed by scanning electron microscopy (SEM), and the operation of strain activation and preparation of bacterial suspension was the same as above. After adding different concentrations of T-4-O (1MIC, 2MIC and 3MIC), the bacterial suspension was incubated in a shaker at 180r/min, and at the same time, distilled water was used as a control, and after incubation at 37 °C for 1h, 2mL of bacterial suspension was taken out from each sample and centrifuged at a speed of 4000r/min for 10min. The collected cells are then washed 2 times with 50% ethanol/aqueous solution. Then 20 μL of the suspension was coated on the coverslip, and 100 μL of glutaraldehyde solution containing a volume fraction of 2.5% (diluted with phosphate buffer) was added to the coverslip for fixation for 2~3h, and then gold spraying and SEM observation were performed.

对菌株细胞壁的影响测定：正常情况下AKP（Alkaline phosphatase）不能透过细胞壁渗出至细胞外，检测不到其活性，因而其活性可以作为反映革兰氏阳性菌株细胞壁是否完整的依据。碱性磷酸酶检测试剂盒可以与细胞壁内物质结合染色而在分光光度计下被检测到，本试验中金黄色葡萄球菌菌种活化及菌悬液制备的操作同上，依据碱性磷酸酶检测试剂盒的说明方法进行加样。通过添加不同浓度T-4-O（1MIC、2MIC和3MIC），同时以蒸馏水为对照，每隔一段时间使用酶标仪在520nm下进行检测，以APK活性的变化作为细胞壁完整性的依据从而反映药液对细菌细胞壁的影响。
Determination of the effect on the cell wall of the strain: under normal circumstances, AKP (Alkaline phosphatase) cannot penetrate the cell wall to the outside of the cell, and its activity cannot be detected, so its activity can be used as a basis to reflect whether the cell wall of the Gram-positive strain is complete. The alkaline phosphatase detection kit can be detected under spectrophotometer by combining staining with the substances in the cell wall, and the activation of Staphylococcus aureus strains and the preparation of bacterial suspension in this test are the same as above, and the sampling is carried out according to the instructions of the alkaline phosphatase detection kit. By adding different concentrations of T-4-O (1MIC, 2MIC and 3MIC), distilled water was used as the control, and the microplate reader was used at intervals to detect at 520 nm, and the change of APK activity was used as the basis for the integrity of the cell wall, so as to reflect the effect of the liquid on the bacterial cell wall.

（2）对菌株细胞膜及物质泄露的影响
(2) Effect on cell membrane and substance leakage of strains

对菌株细胞膜的影响：菌种活化及菌悬液制备的操作同上。将菌液与不同浓度T-4-O（1MIC、2MIC和3MIC）吹打混匀，分别取300μL于96孔板中，以LB肉汤培养基作为空白对照，37℃恒温培养36h。之后吸弃96孔板中的液体，每孔加入200μL甲醇静置15min。吸弃甲醇，每孔加入30μL无菌PBS（Phosphate buffer saline）进行清洗，重复3次。PBS清洗后，待其自然晾干，每孔加入250μL 10g/L结晶紫溶液，静置15min。吸弃结晶紫溶液，用无菌PBS清洗3次，每孔加入250μL体积分数30%乙酸溶解，30min后，570nm波长下测其OD，以结晶紫染色法检测生物膜量的变化分析T-4-O对金黄色葡萄球菌生物膜的影响。将菌液同样精油处理后离心弃去上清液，用戊二醛液低温冷冻4h，之后乙醇逐级脱水，再用乙酸异戊酯完全置换，逐级冷冻后使用冷冻干燥机升华干燥，制作压片在傅里叶红外光谱下扫描，吸收峰强度的变化将代表特定信息区成分含量的变化，以此显现出T-4-O对细胞膜成分的影响。
Effect on the cell membrane of the strain: the operation of strain activation and preparation of bacterial suspension is the same as above. The bacterial solution was mixed with different concentrations of T-4-O (1MIC, 2MIC and 3MIC), 300μL was taken in a 96-well plate, LB broth medium was used as a blank control, and incubated at a constant temperature of 37 °C for 36 h. After that, the liquid in the 96-well plate was aspirated, and 200 μL of methanol was added to each well and allowed to stand for 15 min. Aspirate the methanol and add 30 μL of sterile PBS (Phosphate buffer saline) per well for washing, repeat 3 times. After PBS washing, let it dry naturally, add 250 μL of 10g/L crystal violet solution to each well, and let it stand for 15min. The crystal violet solution was aspirated, washed with sterile PBS for 3 times, 250 μL of 30% acetic acid was added to each well to dissolve, and after 30 min, its OD was measured at a wavelength of 570 nm, and the change of biofilm amount was detected by crystal violet staining to analyze the effect of T-4-O on Staphylococcus aureus biofilm. After the bacterial solution was treated with the same essential oil, the supernatant was discarded by centrifugation, and the glutaraldehyde solution was frozen at low temperature for 4h, and then the ethanol was dehydrated step by step, and then completely replaced with isoamyl acetate, and then sublimated and dried with a freeze dryer after freezing step by step, and the tablets were made to be scanned under the Fourier transform infrared spectrum, and the change in the intensity of the absorption peak will represent the change in the content of the components in the specific information region, so as to show the effect of T-4-O on the components of the cell membrane.

细胞溶出物泄露测试：菌种活化及菌悬液制备的操作同上。将菌悬液以4000r/min离心10min，收集菌体，用无菌生理盐水洗涤3次，然后重悬于初始体积的无菌生理盐水中。将体积为25mL的细菌悬浮液的等分试样分别加入3种浓度的T-4-O（1MIC、2MIC和3MIC），同时以蒸馏水为对照，在气浴恒温振荡器中37℃培养。分别在0、1、2h将混合物以4000r/min离心10min，使用可见分光光度计在260nm波长条件下测量3mL上清液的吸光度值。在260nm处吸收的物质包括蛋白质和核酸，上清液在260nm的光密度值是细胞膜破裂和内部细胞成分释放的指示，以OD₂₆₀的变化作为反映药液对细菌细胞溶出物泄漏的影响。
Cell lysate leakage test: the operation of bacterial culture activation and bacterial suspension preparation is the same as above. The bacterial suspension was centrifuged at 4000r/min for 10 min, the bacteria were collected, washed 3 times with sterile saline, and then resuspended in the initial volume of sterile saline. An aliquot of 25 mL of bacterial suspension was added with three concentrations of T-4-O (1MIC, 2MIC and 3MIC) and incubated at 37 °C in an air bath thermostatic shaker with distilled water as a control. The mixture was centrifuged at 4000 r/min for 10 min at 0, 1, and 2 h, respectively, and the absorbance value of 3 mL of the supernatant was measured using a visible spectrophotometer at a wavelength of 260 nm. The absorbent at 260 nm includes proteins and nucleic acids, and the optical density value of the supernatant at 260 nm is an indication of cell membrane rupture and the release of internal cellular components, and the ₂₆₀ change in OD is used as a reflection of the effect of the liquid on the leakage of bacterial cell extractables.

细胞内蛋白质泄露测试：菌种活化及菌悬液制备的操作同上。通过测试加入3种浓度的T-4-O（1MIC、2MIC和3MIC）细菌悬浮液上清液的蛋白质含量研究蛋白质泄漏情况。分别在培养1、2h后使用考马斯亮蓝法和紫外可见分光光度计在595nm测定上清液中蛋白质的含量。从标准牛血清白蛋白曲线计算释放的蛋白质的量^[13]。
Intracellular protein leakage test: the procedure for strain activation and bacterial suspension preparation is the same as above. Protein leakage was investigated by testing the protein content of supernatants of bacterial suspensions with 3 concentrations of T-4-O (1MIC, 2MIC, and 3MIC). The protein content in the supernatant was determined by Coomassie brilliant blue method and UV/VIS spectrophotometer at 595 nm after 1 and 2 h of incubation, respectively. Calculate the amount ^{[1 3} of protein released from the standard bovine serum albumin curve.

（3）对细胞生长周期的影响
and (3) effects on the cell growth cycle

取对数期金黄色葡萄球菌菌液，按上述试验结果使得加入T-4-O终浓度为2MIC，混匀后置于37 ℃、150 r/min的摇床中培养1 h。同时，加入等体积的无菌水做空白对照。培养完之后，4 000 r/min离心10 min收集菌体细胞，弃上清液，PBS清洗2 次。接着加入1 mL预冷的70%乙醇充分重悬菌体细胞沉淀，并置于4 ℃冰箱过夜固定。4 000 r/min离心10 min去上清液，PBS清洗2 次，向金黄色葡萄球菌细胞沉淀中加入450 μL PBS重悬，再加入50 μL 1 mg/ml RNase溶液和5 μL 1 mg/mL PI（Propidium iodide）溶液，混匀后置于4 ℃条件下避光孵育15 min，最后使用流式细胞仪检测，数据使用FlowJo软件进行分析^[14]。
The logarithmic phase of Staphylococcus aureus was taken and the final concentration of T-4-O was 2MIC according to the above test results, and it was mixed and placed in a shaker at 37 °C and 150 r/min for 1 h. At the same time, add an equal volume of sterile water as a blank control. After culture, the cells were collected by centrifugation at 4 000 r/min for 10 min, the supernatant was discarded, and washed twice in PBS. Then add 1 mL of pre-chilled 70% ethanol to fully resuspend the cell pellet and place in a 4 °C freezer overnight. Centrifuge at 4 000 r/min for 10 min to remove the supernatant, wash twice in PBS, add 450 μL of PBS to the Staphylococcus aureus cell pellet and resuspend it, then add 50 μL of 1 mg/ml RNase solution and 5 μL of 1 mg/mL PI (Propidium iodide) solution, mix well, and incubate at 4 °C for 15 min in the dark, and finally use flow cytometry to detect the data, and the data were analyzed ^[1 by FlowJo software ⁴ 。

（4）转录组学测序分析
(4) Transcriptomic sequencing analysis

同上述细胞周期的前期培养操作，收集T-4-O处理组和空白对照组的菌体各3份，采用Omege soil RNA Kit提取总RNA，所提取样品均检验合格后送至北京奥维森基因有限公司通过IlluminaHiseq 4000高通量测序平台完成相关的样本文库构建和转录测序工作。所得的测序数据进行RNA-Seq相关性检查。各样品基因表达水平的分析采用HTSeq软件进行，模型为union，用FPKM（Fragments Per Kilobase of exon model per Million mapped fragments）作为表达定量结果，筛选FPKM>1的基因^[15]。采用DESeq进行基因差异表达分析，差异基因筛选的标准为qvalue＜0.05^[16]，所筛选得到的差异表达基因输入GO（Gene Ontology）和KEGG（Kyoto Encyclopedia of Genes and Genomes）数据库进行比对和功能注释分类^[17]。
In the same way as the above-mentioned cell cycle pre-culture operation, 3 copies of cells from T-4-O treatment group and blank control group were collected, and total RNA was extracted by Omege soil RNA Kit, and the extracted samples were sent to Beijing Ovison Gene Co., Ltd. to complete the relevant sample library construction and transcription sequencing work through the IlluminaHiseq 4000 high-throughput sequencing platform. The resulting sequencing data were examined for RNA-Seq correlation. The gene expression level of each sample was analyzed by HTSeq software, the model was union, and FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) was used as the expression quantification result to screen the FPKM>1 gene ^{[1 5} . DESeq was used for differential gene expression analysis, and the standard for differential gene screening was qvalue<0.05 ^[1 , ⁶ and the selected differentially expressed genes were entered into GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases for comparison and functional annotation classification ¹⁷ .

2. 结果
2. Results

2.1 TTO的化学成分分析
2.1 Chemical composition analysis of TTO

通过GC-MS测定TTO的化学成分总离子流图分析结果如图1、表1所示，图中出现20个峰，鉴定出15个化合物（部分峰为柱流出物，非精油成分，占15.46），包括醇类（占38.92%）、烯类（14.66%）、烷烃类（14.96%）、酯类（16.00%）。其中松油烯-4-醇占31.09%，1,8-桉叶素占2.12%，符合《GBT 2651-2011互叶白千层（精）油，松油烯-4-醇型[茶树(精)油] 》中松油烯-4-醇≥30%，1,8-桉叶素≤5%的规定。
As shown in Figure 1 and Table 1, 20 peaks appeared in the figure, and 15 compounds (some peaks were column effluents, non-essential oil components, accounting for 15.46), including alcohols (38.92%), alkenes (14.66%), alkanes (14.96%), and esters (16.00%). Among them, terpinene-4-ol accounted for 31.09% and 1,8-cineolin accounted for 2.12%, which conformed to the provisions of "GBT 2651-2011 Melaleuca alternifolia (refined) oil, terpinene-4-ol type [tea tree (refined) oil] ≥ 30% and 1,8-cineol≤5%.

2.2 T-4-O抑菌性能测定结果
2.2 Determination results of antibacterial performance of T-4-O

2.2.1抑菌圈大小的测定结果
2.2.1 Determination results of the size of the inhibition zone

T-4-O对不同菌株的抑菌能力的测定结果表明，T-4-O对金黄色葡萄球菌的抑菌效果要强于对大肠杆菌和白色念珠菌（图2）。
The results of the determination of the antibacterial ability of T-4-O against different strains showed that T-4-O had a stronger antibacterial effect on Staphylococcus aureus than against Escherichia coli and Candida albicans (Fig. 2).

2.2.2 MIC的测定结果
2.2.2 Measurement results of MIC

T-4-O对不同菌株的MIC、MBC测定结果如表2所示。结果表明，T-4-O对金黄色葡萄球菌作用最强，其MIC为2.12 mg/mL，MBC为8.47 mg/mL。
Table 2 shows the results of MIC and MBC determination of T-4-O for different strains. The results showed that T-4-O had the strongest effect on Staphylococcus aureus, with MIC of 2.12 mg/mL and MBC of 8.47 mg/mL.

2.3 T-4-O对金黄色葡萄球菌抑菌机理研究结果
2.3 Findings on the antibacterial mechanism of T-4-O against Staphylococcus aureus

2.3.1 对菌株生长和结构影响测定结果
2.3.1 Determination results of effects on strain growth and structure

菌株的生长：不同浓度T-4-O对菌株生长的影响如图3A所示。结果表明，与对照组相比，添加T-4-O对金黄色葡萄球菌具有明显的抑制作用，随着添加浓度的增大，菌株生长受到更加明显的抑制，但添加到3MIC浓度精油其抑制作用与添加2MIC影响差别不大。
Growth of strains: The effects of different concentrations of T-4-O on strain growth are shown in Figure 3A. The results showed that compared with the control group, the addition of T-4-O had a significant inhibitory effect on Staphylococcus aureus, and the growth of the strain was more obviously inhibited with the increase of the addition concentration, but the inhibitory effect of essential oil added to 3MIC concentration was not significantly different from that of 2MIC.

菌体的整体结构：不同浓度的T-4-O与金黄色葡萄球菌共培养后，扫描电子显微镜（SEM）对菌株不同培养时间细胞整体形态的观察如图3B所示。结果表明，对照组未经T-4-O处理的金黄色葡萄球菌细胞结构完整且表面光滑、呈圆球状；不同浓度T-4-O处理1h后其细胞膜均受到不同程度的损伤，细胞膜表面出现了褶皱和局部塌陷；且随着浓度的增加，T-4-O对金黄色葡萄球菌细胞膜的破坏程度更大，细胞的塌陷和破裂变得更加严重。
The overall structure of the cell: After different concentrations of T-4-O were co-cultured with Staphylococcus aureus, the overall morphology of the cells at different culture times of the strain was observed by scanning electron microscopy (SEM) as shown in Figure 3B. The results showed that the Staphylococcus aureus cells in the control group without T-4-O treatment had intact structure and smooth surface and spherical shape. After 1 h of treatment with different concentrations of T-4-O, the cell membrane was damaged to varying degrees, and the surface of the cell membrane was wrinkled and partially collapsed. With the increase of concentration, the damage of T-4-O to the cell membrane of Staphylococcus aureus was greater, and the collapse and rupture of the cells became more serious.

菌株的细胞壁：不同浓度T-4-O与金黄色葡萄球菌共培养后，测定的AKP活性变化如图3C所示。结果表明，与对照组相比，加入不同浓度T-4-O与金黄色葡萄球菌共培养后，其APK的活性测定活性明显增加；说明不同浓度T-4-O对菌株细胞壁均有一定程度的破坏作用，使菌体内的碱性磷酸酶释放，从而起到抑菌作用。不同浓度的T-4-O测定AKP活性变化与对照组变化趋势一致，可能与金黄色葡萄球菌的生长周期有关。
Cell wall of the strain: Changes in AKP activity measured after co-culture with Staphylococcus aureus at different concentrations are shown in Figure 3C. The results showed that compared with the control group, the activity of APK was significantly increased after the addition of different concentrations of T-4-O and Staphylococcus aureus co-culture. The results indicated that different concentrations of T-4-O had a certain degree of damage to the cell wall of the strain, which caused the release of alkaline phosphatase in the bacteria, thereby playing a role in inhibiting bacteria. The changes in AKP activity at different concentrations of T-4-O were consistent with those in the control group, which may be related to the growth cycle of Staphylococcus aureus.

2.3.2 对菌株细胞膜和物质泄露影响的测定结果
2.3.2 Measurement results of the effect on the cell membrane and material leakage of the strain

细胞膜：不同浓度T-4-O与金黄色葡萄球菌共培养后，其对金黄色葡萄球菌生物膜的清除作用如图4A所示。结果表明，与对照组相比，添加T-4-O对金黄色葡萄球菌生物膜具有明显的影响，且随着浓度的增加其对生物膜的清除率增强；当添加量为2MIC时其对金黄色葡萄球菌生物膜形成的抑制率达50%左右，与3MIC差异不显著（P>0.05）。不同浓度T-4-O与金黄色葡萄球菌共培养后其菌体的红外光谱分析如图4B所示。1080^-1为糖苷键多糖中的肽聚糖糖苷键结构及构成细胞组分的磷脂、核酸、脂肪糖中的含磷基团；1800～1500^-1为蛋白和多糖的酰胺信息区，3000～2800^-1为脂肪酸信息区。结果表明，随着添加不同浓度的精油，金黄色葡萄球菌上述几个基团的吸收峰明显增强，表明细胞细胞膜、细胞壁中的肽聚糖、蛋白质、脂质等成分被破坏，细胞膜可能发生局部位移，致使细胞膜形成孔洞，一些大分子物质进入细胞，使得菌体细胞内容物发生泄漏，最终导致细胞死亡。
Cell membrane: After different concentrations of T-4-O were co-cultured with Staphylococcus aureus, its clearance effect on Staphylococcus aureus biofilm is shown in Figure 4A. The results showed that compared with the control group, the addition of T-4-O had a significant effect on the biofilm of Staphylococcus aureus, and the clearance rate of the biofilm was enhanced with the increase of concentration. When the addition amount was 2MIC, the inhibition rate of Staphylococcus aureus biofilm formation was about 50%, and there was no significant difference with 3MIC (P>0.05). The infrared spectroscopy analysis of T-4-O after co-culture with Staphylococcus aureus at different concentrations is shown in Figure 4B. 1080 ^-1 is the peptidoglycan glycosidic bond structure in glycosidic bond polysaccharides and the phospholipids, nucleic acids, and phosphorus-containing groups in fatty sugars that constitute cell components; 1800~1500 ^-1 is the amide information area of proteins and polysaccharides, and 3000~2800 ^-1 is the fatty acid information area. The results showed that with the addition of different concentrations of essential oils, the absorption peaks of the above groups of Staphylococcus aureus were significantly enhanced, indicating that the peptidoglycan, protein, lipid and other components in the cell membrane and cell wall were destroyed, and the cell membrane may be partially shifted, resulting in the formation of pores in the cell membrane, and some macromolecular substances entering the cell, causing the cell contents of the cell to leak and eventually lead to cell death.

细胞溶出物泄露：不同浓度T-4-O处理金黄色葡萄球菌0、1、2h后OD₂₆₀值如图4C所示。结果表明，与对照组相比，添加T-4-O后，随着时间和T-4-O浓度的增加，OD₂₆₀的光密度值增加，造成了金黄色葡萄球菌的细胞溶出物的泄漏，整体上处理金黄色葡萄球菌1、2h后2MIC和1MIC之间差异显著（P<0.05），与3MIC之间差异不显著（P>0.05）。
Cell lysate leakage: The OD ₂₆₀ values of Staphylococcus aureus after 0, 1, and 2 h of different concentrations of T-4-O are shown in Figure 4C. The results showed that compared with the control group, the optical density value ₂₆₀ of OD increased with the increase of time and T-4-O concentration after the addition of T-4-O, resulting in the leakage of cell extractables of Staphylococcus aureus, and the overall difference between 2MIC and 1MIC after 1 and 2 h of treatment of Staphylococcus aureus was significant (P<0.05), but not significantly different from 3MIC (P>0.05).

细胞内蛋白质泄露：不同浓度T-4-O处理金黄色葡萄球菌0、1、2h后OD₅₉₅值如图4D所示。结果表明，与对照组相比，添加茶树精油后，随着时间和精油浓度的增加，细胞中蛋白质释放，OD₅₉₅光密度值增加，表明添加茶树精油造成了金黄色葡萄球菌的蛋白质的泄漏，且这一现象随着精油浓度的增加破坏作用更明显，但整体上处理不同时间后，均是2MIC和1MIC之间差异显著（P<0.05），与3MIC之间差异不显著（P>0.05）。
Intracellular protein leakage: The OD ₅₉₅ values of Staphylococcus aureus after 0, 1, and 2 h of different concentrations of T-4-O are shown in Figure 4D. The results showed that compared with the control group, with the increase of time and essential oil concentration, the protein release and OD ₅₉₅ optical density value in the cells increased with the increase of time and essential oil concentration, indicating that the addition of tea tree essential oil caused the protein leakage of Staphylococcus aureus, and this phenomenon was more obvious with the increase of essential oil concentration, but on the whole, there was a significant difference between 2MIC and 1MIC (P<0.05), and no significant difference between 3MIC and 3MIC (P>0.05).

2.3.3 细胞周期影响的测定结果
2.3.3 Measurement results of cell cycle effects

在原核细胞中，细菌的细胞周期分为I、R和D 3个时期，其中R期是细菌DNA复制期，相当于细胞的S期[^18]。流式细胞术结果如图5A和5B所示，正常金黄色葡萄球菌处于R期的细胞数为15.1%，2MICT-4-O作用后，进入R期的细胞数增加至26.67%，T-4-O组在R期金黄色葡萄球菌的数量极显著升高（P<0.01），表明T-4-O能结合金黄色葡萄球菌的DAN，使得更多的菌留在了R期，影响了金黄色葡萄球的DAN的复制。
In prokaryotic cells, the cell cycle of bacteria is divided into three phases: I, R, and D, where the R phase is the bacterial DNA replication phase, which is equivalent to the S phase of the cell [ ¹⁸ . The flow cytometry results were shown in Fig. 5A and 5B, the number of normal Staphylococcus aureus cells in R phase was 15.1%, and the number of cells entering R phase increased to 26.67% after the action of 2MICT-4-O, and the number of Staphylococcus aureus in R phase was extremely significantly increased in the T-4-O group (P<0.01), indicating that T-4-O could bind to the DAN of Staphylococcus aureus, so that more bacteria remained in the R phase and affected the DAN replication of Staphylococcus aureus.

2.3.4 转录组测序分析结果
2.3.4 Transcriptome sequencing analysis results

将所获得的转录组数据进行转化处理后，按筛选原则进行筛选，结果如图6A所示。与对照组相比，T-4-O处理的试验组共筛选出4768个DEGs（Differentially Expressed Genes，差异表达基因），其中2362个基因上调表达，占总差异表达的49.54%；2406个基因下调表达，占比50.46%。对差异基因进行GO富集分析，其中富集最显著的30个GO term进行作图（图6B），在“Biological process”类别中，“Carbohydrate transport”和“Cellular protein metabolic process”富集到的差异基因最多；而在“Cellular component”类别中是“Cellular_component”、“Cell part”和“Cell”；在“Molecular function”类别中，是“Substrate-specific transporter activity”、“Substrate-specifictransmembrane activity”和“Transmembrane transporter activity”。对差异表达基因富集最显著的20 条KEGG代谢通路进行分析（图6C），其中“嘧Pyrimidinemetabalism”、“Nitrogenmetabalism”、“Organelle membrane”、“Integral component of membrane”和“Intrinsic component of membrane”DEGs的富集差异显著（P<0.05），这些代谢途径与细菌生物膜形成密切相关^[19]。
The obtained transcriptome data were transformed and filtered according to the screening principle, and the results are shown in Figure 6A. Compared with the control group, a total of 4768 DEGs (Differentially Expressed Genes) were screened in the T-4-O treatment group, of which 2362 genes were up-regulated, accounting for 49.54% of the total differential expression. 2406 genes were down-regulated, accounting for 50.46%. GO enrichment analysis was performed for differential genes, and the 30 GO terms with the most significant enrichment were mapped (Fig. 6B), and in the "Biological process" category, "Carbohydrate transport" and "Cellular protein metabolic process" were the most enriched. In the "Cellular component" category, it is "Cellular_component", "Cell part", and "Cell"; In the Molecular function category, there are Substrate-specific transporter activity, Substrate-specifictransmembrane activity, and Transmembrane transporter activity. The 20 KEGG metabolic pathways with the most significant enrichment of differentially expressed genes were analyzed (Fig. 6C), including "pyrimidinemetabalism", "nitrogenmetabalism", "Organelle membrane", "Integral component of membrane" and "Intrinsic component of membrane" The enrichment of DEGs was significantly different (P<0.05), and these metabolic pathways were closely related to bacterial biofilm formation ¹⁹ .

3. 讨论
3. Discussion

植物精油作为良好的天然生物活性物质，其抗菌^[20-21]作用已得到了国内外大量研究的试验证实，但由于不同植物精油来源和成分不同，其抑菌的作用效果也不一样。因此，有必要对植物精油的成分进行测定分析，阐明其抑菌的作用机理，对植物精油进行天然药物的开发和应用具有重要的意义。本研究测定了茶树精油的化学成分，从中得到了醇类、烯类、烷烃类和酯类共20个化合物，其中松油烯-4-醇（T-4-O）占31.09%，为主要的物质。研究表明，T-4-O作为植物提取物，其应用时不易产生耐药性，安全性好，在特定领域具有替代抗生素等药物的潜力^[22]。因此，本文以T-4-O为研究对象，从单体角度更合理、科学的探究茶树精油及其主要成分的的抑菌机理。
As a good natural bioactive substance, the antibacterial ^{20 -2 1} effect of plant essential oil has been confirmed by a large number of studies at home and abroad, but due to the different sources and components of different plant essential oils, its antibacterial effect is also different. Therefore, it is necessary to determine and analyze the components of plant essential oils, clarify their antibacterial mechanism, and have great significance for the development and application of natural medicines of plant essential oils. In this study, the chemical composition of tea essential oil was determined, and a total of 20 compounds including alcohols, alkenes, alkanes and esters were obtained, among which terpinene-4-ol (T-4-O) accounted for 31.09%, which was the main substance. Studies have shown that T-4-O, as a plant extract, is not prone to drug resistance when applied, has good safety, and has the potential to ^[2 replace drugs such as antibiotics in specific fields ² . Therefore, this paper takes T-4-O as the research object to explore the antibacterial mechanism of tea essential oil and its main components from the perspective of monomers in a more reasonable and scientific way.

已有相关报道，T-4-O对金黄色葡萄球菌具有明确的抑菌活性^[23-24]，其作用MIC为1.25~2.50 g/L^[25]，与本文报道的MIC 2.12 mg/mL一致。同时，本文还发现T-4-O对金黄色葡萄球菌生长呈浓度依赖性，当添加浓度为2MIC时能基本抑制金黄色葡萄球菌的生长。再进一步研究表明，T-4-O对金黄色葡萄球菌抑菌作用主要是影响其细胞膜结构，转录组测序分析表明T-4-O可能干扰了细菌生物膜形成的代谢途径，如Pyrimidine metabalism”、“Nitrogenmetabalism”、“Organelle membrane”、“Integral component of membrane”和“Intrinsic component of membrane”等与细菌生物膜形成密切相关代谢途径的DEGs在T-4-O处理试验组中富集差异显著（P<0.05）。结合红外光谱分析表明正是细菌生物膜形成代谢途径的改变，使得细菌细胞膜可能发生局部位移，致使细胞膜形成孔洞，菌体细胞内容物发生泄漏。而SEM分析证实，T-4-O作用金黄色葡萄球菌能使其细胞膜发生破裂，进而菌体细胞发生塌陷、死亡；当T-4-O添加量为2MIC对金黄色葡萄球菌生物膜形成抑制率可达50%左右，是发挥抑菌良好抑菌效果的较适添加浓度。此外，研究还发现T-4-O还能影响金黄色葡萄球菌细胞壁的形成，造成其细胞溶出物泄漏、蛋白质泄露。结合流式细胞术细菌细胞周期的测定表明，细胞内物质的泄露导致了金黄色葡萄球菌细胞生长无法正常进行，细胞周期一直停留在R期，影响了DAN的复制，导致死亡，从而发挥了抑菌作用。
It has been reported that T-4-O has definite antibacterial activity ^{23 -} against Staphylococcus aureus ²⁴ , and its MIC is 1.25~2.50 g/L ²⁵ , consistent with the MIC of 2.12 mg/mL reported here. At the same time, it was also found that T-4-O was concentration-dependent on the growth of Staphylococcus aureus, and the growth of Staphylococcus aureus could be basically inhibited when the concentration of 2MIC was added. Further studies showed that the antibacterial effect of T-4-O on Staphylococcus aureus mainly affected its cell membrane structure, and transcriptome sequencing analysis showed that T-4-O may interfere with the metabolic pathways of bacterial biofilm formation, such as Pyrimidine metabalism, Nitrogenmetabalism, Organelle membrane, Integral component of membrane" and " DEGs closely related to the formation of bacterial biofilms, such as "intrinsic component of membrane", were significantly enriched in the T-4-O treatment group (P<0.05）。 Combined with infrared spectroscopy analysis, it was the change of the metabolic pathway of bacterial biofilm formation that caused the local displacement of the bacterial cell membrane, resulting in the formation of pores in the cell membrane and the leakage of cell contents. SEM analysis confirmed that T-4-O could rupture the cell membrane of Staphylococcus aureus, and then the somatic cells collapsed and died. When the amount of T-4-O is 2MIC, the inhibition rate of Staphylococcus aureus biofilm formation can reach about 50%, which is the most suitable addition concentration to exert good antibacterial effect. In addition, it has been found that T-4-O can also affect the formation of the cell wall of Staphylococcus aureus, resulting in the leakage of cell lysates and proteins. Combined with the measurement of the bacterial cell cycle by flow cytometry, the leakage of intracellular substances led to the inability of Staphylococcus aureus cell growth to proceed normally, and the cell cycle stayed in the R phase, which affected the replication of DAN and led to death, thus exerting an antibacterial effect.

4. 结论
4. Conclusion

GC-MS分析表明，茶树精油（TTO）包括醇类、烯类、烷烃类和酯类共15个化合物，其中松油烯-4-醇（T-4-O）占31.09%，为主要的物质。T-4-O对金黄色葡萄球菌具有良好的抑菌效果，MIC和MBC分别为2.12 mg/mL和8.47 mg/mL。当T-4-O浓度为2MIC时，能基本抑制金黄色葡萄球菌的生长，在该浓度下，T-4-O干扰了细菌生物膜形成的代谢途径，对金黄色葡萄球菌生物膜形成抑制率可达50%左右；能使得细菌细胞膜可能发生局部位移，并影响了细胞壁的形成，细胞溶出物、蛋白质等物质发生泄露，菌体无法正常分裂、生长，细胞生长周期一直停留在R期，最终使菌体细胞膜发生破裂、死亡，从而起到了抑菌效果。
GC-MS analysis showed that tea tree essential oil (TTO) included 15 compounds including alcohols, alkenes, alkanes and esters, among which terpinene-4-ol (T-4-O) accounted for 31.09%, which was the main substance. T-4-O had a good antibacterial effect on Staphylococcus aureus, with MIC and MBC of 2.12 mg/mL and 8.47 mg/mL, respectively. When the concentration of T-4-O is 2MIC, it can basically inhibit the growth of Staphylococcus aureus, and at this concentration, T-4-O interferes with the metabolic pathway of bacterial biofilm formation, and the inhibition rate of Staphylococcus aureus biofilm formation can reach about 50%. It can make the bacterial cell membrane shift locally, and affect the formation of the cell wall, the leakage of cell extractables, proteins and other substances, the bacteria can not divide and grow normally, and the cell growth cycle has been stuck in the R phase, and finally the cell membrane of the bacteria is ruptured and died, thus playing a bacteriostatic effect.