Chemical components of volatile oil from Cinnamomum jensenianum Hand Mazz leaf in Yongzhou , and its antibacterial and antioxidant properties

Purpose: To study the chemical components, and in vitro antibacterial and antioxidant properties of volatile oil extracted from Yongzhou Cinnamomum jensenianum Hand. Mazz leaves. Methods: The extraction process of volatile oil in the leaves of Yongzhou Cinnamomum jensenianum Hand. Mazz was optimized with respect to volatile oil yield, and the oil was subjected to gas chromatography-mass spectrometry (GC-MS) analysis. In vitro antimicrobial activities against bacteria and fungi were evaluated by the filter paper method, while in vitro antioxidant potential was determined by assaying its ability to scavenge DPPH radicals. Results: The optimized extraction conditions for volatile oil from the leaves of Yongzhou Cinnamomum jensenianum Hand. Mazz were 3.5 h extraction time, solid-liquid ratio of 1:10, and soaking time of 4 h, which resulted in volatile oil yield of 2.4 ± 0.19 %. A total of 37 components were isolated and identified, accounting for 99.19 % of the volatile oil. The in vitro antimicrobial activities against bacteria and fungi were significant, with minimum inhibitory concentration (MIC) between 10 and 20 uL. At a volatile oil concentration of 0.09 mg/mL, the DPPH scavenging ability of the oil was stronger than that of BHT. Conclusion: There is a high level of extractability of volatile oil from the leaves of Yongzhou Cinnamomum jensenianum Hand. Mazz. The main component is linalool (94.45 %), and it has good antibacterial and antioxidant properties.


INTRODUCTION
Cinnamomum jensenianum Hand.Mazz is a member of the genus Polygonaceae and is distributed in Hubei, Hunan, Guizhou, Sichuan and Jiangxi Province [1][2][3].Its branches and fruits contain aromatic oils that can be used as industrial raw materials.At present, only few studies have been carried out on the chemical components of Cinnamomum jensenianum Hand.Mazz, and there are only two studies on the analysis of the volatile components of the leaves of Yongzhou Cinnamomum jensenianum Hand.Mazz and their antimicrobial activities.
There are significant differences in the anatomical characteristics, tissue structure, and material content of Cinnamomum jensenianum grown in different regions [4][5][6].Therefore, it is necessary to study the extraction process and the in vitro biological activities of Yongzhou Cinnamomum jensenianum Hand.Mazz [7][8][9].
In this study, volatile oil was extracted from Cinnamomum jensenianum by stream distillation, and analyzed by GC-MS.The results were compared with previous data through database search for qualitative and quantitative analyses.The oil was also assessed for antioxidant property using DPPH* scavenging assay, in addition to determination of its in vitro antibacterial activity.

EXPERIMENTAL
Yongzhou Cinnamomum jensenianum Hand.Mazz was purchased from Hunan Health-Guard Bio-tech Inc. (product number = 151109).The product was shade-dried to a constant weight, and then ground, sifted, sealed in a plastic bag, and stored in the refrigerator at 4 °C

Extraction of volatile oil
The plant product (100 g) was soaked in distilled water in a 2,000-mL round-bottomed flask for a period of time, and extracted with a volatile oil extractor.The oily liquid in the volatile oil extractor was dehydrated over anhydrous Na 2 SO 4 .

Estimation of volatile oil yield
The yield of volatile oil was calculated according to Eq 1.
where C (in %) is the yield of volatile oil; m 1 (in g) is weight of the bottle and oil; m 2 (in g) is the weight of bottle; m 3 (in g) is the weight of dried powder of product.

Influence of duration of extraction on volatile oil yield
Five (5) parts (100 g each) of dry powder were placed separately in 2000-mL round bottom flasks, and 1200 mL of distilled water was added.The mixture was allowed to soak for 8 h, and then extracted with a volatile oil extractor for different periods i.e. 2, 3, 4, 5, and 6 h.In each case, the oily liquid in the volatile oil extractor was collected and dried over anhydrous sodium sulfate.The yield of volatile oil was calculated to determine the optimum extraction time.

Influence of powder: liquid ratio on yield of volatile oil
Five (5) parts (100 g each) of dry powder were put separately in 2000-mL round bottom flasks, and 600, 800, 1000, 1200, and 1400 mL of distilled water were added to different flasks.The mixtures were allowed to soak for 8 h, and then extracted with a volatile oil extractor for 4 h.
The oily liquid in the volatile oil extractor was collected and dried over anhydrous sodium sulfate.The yield of volatile oil was calculated to determine the optimum solid-liquid ratio.

Impact of soaking duration on volatile oil yield
Five parts (100 g each) of dry powder were mixed separately with1200-mL portions of distilled in 2000-mL round bottom flasks for different periods of time, ranging from 2 to 10 h.They were then extracted with a volatile oil extractor for 4 h.
The oily liquid in the volatile oil extractor was collected and dried as usual over anhydrous sodium sulfate, and the oil yield was calculated to determine the optimum soaking time.

Orthogonal experiment
In order to determine the optimum extraction conditions for the volatile oil, the extraction time (A), liquid-solid ratio (B) and soaking time (C) were evaluated, and L9 ( 33 ) was used for orthogonal studies on the basis of outcomes of the single factor extraction studies above.The design is shown in Table 1.

Conditions for GC-MS analysis
The volatile oil was subjected to GC-MS analysis in a Perkin Elmer TurboMass GC mass spectrometer at a temperature of 270 ℃ and 5min desorption.An Agilent HP-1 column of dimensions 30 m × 0.25 mm, and stationary phase thickness of 0.25 μm (J & W Scientific) was used, with helium as carrier gas at the rate of 1 mL/min.The injection volume and split ratio were 1 μL and 20:1, respectively, and the precolumn pressure was

Determination of antibacterial activity of volatile oil
Disc diffusion procedure was used to determine the diameter of the zone of inhibition of the volatile oil.The minimum inhibitory concentration (MIC) was estimated as outlined previously [10].

Evaluation of DPPH radical scavenging capacity of volatile oil
Equal volumes of 0.03, 0.06, 0.09, 0.12, 0.15, and 0.18 mg/mL Cinnamomum jensenianum Hand.Mazz volatile oil in different test tubes were incubated with 2 mL of 0.1 mmol / L DPPH solution for 30 min in the dark.The absorbance (A i ) of each tube was measured at 518 nm [11].A mixture of equal volumes of ethanol solution of volatile oil, and anhydrous ethanol was similarly treated, but without DPPH•, and the absorbance (A j ) was measured at 518 nm.In another tube, equal volumes of ethanol and DPPH• solution were also treated similarly, and the absorbance (A 0 ) was determined.A tube containing BHT in place of volatile oil served as control, and the DPPH• scavenging capacity (W) was estimated using the equation 2.

W = {1-[(A
where W (in %) is the DPPH radical scavenging capacity; A i is the absorbance of volatile oil solution and DPPH; A j is the absorbance of oil solution and anhydrous ethanol; and A 0 is the absorbance of the control.

Statistical analysis
The results are presented as mean ± standard deviation (SD).They were analyzed for statistically significant differences using Duncan multiple comparison (SSR method).All analyses were done with Microsoft Office 2007, Design Expert 8. 0.5b and SPSS.Values of p < 0.05 were taken as indicative of significant differences between treatments groups.

Effect of extraction time on the yield of volatile oil
The yield of volatile oil increased as extraction time was increased, and reached maximum when the duration of extraction was 4 h (Figure 2).However, the yield stabilized beyond 4 h and became almost constant, indicating that the volatile oil was already completely extracted.

Effect of powder:liquid ratio on the yield of volatile oil
The effect of powder : liquid ratio on the yield of volatile oil is shown in Figure 3.The yield of volatile oil increased with increase in the amount of distilled water used.The yield of volatile oil reached maximum at powder : liquid ratio of 1:12.However, with further increase in distilled water, the yield of volatile oil showed a downward trend.

Orthogonal optimization
From Table 2, the order of factors that affect the yield of volatile oils was: A > B > C, and the optimum process parameters were A 3 B 2 C 3 , i.e. extraction time of 5 h, solid-liquid ratio of 1:12, and soaking duration of 10 h.

GC-MS results
Thirty-seven compounds were isolated and identified, accounting for 99.19 % of the volatile oil.The main chemical component was linalool (1,6-octadien-3-ol) which accounted for 94.45 %).The other components were present in trace amounts (Table 3).

Antibacterial activity
As shown in Table 4, the volatile oil exerted the strongest antibacterial ability against Aspergillus niger while the effect on Penicillium, B. subtilis, S. aureus, and E. coli was milder.The antibacterial activity against fungi was stronger than that against bacteria.The minimum inhibitory concentration (MIC) of volatile oil was determined for each strain tested.The MIC value for Aspergillus niger was 10 uL; while the MIC for Penicillium and Staphylococcus aureus was 15 uL.For Escherichia coli and Bacillus subtilis, the MIC was 20 uL.

DPPH• scavenging potential
The DPPH• scavenging ability gradually increased with increase in volatile oil and BHT concentrations.When the concentration of volatile oil reached 0.09 mg/mL, the volatile oil scavenged DPPH• more efficiently than BHT.

DISCUSSION
The essential oil produced by the secondary metabolism of Cinnamomum jensenianum Hand.Mazz is the main active component of the plant.Studies have demonstrated variabilities in characteristics, tissue structure, and material contents of the same plant resources grown in different regions [12][13].This necessitated the study of Yongzhou Cinnamomum jensenianum Hand.Mazz.In this study, steam distillation method was used to extract volatile oil from the plant.Optimization of the extraction process showed that extraction duration of 5 h, solidliquid ratio of 1:12, and soaking duration of 10 h resulted in volatile oil yield of 2.4 ± 0.19 %.This yield is higher than what was obtained in previous studies.
The major component of the volatile oil was linalool, which accounted for 94.45 %.Linalool is widely used in flavors and fragrances, health care, synthetic vitamin A, synthetic anticancer drugs, deodorants, dental applications, insect repellents and insecticides.The annual demand of linalool for the formulation of various flavors worldwide is as high as 10,000 tons [13][14][15].The present study has demonstrated that the volatile oil has good antibacterial and antifungal properties.The MICs were in the range 10 -20 uL, which are better than MICs reported in previous studies.At a concentration 0.09 mg / mL, the DPPH scavenging capacity of the oil was stronger than that of BHT, and its antioxidant activity was also better.The antioxidant and antibacterial properties of the oil may be due to linalool, which is consistent with the report of Deepak [16][17][18][19][20]. Future studies will be focused on the composition, antibacterial activities and antioxidant activities of volatile oils of different origins, so as to provide theoretical support for research and comprehensive development of the volatile oils from this plant.

CONCLUSION
The content of volatile oil in Yongzhou Cinnamomum jensenianum Hand.Mazz is relatively high.The method of steam distillation is a simple and efficient method for obtaining volatile oil from this species.The volatile oil of Cinnamomum jensenianum Hand.Mazz has good antibacterial and antioxidant activities.

DECLARATIONS
8.04 psi (55.44 k Pa).The column temperature was maintained for 3 min at 60 o C, raised at 10 o C per min to 280 ℃, and kept constant for 10 min at 280 o C. The mass detector used functioned at 70 eV within 30 -600 m/z in ionization mode, with an interface temperature of 230 ℃.The MS database was NIST05a.5L.

Figure 1
Figure 1 shows the effect of soaking time on the yield of volatile oil.The yield of volatile oil increased with increase in the duration of soaking, and attained a maximum value at a soaking time of 8 h.Beyond 8 h, the yield of volatile oil was almost constant.

Figure 1 :
Figure 1: Effect of soaking time on the yield of volatile oil.Different letters indicate significant differences between groups

Figure 2 :
Figure 2: Effect of extraction time on the yield of volatile oil.Different letters indicate significant differences between groups

Figure 3 :
Figure 3: Influence of solid (powder): liquid ratio on oil yield.Different letters indicate significant differences between groups

Figure 4 :
Figure 4: DPPH• scavenging capacity of volatile oil, relative to that of BHT.Different letters indicate significant differences between the two.□ = Scavenging by volatile oil; △= Scavenging by BHT

Table 1 :
Design used for orthogonal test

Table 2 :
Outcome of orthogonal experiment

Table 3 :
Chemical components of volatile oil from Yongzhou Cinnamomum jensenianum

Table 4 :
Inhibition zone diameter and MIC of volatile oil from Yongzhou Cinnamomum jensenianum