Variations in Essential Oil Yield, Composition, and Antioxidant Activity of Different Plant Organs from Blumea balsamifera (L.) DC. at Different Growth Times

Blumea balsamifera, also named Ainaxiang, is widely used as an ancient medicinal herb in tropical and subtropical Asia. It is rich in essential oils. In this work the essential oils of B. balsamifera from different plant organs and in different months were extracted, and then analyzed by gas chromatography-mass spectrometry. The results showed that essential oil yield of young leaves was the highest (0.65 mL/100 g), followed by mature leaves (0.57 mL/100 g), and the oil yield was higher in October (0.47 mL/100 g) than other months. A total of 44 compounds were identified, representing 92.64%–96.71% of the oil. Eighteen common chemical components were found among the six plant organs, representing >80% of the oil constituents. l-borneol was the main ingredient in leaves, and its content was the highest in senescent leaves and in December. In the essential oils of young shoots and young stems, the main component was dimethoxydurene. Antioxidant activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene bleaching (BCB) assays. The results indicated that the β-carotene bleaching activity was far stronger than the DPPH radical-scavenging capacity, and the young leaves and young shoots showed stronger antioxidant activity. Dimethoxydurene, β-caryophyllene, and α-caryophyllene play a positive role in good antioxidant activity, while β-eudesmol, phytol, and tetradecanal play a negative role. The antioxidant activity revealed in this study might help in developing this promising bioresource for use in the medicinal and cosmetic industries.


Introduction
Blumea balsamifera (L.) DC, also named as Ainaxiang or Dafeng'ai in China, is a perennial shrub which belongs to the Asteraceae family. It is widely distributed in China, Malaysia, Philippines, Thailand, and Vietnam. B. balsamifera is the most important member of the genus Blumea and is an indigenous herb of tropical and subtropical Asia. The whole plant or aboveground portion is

Yields of Essential Oil
The yields of essential oils from different plant organs in different months ranged from 0.13 mL/100 g to 0.80 mL/100 g in terms of dry weight ( Table 1). The young leaves showed the highest yield (0.65 mL/100 g; mean value for four months), followed by mature leaves (0.57 mL/100 g; mean value for four months). No significant difference was noted between them (p > 0.05), but both showed significant differences with the remaining four plant organs (p < 0.05). The young stems showed the lowest yield (0.14 mL/100 g; mean value for four months), and showed a significant difference with the other five plant organs (p < 0.05). The oil yield was higher in October (0.47 mL/100 g), while the oil yield was the lowest in November (0.36 mL/100 g), but there was no significant difference between the four months (p < 0.05).

Chemical Compositions in Essential Oil
The chemical compositions of B. balsamifera essential oils from different plant organs and different months are presented in Table 2. Since the interaction between part of the plant and months is not significant, the average data of the plant and months was used in Table 2 and Figure 1. A total of 44 compounds were identified, representing 92.64%-96.71% of the oil. Eighteen common chemical components were found among the six plant organs, representing more than 80% of the oil. With the exception of the dead leaves, because of their low dimethoxydurene percentage, the mean concentration of the top six ingredients were: l-borneol, β-caryophyllene, dimethoxydurene, α-caryophyllene, 2,2,8-trimethyltricyclo[6.2.2.01,6]dodec-5-ene, and thujopsene-(I2). Figure 1 shows that the plant organs caused higher changes in the six ingredients of B. balsamifera essential oils than the time of growth. l-borneol was the main ingredient in leaves, and its content was the highest in senescent leaves (43.39%). Variations in oil yields did not show the same pattern as the percentages of l-borneol in the essential oil. In the essential oils of young stems and young shoots, the main composition was dimethoxydurene (34.20% and 25.64%, respectively). Except for the young stems, all the remaining five organs contained xanthoxylin, linalool, (´)-guaiol, (˘)-trans-nerolidol, elemol, ledol, etc. The content of xanthoxylin was the highest (1.46%-3.68%). Except for the young leaves, the remaining five organs all contained dehydroaromadendrene, and the content of the young stems was the highest (1.15%). Except for the young shoots and young stems, the remaining four organs all contained 3,3-dimethyl-6-methylenecyclohexene, chrysanthenone, δ-cadinene, etc. 1-Octen-3-ol was only found in the young leaves, mature leaves, and senescent leaves. [(1S,2S,4R)-1,3,3-trimethyl-norbornan-2-yl] acetate and L-(+)-ascorbic acid 2,6-dihexadecanoate were only seen in the young stems, and the content of L-(+)-ascorbic acid 2,6-dihexadecanoate was higher (4.33%). (+)-α-Longipinene, (´)-globulol, linoleic acid, etc. were present only in young shoots and young stems.

Antioxidant Activity
Since the interaction between part of the plant and months was not significant, the mean data of the plant and months was used in Tables 3 and 4. From Table 3, the DPPH radical-scavenging ability of the essential oils from the six plant organs of B. balsamifera can be arranged in the following descending order: young leaves > mature leaves > young shoots > senescent leaves > dead leaves. The young leaves showed the strongest DPPH radical-scavenging ability (IC50: 3.36 ± 0.68), and a significant difference with senescent leaves and dead leaves (p < 0.05), while the dead leaves showed the weakest DPPH radical-scavenging ability (IC50: 14.86 ± 5.92), and a significant difference with the other plant organs (p < 0.05). The β-carotene bleaching inhibition ability of the essential oils from the six plant organs of B. balsamifera is arranged in descending order as follows: young shoots > young leaves > dead leaves > mature leaves > senescent leaves. The young shoots showed the strongest β-carotene bleaching inhibition ability (IC50: 1.81 ± 0.47), followed by young leaves (IC50: 1.84 ± 0.34), and they showed a significant difference with mature senescent and dead leaves (p < 0.05), while the senescent leaves showed the weakest β-carotene bleaching inhibition ability (IC50: 2.59 ± 0.68), and showed a significant difference with young and dead leaves and young shoots (p < 0.05). From Table 4, the DPPH radical-scavenging ability of the essential oils of B. balsamifera in different months can be arranged in the following descending order: December > October > November > September. The essential oils extracted in December showed the strongest DPPH radical-scavenging ability (IC50: 4.83 ± 2.99), and showed a significant difference with the other three months (p < 0.05), while the essential oils extracted in September showed the weakest DPPH radical-scavenging ability (IC50 value: 9.13 ± 7.31), and a

Antioxidant Activity
Since the interaction between part of the plant and months was not significant, the mean data of the plant and months was used in Tables 3 and 4. From Table 3, the DPPH radical-scavenging ability of the essential oils from the six plant organs of B. balsamifera can be arranged in the following descending order: young leaves > mature leaves > young shoots > senescent leaves > dead leaves. The young leaves showed the strongest DPPH radical-scavenging ability (IC 50 : 3.36˘0.68), and a significant difference with senescent leaves and dead leaves (p < 0.05), while the dead leaves showed the weakest DPPH radical-scavenging ability (IC 50 : 14.86˘5.92), and a significant difference with the other plant organs (p < 0.05). The β-carotene bleaching inhibition ability of the essential oils from the six plant organs of B. balsamifera is arranged in descending order as follows: young shoots > young leaves > dead leaves > mature leaves > senescent leaves. The young shoots showed the strongest β-carotene bleaching inhibition ability (IC 50 : 1.81˘0.47), followed by young leaves (IC 50 : 1.84˘0.34), and they showed a significant difference with mature senescent and dead leaves (p < 0.05), while the senescent leaves showed the weakest β-carotene bleaching inhibition ability (IC 50 : 2.59˘0.68), and showed a significant difference with young and dead leaves and young shoots (p < 0.05). From Table 4, the DPPH radical-scavenging ability of the essential oils of B. balsamifera in different months can be arranged in the following descending order: December > October > November > September. The essential oils extracted in December showed the strongest DPPH radical-scavenging ability (IC 50 : 4.83˘2.99), and showed a significant difference with the other three months (p < 0.05), while the essential oils extracted in September showed the weakest DPPH radical-scavenging ability (IC 50 value: 9.13˘7.31), and a significant difference with the other three months (p < 0.05). The β-carotene bleaching inhibition ability of the essential oils from B. balsamifera in different months could be arranged in descending order as follows: September > December > October > November. The essential oils extracted in September showed the strongest β-carotene bleaching inhibition ability (IC 50 : 1.98˘0.44), followed by December (IC 50 : 2.07˘0.36), and they significant differences with October and November (p < 0.05), while the oils extracted in November showed the weakest β-carotene bleaching inhibition ability (IC 50 : 2.43˘0.69), and showed a significant difference with the other three months (p < 0.05).  The results from Figure 2 and Table 2 show that these components including dimethoxydurene (15), β-caryophyllene (16), α-caryophyllene (17), β-eudesmol (33), tetradecanal (37) and phytol (41) are related to the antioxidant activity and are relatively high in the essential oil. significant difference with the other three months (p < 0.05). The β-carotene bleaching inhibition ability of the essential oils from B. balsamifera in different months could be arranged in descending order as follows: September > December > October > November. The essential oils extracted in September showed the strongest β-carotene bleaching inhibition ability (IC50: 1.98 ± 0.44), followed by December (IC50: 2.07 ± 0.36), and they significant differences with October and November (p < 0.05), while the oils extracted in November showed the weakest β-carotene bleaching inhibition ability (IC50: 2.43 ± 0.69), and showed a significant difference with the other three months (p < 0.05).

Overall Impact of Plant Organ and Growth Time on the Essential Oils of B. balsamifera
The results from Figure 2 and Table 2 show that these components including dimethoxydurene (15), β-caryophyllene (16), α-caryophyllene (17), β-eudesmol (33), tetradecanal (37) and phytol (41) are related to the antioxidant activity and are relatively high in the essential oil.   Table 2.

Discussion
The oil yield of this plant has been researched before. Pang et al. obtained an oil yield of 3.2-4.3 mg/g (unpublished data) from plants collected in Hainan, China. He et al. reported that the oil yield is at least 2.5 mg/g in plants from Guizhou, China, and it could be increased to 7.72 mg/g if the plants were fertilized in a certain way [18]. There is no prior research discussing the differences among different plant organs at different times. The results in our research indicated that there were significant differences in oil yields of different plant organs and the same plant organ in different months. The essential oil yield of young leaves was the highest, followed by mature leaves and senescent leaves, and the oil yield was higher in October. Chen et al. found that glandular hairs are the volatile oil generating organ of B. balsamifera, and the density of glandular hairs is the biggest in the young leaves, and with the continuous development of leaves, the density of glandular hairs becomes smaller (unpublished conclusions).This conclusion explains well why the oil yield of young leaves was the highest.
Wang et al. [21] reported that the main constituents in essential oils of B. balsamifera leaves extracted by steam distillation are l-borneol (46.56%), xanthoxylin (8.92%), (E)-caryophyllene (8.75%) and (+)-γ-gurjunene (3.76%), Xia et al. found that there was big difference in the content of components of different regions by selecting B. balsamifera from fourteen different regions to establish a GC chromatographic fingerprint [16]. Except for l-borneol, the main constituents are different in the above studies. This may be because of the different origin, plant organs, different methods of extraction and GC-MS analysis conditions. Antioxidants scavenge free radicals mainly by hydrogen atom transfer and electron transfer [22,23]. Therefore, it is more accurate to evaluate the antioxidant activity of the sample through several testing methods. In this study, the antioxidant activity was evaluated using DPPH and BCB assays, and the results proved that the essential oils of B. balsamifera showed a certain antioxidant activity. By comparing the IC 50 values in the DPPH and BCB tests, it can be concluded that β-carotene bleaching inhibition activity of different plant organs and the same plant organ in different months is far stronger than the DPPH radical-scavenging capacity, which indicates that the essential oils showed stronger antioxidant activity in a lipid system. The young leaves showed stronger antioxidant activity in both the experiments, and showed a significant difference with senescent leaves and dead leaves.
To summarize the original information, for example, the impact of growth time and type of plant organ on the composition of essential oils and antioxidant activity of B. balsamifera plants and to identify the association between the antioxidant effects and the chemical ingredients of the essential oils from B. balsamifera, a PCA was used. The content of β-eudesmol and phytol was high in the essential oils extracted from dead leaves and it also had higher IC 50 values using the DPPH method, which indicates that it possessed lower anti-radical activity. The content of tetradecanal was high in the essential oils from the senescent leaves and was also gave higher IC 50 values using the BCB method, which indicates that it possessed lower antioxidant activity.
The content of dimethoxydurene and α-caryophyllene was high in young shoots, and the content of dimethoxydurene, β-caryophyllene, and α-caryophyllene was also high in young leaves. All of them had lower IC 50 values in the DPPH and BCB methods, which indicate that it possessed higher antioxidant activity. These components might play a synergistic role as a good antioxidant. However, further studies should be performed to confirm the association between the antioxidant effects and the chemical ingredients of the various essential oils from B. balsamifera.

Plant Material
The experiments were performed in the experimental field of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (Danzhou, Hainan, China; localization 19.52˝N, 109.50˝E; altitude 118 m; annual average precipitation 1815 mm; annual average temperature 23.5˝C; the soil characteristics are presented in Table 5). The experimental B. balsamifera plants were one-year old, and were propagated by the seeds collected from B. balsamifera planted in the experimental field of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences. They were planted with a planting spacing of 80 cmˆ80 cm. On the 20th day of each month (from September 2014 to December 2014, which is the traditional harvest time), 30 one-year old B. balsamifera plants were randomly collected. Their young leaves (leaves on young shoots, Figure 3A), mature leaves (leaves which are mature but without yellow spots, Figure 3B), senescent leaves (leaves with yellow spots and those with dark brown leaf tips, Figure 3C), dead leaves (leaves that have turned dark brown, Figure 3D), young shoots (stems from buds to 10-20 cm part without woody parts, Figure 3E), and young stems (green stems and not completely woody, Figure 3F) were collected. These samples were divided into three parts (replicates), dried under shade, and ground to a fine powder (20-mesh sieve), packed in zip-lock bags, and stored in the refrigerator (4˝C) for oil extraction. Voucher specimens were deposited in the Herbarium of Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences.

Plant Material
The experiments were performed in the experimental field of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (Danzhou, Hainan, China; localization 19.52° N, 109.50° E; altitude 118 m; annual average precipitation 1815 mm; annual average temperature 23.5 °C; the soil characteristics are presented in Table 5). The experimental B. balsamifera plants were one-year old, and were propagated by the seeds collected from B. balsamifera planted in the experimental field of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences. They were planted with a planting spacing of 80 cm × 80 cm. On the 20th day of each month (from September 2014 to December 2014, which is the traditional harvest time), 30 one-year old B. balsamifera plants were randomly collected. Their young leaves (leaves on young shoots, Figure 3A), mature leaves (leaves which are mature but without yellow spots, Figure 3B), senescent leaves (leaves with yellow spots and those with dark brown leaf tips, Figure 3C), dead leaves (leaves that have turned dark brown, Figure 3D), young shoots (stems from buds to 10-20 cm part without woody parts, Figure 3E), and young stems (green stems and not completely woody, Figure 3F) were collected. These samples were divided into three parts (replicates), dried under shade, and ground to a fine powder (20-mesh sieve), packed in zip-lock bags, and stored in the refrigerator (4 °C) for oil extraction. Voucher specimens were deposited in the Herbarium of Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences.

Chemicals
Vitamin C and thiobarbituric acid were purchased from Solarbio (Beijing, China).

Extraction of Essential Oil
The essential oils from the six plant organs in four months were extracted using hydrodistillation according to the method noted in Pharmacopoeia of the People's Republic of China [24]. Precisely weighed B. balsamifera powder (200 g) was mixed with distilled water (3000 mL) The mixture was heated, and kept at a low boil for 4.5 h till the amount of oil in the vessel no longer increased, and then heating was stopped. After 1 h, the volume of essential oil was recorded. There were 24 samples and each sample was divided into three parts (replicates) after being collected from the experimental field. The percentage of essential oil yield was calculated using the formula volume of essential oil divided by the weight of sample powder. The volatile extract obtained was kept at 4˝C after drying with anhydrous sodium sulfate. The upper yellow oil was used as the sample for further analysis.

Analysis of Essential Oil
A GCMS-QP2010 Plus Mass Spectrometer (Shimadzu, Kyoto, Japan), equipped with a DB-5 MS capillary column (30.0 mˆ0.25 mm; film thickness, 0.25 µm) and a mass spectrometry (MS) detector, was used for GC-MS analysis. The injector temperature was 250˝C. The oven temperature was programmed from 50˝C (1 min isothermal) to 180˝C at a rate of 5˝C/min and then to 250˝C at a rate of 10˝C/min, and then kept for 6 min. The interface was kept at 280˝C. The mass spectra were obtained at 70 eV. The sector mass analyzer was set to scan from 30 to 550 amu. Helium was used as a carrier gas with a flow rate of 1 mL/min. Essential oil (0.1 mL) of sample was injected (in split mode 20:1). Volatile oil components were calculated as a relative percentage of the total oil using peak area. Retention index of all the components were determined by Kovats method using n-alkanes (C6-C32) as standards. Identification of individual constituents was accomplished by comparing their MS spectra by matching the mass spectral data with those from the NIST (NIST 08, NIST 08s; National Institute of Standards and Technology, Gaithersburg, MD, USA), and by comparison of their MS spectra and GC retention indices with those of standard compounds available in the laboratory (β-caryophyllene, l-borneol and camphor) and also by comparison with some other relevant references [1,9,13,15].

DPPH Radical-Scavenging Assay
First, the essential oils (separately obtained from the five plant organs in four months) and vitamin C, which was a positive control, were formulated into 1, 5, 10, 20, and 40 mg/mL ethanol solutions. The DPPH was formulated as a 1ˆ10´4 mol/L ethanol concentration. The test solution (100 µL) and DPPH solution (200 µL) were placed in a 96-well plate as the test group. The control group contained ethanol (100 µL) and DPPH solution (200 µL), and the blank group contained test solution (100 µL) and ethanol (200 µL). After spotting was completed, the 96-well plate was placed into the Multiscan Spectrum (Multiskan GO 1.00.40, Thermo Fisher Scientific Inc., Waltham, MA, USA), shaken for 30 min, following which its absorbance was measured at 517 nm. The radical-scavenging activities (expressed as percentage inhibition of DPPH) were calculated based on the formula: inhibition percentage (Ip) = [1´(As´Ab)/Ac]ˆ100%, where As, Ab, and Ac are the absorbance values of the test sample, blank sample, and control sample, respectively. The assay was repeated three times for each sample. The antioxidant activity of the essential oils was expressed in terms of half maximal inhibitory concentration (IC 50 ).

β-Carotene Bleaching Test
Antioxidant activity of the essential oils was determined on the basis of slightly modified version of the β-carotene bleaching (BCB) method [25]. β-Carotene (0.1 mg) was dissolved in chloroform (10 mL), followed by the addition of 100 mg Tween 40 and 20 mg linoleic acid. After evaporation to dryness under vacuum at 50˝C using a rotary evaporator, 50 mL oxygenated distilled water was added. The mixture was emulsified for 1 min in a sonificator to form emulsion A. The essential oils which were separately obtained from the five plant organs in four months were formulated into 0.1, 0.5, 1, 2, 3, 4, and 6 mg/mL ethanol solutions as the test samples. Next 200 µL of each sample was mixed with 5 mL of emulsion A in open-capped cuvettes; 200 µL of ethanol and 5 mL of emulsion A were prepared as the control. The absorbance of all samples was measured immediately (t = 0 min) and after 120 min on a spectrophotometer (UV-2102PC/PCS, UNIC (Shanghai) Instrument Co., Ltd., Shanghai, China) at 470 nm. The cuvettes were thermostated at 50˝C between measurements. All determinations were performed in duplicate. Another emulsion (emulsion B) consisting of 20 mg linoleic acid, 100 mg Tween 40, and 50 mL oxygenated water was also prepared. Emulsion B (5 mL) with 200 µL ethanol was used to zero the spectrophotometer. All determinations were performed in duplicate. The inhibition was calculated on the basis of the formula: % inhibition = [(A A(120)Á C(120)) /(A C(0)´AC(120)) ]ˆ100, where A A(120) is the absorbance of the test sample at t = 120 min and A C(0) is the absorbance of the control at t = 0 min. A C(120) is the absorbance of the control at t = 120 min. The antioxidant activity of the essential oils was expressed in terms of IC 50 .

Statistical Analysis
The results of the yield of essential oils and antioxidant activity were expressed as mean valuess tandard deviation. One-way analysis of variance and multiple comparison methods were used to measure significant difference in the oil yield. Double factor variance analysis was used to measure significant difference in the antioxidant activity. Principal component analysis (PCA) was used to find the association between the antioxidant effects and the chemical ingredients of the essential oil which were separately obtained from the five plant organs in four months. Statistical analysis was performed using SPSS version 19.0 software (IBM Company, Chicago, IL, USA). Results were considered statistically significant when p < 0.05.

Conclusions
The results of this study indicate that time of growth and type of B. balsamifera plant organs influence the production of oil, its composition, and antioxidant activity. The essential oil level in the young leaves was the highest, followed by mature leaves and senescent leaves, and the oil content was higher in October. A total of 44 compounds were identified. In the essential oils of leaves, the main ingredient is l-borneol, and the content was the highest in senescent leaves and in December. Variations in oil yields did not show the same pattern as the percentages of l-borneol in the essential oil. In the essential oils of young shoots and young stems, the main composition was dimethoxydurene. Therefore, the time of harvest and type of plant organs should be distinguished based on the different harvesting purposes. To extract the volatile oil, the aboveground parts except stems in October should be chosen for harvest. To get a high content of l-borneol in volatile oil, it is more appropriate to select the leaves in December. The antioxidant activity was evaluated using DPPH and BCB assays in this study, and the results proved that the essential oils of B. balsamifera showed a certain antioxidant activity, and the β-carotene bleaching activity is far stronger than the DPPH radical-scavenging capacity. The young leaves and young shoots showed stronger antioxidant activity due to the high content of dimethoxydurene, β-caryophyllene, and α-caryophyllene. These results indicate that the essential oils of B. balsamifera have the potential to be developed into natural antioxidants for the food and cosmetics industries, while the application range and effect remain to be confirmed by further studies.