Journal of Applied Botany and Food Quality 91, 171 - 179 (2018), DOI:10.5073/JABFQ.2018.091.023 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, China 2 Guangdong Technology Research Center for Traditional Chinese Veterinary Medicine and Natural Medicine, South China Agricultural University, Guangzhou, China 3 Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou, China 4 Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, China Alkaloid content and essential oil composition of Mahonia breviracema cultivated under different light environments Yanqun Li1,2,3, Dexin Kong1, Hui-Ling Liang4, Hong Wu1,2,3,* (Submitted: January 9, 2018; Accepted: May 22, 2018) * Corresponding author Summary Light can affect the yields of alkaloid and essential oil in the syn- thesis of secondary metabolites directly or indirectly through plant growth. Despite Mahonia breviracema being an endemic medicinal species in China, research on the influence of light on production of alkaloid and essential oil is scarce. Thus, this research evaluated the influence of various lighting conditions on alkaloid yields and the composition and yields of the essential oils of M. breviracema. The results revealed significant differences in alkaloid yields, oil yields and chemical characteristics of M. breviracema grown in four dif- ferent light intensities from 10 to 100% full sun shine. The total amount of alkaloids in plants under I30 and I50 was higher than that under I10 and I100 due to the higher biomass of plants. Oil yield of M. breviracema leaf increased linearly with the increase of light incidence. Plants grown under I10 had less plastoglobuli, which co- incided with the lowest oil yield (1.91 g kg-1). The plastoglobuli in chloroplasts increased when the irradiance levels increased, resul- ting in the highest oil yields under I100 (4.53 g kg-1). The principal components in the leaves of M. breviracema were hexadecanoic acid (10.54-72.19%) and α-ionone (1.25-42.39%). The highest hexadeca- noic acid content was obtained under I50, followed by I30, and the highest α-ionone content was obtained under I100. Therefore, it is ne- cessary to control the light environment to obtain raw materials with high quality. Keywords: Mahonia breviracema; Irradiance; Chloroplast; Alka- loids; Essential oil Introduction Mahonia is a genus of about 60 species which are distributed in Southeast and East Asia. A total of 35 species of Mahonia have been found in China, 20 of which are regarded as medicinal plants (HE and MU, 2015). The roots and stems are used as raw materi- als “GongLaoMu” recorded in the Chinese Pharmacopoeia (CHINA PHARMACOPEIA COMMISSION, 2010). It is a medicinal plant traditio- nally used to treat aridity, clear heat, and relieve cough and phlegm (CHINA PHARMACOPEIA COMMISSION, 2010). The leaves of Mahonia plants are the raw material “GongLaoYe” (YE, 2009), which has cu- rative effects of clearing heat, relieving cough, and reducing sputum antioxidant and anti-malignant tumor. The major bioactive compo- nents of “GongLaoMu” are alkaloids, including jatrorrhizine, palma- tine and berberine. “GongLaoYe” contain abundant essential oil (LIU et al., 2010a, 2010b; ZENG et al., 2006). Medicinal plants have been increasingly used, which promotes the loss and exploitation of species. About 40% flora faces the danger of extinction because of human activities, such as excessive collection and extraction of certain species (OLIVEIRA, 2010). Mahonia brevi- racema Y.S. Wang & P.G. Xiao. is genus Mahonia (Berberidaceae.), and it is an endemic genus to Southwest China. M. breviracema is a short shrub mainly distributed in Guangxi, and grows in, coniferous forests, deciduous and evergreen forests. It has been reported that M. breviracema contains high amounts of alkaloids (KONG et al., 2011). In recent years, the number of M. breviracema in wild distri- bution areas has sharply decreased because M. breviracema is not only sold as “GongLaoMu” and M. breviracema but also used as ornamental plants. Plants in the environment different from their natural habitat may show quantitative and/or qualitative changes in their composition in terms of special metabolites. Therefore, explo- ring the influence of irradiation intensity on growth and development of plants, particularly on the accumulation of secondary metabolites, is important to cultivate medicinal plants. Irradiance is an important environmental factor influencing normal physiological functions, plant growth and secondary metabolic pro- duct accumulation (MA et al., 2010). High light intensity enhances the accumulation and synthesis of total flavonoids and phenolics in young Ginger (GHASEMZADEH et al., 2010). However, excessively high light intensity leads to low flavonoid contents and weak pho- tosynthetic capability in Anoectochilus (MA et al., 2010), whereas a decrease in the content of essential oil at low light intensity has been reported in Ocimum basilicum L. (CHANG et al., 2008). The content of sabinene in Origanum vulgare L. ssp. vulgare essential oil is decreased by lower light intensity (DE FALCO et al., 2013). KONG et al. (2016) reported that there were obvious differences in the plas- toglobules of Mahonia bodinieri (Gagnep.) Laferr. leaves at different light intensity conditions, and M. bodinieri leaf oils are complex sys- tems with varying compositions (DONG et al., 2008). The influence of light intensity on the composition of essential oils and the content of alkaloid in M. breviracema has not been reported. Therefore, M. breviracema is used as the material to analyze the content changes of volatile oil and alkaloids under different light conditions through botanical microtechnique, HPLC and GC-MS. Moreover, the opti- mal cultivating condition of M. breviracema is discussed in the pre- sent study. The study result provides technical support and theoreti- cal basis for the breeding and cultivation of M. breviracema. Materials and methods Plant material and growth conditions The experiment was carried out at Guangxi Institute of Botany in Yanshan, Guilin, China. On Marth 15th, 2011, the seeds of M. brevi- racema were sown. Experimental design was performed according to the methods of KONG et al. (2016). On May 15th, 2014, seedlings of M. breviracema with uniform size (40-50 cm in height) were transferred to pots containing peat soil 172 Y. Li, D. Kong, H.-L. Liang, H. Wu and limestone mountain soil (1:1, v/v; pH at 5.8). After one month, the plants were subjected to four treatments of light irradiance (10% (I10), 30% (I30), 50% (I50) and 100% (I100) light-full sun) for 6 months. The full sun treatment in an experiment conducted on the farm was about 2000 ± 20 μmol photons m-2 s-1 at noon, which is determined by a Li-6400 portable photosynthesis system (Li-6400, LICoR, Lin- coln, NE, USA). Light intensity was controlled through the modified method proposed by LIAO et al. (2005). A total of 20 pots included in each treatment were arranged from the north-west to the south-east every day. Biomass Five samples of plants were weighted after being separated into leaves, stems and roots to calculate their total dry biomass. The dry biomass was obtained by using an oven with air ventilation at 60 °C, until constant weight. HPLC analysis Five plants were collected randomly in each treatment to determine the content of alkaloid. The leaves were dried in oven for 20 min at 105 °C to deactivate enzymes and then cooled to 55 °C quickly. Afterwards, stems, roots and leaves were dried for at least 48 h in a oven at 55 °C. The samples were further ground (60 mesh) and preserved in a drying oven. Each sample was accurately weighed as 0.50 g and placed into a 250 mL conical flask; 100 mL of a hydro- chloric acid-methanol solution (1:100, v/v) was added and the mix- ture was heated by reflux for 15 min at 55 °C. After reflux, the mix- ture was extracted with ultrasonication for 45 min; the mixtures were then shaken and cooled to room temperature and filtered. The residue was adjusted to 100 mL of hydrochloric acid-methanol (1:100, v/v), and the above procedure was repeated. Finally, the two filtrates were combined and the mixture was filtered. The filtered fluid was then dried, re-dissolved, and filtered through a 0.22 μm syringe filter prior to HPLC-DAD analysis. An Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, USA) consisting of a vacuum degasser, a G1311A quaternary pump, a G1315A diode array detector (DAD) and a G1329A autosampler was utilized to obtain HPLC chromatograms. The system was controlled by Agilent Chemstation software (Agilent Technologies, Palo Alto, USA). Chromatography was operated on a Gemini C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) maintained at 25 ℃. The mobile phase was performed by using solvent A (0.05 mol L-1 KH2PO4 buf- fer solution, pH 3.0 adjusted with H3PO4) and B (acetonitrile) (72:28, v/v) at a flow rate of 1.0 mL min-1. The pump of the HPLC equipment was carefully washed by 10 % isopropyl alcohol for 10 min before determining the samples, and 10 min was required to equilibrate the column by mobile phase after each sample. The UV spectra were recorded at 265 nm. Identification of jatrorrhizine, palmatine and berberine was performed by comparing the retention times of chro- matographic signals between detected samples and those of the three alkaloids standards under the same experimental conditions. Twenty μL of each sample was injected and HPLC analyses were performed in triplicate for statistical analysis. Preparation of samples and extraction of essential oil Fresh leaves were randomly collected from five plants of M. brevi- racema in each sample. Leaves were dried in shade for 15 days at room temperature. The dried samples were also ground (60 mesh- es) and preserved in a drying oven. The essential oil was extracted through hydrodistillation according to the methods recorded in the Pharmacopoeia of the People’s Republic of China (CHINA PHARMA- COPEIA COMMISSION, 2010). The leaves (15 g each) were conducted with hydrodistillation in a sealed vessel for 5 hours (LI et al., 2013). We determined the yield of essential oils in triplicate, and the mean values were expressed as the results. The volatile oils were kept at 4 °C for further analysis. The average concentration of essential oil was calculated as the weight of oil (g) per 1 kg of dry leaf biomass and essential oil yield per plant (mg plant-1). The plots were analyzed through Origin software. GC–MS analysis The essential oil was analyzed by gas chromatograph (Finnigan Trace GC-2000)-mass spectrometer (Thermo Finnigan, American). A 30 m × 0.25 mm IDHP-1 bonded-phase fused-silica capillary column with a film thickness of 1 μm was used. The temperature of the injector was 150 °C. The initial temperature was maintained at 100 °C for 4 min and then increased to 130 °C at the rate of 5 °C/ min; Afterwards, the temperature was maintained at 130 °C for 20 min. Linear velocity of the helium carrier gas was 1.2 mL.min-1 at the split ratio of 30:1; EI was used as the ion source at 230 °C. Sector mass analyzer scanned from 30 amu to 550 amu. Diluted samples (25 μg/mL) were prepared through methylene dichloride, and 0.4 μL samples were injected. The chemical constituents were identified by comparing with the National Institute of Standards and Technology (NIST05.LIB) library spectra and the literature (ADAMS, 2001). The retention indices were calculated by injecting a series of n-alkanes in the same conditions. Light microscopy and transmission electron microscopy The tissue of leaves was cut into pieces with the size of 1 mm × 1 mm × 0.5 mm. The specimens were processed through the method proposed by LIU et al. (2012). Sections (1 μm thick) were stained with 0.5% Toluidine Blue O and photographed through a Leica EM UC6 ultramicrotome (Leica, Germany). The chloroplasts number per mm2 was counted and averaged within 15 1000 × 1000 mm2 squares randomly selected from the cross sections. For the ultrastructural ob- servations, ultrathin sections (70-90 nm in thickness) were stained with lead citrate and uranyl acetate. A Philips FEI-Technai 12 micro- scope was used to observe the sections. Statistical analysis Data were reported as mean ± standard deviation of at least three ex- periments. One-way analysis of variance (ANOVA) was subjected to using SPSS version 18.0 (SPSS Inc., Chicago, USA). Duncan’s mul- tiple range test was performed to detect differences between treat- ments on each variable (p < 0.05). Results Biomass The results show that light conditions can significantly influence the biomass of root, stem and leaf and total biomass (Fig. 1), which were obviously higher under I30, followed by I50. There was no significant difference between I30 and I50. However, the total biomass under both I30 and I50 was statistically higher than that under I10 and I100. Alkaloids analyses In roots and stems, higher contents of jatrorrhizine and palmatine in M. breviracema were observed under moderate light intensity. Plants grown under I30 and I50 had higher concentrations of jatrorrhizine and palmatine in roots, i.e., 10.83 and 10.84 mg·g-1, and 2.04 and 1.86 mg·g-1, respectively. Higher concentrations of jatrorrhizine and palmatine in the stems were observed under I50, (15.71 and 10.58 mg·g-1, respectively), followed by I30 (13.49 and 10.40 mg·g-1) (Fig. 2, Influence of light on alkaloid content and essential oil composition in Mahonia breviracema 173 Fig. 1: Dry biomass of M. breviracema plant grown under different light intensities. The values are the average ± SE, and different letters indicate that there are obvious differences in the shade treatments (P<0.05). A and C). The highest concentration of berberine were obtained under I10 (11.80 mg·g-1) followed by I50 (11.21 mg·g-1) in the roots (Fig. 2A). While the concentration of berberine in the stems de- creased with the increase of light irradiance, the highest values of berberine were up to 10.83 mg·g-1 under I10 (Fig. 2C). We estimated the changes in total amount of alkaloid in root, stem, leaf and each plant further study the data. The variation of total amount of alka- loids with higher production in the roots and stems was obtained under the conditions of intermediate irradiance (I30 and I50), which showed low values under I10 and I100 (Fig. 2, B and D), although it was not statistically significant among the various light intensities for roots. Moreover, the concentrations of jatrorrhizine and berberine were obviously higher than palmatine in the roots, while palmatine content in the stems was significantly higher than that in the roots. In particular, the concentrations of palmatine under I30 and I50 were higher than berberine (Fig. 2, A and C). The concentrations of jatrorrhizine (0.65-0.35 mg·g-1), palmatine (0.66-0.36 mg·g-1), and berberine in leaves were significantly lower compared to those in the roots and stems (Fig. 2E). Jatrorrhizine was only detected under I50 and I100 (0.65 and 0.35 mg·g-1, respectively), and berberine was not detected under all light treatments (Fig. 2E). Leaf structure There were notable changes taking place in leaf anatomical charac- teristics induced by light intensity. The results showed that the thick- ness of the entire lamina, palisade parenchyma and spongy paren- chyma increased with the increase of light incidence (Tab. 1; Fig. 3). At the same time, chloroplast sizes and numbers in the palisade parenchyma decreased with increasing light intensity (Tab. 2). To study the distribution and accumulation of oil in M. breviracema leaves, we investigated the microstructure of the leaves under differ- ent light intensities. Chloroplast structure of M. breviracema were obviously influenced by light intensity (Fig. 4). Most chloroplasts in leaves grown under I10 and I30 had large size and showed normal ultrastructural organization, with a typical arrangement of stroma and grana thylakoids (Fig. 4, C and F). Few plastoglobules were ob- served under I10 (Fig. 4, A and B). There were more electron-trans- parent plastoglobules in the chloroplasts under I30 (Fig. 4, D and E). Abnormal chloroplast structure with irregular and blurring grana lamellae arrangement was found under I50 (Fig. 4I). Electron-dense plastoglobules was significantly increased for I50 (Fig. 4, G and H). Under I100, the grana were totally ruptured (Fig. 4, K and L), but the chloroplasts were filled with abundant plastoglobules (Fig. 4, J and K). Yield of essential oil The content of essential oil in leaves of M. breviracema grown un- der different light conditions changed significantly. The contents and yields of essential oil in M. breviracema leaves were sensitive to ir- radiance with a rising linear behavior, with the highest values found in plants grown under I100 (4.53 g kg-1), followed by I50 (3.12 g kg-1). Plants grown under I10 had the lowest essential oil yield (1.91 g kg-1) (Fig. 5A). The total amount of essential oil also followed a similar trend (Fig. 5B). Composition of essential oils The irradiance affected the essential oil composition of M. brevi- racema eliciting a variation of 29, 31, 28 and 28 identified compo- nents at I10, I30, I50 and I100, respectively, representing 91.97-97.34% of total essential oils and 26 common peaks (Tab. 3). The study showed that hexadecanoic acid (72.19-10.54%) and α-ionone (1.25-42.39%) were found in greater quantities in M. breviracema leaf oils. The content of hexadecanoic acid was the largest under I50, followed by I30, and the lowest content was found under I100, i.e., only 10.54% of hexadecanoic acid. Interestingly, the α-ionone constituents were significantly increased under I100, leading to a significant increase in the α-ionone content (42.39%); the other treatments only showed 1.25-2.32%. The contents of hexadecanoic acid, methyl ester and hexadecanoic acid, ethyl ester under I10 and I100 (1.48 and 1.83%, 1.20 and 1.77%, respectively) were higher than that of I30 and I50 (0.46 and 0.60%, 0.72 and 0.79%, respectively). Moreover, the changes in the contents of octadecanoic acid, ethyl ester and methyl linolenate with a higher production under intermediate irradiance (I30 and I50) demonstrated that this component was also influenced by irradiance. Nerolidol increased in the treatments with 50% and 100% light. These results showed that the 4 samples mainly contained large le- vels of monoterpenes (2.69-42.95%) and oxygenated sesquiterpenes (13.27-73.33%). Interestingly, the result showed that the variation of monoterpenes and oxygenated sesquiterpenes were inversely related, i.e. a rise of the monoterpenes content accompanied the decrease in the content of oxygenated sesquiterpenes. The leaf oils had the high- est content of monoterpenes (42.95 %) under I100, while the highest oxygenated sesquiterpenes content (73.33 %) was observed under I50. Discussion Irradiance is essential for plant growth, since it affects the primary metabolism providing energy for photosynthesis and generating sig- nals that regulate their development and even interfere with the trans- location of assimilates among different plant organs (LIMA et al., 2010). Plants grown in full light conditions absorb much more light energy in the leaves than the plants require and utilize for photosyn- thetic CO2 fixation (WILHELM and SELMAR, 2011). High irradiation often is co-occurring with water deficiencies (KLEINWÄCHTER and SELMAR, 2015). Therefore, high light levels may reduce plant growth (TANG et al., 2015). The foliar thickness, the ratio of palisade and spongy tissue thickness, as well as palisade and spongy tissue thick- ness increased in M. breviracema with increasing irradiance to cope with these light stresses (ALERIC and KIRKMAN, 2005). The adapta- tion of plant morphology, anatomy to the changes in light intensity may influence the accumulation of secondary metabolites (MA et al., 2010). Similarly, irradiance can influence the production of alkaloids 174 Y. Li, D. Kong, H.-L. Liang, H. Wu Fig. 2: Levels of three alkaloids in M. breviracema roots (A), stems (C) and leaves (E) at various light levels. Estimated total amounts of alkaloid in the roots (B), stems (D) and leaves (F) of a plant under different light intensities (g per plant). The values are the average ± SE. Different letters demonstrate that there are obvious differences in the shade treatments (P<0.05) Tab. 1: The changes of anatomical characteristics of M. breviracema leaves under different light intensity. Light intensity Lamina thickness (μm) Palisade tissue (μm) Spongy parenchyma (μm) Palisade/spongy I10 525.39±16.05d 65.09±2.05d 413.50±2.11d 0.157±0.02c I30 576.78±9.61c 76.09±2.13c 459.05±10.45c 0.166±0.01bc I50 682.56±4.53b 91.50±0.91b 503.09±4.49b 0.182±0.01b I100 742.90±2.14a 131.02±2.57a 571.50±9.87a 0.229±0.01a The values represent the mean ± SE, and the same letters indicated no significant differences in four shade treatment (P<0.05) Influence of light on alkaloid content and essential oil composition in Mahonia breviracema 175 both directly and indirectly by increasing plant biomass (KONG et al., 2016). LI et al. (2009) reported that the contents of berberine, jatror- rhizine and palmatine increased with the light incidence for Amur corktree, while the production of biomass was the highest at 75% full-sunlight treatment. In this study, we reveled that the irradiance levels significantly affected the plant biomass and alkaloid content in different plant organs of M. breviracema. Notably, roots and stems showed higher contents of jatrorrhizine, palmatine and berberine, which were very low or even undetected in the leaves. Jatrorrhizine, palmatine and berberine showed similar trends in roots and stems. However, the content of palmatine in stems was much higher than that in roots. In addition, jatrorrhizine was not detected under I10 and I30 in leaves, which indicated that higher light intensity promotes the synthesis of alkaloids (WILHELM and SELMAR, 2011). The highest contents of jatrorrhizine and palmatine in roots and stems were all observed under I30 and I50, and the content of berberine in roots and stems under I10 was the highest. However, the plant biomass was sig- nificantly higher under I30 and I50 than that under I10 and I100. Thus, the total amount of jatrorrhizine, palmatine and berberine, and the total alkaloid yields in stems, roots and each plant under I30 and I50 were much higher compared with other treatments. In many cases excessive light under nutrient or water limitation stimulates the synthesis of secondary metabolites (WILHELM and SELMAR, 2011). Research on the yield of essential oil under different shade conditions indicates that species have different responses to light intensity, like Lippia sidoides Cham. (SOUZA et al., 2007) and Ocimum basilicum (FIGUEIREDO et al., 2008) showed the increase of essential oil yields when grown at high light intensities. KONG et al. (2016) revealed that plastoglobules in the chloroplasts were sig- nificantly increased at higher light intensity for M. bodinieri. This study showed that not only the numbers of grana lamellae, grana and chloroplasts decreased with the increase of light irradiance intensity, but also the plastoglobules were obviously affected by the light inten- sity for M. breviracema. Notably, we revealed a positive correlation between the number of plastoglobules with the yield of essential oil at different growth stages in M. breviracema. The smaller amounts of plastoglobules were accumulated under I10, which was accordance with a lower yield of essential oil. Our research showed significant chloroplast structural damage and increased plastoglobules with in- creasing light intensity. In particular, the grana totally ruptured and disappeared, but the chloroplasts were filled with abundant plasto- globules. The yield of essential oil rose sharply to 4.53 g kg-1 under I100. These results are in accordance with that of SOUZA et al. (2007) and FIGUEIREDO et al. (2008) in that a higher light intensity enhan- ced the accumulation and synthesis of essential oils. Accumulation and synthesis essential oils in M. breviracem mainly occur in chloro- plasts. Furthermore, these results further confirmed that the increase of plastoglobules were closely related to the reduced chloroplast function and senescence under high light intensity (BISWAL, 1995). In general, the composition of essential oils is very sensitive and can suffer numerous reactions under stress factors (SELMAR and KLEIN- WÄCHTER, 2012; KLEINWÄCHTER and SELMAR, 2015). In the current study, the major and most representative component in all analyzed samples is the saturated fatty acid hexadecanoic acid which is ex- hibited in high percentage in the four samples (10.54-72.19%). This result is similar to that obtained in M. bodinieri (LIU et al., 2010a). Thereinto, the intermediate irradiance (I50) was beneficial for in- creasing hexadecanoic acid. Moreover, in all these samples hexade- canoic acids were found in various forms such as ethyl ester, methyl ester. Hexadecanoic acid isolated from a marine red alga may be a lead compound of anticancer drugs (HARADA et al., 2002) and pre- sents important anti-inflammatory and analgesic activities properties (APARNA et al., 2012; HAMDI et al., 2018). Indeed, hexadecanoic acid derivatives showed and anti-nociceptive anti-inflammatory activities (ZITTERL-EGLSEER et al., 1997; DECIGA-CAMPOS et al., 2007). Such as, hexadecanoic acid, methyl ester is responsible for various phar- macological actions like antimicrobial and antioxidants activities (TAPIERO et al., 2002). The presence of phytochemicals could be con- sidered as sources of quality raw materials for food and pharmaceuti- cal industries. Interesting, we found that α-ionone, another important composition in the oil of M. breviracema, showed highest content in full light conditions. Ionone has been reported as a product of oxi- dative rupture of β-carotene (SÁNCHEZ-CONTRERAS et al., 2000). It is possible to increase the leaf temperature in the high light condi- tions, which is beneficial to the accumulation of carotenoids, while the high temperature stimulates the degradation of carotenoids and the accumulation of aromatic substances (ionone) (KAWAKAMI and KOBAYASHI, 2002; ZEPKA and MERCADANTE, 2009; ZHAN et al., 2012; RAMEL et al., 2012). Carotenoids are sources of essential pre- cursors for the biosynthesis of bioactive compounds in plants when oxidative cleavages occur to form carotenoids derivatives. These compounds serve as signal molecules (RAMEL et al., 2012) and they have been implicated in the interactions of plants with their envi- ronment (light and temperature) (WALTER and STRACK, 2011; NISAR Tab. 2: Changes in number of mesophyll chloroplast (n mm-2) and structural characteristics of chloroplast in leaves of M. breviracema in various light conditions. Light Chloroplast Chloroplast Chloroplast intensity length (μm) width (μm) number (n/mm2) I10 7.14±0.23a 4.27±0.26b 5293±109.41a I30 7.96±0.14a 3.68±0.29b 4403±232.38b I50 7.80±0.47a 3.44±0.68b 3227±186.19c I100 8.18±0.30a 2.62±0.46b 2796±172.05c The values indicate the average ± SE. The same letters show there is no ob- vious difference in the four shade treatments (P<0.05) Fig. 3: Light micrographs of characteristic semi-thin cross-sections in the leaves of M. breviracema at different light intensities. I10 (A), I30 (B), I50 (C), I100 (D). 176 Y. Li, D. Kong, H.-L. Liang, H. Wu Fig. 4: Ultrastructure of chloroplast in the leaves of M. breviracema at different light intensities. I10 (A–C), I30 (D–F), I50 (G–I), I100 (J–L). DP: Electron-dense plastoglobules, G: Granum, TP: Electron-transparent plastoglobules. et al., 2015; BRIARDO et al., 2016). Thereby, carotenoids play an im- portant role on sensing and signalling oxidative stress, as chemical oxidation of b-carotene by 1O2, forming a wide variety of products, such as b-Cyc b-ionone and a-ionone (RAMEL et al., 2012). Hexadecanoic acid and α-ionone are the major components of M. breviracema leaf oils, suggesting a chemotype different from that described by LIU et al. (2010b), where the main component is 4-ter- pineol (43.73%) for Mahonia duclouxiana Gagnep. Hexadecanoic acid is used to treat blood lipids and cardiovascular diseases (CON- SULTATION, 2003). α-ionone is an important material in the flavors and fragrances industry (SELL, 2006) Therefore, M. breviracema has important development potential and high medicinal value. More- over, M. breviracema is a good ornamental plant. Furthermore, the leaf oils also contained high levels of nerolidol and octadecanoic acid. It was reported that sesquiterpene indole (the main volatile of nerolidol) provided indirect plant defense against various herbivores (PACHECO et al., 2016). Octadecanoic acid is an inducible plant defense molecule against insects (MARKO-VARGA et al., 2008). Thereby, the increase of these compounds in leaves un- der high light intensity can indicate that there is an effective defense system for the adverse environmental conditions. Conclusion This research explores the significant differences in the content of alkaloid and chemical characteristics of leaf oil of M. breviracema grown under various light intensities. The contents of jatrorrhizine, palmatine and berberine were notably higher in root and stem sam- ples compared to the leaf samples. By analyzing the variations of alkaloid contents and biomass, I30 and I50 are determined as the best Influence of light on alkaloid content and essential oil composition in Mahonia breviracema 177 Tab. 3: Chemical constituents (%) of essential oil in leaves of M. breviracema under different light intensities. No Compound RIa Relative content (%)b Identification I10 I30 I50 I100 1 2,2,4,6,6-Pentamthyl heptanes 1030 0.23±0.05b 0.43±0.05b 0.17±0.02b 10.37±1.17a GC–MS, RI 2 2,6,8-Trimethyldecane 1121 0.39±0.05b 0.06±0.02c 0.06±0.06c 1.52±1.52a GC–MS, RI 3 α-ionone 1366 1.92±0.18b 2.32±0.21b 1.25±0.16b 42.39±1.65a GC–MS, RI, Co 4 2,6,10-Trimethyl dodecane 1429 0.18±0.03b 0.26±0.01a 0.05±0.01c - GC–MS, RI 5 Geranyl acetone 1434 0.22±0.02a 0.11±0.01b 0.04±0.01c Tr GC–MS, RI 6 2,6,10-Trimethyltetradecane 1555 0.90±0.37a 0.1±0.04c 0.16±0.03bc 0.51±0.02ab GC–MS, RI 7 Nerolidol 1567 0.75±0.06c 0.54±0.03c 1.14±0.09b 2.75±0.20a GC–MS, RI 8 Hexadecane 1600 2.71±0.29a 0.52±0.11d 0.91±0.11c 1.47±0.12b GC–MS, RI 9 1,2,3-Popanetricarboxylic acid, 2-hydroxy-, 1655 1.19±0.05a 0.26±0.04d 0.42±0.10c 0.63±0.07b GC–MS, RI triethyl ester 10 Phytane 1809 0.91±0.02a 0.53±0.11b 0.66±0.08b 0.93±0.10a GC–MS, RI 11 6,10,14-Trimethyl-2-Pentadecanone 1843 - 1.43±0.11a Tr 0.07±0.03b GC–MS, RI 12 Hexadecanoic acid, methyl ester 1908 1.48±0.10a 0.46±0.08d 0.72±0.08c 1.20±0.20b GC–MS, RI 13 α-Farnesylacetone 1914 2.46±0.08a 0.56±0.08d 0.96±0.10c 1.60±0.30b GC–MS, RI 14 Methyl hexadecanoate 1924 2.50±0.04a 0.38±0.05d 0.64±0.04c 0.79±0.11b GC–MS, RI 15 Hexadecanoic acid, ethyl ester 1968 1.83±0.16a 0.60±0.30b 0.79±0.09b 1.77±0.21a GC–MS, RI 16 Hexadecanoic acid 1978 52.23±2.27b 60.05±7.02b 72.19±5.89a 10.54±2.74c GC–MS, RI, Co 17 Octadecanoic acid 2002 6.54±0.88a 1.07±0.09c 1.44±0.21c 3.73±0.65b GC–MS, RI 18 Isochiapin B 2005 1.01±0.03a 0.45±0.12b 0.50±0.10b 0.54±0.10b GC–MS, RI 19 Eicosane 2008 0.41±0.09a 0.12±0.06c 0.12±0.08c 0.27±0.05b GC–MS, RI 20 Phytol 2042 0.28±0.07a 0.27±0.05a - 0.11±0.04b GC–MS, RI 21 9,12,15-Octadecatrienoic acid, Methyl ester 2051 - 1.35±0.19a 1.01±0.19b 0.96±0.12b GC–MS, RI 22 Methyl linoleate 2062 0.21±0.05b 0.44±0.56a 0.11±0.03b 0.10±0.02b GC–MS, RI 23 9-Octadecenoic acid (Z)-, Methyl ester 2078 0.81±0.17a 0.18±0.06b Tr 0.56±0.18a GC–MS, RI 24 Octadecanoic acid, ethyl ester 2083 1.05±0.07b 12.94±3.01a 1.39±0.19b 0.76±0.22b GC–MS, RI 25 Methyl linolenate 2102 - 0.33±0.11a 0.12±0.07b Tr GC–MS, RI 26 Ethyl linoleate 2165 1.81±0.38a 1.24±0.16b 0.71±0.29c 1.15±0.15bc GC–MS, RI 27 Linolenic acid ethyl ester 2173 0.32±0.06a Tr Tr 0.22±0.01b GC–MS, RI 28 3,7,11,15-tetramethyl-, 2201 6.74±1.14b 9.01±0.63a 6.46±0.14b 3.07±0.18c GC–MS, RI [R-(R*,R*-(E))]-2-Hexadedcen-1-ol 29 Docosane 2208 0.55±0.10b 0.42±0.14b 0.36±0.04b 0.88±0.10a GC–MS, RI 30 Carvacrol 2214 0.55±0.13a 0.25±0.10b 0.33±0.03b 0.56±0.02a GC–MS, RI 31 Tricosane 2300 2.04±0.24a 0.66±0.23b 2.43±1.01a 1.93±0.17a GC–MS, RI Monoterpenes 2.69 2.68 1.62 42.95 Sesquiterpenoids 6.72 1.33 1.49 3.37 Oxygenated sesquiterpenes 52.98 60.6 73.33 13.29 Hydrocarbons 6.62 2.25 4.48 5.48 Others 22.98 30.48 14.22 26.88 Total 91.99 97.34 95.14 91.97 a RI=Retention indices on the basis of a homologous series of normal alkanes. b Data are represented as the average ± SD. Values sharing the same small let- ter within a line are not obviously different at P<0.05; GC–MS, gas chromatography – mass spectrometry; Co, co-injection with authentic compounds; –, not detected; Tr (Trace), relative content <0.1%. Fig. 5: The content of essential oil (A) and the total amount of essential oil (B) in leaves of M. breviracema under different light intensities. The columns with different uppercase letters show obvious differences (P<0.05). 178 Y. Li, D. Kong, H.-L. Liang, H. Wu DOI: 10.1002/ffj.1875 GHASEMZADEH, A., JAAFAR, H.Z.E., RAHMAT, A., WAHAB, P.E.M., HALIM, M.R.A., 2010: Effect of different light intensities on total phenolics and flavonoids synthesis and anti-oxidant activities in Young Ginger varie- ties (Zingiber officinale Roscoe). Int. J. Mol. Sci. 11, 3885-3897. 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Higher irradiance is conductive to increasing the yield of essential oil. The essential oil extracted from leaves was rich in hexadecanoic acid and α-ionone. Moderate light conditions (I30 and I50) were suitable for accumulation and synthesis of hexadecanoic acid, and high light intensity (I100) was beneficial for the accumula- tion and synthesis of α-ionone. By analyzing the changes in oil yields and compositions, I50 and I100 are determined the optimal growth environment to get the highest of leaf oils yield or high quality hexa- decanoic acid and α-ionone. These results can be used as a reference for industrial exploiters of M. breviracema essential oils. Acknowledgements This study was supported by the Nature Science Foundation of China (31500261), the Science and Technology Innovation Fund Project on Forestry of Guangdong Province (2017KJCX006), and the Youth Foundation of College of Forestry and Landscape Architecture of South China Agricultural University (201603). 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ZITTERL-EGLSEER, K., SOSA, S., JURENITSCH, J., SCHUBERT-ZSILAVECZ, M., DELLA LOGGIA, R., TUBARO, A., BERTOLDI, M., FRANZ, C., 1997: Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.). J. Ethnopharmacol. 57, 139-144. DOI: 10.1016/S0378-8741(97)00061-5 Address of the corresponding author: H. Wu, E-mail: wh@scau.edu.cn © The Author(s) 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).