Solvent effects on terpenoid compositions and antioxidant activities of Cinnamomum camphora (L.) J. Presl extracts and the main antioxidant agent evaluation through in vitro and in vivo assay

Plant secondary metabolites can protect organisms against oxidative stress caused by adverse environmental conditions. Cinnamomum camphora (L.) J. Presl contains plentiful terpenoids and is subdivided into 5 chemotypes. To develop


Introduction
In organisms, reactive oxygen species (ROS) and free radicals are produced during the normal metabolic processes, with an equilibrium in the production and scavenging [1].However, the exposure to some environmental factors such as pollution, O 3 , industrial chemicals and stress conditions promotes the accumulation of massive ROS, leading to secondary oxidative stress [2,3].Oxidative stress not only damages protein, lipid and DNA biomolecules [3,4], but could be connected with several diseases in human bodies, such as aging, diabetes, cancer, inflammation, cardiovascular diseases, Alzheimer's and Parkinson's diseases [5,6].Therefore, the effective reduction of ROS and free radicals is crucial to maintaining human health and promoting other organisms tolerating adverse environmental conditions.
Plant secondary metabolites can be considered natural antioxidants, and are being utilized in the production of medicines, cosmetics, foods, etc. [7,8].Among plentiful plant sources, medicinal plants should have the maximum developing potential for their abundant secondary metabolites.Based on this reason, the antioxidant abilities of essential oils and extracts from some medicinal plants have been detected, such as Hertia cheirifolia (L.) Kuntze [9], Berberis calliobotry Bien ex.Koehne [10], Pistacia lentiscus L. [11], Scutellaria baicalensis Geor.[12], Bacopa monnieri (L.) Pennell [13] and Mimosa acutistipula (Mart.)Benth [14].Meanwhile, different solvents led to various extraction effects and antioxidant abilities, e.g., methanol (MeOH) extracts had stronger antioxidant abilities for containing more secondary metabolites in contrast to water extracts [15,16].When Amygdalus communis L. was extracted with water, MeOH and ethyl acetate (EA), the maximum phenolic content was found in EA extracts, while the maximum flavonoid content was detected in MeOH extracts [17].However, the total phenolic content in EA extracts from Onosma pulchra Riedl.was lower than that in MeOH extracts [18].For 7 solvents (water, MeOH, acetone, ethanol, EA, chloroform, and n-hexane (Hex)), the maximum flavonoids and total phenolics were extracted by MeOH and water from Erigeron annuus (L.) Desf.flower, respectively, and MeOH extracts showed the strongest antioxidant ability [19].
Cinnamomum camphora (L.)J. Presl belongs to the genus Cinnamomum in Lauraceae.It is an excellent tree species with producing a wide spectrum of terpenoids to repel insects and is also used for treating diseases as an important Chinese herbal medicine [32,33].According to the typical terpenoid, 5 main chemotypes are distinguished, including linalool, eucalyptol, borneol, camphor and iso-nerolidol chemotype [34,35].In previous study, more terpenoids were extracted by ethanol from the fresh and fallen C. camphora leaves in contrast to water [36,37].The ethanol extracts from the former 4 chemotypes exhibited in vitro antioxidant abilities.Among a year, the extracts in summer showed the strongest antioxidant ability, and linalool and eucalyptol chemotype extracts had the strongest antioxidant ability among the 4 chemotype extracts [38].
In this study, the terpenoid composition and antioxidant abilities in MeOH, EA, Hex and petroleum ether (PE) extracts from linalool and eucalyptol chemotypes in summer were investigated, and the solvent effects on in vitro antioxidant activity of 8 main terpenoids were analyzed.Meanwhile, the in vivo effects of two strong antioxidant terpenoids were evaluated.These findings uncovered the suitable solvent for terpenoid extraction from C. camphora and identified two strong antioxidant agents, which were beneficial to development and utilization of the plant terpenoids as natural antioxidants.

Preparation of extracts from C. camphora
In July, the healthy mature leaves were randomly collected from linalool and eucalyptol chemotypes of C. camphora with height of 10-12 m, and the growth conditions of the trees have been described in previous study [35].In each chemotype, a random selection of 4 plants was carried out for leaf collection, with each as a repeat.These leaves were dried with a freeze dryer and smashed to powder using a pulverizer.The powder of 10 g was separately added into 100 mL MeOH, EA, Hex and PE to extract terpenoids at room temperature.After 48 h, the mixture was centrifuged at 7000 g, and the supernatant (100 mg•mL −1 ) was collected and used for terpenoid composition analysis and antioxidant activity measurement.

Analysis of terpenoid composition in the extracts
The terpenoid compositions in MeOH, EA, Hex and PE extracts were analyzed with a gas chromatography-mass spectrometry (GC-MS), and the operating conditions were described in a previous study [39].Briefly, the GC (7890B, Agilent Technologies, Santa Clara, CA, USA) was run with the column temperature raising from 50 to 180 °C at a rate of 20 °C•min −1 , and from 180 to 220 °C at a rate of 10 °C•min −1 .For MS (5977B, Agilent Technologies), the ionization energy was set at 70 eV, and quadrupole temperature and source temperature were set at 150 °C and 230 °C, respectively.The qualitative analysis of the GC-MS data was performed by searching NIST Library (NIST 14, Gaithersburg, USA).The quantitative analysis of the corresponding terpenoids in the extracts was performed using the standards, including linalool, eucalyptol, ocimene, α-pinene, D-limonene, terpinene, β-pinene and longifolene (Aladdin, Shanghai, China).For other monoterpenoids and sesquiterpenoids, their content was calculated referring to D-limonene and longifolene, respectively.

Measurement of free radical-scavenging activities of the extracts
MeOH, EA, Hex and PE extracts were diluted to 0.25, 0.5, 1, 1.5 and 2 mg•mL −1 using the same solvent.DPPH free radical solution of 0.2 mM was also correspondingly prepared with MeOH, EA, Hex and PE, respectively.According to the description of Burits and Bucar [40], the diluted extracts of 2 mL were added into DPPH free radical solution of 2 mL following a rule of the same solvent, and the optical density at 517 nm (OD 517 ) was recorded after 30 min at 25 °C.For the scavenging activity, it was calculated following the formula, scavenging activity (%) = (Ac -As)/ Ac × 100%, where Ac and As represented the OD value of the control and sample, respectively.The extract concentration with 50% scavenging ability (IC 50 ) was evaluated according to the plot of percentage scavenging ratio against extract concentration.
Following the procedure of Petretto et al. [41], ABTS free radical solution was prepared by using a mixture of potassium persulfate (2.45 mM) and ABTS (7 mM) in MeOH, EA, Hex and PE, respectively.The diluted extracts of 150 µL were mixed with the ABTS free radical solution of 3 mL following the rule of the same solvent, and the OD 734 was recorded after 30 min at 25 °C.The ABTS free radical-scavenging activity was evaluated following the above formula, and the IC 50 was also calculated.

Determination of free radical-scavenging activities of the main terpenoids
The two free radical-scavenging activities of 8 main terpenoids such as linalool, eucalyptol, ocimene, α-pinene, D-limonene, terpinene, β-pinene and longifolene in the two chemotype extracts were determined.These terpenoid solutions of 500 mM were separately prepared with the 4 solvents, including MeOH, EA, Hex and PE.For assaying the DPPH free radical-scavenging activity, a certain amount of terpenoids was added into 2 mL DPPH free radical solution following the rule of the same solvent and supplemented the same solvent to 4 mL.For assaying the ABTS free radical-scavenging activity, a similar operation was performed in a 3.15 mL reaction system, with the ABTS free radical solution of 3 mL.The content of the terpenoids was from 0.015 to 1.5 mmol and 0.015 to 0.3 mmol, respectively, in scavenging DPPH and ABTS free radicals (detailed content of each terpenoid in Additional file 2: Tables S1, S2).

In vivo antioxidant activity assay of ocimene and longifolene
Chlamydomonas reinhardtii, a single-celled model organism, was used to assay the in vivo antioxidant activities of ocimene and longifolene.C. reinhardtii cells were kept in tris-acetate-phosphate (TAP) medium under the condition of 16-h light (30 μmol•m −2 •s −1 ) and 8-h dark at 25 °C.The algal cell cultures of 25 mL in a conical flask (about 5 × 10 6 cells•mL −1 ) were pretreated with 1, 5 and 10 μM ocimene and longifolene for 1 h, respectively, and then they were treated with 4.8 mM H 2 O 2 .After 12 and 24 h, the cell density was determined with a hemocytometer (25 × 16), and the ROS levels were determined during the 24-h treatment.
For measuring the ROS levels, C. reinhardtii pellets were collected by centrifugation at 4000 g, and incubated with 2´,7´-dichlorofluorescein diacetate (H 2 DCF-DA) of 100 μM for 40 min.The non-fluorescent probe of H 2 DCF-DA entering the cells was hydrolyzed to generate 2´,7´-dichlorodihydrofluorescein (DCFH) without fluorescence.However, the DCFH was oxidized by ROS to form fluorescent 2´,7´-dichlorofluorescein (DCF) in the cells.Then, the fluorescence was observed using a fluorescence microscope (Olympus BX51, Japan), and the fluorescence (about 530 nm) intensity was recorded with a flow cytometry (BD Accuri ™ C6 Plus, USA) [42].

Statistical analysis
There were 4 replicates in each measurement, and the statistical analyses were carried out with Origin 8.0 following the Tukey test in one-way ANOVA.

Free radical-scavenging activities of C. camphora extracts
For linalool chemotype of C. camphora, the DPPH and ABTS free radical-scavenging activities gradually enhanced with raising the concentration of MeOH, EA, Hex and PE extracts.Among the 4 extracts, MeOH extracts showed the strongest scavenging activity against DPPH and ABTS free radicals (Fig. 1A and C), with the lowest IC 50 (0.17 and 0.49 mg•mL −1 ) (Fig. 1B  and D).
For the most of terpenoids, their higher content was always detected in the MeOH extracts, while their lower content in the PE extracts (Table 1).For the MeOH extracts, the total content of terpenoids was 16.0% (P < 0.05), 21.4% (P < 0.05) and 28.7% (P < 0.05) higher than that in EA, Hex and PE extracts, respectively (Fig. 3A).

Free radical-scavenging activities of the main terpenoids in different solvents
When MeOH, EA, Hex and PE were used as the reaction media, the DPPH free radical-scavenging activity gradually enhanced with increasing the concentration of the 8 main terpenoids, including linalool, eucalyptol, ocimene, α-pinene, D-limonene, terpinene, β-pinene and longifolene.All terpenoids exhibited the optimum scavenging activity in MeOH among the 4 reaction media (Fig. 4), even if in ocimene this scavenging activity was slightly stronger than that in others reaction media only at concentration ≥ 0.225 mM.Also the ABTS free radical-scavenging activity was highest in MeOH compared to the other media (Fig. 5).

Free radical-scavenging activity differences of the main terpenoids
In MeOH reaction medium, ocimene showed the strongest scavenging activity against DPPH free radical, and the scavenging ratio reached to 86.5% when ocimene content was 0.375 mmol.For longifolene, its scavenging activity was second to ocimene, but was stronger than other 6 terpenoids (Fig. 6A).Similarly, ocimene and longifolene in EA, Hex and PE also exhibited strong scavenging activities against DPPH free radical (Fig. 6B-D).
For ABTS free radical, longifolene showed the strongest scavenging activity in the 4 reaction media, while the scavenging activity of ocimene was second to longifolene.For ABTS free radical, also α-pinene showed a good scavenging activity.For the other terpenoids, the activities were similar and much lower than that of aforementioned terpenoids (Fig. 7).

Effects of ocimene and longifolene on C. reinhardtii growth under H 2 O 2 stress
H 2 O 2 stress significantly (P < 0.05) inhibited C. reinhardtii growth, and the cell density was reduced by 23.0% and 50.0%, respectively, after 12 and 24 h.In the pretreatments with 1, 5 and 10 µM ocimene (O1 + H 2 O 2 , O5 + H 2 O 2 , and O10 + H 2 O 2 ), C. reinhardtii improved the tolerance to the H 2 O 2 stress, and the cell density gradually increased with raising the ocimene concentration (Fig. 8A).The same result but more pronounced was

Effects of ocimene and longifolene on the ROS levels in C. reinhardtii under H 2 O 2 stress
Under H 2 O 2 stress, the ROS in C. reinhardtii cells were accumulated to the highest level after 1 h, and the cells showed strongest fluorescence intensity.However, the pretreatments with ocimene and longifolene remarkably lowered the ROS levels, and the lowering effects gradually enhanced with raising the compound concentration.At the same concentration, longifolene showed stronger lowering effect with respect to ocimene (Fig. 9).

Discussion
Plants synthesize a wide spectrum of secondary metabolites, and different solvents showed various extracting effects on these compounds.Compared with water extracts, MeOH extracts from Gynostemma pentaphyllum Thunb contained more phenolic compounds and showed higher antioxidant ability [15].For grapevine leaves, the types of secondary metabolites in MeOH extracts were 2 folds of those in water extracts, and the terpenoid content in MeOH extracts was 3.9 folds of that in water extracts [39].Among MeOH, water and EA, MeOH exhibited the maximum effect on extracting secondary metabolites from Amygdalus Communis L. hulls [17].Among water, MeOH, acetone, ethanol, EA, chloroform and Hex, the maximum flavonoids were extracted by MeOH from Helianthus annuus L. flower, and the maximum phenolics were extracted by water [19].Compared with ethanol, EA can extract more flavonoids from Amomum compactum Sol.ex Maton fruits [43].
In the present study, the 4 solvents also showed different extracting effects on the terpenoids from linalool and eucalyptol chemotypes of C. camphora, and the maximum terpenoids content was detected in MeOH extracts (Fig. 3).For each terpenoid compound, MeOH always exhibited the maximum extracting effect among the 4 solvents (Tables 1, 2).These results indicate that MeOH is an optimum solvent for extracting plant secondary metabolites from the aspect of extraction efficacy.
For the two chemotypes of C. camphora, the monoterpenoid content in MeOH, EA, Hex and PE extracts was much higher than sesquiterpenoid content (Tables 1, 2).These results were coincident with previous findings in the ethanol extracts [38] and volatile organic compounds (VOCs) [35] from C. camphora.Among the terpenoids, linalool and eucalyptol were typical monoterpenes in the corresponding chemotype [38] and exhibited the highest content in the extracts from corresponding chemotype (Tables 1, 2).For the two chemotypes, there were  obvious differences in the types and content of terpenoids (Tables 1, 2).For the monoterpenoids, their differences were caused by different expression of the genes in methylerythritol-4-phosphate (MEP) pathway and monoterpene synthases, while the sesquiterpenoid differences were caused by different expression of the genes in mevalonate (MVA) pathway and sesquiterpene synthases [35].
For plant secondary metabolites, they can be extracted with different solvents, which result in various antioxidant abilities.In contrast to water extracts, the MeOH extracts from Gynostemma pentaphyllum (Thunb) Makino [15] and Stachys cretica L. [44] showed stronger antioxidant activity, due to their higher content of phenolic and flavonoid compounds.The similar stronger antioxidant activity was also found in ethanol extracts from Sida linifolia Juss ex.Cav.compared with water extracts, due to higher content of secondary metabolites, including terpenoids, flavonoids, phenolics, steroids, alkaloids, tannins and saponins [45].When E. annuus flower was extracted with 7 solvents, the MeOH extracts showed the strongest activity in quenching DPPH free radical and reducing Cu 2+ and Fe 3+ , as well as the strongest protective effect on TM3 mouse Leydig cells against   , 2), and showed stronger antioxidant ability compared with previous ethanol extracts [38], which was associated with the higher terpenoids content in MeOH extracts (Fig. 3).
In the 4 extracts from C. camphora, the terpenoids content was disproportionate with the antioxidant ability (Figs. 1, 2and3).For 8 main terpenoids, they always showed the strongest antioxidant ability with MeOH as the reaction medium (Figs. 4, 5), which might be caused by the strongest polarity of MeOH among the 4 reaction media [30].These results demonstrate that MeOH can provide an appropriate reaction condition for activating terpenoid unsaturated bonds to quench free radicals and ROS, which might also contribute to MeOH extracts exhibiting the strongest antioxidant ability.This might also lead to the stronger antioxidant ability of MeOH extracts compared with previous ethanol extracts from C. camphora [38].
Although terpenoids, phenolics and flavonoids have the potential for developing as natural antioxidants, limited specific compounds are identified as the functional agents.In terpenoids, astaxanthin, belongs to tetraterpenes, is considered as the strongest natural antioxidant, and has been commercially produced but with very limited yield [28,29]. Limonene, linalool, geraniol and camphor were the main monoterpenoids in essential oils from coriander seeds, which were regarded as the main antioxidant compounds without antioxidation evaluation [47].In citrus, limonene was the typical monoterpene, and was identified as the main antioxidant agent by scavenging DPPH free radical [48].In addition, eucalyptol, linalool, α-pinene, γ-terpinene, β-pinene, terpinene-4-ol, myrcene and α-phellandrene have been identified as the antioxidant agents through in vitro assay by quenching DPPH and/or ABTS free radicals [8,30,31].There are different numbers of C = C unsaturated bonds in terpenoids, which are response for the antioxidant activities of these compounds [30].For example, γ-terpinene exhibits strong antioxidant activity for easily losing the H at the 3-and 6-positions in the two C = C unsaturated bonds, and α-terpinene loses the H at the 5-and 6-positions [49].
In this study, linalool, eucalyptol, ocimene, α-pinene, D-limonene, terpinene, β-pinene and longifolene exhibited different scavenging effects on DPPH and ABTS free radicals, with the strongest antioxidant ability with MeOH as the reaction medium (Figs. 4, 5).This result might be due to the strong polarity of MeOH, that easily causing the activation of C = C unsaturated bonds in terpenoids.Among the 8 main terpenoids, ocimene and longifolene showed strong antioxidant abilities, with the strongest scavenging effect on DPPH and ABTS, respectively (Figs. 6, 7).There are 3 C = C unsaturated bonds in ocimene with one conjugated double bond [30], which might lead to its strong antioxidant activity.Longifolene is a tricyclic sesquiterpene with 1 C = C unsaturated bond, of which 1,4-cyclohexadiene moiety might enhance the scavenging activity against ABTS free radical [30].
For in vivo evaluating the antioxidant effects of plant extracts, several organisms and cells have been used as testing materials, e.g., mice fed with the extracts from linden bee pollen [50], Atractylis gummifera L. (Less.)[51], Ganoderma lucidum L. [52] and Artemisia brevifolia L. [53] can improve antioxidant enzyme activities and decline ROS content under stress conditions, and eucalyptus extracts showed the similar effects on chicken [54].When Caenorhabditis elegans and Escherichia coli pretreated with Rosa roxburghii Plena extracts were exposed to paraquat, a decrease was detected in the ROS levels [55].The similar decrease was also found in C. elegans treated with Warburgia salutaris (Bertol.f.) Chiov.bark extracts under H 2 O 2 stress [56].Under H 2 O 2 stress, Schinus terebinthifolius Raddi fruit [57] and Plantago australis Lam.[58] extracts showed obvious effects on reducing ROS levels in yeast cells.However, there are Stress conditions can cause ROS accumulation in organisms and burst in short time.For example, ROS burst was detected in C. reinhardtii cells exposed to NaCl and Na 2 CO 3 [59] as well as cyanobacterial VOCs β-cyclocitral [60] for 0.5 h.In this study, ROS burst was also detected in C. reinhardtii treated with H 2 O 2 for 1 h, and ocimene and longifolene effectively declined the ROS accumulation with dose-dependent (Fig. 9), which resulted in the corresponding protective effects on the cell growth (Fig. 8).Meanwhile, the single treatment with 10 µM ocimene or longifolene did not impact the algal growth (Fig. 8).Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) are essential antioxidant enzymes in scavenging ROS, whose activities can be induced to improve by ROS accumulation [3,61].In this study, the activities of the three antioxidant enzymes significantly (P < 0.05) improved after 24-h H 2 O 2 treatment, but gradually decreased with raising the concentration of ocimene and longifolene, due to the gradual decrease of ROS levels (Additional file 1: Fig. S3).In cells, ROS can attack biomacromolecules, leading to oxidative damage to nucleic acids, membrane lipids and proteins [3,4], and the oxidation of membrane lipids leads to the generation of malondialdehyde which is indicated by thiobarbituric acid reactive substance (TBARS).In the treatment with H 2 O 2 for 24 h, the TBARS content was remarkably higher than the control, but it gradually declined with raising the concentration of ocimene and longifolene, due to the gradual decrease of ROS levels (Additional file 1: Fig. S4).These results indicate that ocimene and longifolene have the potential for developing as natural antioxidant agents without cell toxicity.

Conclusion
For linalool and eucalyptol chemotypes of C. camphora, MeOH extracts exhibited the strongest antioxidant ability among the 4 extracts, due to their highest terpenoid content, indicating that MeOH was the optimum solvent for extracting terpenoids from C. camphora.Meanwhile, MeOH might activate terpenoid unsaturated bonds to improve free radical-scavenging activity, which also contributed to the strongest antioxidant ability of MeOH extracts.Among the 8 main terpenoids in the plant extracts, ocimene and longifolene exhibited strong antioxidant abilities, and can effectively decline ROS levels in in vivo assay without cell toxicity, demonstrating that the two monoterpenoids have the potential for developing as natural antioxidant agents.

Fig. 1
Fig. 1 Scavenging activities of different extracts from linalool chemotype of C. camphora against DPPH (A and B) and ABTS (C and D) free radicals.MeOH Methanol, EA Ethyl acetate, Hex n-Hexane, PE Petroleum ether.Different lowercase letters indicate the significant difference at P < 0.05.Means ± SE (n = 4)

Fig. 2
Fig. 2 Scavenging activities of different extracts from eucalyptol chemotype of C. camphora on DPPH (A and B) and ABTS (C and D) free radicals.MeOH Methanol, EA Ethyl acetate, Hex n-Hexane, PE Petroleum ether.Different lowercase letters indicate the significant difference at P < 0.05.Means ± SE (n = 4)

Table 1
The main terpenoids in different extracts from the linalool chemotype of C. camphora MeOH Methanol, EA Ethyl acetate, Hex n-Hexane, PE Petroleum ether.Means ± SE (n = 4)

Table 2
The main terpenoids in different extracts from the eucalyptol chemotype of C. camphora MeOH Methanol, EA Ethyl acetate, Hex n-Hexane, PE Petroleum ether.Means ± SE (n = 4)