Chemicals in Essential Oils of Lamiophlomis rotata (Benth.) Kudo and Their Antioxidant Activities

1. Introduction

Lamiophlomis rotata (Benthl) Kudo (L. rotata), a medicinal herb, is the sole member of the Lamiophlomis Kudo of Lamiaceae, which grows at the high altitudes in China [1]. The portion that grows above the ground is collected as the “Duyiwei” (Lamiophlomis herba) with slight fragrance in Chinese materia medica (CMM) [1,2], also known as “Daba” and “Dabuba” in the traditional Tibetan system (TTS) [3]. It has been used to treat rheumatic arthritis and grasserie for more than 2000 years in TTS. According to the theory of Chinese medicine, the tropism of “Duyiwei” is sweet and bitter [2,4].

Due to the low content of volatile components, the researches are mainly focused on the involatile compounds of L. rotata, and some efficacious ingredients such as iridoids, flavonoids, phenylethanoids, have been found [5,6,7,8,9]. However, the petroleum ether extracted part of L. rotata was reported to have the anti-tumor activity, which means its essential oil (EO) may have such activity [10]. Up to now, there is only one paper reported the chemicals in such EOs, and another paper reported the lipophilic composition in the CH₂Cl₂ extracted part, and no-evaluation of their antioxidant activities, to the best of our knowledge [11,12]. In previous report, EOs with light yellow color, yields as 0.1% and 0.23% (volume mL/weight g) had been extracted by steam distillation from the above and below the ground components of L. rotata, respectively. A total of 17 components were identified and quantified, in which, 16 and 13 ones (92.9% and 95.5%) were identified in the aboveground and underground portions, respectively. The main compounds were fatty acids (FAs), especially long-chain FAs (LCFAs, when the chain is not less than ten crabons), n-Hexadecanoic acid (palmitic acid) (50.1% and 34.5%), oleic acid (13.4% and 11.1%), linoleic acid (7.6% and 23.9%), and linoleic acid ethyl ester, which identification is debatable considering its linear retention index (LRI) value, (1.7% and 14.4%) are the four prominent compounds in the aboveground and underground parts of the herb, respectively [11]. A total of 67 components were qualified and quantified in the CH₂Cl₂ extracted part of flower, leaf, and root of L. rotata collected from Tibet, Yunnan, and Qinghai of China. Among them, only 4 FAs accounting for 13.66-46.27%, 4 esters of FAs accounting for 8.77-20.8%, and 35 alkanes ranged from 6.1% to 37.84% were detected. Palmitic acid (PA) (7.08-18.54%), linoleic acid (2.75-19.11%), linolenic acid, methyl ester (8.77-20.8%), and β-sitosterol (13.05-18.00%) were the four prominent compounds [12].

By now, there is no evaluation on the antioxidant activities (AAs) of EOs from L. rotata. However, supplemental FAs have important meaning for keeping the human health since they can keep the oxidant and antioxidant balance in cells [13,14,15,16,17,18,19,20]. The effects of FAs on oxidant injury appears to be related to the degree of FAs unsaturation[14]. Saturated fatty acids (SFAs) such as PA can increase oxidative stress in angiogenic mononuclear cells in a concentration dependant manner [21], but octadecanoic acid was reported to protect pulmonary artery endothelial cell from oxidant injury. Usually, polyunsaturated fatty acids (PUFAs) can reduce oxidant injury [15 +4, 16 +6-19 +9], but there also has exception [14]. The relationship between the degree of FAs unsaturation and susceptibility to oxidant injury remains controversial [13].

In order to study the chemicals in EOs of L. rotata further and try to explore the AAs of such EOs and its representative chemical PA, we have first done an exhaustive exploration on the chemicals present in these EOs and analyzed their AAs through DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), ABTS ((2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), and FRAP (ferric reducing/antioxidant power) assays based on the previously investigation [9], to the best of our knowledge..

2. Results

2.1. Chemicals in the EOs of L. ratata

A light yellow EO with fresh and elegant smell like the lavender flavour was obtained from three samples named L8, L9, and L10 of L. rotata, respectively. The average yield (0.11%) was close to the yield (0.1%) reported previously [11]. In total, 55 compounds were qualified and quantified (Table 1, Figure 1).

The mass spectra of compounds 40, 41, and 42 were highly similar with those of linoleic acid, oleic acid, and octadecanoic acid, respectively, whereas their LRI^c values of 2884, 2770, and 2700, respectively, were significantly different from the corresponding LRI^d values of 3164, 3173, and 3136, respectively. Considering the MS oven temperature program of FFAP, the max calculated LRI^c was 2984, and the chemicals with LRIs^d higher than 2984 such as linoleic acid, oleic acid, and octadecanoic acid, would not be eluted in the employed analytical conditions and would be eluted in the next chromatogram, which would significantly change their LRI^c values. In such a scenario, the compounds 40, 41, 42 were still identified as linoleic acid, oleic acid, and octadecanoic acid, respectively, which were also reported previously [11,12].

In addition, four compounds detected in the total ion chromatograms (TICs), which characteristic ion peaks could be seen in Table 2, could not be elucidated by mass spectra and LRI values, respectively, based on the NIST 14, 17 or other database [22].

Unknown-1 should be an analogue of phytol acetate according to its characteristic ion peaks (Table 2, Figure 2) and LRI value.

Unknown-2 should be an unsaturated long-chain fatty acid (ULCFC) or the corresponding ester based on its characteristic ion peaks (Table 2, Figure 3) and LRI value.

The most suitable match for unknown-3 was 1-cyclohexenylacetic acid, which has a M_w (molecular wight) of 140 (Figure 4), whereas its M_w should be beyond 140 because of the m/z 149 displayed as one of its characteristic ion peaks, which demonstrates that unknown-4 should be a derivative of 1-cyclohexenylacetic acid. Previously, cyclohexenylacetic acid was reported as a compound in the CH₂Cl₂ extract of L. rotata, which should be corresponding to unknown-3 in this study [12].

The most suitable match of unknown-4 was palmitoleic acid (Figure 5), whereas its LRI^c 2975 was different from the LRI^d 2926 of palmitoleic acid to some extent. Meanwhile, palmitoleic acid was identified as compound 29 with LRI^c 2926, and 9E-hexadecenoic acid was identified as compound 28 with LRI^c 2935. Therefore, this compound should be an analogue of palmitoleic acid and not 9E-hexadecenoic acid.

It should be noted that only eight compounds including hexanal, 1-octen-3-ol, limonene, linalool, α-terpineol, hexahydrofarnesyl acetone, methyl hexadecanoate and n-hexadecanoic acid were detected by MS using DB-5 because of the low concentration of samples. Among them, the contents of limonene, α-terpineol, and n-hexadecanoic acid were relatively high. Whereas, limonene and α-terpineol were undetected previously [11,12]. Considering the EOs extracted from the peels of Citrus reticulata Blanco such as Nanfengmiju (C. kinokuni Hort. ex Tanaka) and C. reticulata ‘Dahongpao’ were also studied at the same time, and limonene and α-terpineol were the major compounds of such EOs, their has the possibility that these compounds were introduced from such EOs [23]. In such scenario, the quantitation results were based on the contents gotten from MS detected with FFAP column.

Twenty-one compounds including hexanal, β-pinene, 1-octen-3-ol, p-cymene, limonene, γ-terpinene, cis-linalool oxide, trans-linalool oxide, α-terpineol, n-tridecane, farnesyl acetone, hexahydrofarnesyl acetone, methyl hexadecanoate, palmitoleic acid, hexahydrofarnesyl acetone, methyl hexadecanoate, unknown-1, methyl stearate, phytol acetate, methyl 5,6-octadecadienoate, unknown-2 and unknown-4 were first reported from the EOs of L. rotata.

The EOs, crystals, and EOs removed crystals were mainly consisted of FAs (62.2-72%, 78.4-82.1%, 50.9-58.7%; the content in sequence were denoted for EOs, crystals, and EOs removed crystals, the same for following) including 11 compounds, especially LCFAs (62-72%, 78.1-81.9%, 50.3-58.1%) inclduing 9 compounds, and their esters (6.6-14.8%, 3.2-7.6%, 12.2-15.3%) including 7 compounds. According to the unsaturation, SFAs (52.4-64.7%, 72.7-77.7%, 27.2-54.2%) including 7 compounds were major. n-Hexadecanoic acid (47.1-60.8%, 61.3-69.2%, 17.8-44.8%) is the most outstanding one, which was in line with the reported results [11,12]. Then, tetradecanoic acid (2.8-3.9%, 4.4-5.6%, 5.4-6.4%) was also reported previously [11,12]. In addition, pentadecanoic acid (0-0.5%, 0.7-0.8%, 0.6-0.8%) with odd carbons detected in this study was reported to have anti-tumor activities [24]. MUFAs (0-5.6%, 4-5.2%, 1.5-14%) including 9E-hexadecenoic acid, palmitoleic acid and oleic acid, and PUFAs (2.7-7.7%, 0.1-1.1%, 0-9.7%) only including linoleic acid were minor. The prominent ones were oleic acid (0-3.7%, 2.9-4.0%, 0-10.0%) also reported previously [11], and linoleic acid (2.7-7.7%, 0.1-1.1%, 0-9.7%) also reported previously [11,12].

The content of PA is relatively higher in crystal, but relatively lower in EO removed crystal compared with that in the corresponding EO, whereas the content of MUFAs or PUFAs is usually relatively lower in crystal, but relatively higher in EO removed crystal compared with that in the corresponding EO.

Among the 7 esters of FAs, methyl linolenate (1.8-3.4%, 0%, 0-1.2%) was reported as the main component previously [12]. The other six esters were first reported, in which, methyl hexadecanoate (1.5-3.9%, 1.6-4.1%, 4.1-8.3%) was prominent. In addition, hexahydrofarnesyl acetone (2-3%, 2.3-3.5%, 5.6-7%) was prominent.

The n-alkanes (0-1.3%, 0.7-1.8%, 2.6-5.5%) with the largest number include 17 ones (C₁₃-C₂₉). Among them, tridecane (0%, 0%, 0-tr%) was first reported in the EOs of L. rotata. Previously, 22 n-alkanes (C_11-C₁₂ and C₁₄-C₃₃) with content 4.6-37.8%, and 13 branched alkanes with content 0.55-6.75% were detected [12]. Two prominent ones were tricosane (0-0.2%, 0.2%, 0.4-0.6%), and pentacosane (0-0.1%, 0.2-0.3%, 0-0.6%). HMs (1-3.7%, 0.1-3.4%, 0.3-0.9%) including four compounds such as β-pinene, p-cymene, limonene, and γ-terpinene were detected. Previously, only α-pinene was detected in flower and leaf of L. rotata with content 0-0.2% and 0-0.2%, respectively [11,12]. AMs (5.2-8.3%, 1.8-3.6%, 6.7-9.4%) including four compounds such as cis-linalool oxide, trans-linalool oxide, linalool and α-terpineol were detected, in which, α-terpineol (2.8-4.1%, 1.1-1.3%, 2.0-4.7%) was prominent, and only linalool was detected in leaf of L. rotata collected in Tibet with content 0.2% previously [12].

The 1-octene-3-ol (0-1.8%, 0-0.7%, 0-1.1%) was reported to have a typical mushroom flavor [25].

2.2. AAs of these EOs and their representative chemicals

Nine samples including E8, E9, E10, C8, C9, C10, RC8, RC9 and RC10 and two chemical standards such as n-hexadecanoic acid and α-terpineol were tested the AAs. First, α-terpineol was chosen as a representative chemical due to its high content detected by GC-MS using DB-5, whereas, it should not be a compound with high content as the analyses above. The data of α-terpineol was still listed. In beginning, the samples and chemical standards in three different concentrations such as 5, 15, and 25 μg·mL^-1 were tested the DPPH free radical scavenging ability, respectively. However, the results showing that the clearance rates of these samples and standards at these concentrations were minute. Following, the concentrations of samples and chemical standards were increased and the volume of DPPH, ABTS and FRAP working solution was reduced accordingly. The IC₅₀ (inhibitory concentration of 50% radical scavenging activity (RSA)) of each sample detected or deduced by DPPH, ABTS, and FRAP, respectively, can be seen in Table 3.

The DPPH results (Figure 6) showed that all samples presented a certain DPPH free radical scavenging rate. Among them, the RC10 showed antagonistic effect with C10, and their clearance rate was higher when they existed alone than when they coexisted.

However, the C8 and C9 demonstrated a synergistic effect with the corresponding RC8 and RC9. The highest DPPH RSA value was 14.7% found in the RC10 at 110 μg·mL^-1, and the IC₅₀ of ascorbic acid was 7.7 μg·mL^-1. Interestingly, the clearance rate of C8, C9 and C10 was minimum when the concentration was 80 μg·mL^-1, respectively, which indicated that some compounds in the crystals acting as a pro-oxidation function may reach the effective concentration. The clearance rate of α-terpineol was only 8.21% at 90 mg·mL^-1. The highest clearance rate of PA was 4.1% at 1.5 mg·mL^-1, and was gradually decreased with the increase of concentration.

The RSA values of the samples detected by ABTS (Figure 7) showed that the crystals may contain more substances to promote oxidation. After removing the crystals, the scavenging rate was increased to some degrees. The highest clearance rate was 23.89% still from RC10 at 110 μg·mL^-1. It is worth noting that most of the samples showed better AAs compared with those in the DPPH experiment, which should be due to the higher reactivities of ABTS radical cations [26]. The scavenging rate of PA was negative in the ABTS experiment. The maximum scavenging rate of α-terpineol was 27.73% at 110 μg·mL^-1.

The FRAP values of the samples and the α-terpineol were nearly the same as that of ascorbic acid at 5 μg·mL^-1 (Figure 8), which indicated that the tested samples had partial electron transfer ability. Interestingly, the PA had a stronger FRAP value compared with that of ascorbic acid. However, the mixture solution of PA with the FRAP working solution is milky white turbid liquid, and there is no dark blue unique to ferrous ions. Since the FRAP working solution was mainly composed of pure water, and the solubility of PA in water is relatively less, it would result in the partial precipitation of PA.

Three kinds of EOs and EOs removed crystals present some AAs, respectively, but not so strong compared with that of ascorbic acid. It should be noteworthy that the crystals usually present weaker AAs compared with that of EOs or EOs removed crystals, and sometimes even present pro-oxidant activities. The PA usually presents pro-oxidant activities and in a concentration dependent manner. At the same time, the EOs removed crystals usually present some stronger AAs compared with that of the corresponding EOs.

3. Discussion

The EOs were mainly composed of LCFAs and their ester, which was in agreement with the previous report [11,12]. The crystals were mainly composed of PA, but had relatively higher content compared with that of EOs. The chemicals, which have high boiling point (BP) such as FAs and their ester, lead to the lower extraction rate compared with that of the EOs from other plants, such as Citrus L. Twenty-one compounds were first reported from the EOs of L. rotata. Seven compounds including n-hexadecanoic acid, tetradecanoic acid, linoleic acid, oleic acid, methyl hexadecanoate, hexahydrofarnesyl acetone, and phytol, were identified as the major chemicals in these EOs, which could be chosen as the chemical markers in such EOs. The EOs extracted from L. rotata presented some similarities with the EOs extracted from M. sylvestris [27], Cirsium japonicum var. ussurience Kitamura, Ixeris dentate and I. stolonifera [28,29], since they were all represented with the high BP compounds such as n-hexadecanoic acid, hexahydrofarnesyl acetone, etc., as the major components.

Besides limonene and α-terpineol, there also has the possibility that three HMs including β-pinene, p-cymene, and γ-terpinene and two AMs including cis-linalool oxide and trans-linalool oxide were introduced from the peels EOs of Nanfengmiju (C. kinokuni) and C. reticulata ‘Dahongpao’ [23].

As for AAs assays, small differences in the experiment process may lead to large differences in results. The results of three detection methods are closely related to the environment such as the ratio of working solution to sample solution, the concentration of the samples, and the intrinsic reactivity to free radicals and other reactive oxygen species (ROS) of an antioxidant [30]. It is hardly to get the same result under the “equal condition” since the result was also effected by environmental factors such as the climate, temperature, etc.. Only the data obtained from the environment at that time can be used to draw conclusions after comparing with those of the positive reference substance.

FAs may constitute an important strategy for protecting cells against oxidant injury [13]. The oxidant injury can be alternately enhanced or reduced by supplemental FAs, depending on their degree of unsaturation [14]. Some investigators have shown that enrichment with SFAs enhances oxidant injury [16,17,18,19]. For example, PA increases oxidative stress in cells in a concentration dependent manner [21,32]. PA can react with cells to generate ROS, reduce the content of NO, and make cells more prone to oxidative stress[32]. Usually, PUFAs can reduce oxidant injury[15,16,17,18,19], since the ROS tend to react with the loosely bound electrons of carbon double bonds found in abundance in the fatty acyl chains of cell membrane lipid bilayers [13,20]. In addition, MUFAs such as cis-vaccenic acid (l8:1, n-7) can also decrease the susceptibility of cultured endothelial cells to oxidant injury [13]. It can be deduced from the result in this study that the MUFAs and PUFAs also have AAs.

However, another study showed that SFAs such as octadecanoic acid protected pulmonary artery endothelial cell from oxidant injury, but PUFAs such as linolenic acid (l8:3, n-6) and eicosatrienoic acid (20:3, n-3) enhanced oxidant injury [14].

The effects of FAs on oxidant injury appear to be related to the degree of FAs unsaturation rather than fatty carboxyl chain length or the position of the double bond system[14]. The relationship between the degree of FAs unsaturation and susceptibility to oxidant injury remains controversial [13].

4. Materials and Methods

Three populations of the aboveground portion of L. rotata, named L8, L9, L10, which were corresponding to the same No. samples in previous research [9], were collected from the BianBa, LeiWuQI, and NaQu counties of Tibet, at the GPS coordinates E:93° W:31°. The collected populations were authenticated by Professor Yi Zhang (Chengdu University of Traditional Chinese Medicine (CUTCM), Chengdu, China) and internal transcribed spacer 2 (ITS2) DNA barcodes in our previous study [9], and voucher samples L8, L9, L10 were deposited in the College of Ethnic Medicine (CUTCM, Chengdu, China) and the Chongqing Academy of Chinese Materia Medica (Chongqing, China). Some non-volatile components of these samples were analyzed previously [9].

n-Hexane for high-performance liquid chromatography (HPLC), linalool (98%+), p-cymene (99%+), α-terpineol (98%+), and nonane (98%) were produced by Adamas Reagent Company Ltd. d-Limonene (96%) was produced by Acros organics, USA. γ-Terpinene (97%) was produced by Wako pure chemical industries, Ltd., Japan. n-Hexadecanoic acid was produced by CATO. n-Alkanes standard solution of C₁₀–C₂₅, produced by Dr. Ehrenstorfer Inc, Germany, and n-octacosane (99%) produced by Aldrich, were used to determine LRIs. The above reagents, and chemicals were all supplied by Shanghai Titan Scientific Co.,Ltd., China.

DPPH, Ascorbic acid, ABTS powder, potassium persulfate (K₂S₂O₈), were all supplied by Shanghai Titan Scientific Co.,Ltd., China.

4.1. Extraction and Separation

The L8, L9, and L10 were crushed to a powder, and weighed 315 g, respectively. The powders were swollen with 3150 mL of pure water (10 volumes) in a round-bottomed flask, and were soaked for 0.5 h at 40 °C, respectively. The EOs were extracted thrice from each of the powders for 5 h by hydrodistillation through Clevenger-type apparatus with n-hexane as the collecting solvent. The water in the light yellow EOs was removed using anhydrous Na₂SO₄. A total of 0.29, 0.26, and 0.19 g, corresponding to 418, 405, and 238 μL, with densities of 0.69, 0.64, and 0.80, yields as 0.13, 0.13, and 0.08 (%, v/w) of the EO was obtained from L8, L9, and L10, respectively.

The EOs of L8, L9, and L10 were stored at 4, -4, and -80 °C, respectively, to evaluate crystallization. Crystals were obtained at 4 and -4 °C for L8, L9, and L10, respectively. At -80 °C, the EOs removed crystals in 4, and -4 °C were all being solid state. As a result, there were three samples as EO, crystal, and EO removed crystal for L8, L9, L10, respectively, corresponding to E8, E9, E10, C8, C9, C10, and RC8, RC9, RC10. The samples were stored in separate screw-capped vials at 4 °C.

4.2. Sample Preparation

The samples of E8, E9, E10, C8, C9, C10, and RC8, RC9, RC10 were diluted in the ratio V_sample: V_n-hexane (HPLC) 1: 1000 (0.1%) for the GC-FID (Flame Ionization Detector) and GC-MS detection using the DB-5 column (30 m×0.25 mm i.d., 0.25 μm film thickness), and were diluted in the ratio V_sample: V_n-hexane (HPLC) 1: 250 (0.4%) for GC-MS detection using a FFAP column (30 m × 0.32 mm×0.5 μm).

First, the samples of E8, E9, E10, C8, C9, C10, RC8, RC9, RC10, and chemical standards such as α-terpineol and n-hexadecanoic acid, were diluted in methanol to the concentrations such as 5, 15, and 25 μg·mL^-1 for DPPH, ABTS, and FRAP detection, respectively. Then, the samples of E8, E9, E10, C8, C9, C10, and RC8, RC9, RC10, were diluted in methanol to the concentrations such as 50, 80, and 110 μg·mL^-1 for DPPH, ABTS, and FRAP detection, respectively. The samples of chemical standards such as α-terpineol was diluted in methanol to the concentrations such as 30, 60, and 90 mg·mL^-1 for DPPH, ABTS, and FRAP detection, respectively. The samples of chemical standards such as n-hexadecanoic acid was diluted in methanol to the concentrations such as 1.5, 3, and 4.5 mg·mL^-1 for DPPH, ABTS, and FRAP detection, respectively. The samples of ascorbic acid were diluted in methanol to the concentrations such as 5, 10, and 15 μg·mL^-1 for DPPH, ABTS, and FRAP detection, respectively.

4.3. GC Analyses

GC–FID analyses were obtained on a GC-2010 (Shimadzu, Japan) with a DB-5 column. The oven temperature was programmed from 60 (3-min hold) to 250 °C at 2.5 °C·min^-1, and then held for 2 min. The carrier gas was nitrogen at a constant flow of 1.7 mL·min^-1. The injector and detector were maintained at 250 °C both. The splitting ratio was 5: 1, and the injection volume was 1 μL.

GC–MS analyses were carried out using a GCMS-TQ8040 (Shimadzu, Japan) matched with a NIST 14 MS database, a DB-5 column, and an FFAP column, respectively. The oven temperature for DB-5 was programmed from 60 (3-min hold) to 280 °C at 2.5 °C·min^-1, and then held for 2 min. The oven temperature for FFAP was programmed from 60 (3-min hold) to 230 °C at 2.5 °C·min^-1, and then held for 2 min. The following parameters were the same for DB-5 and FFAP. The carrier gas was helium, at a constant flow of 1 mL min^-1. The splitting ratio was 100: 1. The solvent delay was 3.0 min. The injector, ion-source, and interface were maintained at 250, 200, and 250 °C, respectively. Electron impact mass spectra were acquired at 70 eV at a scan rate of 3.9 scans·s⁻¹ from m/z 25-450 amu. The injection volume was 1 μL.

4.4. Identification and Quantitation

4.4.1. Identification

The peaks in the TICs obtained by GC-MS were identified by probability-based matching (PBM) first. Since overlapped and embedded peaks typically exist in the TICs, the identification results may be incorrect. In such situations, the characteristic ion peaks were selected and compared with the NIST (National Institute of Standards and Technology) 14 or 17 database or the mass spectra of the standards.

The LRIs were calculated relative to the retention time (t) of the n-alkanes (C₁₀-C₂₅, C₂₈) (t_n, t_n+1) and detected compound x (t_x, t_n≤t_x≤t_(n+1)) by the equation proposed by Van Den Dool and Kratz [33,34].

LRI=100n+100[(t_x-t_n)/(t_(n+1)-t_n)]

(1)

The calculated LRIs were compared with the LRIs of the corresponding chemicals provided in the NIST 17 database, literatures, or standards.

4.4.2. Quantitation

The peak area normalization was used to calculate the relative area percentage of each compound.

4.5. Antioxidant activities

4.5.1. DPPH Assay

A slight improvement was made according to the literature method [35]. Samples 100 μL at different concentrations diluted in methanol (MeOH) were placed in a 96-well microplate and then supplemented with 100 μL of DPPH (100 μmol·L^-1) solution also diluted by MeOH. After incubation for 30 min in the dark at room temperature, the absorbance was measured at 517 nm using a microplate reader. Each sample and standard set up 3 holes. Methanol was served as the blank control. Radical-scavenging activity was calculated as a percentage of DPPH discoloration using the following equation:

RSA (%)=[(A_Blank-A_Sample)/A_Blank]*100.

(1)

In this equation, A_Sample is the absorbance of the reaction mixture containing the sample, and A_Blank is the absorbance of the blank control. Ascorbic acid was used as the positive control. Calculated the inhibition rate in a series of concentration (50-110 μg·mL^-1, diluted in methanol).

4.5.2. ABTS Assay

A slightly modification was made based on the literature description method [36]. The ABTS radical cation (ABTS^•+) solution was prepared by reaction of 5 mL of a 7 mM aqueous ABTS solution and 88 µL of a 140 mM (final concentration 2.45 mM) potassium persulfate (K₂S₂O₈) aqueous solution, which was kept in darkness at room temperature for 16 h. Then, radical cation was diluted with methanol (about 30-50 times) to absorbance value as 0.7±0.02 at 734 nm. Samples 100 μl dissolved in methanol (50–110 μg·ml^-1) was added to 100 μl of ABTS radical solution and mixed totally at room temperature for 6 min, and then, the absorbance at 734 nm was measured through using the microplate reader. Ascorbic acid (5–15 μg·ml^-1) was used as the standard control. The calculation method of radical scavenging activity was consistent with DPPH assay.

4.5.3. FRAP Assay

A slight modification was made based on the literature method [35]. Sample solution 0.1 ml dissolved by methanol (50-110 μg·ml^-1) was added to 0.1 ml of FRAP working solution, which was consisted of acetic acid buffer (0.3 mol·L^-1), TPTZ solution (10 mM), and FeCl₃ (20 mM) solution in this order at a volume ratio of 10: 1: 1 corresponding to 25, 2.5, 2.5 mL, respectively. The mixture was left in the darkness at 37 ℃ for 30 min, which was then immediately placed in a microplate reader to measure the increase of absorbance value at 593 nm.

A calibration curve was found through mixing the obtained 0.1 ml Fe(II) aqueous solutions in concentration range 0.01-0.2 mM with 0.1 ml FRAP reagent. In this measuring system, the total antioxidant capacity was calculated by the Fe (II) equivalents. The concentration (mmol·L^-1) of FeSO₄ was calculated by the absorbance value after reaction demonstrated in the standard curve, which was denoted as the value of FRAP. The higher FRAP value means the stronger antioxidant activity.

5. Conclusions

This study first analyzed the chemcials in EOs, crystals separated from the EOs, and EOs removed crystals of L. rotata and made an evaluation of the AAs of these samples and the representative compound PA, to the best of our knowledge. The EOs and separated crystals were both mainly composed by PA, whereas the latter had higher content. PA presents pro-oxidant activity in a concertration dependence manner, which is in accordance with the former research [21,32]. FAs have important meaning for keeping the oxidant and anti-oxidant balance in cells. This study can give some hints for the full usage of such EOs abundance in FAs.