Prerpints.org logo
Preprint
Article

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

This version is not peer-reviewed.

Submitted:

25 July 2023

Posted:

25 July 2023

Read the latest preprint version here

Abstract
Due to the low content, very few studies were focused on the essential oils (EOs) of Lamiophlomis rotata (Benth.) Kudo (L. rotata), which has been used to treat rheumatic arthritis and grasserie in China. However, such EOs may have important pharmacological activities such as anti-cancer. To explore the potential of such EOs, we firstly conducted a thoroughly investigation on the chemicals in the EOs and their antioxidant activities (AAs), to the best of our knowledge. Light yellow EOs with fresh and elegant smell were obtained by hydro-distillation with average yield as 0.11% (volume mL/weight g). The crystal was separated from the EO through cryoprecipitation, respectively. Therefore, the EOs, crystals, and EOs removed crystals were studied further. A total of 55 components were qualified and quantified, in which 21 ones were first reported in these EOs. In the EOs, crystals and EOs removed crystals, the main compounds were long-chain fatty acids (LCFAs) and their esters, whereas the crystals presented relatively higher content of LCFAs and the EOs removed crystals presented relatively lower content of LCFAs compared with that of corresponding EOs. The most abundant compound n-hexadecanoic acid (palmitic acid), a kind of LCFAs, presented 47.1-60.8%, 61.3-69.2% and 17.8-44.8%, in the EOs, crystals and EOs removed crystals, respectively. Their AAs of three kinds of EOs, crystals and EOs removed crystals and a chemical marker as n-hexadecanoic acid were evaluated through DPPH (1,1-Diphenyl-2-picrylhydrazy1 radical), ABTS ((2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), and FRAP (ferric reducing/antioxidant power) assays, respectively. Three kinds of EOs and EOs removed crystals presented some AAs, but not so strong compared with that of ascorbic acid. Usually, the EOs removed crystals showed some stronger AAs compared with that of the corresponding EOs. It should be noteworthy that the crystals showed nearly non AAs and even pro-oxidant activities. The palmitic acid (PA) also presented pro-oxidant activities in a concentration dependant manner. It can be deduced that the monounsaturated FAs (MUFAs) including 9E-hexadecenoic acid, palmitoleic acid and oleic acid, and polyunsaturated FAs (PUFAs) only including linoleic acid detected in this study had some AAs. The FAs have important meaning for keeping the balance between oxidant and antioxidant which depends on their unsaturation extent. This study will give some hints for the full usage of such EOs with lower extracted rate and mainly composed of FAs such as PA.
Keywords: 
Subject: 
Medicine and Pharmacology  -   Pharmacy
Bioactive Phytochemicals from Plant Essential Oils

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 LRIc values of 2884, 2770, and 2700, respectively, were significantly different from the corresponding LRId values of 3164, 3173, and 3136, respectively. Considering the MS oven temperature program of FFAP, the max calculated LRIc was 2984, and the chemicals with LRIsd 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 LRIc 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 Mw (molecular wight) of 140 (Figure 4), whereas its Mw 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 LRIc 2975 was different from the LRId 2926 of palmitoleic acid to some extent. Meanwhile, palmitoleic acid was identified as compound 29 with LRIc 2926, and 9E-hexadecenoic acid was identified as compound 28 with LRIc 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 (C11-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 Vsample: Vn-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 Vsample: Vn-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) (tn, tn+1) and detected compound x (tx, tn≤tx≤t(n+1)) by the equation proposed by Van Den Dool and Kratz [33,34].
LRI=100n+100[(tx-tn)/(t(n+1)-tn)]
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 (%)=[(ABlank-ASample)/ABlank]*100.
In this equation, ASample is the absorbance of the reaction mixture containing the sample, and ABlank 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.

Author Contributions

Conceptualization, Z.P. and J.W.; methodology, J.W.; software, C.X., X.Y. and J.W.; validation, Z.P., C.X., X.Y., Y.S., A.U.H. and J.W.; investigation, C.X. and J.W.; resources, Z.P.; data curation, C.X., X.Y. and J.W.; writing—original draft preparation, C.X., X.Y. and J.W.; writing—review and editing, Z.P., C.X., X.Y., Y.S., A.U.H. and J.W.; supervision, Z.P. and J.W.; project administration, J.W.; funding acquisition, Z.P. and J.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 81973567), Chongqing Science and Technology Bureau (Grant No. cstc2020jcyj-msxmX0310), and Chongqing Municipal Health Commission (Grant No. 2020ZY023793; ZY201602104).

Acknowledgments

The authors thank the undergraduates, Hang Shi, Qin Duan, Meiying Luo, Shanshan Jiang, and Churui Xiao, for their contributions.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. The Chinese materia medica; Shanghai Science and Technology Press: Shanghai, China, 1999; Volume 7, p. 848.
  2. Pharmacopoeia committee of the People’s Republic of China. Pharmacopoeia of the People’s Republic of China; China Medical Science and Technology Press: Beijing, China, 2020; Volume Ⅰ, p. 274. [Google Scholar]
  3. Yang, Y.C. Flora of Tibetan Medicine; Qinghai people’s Publishing House: Xining, China, 1991; p. 119. ISBN 7225004263. [Google Scholar]
  4. Zhao, C.X.; Zeng, Y.X.; Wan, M.Z.; Li, R.X.; Liang, Y.Z.; Li, C.Y.; Zeng, Z.D.; Chau, F.T. Comparative analysis of essential oils from eight herbal medicines with pungent flavor and cool nature by GC-MS and chemometric resolution methods. J. Sep. Sci. 2009, 32, 660–670. [Google Scholar] [CrossRef] [PubMed]
  5. Yi, J.H.; Zhang, G.L.; Li, B.G.; Chen, Y.Z. Phenylpropanoid glycosides from Lamiphlomis rotata. Phytochemistry 1999, 51, 825–828. [Google Scholar] [CrossRef]
  6. Yue, H.L.; Zhao, X.H.; Wang, Q.L.; Tao, Y.D. Separation and purification of water soluble iridoid glucosides by high speed counter-current chromatography combined with macroporous resin column separation. J. Chromatogr. B. 2013, 936, 57–62. [Google Scholar] [CrossRef] [PubMed]
  7. Zhang, F.; Wu, Z.J.; Sun, L.N.; Wang, J.; Tao, X.; Chen, W.S. Iridoid glucosides and a C13-norisoprenoid from Lamiophlomis rotata and their effects on NF-κB activation. Bioorg. Med. Chem. Lett. 2012, 22, 4447–4452. [Google Scholar] [CrossRef] [PubMed]
  8. Pan, Z.; Fan, G.; Yang, R.P.; Luo, W.Z.; Zhou, X.D.; Zhang, Y. Discriminating Lamiophlomis rotata according to geographical origin by 1H-NMR spectroscopy and multivariate analysis. Phytochem. Anal. 2015, 26, 247–252. [Google Scholar] [CrossRef] [PubMed]
  9. Wang, J.; Gao, Y.L.; Chen, Y.L.; Chen, Y.W.; Zhang, Y.; Xiang, L.; Pan, Z. Lamiophlomis rotata identifification via ITS2 barcode and quality evaluation by UPLC-QTOF-MS couple with multivariate analyses. Molecules 2018, 23, 3289. [Google Scholar]
  10. Jia, Z.P.; Li, M.X.; Zhang, R.X.; Wang, J.H.; Wang, M.; Guo, X.N.; Shen, T. Vitro screening of the effective antitumor components of Herba Lamiophlomis rotata. Med. J. Nation. Defend Force Northwest Chin. 2005, 26, 173–175. [Google Scholar]
  11. Liu, H.F.; Li, X.; Deng, Y.; Song, X.; Li, H. Study on the chemical constituents of the essential oil from Lamiophlomis rotata. Chin. J. Pharm. Anal. 2006, 26, 1794–1796. [Google Scholar]
  12. Liu, J.; Nan, P.; Wang, L.; Wang, Q.; Tsering, T.; Zhong, Y. Chemical variation in lipophilic composition of Lamiophlomis rotata from the Qinghai-Tibetan plateau. Chem. Nat. Compd. 2006, 42, 525–528. [Google Scholar] [CrossRef]
  13. Hart, C.M.; Tolson, J.K.; Block, E.R. Fatty acid supplementation protects pulmonary artery endothelial cells from oxidant injury. Am. J. Respir. Cell Mol. BioI. 1990, 3, 479–489. [Google Scholar] [CrossRef]
  14. Hart, C.M.; Tolson, J.K.; Block, E.R. Supplemental fatty acids alter lipid peroxidation and oxidant injury in endothelial cells. Am. J. Physiol. 1991, 260, L481–L488. [Google Scholar] [CrossRef] [PubMed]
  15. Dormandy, T.L. Biological rancidification. Lancet 1969, 2, 684–688. [Google Scholar] [CrossRef]
  16. Kehrer, J.P.; Autor, A.P. The effect of dietary fatty acids on the composition of adult rat lung lipids: relationship to oxygen toxicity. Toxicol. Appl. Pharmacol. 1978, 44, 423–430. [Google Scholar] [CrossRef]
  17. Kennedy, J.I.; Chandler, D.B.; Fulmer, J.D.; Wert, M.B.; Grizzle, W.E. Dietary fish oil inhibits bleomycin-induced pulmonary fibrosis in the rat. Exp.Lung Res. 1989, 15, 315–329. [Google Scholar] [CrossRef] [PubMed]
  18. Sosenko, I.R.S.; Innis, S.M.; Frank, L. Polyunsaturated fatty acids and protection of newborn rats from oxygen toxicity. J. Pediatr. 1988, 112, 630–637. [Google Scholar] [CrossRef] [PubMed]
  19. Sosenko, I.R.S.; Innis, S.M.; Frank, L. Menhaden fish oil, n-3 polyunsaturated fatty acids, and protection of newborn rats from oxygen toxicity. Pediatr. Res. 1989, 25, 399–404. [Google Scholar] [CrossRef]
  20. Horton, A.A.; Fairhurst, S. Lipid peroxidation and mechanisms of toxicity. CRC Crit. Rev. Toxicol. 1987, 18, 27–79. [Google Scholar] [CrossRef]
  21. Favre, J.; Yıldırım, C.; Leyen, T.A.; Chen, W.J.Y.; Genugten, R.E.; Golen, L.W.; Garcia-Vallejo, J.J.; Musters, R.; Baggen, J.; Fontijn, R.; Pouw Kraan, T.; Serné, E.; Koolwijk, P.; Diamant, M.; Horrevoets, A.J.G. Palmitic acid increases pro-oxidant adaptor protein p66Shc expression and affects vascularization factors in angiogenic mononuclear cells: Action of resveratrol. Vasc. Pharmacol. 2015, 75, 7–18. [Google Scholar] [CrossRef]
  22. Adams, R.P. Identification of essential oil components by gas chromatography/mass spectrometry, ed. 4.1; Allured publishing: Illinois, America, 2017; pp. 1–804. [Google Scholar]
  23. Wang, J. Alkanes and chemical markers identified in the essential oil from pericarp of Nanfengmiju (Citrus kinokuni Hort. ex Tanaka). J. Mex. Chem. Soc. 2023, 67, 82–93. [Google Scholar] [CrossRef]
  24. Feng, X.Q. Fatty acids and volatile compounds of meat from Cervus elaphus subspecies hybrid offspring; Gansu agricultural university: Gansu, China, 2008. [Google Scholar]
  25. Zhao, Q.Y.; Yousaf, L.; Xue, Y.; Shen, Q. Changes in flavor of fragrant rice during storage under different conditions. J. Sci. Food Agric. 2020, 100, 3435–3444. [Google Scholar] [CrossRef]
  26. Teow, C.C.; Truong, V.D.; Mcfeeters, R.F.; et al. Antioxidant activities, phenolic and β-carotene contents of sweet potato genotypes with varying flesh colours. Food Chem. 2007, 103, 829–838. [Google Scholar] [CrossRef]
  27. Usami, A.; Kashima, Y.; Marumoto, S.; Miyazawa, M. Characterization of aroma-active compounds in dry flower of Malva sylvestris L. by GC-MS-O analysis and OAV calculations. J. Oleo Sci. 2013, 62, 563–570. [Google Scholar] [CrossRef] [PubMed]
  28. Choi, H.S. GC-MS analyses of the essential oils from Ixeris dentate (Thunb.) Nakai and I. stolonifera A. Gray. Korean J. Food Nutr. 2012, 25, 274–283. [Google Scholar] [CrossRef]
  29. Choi, H.S. Chemical composition of Cirsium japonicum var. ussurience Kitamura and the quantitative changes of major compounds by the harvesting season. Korean J. Food Nutr. 2016, 29, 327–334. [Google Scholar] [CrossRef]
  30. Munteanu, I.G.; Apetrei, C. Analytical methods used in determining antioxidant activity: A review. Int. J. Mol. Sci. 2021, 22, 3380. [Google Scholar] [CrossRef] [PubMed]
  31. Wolosiak, R.; Druzynska, B.; Derewiaka, D.; et al. Verification of the conditions for determination of antioxidant activity by ABTS and DPPH assays-A practical approach. Molecules 2022, 27, 50. [Google Scholar] [CrossRef] [PubMed]
  32. Ke, J.; Wei, R.; Liu, Y. Metformin combined with liraglutide has a synergistic protective effect on palmitic acid-induced oxidative damage of endothelial cells. Chin. J. Diabetes Mellitus 2014, 5. [Google Scholar]
  33. Van Den Dool, H.; Kratz, P.D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. 1963, 11, 463–471. [Google Scholar] [CrossRef] [PubMed]
  34. Zhao, C.; Liang, Y.; Hu, Q.; Zhang, T. Review on gas chromatographic retention index. Chinese J. Anal. Chem. 2005, 33, 715–721. [Google Scholar]
  35. Zengin, G.; Sarikurkcu, C.; Uyar, P.; et al. Crepis foetida L. subsp. rhoeadifolia (Bieb.) Celak. as a source of multifunctional agents: Cytotoxic and phytochemical evaluation. J. Funct. Foods 2015, 17, 698–708. [Google Scholar] [CrossRef]
  36. Re, R.; Pellegrini, N.; Proteggente, A.; et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio. Med. 1999, 26, 1231–1237. [Google Scholar] [CrossRef] [PubMed]
Preprints 80453 g001
Figure 1. TIC of E10 detected by Gas Chromatography-Mass Spectrometer (GC-MS) using a FFAP column. Note: Compounds were listed by the corresponding numbers in Table 1. The denoted main compounds were 6 limonene, 3 1-octen-3-ol, 10 linalool, 12 α-terpineol, 23 hexahydrofarnesyl acetone, 27 methyl hexadecanoate, 34 methyl oleate, 33 methyl linoleate, 35 methyl linolenate, 37 phytol, 21 tetradecanoic acid, 31 n-hexadecanoic acid.
Figure 1. TIC of E10 detected by Gas Chromatography-Mass Spectrometer (GC-MS) using a FFAP column. Note: Compounds were listed by the corresponding numbers in Table 1. The denoted main compounds were 6 limonene, 3 1-octen-3-ol, 10 linalool, 12 α-terpineol, 23 hexahydrofarnesyl acetone, 27 methyl hexadecanoate, 34 methyl oleate, 33 methyl linoleate, 35 methyl linolenate, 37 phytol, 21 tetradecanoic acid, 31 n-hexadecanoic acid.
Preprints 80453 g001
Preprints 80453 g002
Figure 2. The mass spectrum of unknown-1 from RC8.
Figure 2. The mass spectrum of unknown-1 from RC8.
Preprints 80453 g002
Preprints 80453 g003
Figure 3. The mass spectrum of unknown-2 from RC9.
Figure 3. The mass spectrum of unknown-2 from RC9.
Preprints 80453 g003
Preprints 80453 g004
Figure 4. The mass spectra of unknown-3 from RC10 and the corresponding match 1-cyclohexenylacetic acid from NIST 14 library.
Figure 4. The mass spectra of unknown-3 from RC10 and the corresponding match 1-cyclohexenylacetic acid from NIST 14 library.
Preprints 80453 g004
Preprints 80453 g005
Figure 5. The mass spectra of unknown-4 from C10 and the corresponding match palmitoleic acid from NIST 14 library.
Figure 5. The mass spectra of unknown-4 from C10 and the corresponding match palmitoleic acid from NIST 14 library.
Preprints 80453 g005
Preprints 80453 g006
Figure 6. The results of DPPH assay. (a) RSA of EO at 50-110 μg·mL–1; (b) RSA of EO removed crystal at 50-110 μg·mL–1; (c) RSA of Crystal at 50-110 μg· mL–1; (d) RSA of PA at 1.5-4.5 mg·mL–1; (e) RSA of α-terpineol at 30-90 mg·mL–1; (f) RSA of ascorbic acid at 5-15 μg·mL–1..The same sequence and concentration for Figure 7 and Figure 8.
Figure 6. The results of DPPH assay. (a) RSA of EO at 50-110 μg·mL–1; (b) RSA of EO removed crystal at 50-110 μg·mL–1; (c) RSA of Crystal at 50-110 μg· mL–1; (d) RSA of PA at 1.5-4.5 mg·mL–1; (e) RSA of α-terpineol at 30-90 mg·mL–1; (f) RSA of ascorbic acid at 5-15 μg·mL–1..The same sequence and concentration for Figure 7 and Figure 8.
Preprints 80453 g006
Preprints 80453 g007
Figure 7. The RSA of ABTS assay.
Figure 7. The RSA of ABTS assay.
Preprints 80453 g007
Preprints 80453 g008
Figure 8. The results of FRAP assay.
Figure 8. The results of FRAP assay.
Preprints 80453 g008
Table 1. The compounds qualified and quantified in EOs from L. rotata.
Table 1. The compounds qualified and quantified in EOs from L. rotata.
No. Compounds CAS LRIsb, d LRIsa LRIsc E8 C8 RC8 E9 C9 RC9 E10 C10 RC10
FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP FID DB-5 FFAP
1 Hexanal 66-25-1 800, 1083 - - 0.1 nd nd 0.3 2 0.1 0.6 3.2 0.2 0.3 nd nd 0.3 nd 0.1 0.6 7.5 0.2 0.3 nd tr 0.3 nd 0.1 0.5 4.7 0.1
2 β-Pinene 127-91-3 970, 1112 - 1114 nd nd tr 0.5 nd nd 1.1 nd nd 4.3 nd nd 1.3 nd nd 2.8 nd nd 4.9 nd nd 1.5 nd nd 2.6 nd nd
3 1-Octen-3-ol 3391-86-4 980, 1450 980 1454 nd nd nd 0.2 nd nd 0.3 nd nd nd nd 1.8 0.3 nd 0.7 0.7 nd 1.1 nd nd 1.6 0.3 3.7 0.6 0.7 nd 1.1
4 Hexanoic acid (6:0) 142-62-1 990, 1846 - 1838 nd nd nd nd nd 0.1 tr nd 0.3 nd nd nd 0.1 nd 0.2 0.2 nd 0.4 0.2 nd 0.1 0.1 nd 0.2 0.1 nd 0.4
5 p-Cymene 99-87-6 1011, 1272 - 1272 1.4 nd 0.2 0.3 nd 0.3 0.3 nd 0.1 1.2 nd nd 0.1 nd tr 0.3 nd tr 0.4 nd 0.1 0.2 nd nd 0.3 nd tr
6 Limonene 138-86-3 1030, 1200 1026 1203 12.8 nd 3.4 4.6 36.4 3 2.3 5.8 0.8 12 nd 1.5 1.0 nd 0.2 3.0 8.1 0.5 3.2 7.3 0.9 1.3 nd 0.1 2 nd 0.3
7 γ-Terpinene 99-85-4 1053, 1246 - 1247 1.6 nd 0.1 0.5 nd 0.2 0.5 nd nd 1.3 nd nd 0.8 nd nd 0.8 nd nd 3.2 nd nd 1.4 nd nd 0.7 nd nd
8 cis-Linalool oxide 5989-33-3 1074, 1444 - 1441 nd nd nd 0.9 nd nd 1.2 nd nd 0.5 nd nd 1.1 nd 0.6 3 nd 1.4 0.5 nd 0.2 1.5 nd 0.6 3.0 nd 1.5
9 trans-Linalool oxide 34995-77-2 1102, 1452 - 1466 nd nd nd 0.8 nd nd 1.0 nd 0.7 0.4 nd nd 1.0 nd 0.5 2.5 nd 1.2 0.4 nd 0.2 1.3 nd 0.6 2.7 nd 1.1
10 Linalool 78-70-6 1082, 1547 1098 1552 0.2 nd 2.4 2.2 6.6 0.7 6.8 20.9 4.0 9.1 nd 4.2 2.9 11.7 1.1 5.5 18.0 2.1 11.0 22.3 4 3.4 22.3 1.2 5.7 22.3 2
11 Caprylic acid (8:0) 124-07-2 1180, 2060 - 2053 nd nd nd nd nd 0.1 nd nd 0.3 nd nd nd nd nd 0.1 nd nd 0.2 nd nd 0.1 nd nd 0.1 nd nd 0.2
12 α-Terpineol 98-55-5 1189, 1697 1185 1690 71.2 100 2.8 5.4 17.5 1.1 12.8 56.5 4.7 17.8 100 4.1 5.9 30.6 1.3 11.4 47.6 2 14.8 70.4 3.4 4.8 27 1.2 8.5 41.4 2
13 Tridecane 629-50-5 1300, 1300 - 1300 nd nd nd nd nd nd nd nd tr nd nd nd 0.2 nd nd 0.1 nd nd nd nd nd nd nd nd nd nd nd
14 Tetradecane 629-59-4 1400, 1400 - 1400 nd nd nd tr nd nd 0.2 nd 0.1 nd nd nd nd nd nd 0.1 nd nd 0.1 nd tr nd nd nd 0.1 nd tr
15 β-Caryophyllene 87-44-5 1419, 1595 - 1583 1.5 nd 0.1 0.2 nd 0.1 0.3 nd 0.1 0.6 nd nd 0.2 nd tr 0.4 nd tr 0.4 nd 0.1 0.2 nd tr 0.4 nd tr
16 Pentadecane 629-62-9 1500, 1500 - 1500 nd nd tr 0.1 nd tr 0.2 nd 0.1 nd nd nd tr nd tr 0.2 nd 0.1 0.2 nd 0.1 tr nd tr 0.1 nd 0.1
17 Dodecanoic acid (12:0) 143-07-7 1556, 2498 - 2474 nd nd 0.7 1.1 nd 0.9 1.2 nd 1.6 1 nd 1.1 1.3 nd 1.4 2.1 nd 2.5 0.7 nd 1.2 1 nd 1.3 1.7 nd 2
18 Cedrol 77-53-2 1598, 2116 - 2086 un nd tr un nd 0.1 un nd 0.2 un nd nd un nd 0.1 un nd 0.2 un nd 0.1 un nd 0.1 un nd 0.1
19 Hexadecane 544-76-3 1600, 1600 - 1600 nd nd 0.1 0.2 nd 0.1 0.3 nd 0.1 nd nd nd 0.1 nd tr 0.3 nd 0.1 0.3 nd 0.1 0.2 nd 0.1 0.5 nd 0.2
20 Heptadecane 629-78-7 1700, 1700 - 1700 nd nd 0.1 0.1 nd 0.1 0.2 nd 0.2 nd nd nd tr nd 0.1 0.2 nd 0.2 0.1 nd 0.1 0.1 nd 0.1 0.7 nd 0.3
21 Tetradecanoic acid (14:0) 544-63-8 1748, 2694 - 2685 nd nd 3.9 5.1 nd 5.6 3.6 nd 6.4 1.9 nd 2.8 3.5 nd 4.4 3.9 nd 5.4 2.3 nd 3.2 4.0 nd 5.3 4.7 nd 6.1
22 Octadecane 593-45-3 1800, 1800 - 1800 nd nd tr 0.1 nd tr nd nd nd nd nd nd tr nd nd 0.1 nd 0.1 0.1 nd nd 0.1 nd nd 0.1 nd 0.2
23 Hexahydrofarnesyl acetone 502-69-2 1842, 2131 1843 2119 nd nd 2 3.5 nd 2.7 5 7.1 6.2 2.7 nd 2 2.5 nd 2.3 6.4 nd 5.6 3.7 nd 3.0 3.8 1.9 3.5 7.9 17 7
24 * Pentadecanoic acid (15:0) 1002-84-2 1823, 2822 - 2790 nd nd 0.5 0.5 nd 0.7 0.2 nd 0.8 0.2 nd nd 0.4 nd 0.8 0.3 nd 0.6 0.1 nd 0.4 0.4 nd 0.7 0.3 nd 0.7
25 Nonadecane 629-92-5 1900, 1900 - 1900 nd nd 0.1 nd nd tr nd nd 0.1 nd nd nd 0.1 nd nd 0.3 nd nd nd nd nd nd nd tr nd nd 0.1
26 Farnesyl acetone 1117-52-8 1919, 2384 - 2362 nd nd 0.7 nd nd 0.1 0.4 nd 0.9 0.8 nd nd tr nd 0.1 nd nd 0.1 0.9 nd 0.8 tr nd 0.1 0.1 nd 0.2
27 Methyl hexadecanoate 112-39-0 1926, 2208 1924 2214 0.2 nd 1.5 1.8 nd 1.6 2.8 6.5 4.1 2.9 nd 2.8 2.5 nd 3 6.7 11.1 7.1 3.8 nd 3.9 3.6 3.3 4.1 7.8 18.4 8.3
28 9E-Hexadecenoic acid (16:1, n-7) 2091-29-4 1942, 2954 - 2935 un nd 0.9 un nd 0.8 un nd 2.7 un nd nd un nd nd un nd 0.4 un nd 0.3 un nd 0.3 un nd 0.4
29 Palmitoleic acid (16:1, n-7) 373-49-9 1951, 2926 - 2926 nd nd 1.8 0.6 nd 1.5 1.4 nd 4.3 0.3 nd nd 0.4 nd 0.7 0.7 nd 1.3 0.6 nd 0.7 0.5 nd 0.8 0.9 nd 1.1
30 Dibutyl phthalate 84-74-2 1965, 2680 - 2675 nd nd nd 0.1 nd 0.3 0.2 nd 0.8 nd nd nd 0.1 nd nd 0.2 nd 0.4 nd nd 0.2 0.2 nd 0.2 0.3 nd 0.5
31 n-Hexadecanoic acid (16:0) 57-10-3 1972, 2931 1960 2894 0.9 nd 51.9 60.3 37.5 64.1 10.0 nd 17.8 23.8 nd 60.8 65.0 57.7 69.2 31.9 7.7 35.7 24.2 nd 47.1 62.1 41.8 61.3 31.7 2.1 44.8
32 Eicosane 112-95-8 2000, 2000 - 2000 0.1 nd tr 0.1 nd tr 0.3 nd 0.1 nd nd nd nd nd nd 0.1 nd 0.1 nd nd nd 0.1 nd tr 0.2 nd 0.1
33 Methyl linoleate 112-63-0 2092, 2482 - 2485 un nd 1.9 un nd 0.3 un nd 2.8 un nd 4.4 un nd 0.6 un nd 0.9 un nd 4.3 un nd 0.4 un nd 0.6
34 Methyl oleate 112-62-9 2091, 2434 - 2439 0.2 nd 0.9 1 nd 0.7 3.2 nd 2.5 nd nd 2.0 1.7 nd 1.9 4.1 nd 4.8 nd nd 2.2 1.9 nd 2.1 4.2 nd 4.4
35 Methyl linolenate 301-00-8 2098, 2571 - 2552 un nd 1.8 un nd nd un nd 1.2 un nd 2.8 un nd nd un nd nd un nd 3.4 un nd nd un nd nd
36 Heneicosane 629-94-7 2100, 2100 - 2100 0.6 nd nd 1.1 nd tr 3.5 nd 0.1 5.7 nd nd 1.7 nd nd 4.3 nd 0.1 6.5 nd 0.1 2 nd tr 4.3 nd 0.1
37 Phytol 150-86-7 2104, 2622 - 2607 un nd 5.8 un nd 1.5 un nd 7 un nd 2 un nd 0.7 un nd 1.3 un nd 4.4 un nd 1.3 un nd 2
38 Unknown-1 - -, - - 2476 un nd 0.2 un nd 0.3 un nd 2.8 un nd nd un nd 0.2 un nd 0.3 un nd 0.3 un nd 0.2 un nd 0.5
39 Methyl stearate 112-61-8 2128, 2418 - 2420 nd nd 0.2 0.2 nd 0.3 0.2 nd 0.7 0.8 nd nd 0.6 nd 0.3 1.4 nd 0.7 0.4 nd 0.4 0.2 nd 0.4 0.6 nd 0.9
40 Linoleic acid (18:2, n-6) 60-33-3 2133, 3164 - 2884 un nd 7.7 un nd 1.1 un nd 9.7 un nd 2.7 un nd 0.1 un nd 0.7 un nd 5.1 un nd 0.6 un nd nd
41 Oleic acid (18:1, n-9) 112-80-1 2141, 3173 - 2770 un nd 2.9 un nd 2.9 un nd 7 un nd nd un nd 3.3 un nd 10.0 un nd 3.7 un nd 4 un nd nd
42 Octadecanoic acid (18:0) 57-11-4 2172, 3136 - 2700 un nd 1.7 un nd 4.3 un nd nd un nd nd un nd 1.6 un nd 1.5 un nd 0.3 un nd 3.8 un nd nd
43 Docosane 629-97-0 2200, 2200 - 2200 1 nd 0.1 0.2 nd 0.1 2.8 nd 0.1 nd nd nd 0.1 nd nd 0.2 nd 0.2 0.2 nd nd 0.1 nd 0.1 0.4 nd 0.1
44 Phytol acetate - -, - - 2512 un nd 0.1 un nd 0.2 un nd 0.6 un nd nd un nd 0.1 un nd 0.4 un nd 0.2 un nd 0.1 un nd 0.2
45 Tricosane 638-67-5 2300, 2300 - 2300 1.5 nd 0.2 0.3 nd 0.2 5.1 nd 0.5 0.3 nd nd 0.3 nd 0.2 0.6 nd 0.6 0.3 nd 0.2 0.3 nd 0.2 0.8 nd 0.4
46 Tetracosane 646-31-1 2400, 2400 - 2400 1.9 nd nd 0.2 nd 0.1 6.8 nd 0.3 0.1 nd nd 0.6 nd 0.1 0.3 nd 0.3 0.2 nd nd 0.1 nd 0.1 0.5 nd 0.2
47 Pentacosane 629-99-2 2500, 2500 - 2500 1.5 nd 0.1 0.3 nd 0.3 6.4 nd 0.6 0.3 nd nd 0.3 nd 0.2 0.5 nd 0.6 tr nd 0.1 0.2 nd 0.2 0.6 nd nd
48 Methyl 5,6-octadecadienoate -, - - 2515 un nd 0.2 un nd 0.1 un nd 0.4 un nd 0.5 un nd 0.7 un nd 1.4 un nd 0.4 un nd 0.4 un nd 0.9
49 Hexacosane 630-01-3 2600, 2600 - 2600 1.2 nd 0.2 0.1 nd 0.1 5.2 nd 0.3 0.1 nd nd 0.1 nd tr 0.1 nd 0.1 nd nd 0.3 tr nd 0.1 0.3 nd 0.1
50 Heptacosane 593-49-7 2700, 2700 - 2700 nd nd 0.2 nd nd 0.4 nd nd nd nd nd nd nd nd nd nd nd 1.5 nd nd 0.2 nd nd 0.2 nd nd 0.4
51 Octacosane 630-02-4 2800, 2800 - 2800 nd nd 0.2 nd nd 0.3 nd nd nd nd nd nd nd nd nd nd nd 0.4 nd nd nd nd nd 0.1 nd nd 0.3
52 Unknown-2 - 2817 un nd 0.9 un nd 1 un nd 3 un nd nd un nd 0.8 un nd 1.7 un nd 0.8 un nd 0.8 un nd 1.4
53 Nonacosane 630-03-5 2900, 2900 - 2900 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 1.1 nd nd nd nd nd nd nd nd nd
54 Unknown-3 - 2952 un nd nd un nd nd un nd nd un nd 4.5 un nd 1 un nd 0.5 un nd nd un nd 0.8 un nd 4.5
55 Unknown-4 - 2975 un nd 1.4 un nd 1.4 un nd 3.1 un nd nd un nd 1.2 un nd 1.9 un nd 1.5 un nd 1.4 un nd 1.9
Total (55) 98.3 100 100 99.6 100 100 98.8 100 100 100 100 100 99.8 100 100 99.9 100 100 97.4 100 100 100 100 100 99.4 100 100
HMs (4) 15.8 0 3.7 5.9 36.4 3.4 4.3 5.8 0.9 18.7 0.0 1.5 3.3 0 0.2 7.0 8.1 0.6 11.7 7.3 1.0 4.4 0 0.1 5.6 0 0.3
AMs (4) 71.4 100 5.2 9.5 24.1 1.8 21.7 77.4 9.4 27.8 100 8.3 10.9 42.3 3.5 22.4 47.6 6.7 26.8 92.7 7.8 11.1 49.4 3.6 20 57.7 6.7
HSs (1) 1.5 0 0.1 0.2 0 0.1 0.3 0 0.1 0.6 0 0 0.2 0 0 0.4 0 0 0.4 0 0.1 0.2 0 0 0.4 0 0
ASs (1) 0 0 0 0 0 0.1 0 0 0.2 0 0 0 0 0 0.1 0 0 0.2 0 0 0.1 0 0 0.1 0 0 0.1
ADs (1) 0.3 0 5.6 1.7 0 1.2 4.2 0 4.2 2.1 0 2.0 0.7 0 0.5 1.3 0 1.0 4.3 0 4.1 1.1 0 1.1 2.1 0 1.5
Aldehydes & ketones (3) 0.1 0 2.7 3.7 2 2.8 6 10.3 7.3 3.8 0 2 2.8 0 2.5 7 7.5 5.9 4.9 0 3.8 4.2 1.9 3.6 8.4 21.7 7.4
FAs (11) 1 0 72 67.6 37.5 82.1 16.4 0 50.9 27.2 0 67.4 70.7 57.7 81.8 39.1 7.7 58.7 28.1 0 62.2 68.1 41.8 78.4 39.4 2.1 55.7
LCFAs (9) 1 0 72 67.6 37.5 81.9 16.4 0 50.3 27.2 0 67.4 70.6 57.7 81.5 38.9 7.7 58.1 27.9 0 62 68 41.8 78.1 39.3 2.1 55.1
SCFAs (2) 0 0 0 0 0 0.2 0 0 0.6 0 0 0 0.1 0 0.3 0.2 0 0.6 0.2 0 0.2 0.1 0 0.3 0.1 0 0.6
SFAs (7) 0.9 0 58.7 67 37.5 75.8 15 0 27.2 26.9 0 64.7 70.3 57.7 77.7 38.4 77 46.3 27.5 0 52.4 67.6 41.8 72.7 38.5 2.1 54.2
MUFAs (3) 0 0 5.6 0.6 0 5.2 1.4 0 14 0.3 0 0 0.4 0 4 0.7 0 11.7 0.6 0 4.7 0.5 0 5.1 0.9 0 1.5
PUFAs (1) 0 0 7.7 0 0 1.1 0 0 9.7 0 0 2.7 0 0 0.1 0 0 0.7 0 0 5.1 0 0 0.6 0 0 0
Esters (8) 0.4 0 6.6 3.1 0 3.5 6.4 6.5 13 3.7 0 12.5 4.9 0 6.6 12.4 11.1 15.7 4.2 0 15.0 5.9 3.3 7.8 12.9 18.4 15.7
Phthalate (1) 0 0 0 0.1 0 0.3 0.2 0 0.8 0 0 0 0.1 0 0 0.2 0 0.4 0 0 0.2 0.2 0 0.2 0.3 0 0.5
Esters of FAs (7) 0.4 0 6.6 3 0 3.2 6.2 6.5 12.2 3.7 0 12.5 4.8 0 6.6 12.2 11.1 15.3 4.2 0 14.8 5.7 3.3 7.6 12.6 18.4 15.2
TOCs (29) 73.8 100 92.5 91.8 63.6 91.9 66.6 94.2 87.6 79.9 100 94.0 94.3 100 95.7 89.4 73.8 89.5 83.9 92.7 95 94.1 100 95.4 89.1 100 88.8
n-Alkanes (17) 7.8 0 1.3 2.8 0 1.8 31 0 2.6 6.5 0 0 3.5 0 0.7 7.4 0 5.5 8 0 1.2 3.2 0 1.2 8.6 0 2.6
Unknowns (4) 0 0 2.5 0 0 2.8 0 0 8.9 0 0 4.5 0 0 3.3 0 0 4.4 0 0 2.6 0 0 3.3 0 0 8.2
1 Note: nd means not detected; un means uncertain; tr means the content is less than 0.05%. Unknown means the compounds can’t be elucidated by its mass spectrum. The same for the following Tables. LRIsb, d detected by semi-standard apolar or polar column were gotten from NIST (National Institute of Standards and Technology) 17 library, respectively, LRIsa and LRIsc detected by DB-5 and free fatty acid phase (FFAP) were gotten in this experiment, respectively. The HMs, AMs, HSs, ASs, ADs, TOCs, SCFAs equal to hydrocarbon monoterpenes, alcohol monoterpenes, hydrocarbon sesquiterpenes, alcohol sesquiterpenes, alcohol diterpenes, total oxygenated compoundss, short-chain FAs (when the chain is less than 10 carbons), respectively. The compounds denoted with red color means they were identified and quantified both in this study and previous literature. FAs are represented by number of carbon atoms in fatty carboxyl chain:number of double bonds. The n- designates the location of the double bond nearest the methyl terminus.
Table 2. The characteristic peaks of unknown compounds.
Table 2. The characteristic peaks of unknown compounds.
Characteristic Ion Peaks (M/W, %) Compounds
123 (100), 57 (97), 81 (90), 43 (81), 69 (81), 95 (80), 68 (77), 55 (76), 82 (68), 278 (6). Unknown-1
55 (100), 41 (77), 69 (76), 43 (74), 83 (73), 97 (59), 57 (57), 96 (56), 84 (56), 222 (11) Unknown-2
80 (100), 140 (59), 81 (45), 94 (33), 79 (33), 122 (30), 67 (28), 41 (27), 43 (25), 149 (3). Unknown-3
43 (100), 55 (81), 57 (80), 83 (67), 41 (65), 69 (62), 97 (58), 96 (45), 194 (8), 236 (8). Unknown-4
Table 3. The IC50 and Ferric reducing ability of each sample. Ferric reducing ability: FRAP value of each sample in the maximum concentration. ND: not determined.
Table 3. The IC50 and Ferric reducing ability of each sample. Ferric reducing ability: FRAP value of each sample in the maximum concentration. ND: not determined.
Samples IC50 (mg·mL– 1) Ferric reducing ability
(mmol·L– 1)
DPPH ABTS
E8 ND 133.1 0.023
E9 764.96 ND 0.02
E10 ND 0.227 0.025
RC8 0.629 0.323 0.023
RC9 ND 0.541 0.019
RC10 0.344 0.293 0.028
C8 ND ND 0.027
C9 ND ND 0.024
C10 ND ND 0.026
Palmitic acid ND ND 0.025
α-Terpineol 747.9 197.65 0.018
Ascorbic acid 0.0077 0.0127 0.098
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Alerts
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2025 MDPI (Basel, Switzerland) unless otherwise stated