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Article

Comparative Biochemical and Pharmacodynamic Analyses of Asarum heterotropoides Fr. Schmidt var. Mandshuricum (Maxim) Kitag and Asarum sieboldii Miq var. Seoulense Nakai Roots

by
Huiling Li
1,
Zhiqing Wang
1,*,
Guangyuan Zhao
1,
Yanhong Wang
2,
Xuanwei Xu
2,
Yingping Wang
3,
Ze Zhang
1 and
Guanghui Wang
1
1
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China
2
Ginseng and Antler Product Quality and Safety Risk Assessment Laboratory, Ministry of Agriculture and Rural Affairs, Jilin Agricultural University, Changchun 130118, China
3
State & Local Joint Engineering Research Center of Ginseng Breeding and Application, College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2024, 17(10), 1301; https://doi.org/10.3390/ph17101301
Submission received: 24 July 2024 / Revised: 26 August 2024 / Accepted: 18 September 2024 / Published: 30 September 2024
(This article belongs to the Section Natural Products)
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Abstract

:
Background:Asarum heterotropoides and Asarum sieboldii are commonly used in traditional Chinese medicine. However, little is known about how they differ in terms of essential oil (EO) and ethanol extract (EE) content and composition. Moreover, the effect of various geographical locations on the essential oil (EO), ethanol extract (EE), and asarinin content of different Asarum samples remains unknown. We tested four root-drying methods, i.e., soil removal and shade drying (P1), water washing and shade drying (P2), and water washing and drying at 30 °C (P3) and 40 °C (P4). We used LC-MS and GC-MS to investigate these differences. We also investigated the pharmacodynamic effects of EO and EE. Results: Overall, the EO, EE and asarinin contents of the analysed samples were 19.21–51.53 μL.g−1, 20.00–45.00 μL.g−1, and 1.268–2.591 mg.g−1, respectively. P1 treatment yielded the lowest volatile oil content compared to the other three treatments. GC-MS analysis revealed 78 EO components. Among the six major EO components, eucarvone, 3,5-dimethoxytoluene, and methyl eugenol were higher in A. heterotropoides than in A. sieboldii. However, the latter had a higher myristicin content. LC-MS analysis identified 888 EE components in roots and leaves of A. heterotropoides and A. sieboldii; 317 differentially accumulated metabolites were identified. EO and EE showed a dose-dependent reduction in the degree of swelling and an increase in the inhibition rate of drug concentration on acetic acid writhing in mice. Asarum EO proved to be more effective than EE in the pharmacodynamic study. Conclusions: We conclude that Asarum species show inter- and intra-specific differences in EO and EE content and composition, which may influence the pharmacodynamics of Asarum root extracts.

1. Background

Asarum originates from Asia, but its members are naturally distributed throughout the temperate zones of the northern hemisphere. Among the members of the genus Asarum, Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag and Asarum sieboldii Miq var. seoulense Nakai are of particular importance because of their frequent use in traditional Chinese medicine (TCM) [1,2].
The roots of both species are listed as “xixin” in the Chinese Pharmacopoeia. Xixin has anti-inflammatory, anticancer, antibacterial, and analgesic effects [3], and is commonly used in TCM to treat colds, headaches, toothaches, rhinorrhoea, wheezing, inflammation, and others [4]. Apart from its use in TCM, Asarum essential oils (EOs) have been reported to have activities against phytopathogens and insects [5,6]. The health-promoting properties and activities against insects and pests of crops are due to the composition of their metabolomes, in particular, the EOs, monoterpenes, lignans, alkaloids, alcohols, asarinin, and amides [7,8]. Asarinin from Asarum species has strong bioactivity such as anticancer [9], antihypertensive, and antiangiogenic activities [10]. Studies on the composition of Asarum oil have shown that methyl eugenol, sesamin, safrole, N-isobutyl-(2E,4Z,8Z,10E)-dodecatetraenamide, pentadecane eucarvone, 3,5-dimethoxytoluene, safrole, myristicin, and elemicin are present in higher percentages [11,12]. Meanwhile, the ethanolic extracts consist of lignans, monoterpenes [11], phenanthrene derivatives, isobutyle amides, and pehnylpropanoids [13,14]. However, the content and composition have been reported to be influenced by extraction methods, species and cultivar, growing conditions, and geography [11,14].
Within China, Asarum is a particularly famous herb in the northeast of the country, such as Xinbin and surrounding areas in Liaoning Province, and in eastern mountainous areas such as Tonghua County, Liuhe County, Ji’an County, and Fusong County in Jilin Province. As the main growing area, wild Asarum is mainly found in the humid and fertile understorey of the Changbai Mountains or in mid-mountainous areas [14,15]. Such a wide area highlights the need to investigate how the same cultivar grown in different areas would affect the EO content (EOC) and composition of ethanol extracts (EEs). Previous work in this regard on China has reported the presence of 48–77 volatile oil compounds. The authors also reported the presence of significant variation in the active constituents of A. heterotropoides var. mandshuricum [14]. Therefore, Asarum grown in different areas needs to be further investigated for the effect of area of cultivation on the content of active ingredients. In addition to the area of cultivation, the way in which the Asarum roots are processed prior to extraction of volatiles or EOs is one of the most important factors. It has been shown for several medicinal plant species that the way the roots (or other useful tissues) are processed can influence the yield of EOs. For example, an increase in menthone and pulegone and a decrease in limonene was observed when aerial parts of Moroccan Mentha pulegium L. were dried compared to fresh samples [16]. Similarly, different drying methods were found to affect phenol, total flavonoids, liquiritin, and glycyrrhizic acid in Glycyrrhiza uralensis (liquorice) [17]. Thus, processing of Asarum roots may or may not lead to changes in the content of EOs, ethanolic extracts, and asarinin. In addition, most of the literature on EO, EE, and asarinin content (AsC) showed that there are differences within species. However, limited work has highlighted the differences between A. heterotropoides and A. sieboldii.
The objective of this study was to compare the effect of four different Asarum root-drying methods on EO, EE, and AsC. We also aimed to compare the EE as well as metabolome profiles of the A. heterotropoides and A. sieboldii samples. Finally, we tested the pharmacodynamic effects of Asarum volatile oil and EE. For these objectives, first, we determined the effect of four different processing methods, i.e., soil + shade drying (P1), water washing + shade drying (P2), water washing + drying at 30 °C (P3), and water washing + drying at 40 °C (P4), on EOC, EE content (EEC), and AsC. We then determined the EO and EE composition of 36 root samples. After that, we investigated the differential metabolomic profiles of both species. Finally, we investigated the anti-inflammatory and analgesic effects of extracts from both species. Our results are valuable for TCM (and the pharmaceutical industry) as well as for Asarum growers to choose the appropriate processing method for Asarum drying.

2. Results

2.1. Effect of Asarum Root-Drying Methods on Biochemical Content Yield

First, we investigated whether the four processing methods showed any variation in volatile oil content. To do this, we processed the Asarum samples by removing the soil + drying in the shade (P1), washing with water + drying in the shade (P2), washing with water + drying at 30 °C (P3), and washing with water + drying at 40 °C. The results showed that the volatile oil content of P1 was lower than the other three methods. The samples washed with water and dried in the shade or in the oven at 30 °C and 40 °C had no significant differences among the 36 Asarum samples (Figure 1A; Table S2).
Overall, the EOC of the analysed samples ranged from 19.21 to 51.53 μL.g−1. The content of EO in 90.44% of the samples was 20.00~45.00 μL.g−1.
The EOC of cultivated (19.21–47.65 μL.g−1) and wild (30.14–51.53 μL.g−1) samples showed visible differences, with the latter having 8.73–25.16% higher contents. Similarly, the EOC of Asarum from different regions was variable, such that the highest levels were found in samples from Jian (30.10–51.53 μL.g−1), followed by Fusong (36.02–47.65 μL.g−1), Lyeyang (34.77–43.65 μL.g−1, Ningqiang (33.73–43.76 μL.g−1), Wang Qing (33.21–40.65 μL.g−1), Xinbin (27.69–33.95 μL.g−1), Tonghua (20.23–33.37 μL.g−1), and Liuhe (19.21–31.74 μL.g−1) (Table 1). These observations suggest that the place of origin of Asarum may be a possible factor influencing EOC (the growth years and different sources influence the EOC).
Regarding the content of ethanolic extracts, the processing methods did not show any significant effect (Figure 1A; Table S2). In general, the EEC in Asarum roots was 80.18–211.06 mg.g−1. Overall, 83.09% of the Asarum samples had an EEC in the range of 90–170 mg.g−1, with wild (90.03–144.36 mg.g−1) having relatively lower contents than the cultivated varieties (80.18–211.06 mg.g−1). Regarding the origin of Asarum cultivars and wild types, the EEC content range was different (Figure 1A; Table S2), with the maximum in samples from Tonghua (91.14–211.06 mg.g−1) and the minimum in samples from Xinbin (105.88–136.01 mg.g−1) (Table 1).
The AsC in the 36 samples analysed ranged from 1.268 to 2.591 mg.g−1; 80.88% of the samples contained 1.500–3.500 mg.g−1. The effect of the processing method was similar to that of EOC and EEC, i.e., P1 had the lowest content. In general, P3 and P4 had a higher AsC. Jian (2.41–4.59 mg.g−1) had the highest maximum AsC, followed by Fusong (2.61–4.07 mg.g−1), Wangqing (1.59–3.45 mg.g−1), Xinbin (2.19–3.35 mg.g−1), Liuhe (1.27–2.79 mg.g−1), Lyeyang (1.77–2.57 mg.g−1), and Ningqiang (1.74–2.57 mg.g−1).
Finally, the A. heterotropoides samples on average exhibited lower average EOC (3.19 vs. 3.37 μL.g−1), EEC (3.26 vs. 3.37 mg.g−1), and asarinin (3.294 vs. 3.40 mg.g−1) compared to A. sieboldii samples. However, when compared the highest content of each of EOC, EEC, and asarinin, A. heterotropoides samples had the maximum contents.
These results indicate that the biochemical yield (EOC, EEC, and asarinin) differs between cultivars and wild-type Asarum collected from different regions. Moreover, it can be understood that washing and drying in an oven may be a useful processing method to obtain higher EOC, EEC, and asarinin.

2.2. Essential Oil Composition of 36 Asarum Samples

Next, we determined the EO composition of the 36 Asarum samples processed by the P4 method (washing with water and drying at 40 ℃) by using GC-MS analysis. A total of 78 compounds were identified (Table S3). These compounds belonged to nine compound classes (Figure 1B). The highest number of compounds were classified as terpenoids (57), followed by phenylpropanoids (07), alkanes (05), ethers (3), benzene alkyl (02), aldehyde (01), alkaloids (01), phenols (01), and others. The highest content in most of the examined samples was of methyl eugenol, 1,3-benzodioxole, 5-(2-propenyl)-, 1,3-dimethoxy-5-methylbenzene, 2,4-cycloheptadien-1-one,2,6,6-trimethyl-, and 1,2,3-trimethoxy-5-methylbenzene. Considering the origin of the samples, we observed that Fusong, Jiang, Liuhe, Tonghua, Wangqing, Xinbin, Ningqiang, and Lyeyang samples (1–2) had 61, 61, 76, 75, 49, 68, 46, and 45 metabolites. These results show that the Asarum samples from different regions have different EO compositions (Table S3).
Furthermore, we quantified the six major EO components, i.e., eucarvone, 3,5-dimethoxytoluene, safrole, methyl eugenol, myristicin, and elemicin (Figure 1C). The contents of eucarvone, 3,5-dimethoxytoluene, and methyl eugenol in the EO of A. heterotropoides were higher than A. sieboldii Miq. The contents of these six EC components ranged from 48.2 to 68.4 μL.μL−1. In addition, the total content of these six essential components in wild samples was higher than that in cultivated samples. The most significantly higher component in wild compared to cultivated samples was elemicin, which was significantly higher. Notably, the contents of myristicin in the EO of A. sieboldii Miq were higher than A. heterotropoides, and the content of the six components is less than 45 μL.μL−1 (Figure 1C).

2.3. Comparative Metabolome Analysis of A. heterotropoides and A. sieboldii Roots and Leaves

Based on the EECs, we selected two Asarum samples, i.e., 24 (A. heterotropoides) and 32 (A. sieboldii), and analysed the extracts by LC-MS (Figure 2). A total of 888 compounds were identified (Table S4). By comparing samples 24 and 32, we screened 317 differentially accumulated metabolites (DAMs): 183 in leaves, 105 in roots, and 29 commonly accumulated metabolites (Figure 3A,B). Within A. heterotropoides, the relative percentage content of 32 major compounds exhibited marked differences. Notably, 3,4-dimethoxycinnamic acid, DL-arginine, glutamic acid, (2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide, N-desmethylvenlafaxine, acetyl-trans-resveratrol, L-pyroglutamic acid, (2E,4E)-N-(2-methylpropyl)dodeca-2,4-dienamide, 5-methyluridine, and several others were present in higher quantities in roots compared to leaves, whereas leaves had markedly higher contents of (3,4,5-trihydroxy-6-methyloxan-2-yl)2-(methylamino)benzoate, saxitoxin, 4-methoxycinnamaldehyde, salsolinol, choline, normorphine, and others (Table 1). The DAMs clearly separated the samples (Figure 3C). They were enriched in various KEGG pathways including phenylpropanoid, flavonoid, unsaturated fatty acid, and monoterpenoid biosynthesis and tyrosine, purine, galactose, and fatty acid metabolism (Figure 3D). Lipid mass annotation highlighted that the DAMs are classified as fatty acyls, glycerophospholipids, polyketides, prenol lipids, sphingolipids, and sterol lipids (Figure 3E).

2.4. Pharmacodynamic Evaluation of Essential Oil and Ethanol Extracts of Asarum Roots

The results clearly showed that different doses of volatile oil extracts of Asarum roots significantly reduced the degree of ear swelling (Table 2). The degree of swelling decreased as the dose increased, i.e., from low to high. In the case of mice treated with EE, the degree of swelling generally decreased with increasing dose. The drug inhibition rate of EO ranged from 29.82% to 56.32%, whereas the drug inhibition rate of alcohol-soluble extract ranged from 17.72% to 38.60%. It was observed that under the same drug, the higher the dose, the higher the inhibition rate. In comparison, the inhibition rate of volatile oil extract was significantly higher than the inhibition rate of ethanol-soluble extract at the same dose level.
The inhibition rate of drug concentration on acetic acid writhing in mice showed an effect in the order of E2low < V2low < E2mild < E8low < V2mild < V8low < ibuprofen < E2high < E8mild < V2high < V8mild < E8high < V8high. The drug inhibition rate of the volatile oil ranged from 31.07% to 84.95%, and the drug inhibition rate of the alcohol-soluble extract ranged from 24.27% to 72.33%. For the same drug, the higher the dose, the higher the inhibition rate. At the same dose, the volatile oil extract showed a slightly higher inhibition rate than the alcohol-soluble extract (Table 3).

3. Discussion

Asarum species are rich in compounds with biological activities and are therefore used in TCM. Two species, i.e., A. heterotropoides and A. sieboldii, are commonly consumed as xixin in China. The content and composition of the EOs, EEs, and asarinin may differ depending on the drying method. We tested four drying methods, i.e., P1, P2, P3, and P4. Generally, the samples which were washed and then dried (shade, 30 °C, or 40 °C) had relatively higher EOC than those from which just soil was removed followed by drying (Table S2). The limited effect of these methods on EEC is a useful observation when considering large-scale drying and extraction with ethanol. Previously, air-dried methods have been reported to be useful for the study of EEs and identification of new phenanthrene derivatives [18]. The results that AsC was also lowest in P1 compared to the methods in which roots were washed prior to drying indicate the usefulness of the washing and drying. The non-significant effects of drying at 30 or 40 °C suggests just washing with water and shade drying is sufficient without the need of an oven as used in several other methods [19].
The fact that wild samples of A. heterotropoides had relatively higher EOC and lower EEC than cultivated samples is an important observation. Though, in this regard, limited knowledge is available in Asarum species, work on other species such as Foeniculum vulgare [20], Thymus pannonicus [21], Salvia officinalis [22], etc., has highlighted the existence of such differences. There could be several factors affecting this, such as the age of the plant, environment, genetic differences, etc., as reported in Lavandula angustifolia [23], Cymbopgon martini [24], and in Asarum species [19,25]. The results that samples from different growing regions of China showed content variation (Table 1) are consistent with earlier work that altitude can affect the metabolites of Asarum [19]. Further research on the effect of altitude between the same species under similar agronomic practices should be carried out to reveal the ideal altitude in relation to the location’s climate. Moreover, the difference between A. heterotropoides and A. sieboldii in terms of EOC, EEC, and AsC is understandable from a genetic differences point of view. This is consistent with the earlier reports that interspecific differences exist in terms of active compounds, e.g., in Launaea [26], Ocimu [27], and many others. Nevertheless, the highest contents of both EOC and EEC in A. heterotropoides indicates that it should be a preferred source of xixin when choosing between the two species.
The EO of A. heterotropoides has been previously explored for its composition, and >70 components have been reported [11]. Among them, 15 major components have been widely reported [12]. Haque, Moon, Saravana, Tilahun, and Chun [11] reported 44 EO components based on GC-MS; major components were 3,5-dimethoxytoluene, safrole, and myristicin. Similarly, Xiao et al. [28] reported 42 EO components by using micro-wave-assisted steam distillation followed by GC-MS. Our results that 78 compounds were identified are consistent with earlier work [12]. The presence of components with the highest proportions is also consistent with the previous works. The geographical variations were evident from the number of EO components. Work on different medicinal plants has revealed that geography can have significant effect on the EO content as well as composition [29]. Thus, we can conclude that Asarum species also exhibit EO content and compositional variations. Particularly, when considering the samples from the regions included in our work, Liuhe and Tonghua can be preferred. However, the sampling method, age of the plants, and other agronomic conditions should also be considered [19,25]. The observed higher quantities of safrole, elemicin, and myristicin in A. heterotropoides are noticeable variations compared to the higher presence of 3,5-dimethyoxytoluene in A. sieboldii. Thus, for their use as xixin, the relative content of each of the components should be given due consideration. Earlier work on A. sieboldii has shown the ten most important components of EO, including eucarvone, 3,5-dimethoxytoluene, isosafrole, methyl eugenol, and myristicin, together with five others [30]. However, our results based on EOC, EEC, asarinin, and six components of EO clearly indicate that A. heterotropoides is a better choice for xixin.
The composition of the EEs showed a more diverse range of components compared to the EO (Figure 2). The 317 DAMs identified between the roots and leaves of Asarum species provide several reference metabolites for medicinal use. Consistent with our results, previous studies have reported the presence of lignans, monoterpenes, flavonoids, alkaloids, phenols, amino acids, and others [31]. The observation that both species had differences in EE content is consistent with the EEC results (Figure 1A; Table S2). Li, Cui, and Zheng [13] highlighted that the EE composition of A. sieboldii was different from other Asarum species including A. heterotropoides [18], Asarum himalaicum [32], Asarum ichangse [33], and members of the genus Aristolochiaceae [34]. However, the identification of 888 EE components is a valuable addition to the existing compound databases of the two species. The observation that several EE components were specific to each of the two species as well as in leaves or roots further proposes the specific uses of leaves and roots for targeted diseases. In this regard, the results of pharmacodynamic evaluation tests indicated that the EE as well as EO extracts significantly decreased the degree of swelling and increased inhibition rate of writhing in mice (Table 2 and Table 3). Although the EE showed a higher number of components, the increased drug inhibition rates of EO compared to EE highlights that the former is better. Most of the pharmacodynamic evaluation studies involving EE from A. sieboldii [35] and A. heterotropoides [36] have reported inhibition of writhing times of mice, increased pain threshold of thermal stimulation, and inhibition of xylene-treated ear swelling. As for EO of A. seiboldii, it has been reported that it could significantly reduce the amount of nasal secretions, sneezing, and the degree of nasal scratching in Sprague Dawley male rats [35]. A. heterotropoides EO can also induce antinociceptive and anti-inflammatory effects [36]. Several components of EOs from plants have been reported for their anti-inflammatory function via a range of metabolic regulatory pathways. For example, α-pinene, 3-carene, and limonene can regulate COX-2 expression and reduce inflammation [37]. Furthermore, EOs can also release anti-inflammatory mediators, which can regulate the expression of key genes related to reactive oxygen species scavenging, JAK/STAT signalling pathways, and interleukin and tumour necrosis, etc. [38,39]. Thus, our data are significantly important for choosing the Asarum variety and dose for future studies on characterization of individual EO components for their anti-inflammatory effects.

4. Methods

4.1. Plant Material

A total of 36 Asarum samples were included in the study; Samples 1–31 were Asarum heterotropoides Fr. var. mandshuricum (Maxim.) Kitag, and Samples 32–36 were Asarum sieboldii Miq var. seoulense Nakai. Both species used in our study are sources of the traditional Chinese medicine xixin in the Chinese Pharmacopoeia. Samples were collected from Fusong, Jian, Liuhe, Tonghua, and Wangqing in Jilin Province, Xinbin in Liaoning Province, and Anguo in Hebei Province in China. Root and leaf samples were collected and then dried (Table 4; Figure S1). No permission is required to work on this species. Voucher specimens are available in the genebank herbarium of Jilin Agricultural University, China, under the number JINX760552-87. Official identification of the plant material was conducted by Prof Guanghui Wang.
All reagents were of analytical grade and were purchased from ThermoFisher Scientific, Dreieich, Germany; Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China; Chengdu Alfa Biotechnology Co., Ltd., Chengdu, China; and Sichuan Weikeqi Biological Technology Co., Chengdu, China.

4.2. Sample Drying

Asarum roots were harvested and processed using four different methods. Each drying method included five replicates. In the first method, the soil was removed from the Asarum roots followed by drying in the shade for 7 days (P1). In the second method, the root samples were washed with water and dried in the shade for 7 days (P2). In the third method, the root samples were washed and dried for 48 h at 30 °C (P3). In the fourth method, the root samples were washed with water and dried for 48 h at 40 °C (P4).

4.3. Essential Oil Extraction and GC-MS Analysis

The EO in the root of Asarum was extracted by steam distillation. For extraction, the samples were ground on a stainless-steel mesh (40 mm, 0.014-inch wire diameter). The mesh samples (20 g) were hydrodistilled for three hours until no more oil was extracted according to the standard method of the Chinese Pharmacopoeia [40]. The EO was collected and stored at −20 °C. Then, 100 μL of EO was dried over anhydrous sodium sulphate and diluted 50 times with ethyl acetate. An aliquot (1 μL) of the sample was analysed by GC-MS using the HP-5 MS quartz capillary chromatographic column (30 m × 0.25 mm × 0.25 μm) (19091S-433UI, Agilent Technologies, Andover, MA, USA) on an Agilent 6890N-5973MSD gas chromatography–mass spectrometer (GC-MS, Agilent, MA, USA). The analytical conditions were as follows: 40 °C (for 2 min), increasing the temperature from 40 °C to 160 °C at a rate of 2.5 °C min−1, from 160 °C to 280 °C at a rate of 8 °C min−1, and finally maintaining a constant temperature of 280 °C for 10 min. The injection temperature was set at 280 °C. Helium was used as the carrier gas at a flow rate of 1.0 mL.min−1. An injection volume of 1 μL was used with a pulse without shunt. The mass spectrometer operated in an electron impact (EI) mode at 70 eV with scan ranges between 30 and 550 amu. The voltage of the multiplier tube was 0.98 kv. The ion source temperature was maintained at 230 °C and the quadrupole at 150 °C. A series of standard solutions were prepared by adding ethyl acetate to the mixed solution (1, 2, 5, 10, 20, 50, and 100 μg·mL−1). The injection volume was 1 μL, and the standard curve was plotted with the concentration as the abscissa (x) and the chromatographic peak area as the ordinate (y) (Table S1).
The total ion current chromatograms obtained were searched and compared with the spectrum in the NBS mass spectrometry database to determine the chemical components in the EO of each material.

4.4. Preparation of EEs and LC-MS Analysis

The leaf and root samples of the selected Asarum genotypes were ground into powder over a stainless-steel (ISO3310) mesh (#2 mesh in the Chinese Pharmacopoeia; Xinxiang Zhentai Mechanical Co., Ltd., Xinxiang, China) and weighed accurately. The EE was extracted by hot ethanol leaching. Briefly, the powdered sample (2~4 g) was placed in a 250 mL stoppered flask, and 100 mL of ethanol (95%) was added and weighed. The samples were extracted for one hour, then connected to a condensation tube and heated (100~150 °C) to boiling point for one hour. The extract was then allowed to cool to room temperature for one hour. After that, the weight loss was compensated by the addition of ethanol. A volume of 20 mL of the filtrate was placed in a dry and weighed evaporating dish (60 mm, Shengxing Chemistry Porcelain Factory, Tangshan, China) and evaporated in a water bath (HH-8, Yineng Changzhou, China). After evaporation, the extract was dried in a dryer (GZX-9070MBE, Boxun, Shanghai, China) for 30 min. The dried extract was dissolved in 2 mL of methanol, sealed, and stored in a freezer (−20 °C).
Next, 100 μL of sample was taken into an Eppendorf tube, and 400 μL of 80% methanol was added and mixed by vortexing. The Eppendorf tube was kept in an ice bath for five minutes and centrifuged at 15,000× g at 4 °C for 20 min in a D3024R low-temperature centrifuge (Scilogex Company, Rocky Hill, CT, USA). The supernatant was collected and diluted with mass-spectrometry-grade water until the methanol content was 53%. The extract was then analysed on a Hyposil gold (C18) column (100 × 2.1 mm, 1.9 μm) (Thermo Fisher Company, Dreieich, Germany) and on an H-class ultra-high-performance liquid chromatograph (Waters Corporation, Milford, MA, USA). The analytical conditions were as follows. The column temperature and flow rate were 40 °C and 0.2 mL/min, respectively. Mobile phases A and B were water with 0.1% formic acid and methanol, respectively. Negative mode: mobile phase A was 5 mm ammonium acetate (pH = 9.0), and mobile phase B was methanol. Gradient elution procedure: 0 min: 98% (A) and 2% (B); 1.5 min: 98% (A) and 2% (B); 3 min: 0% (A) and 85% (B); 10 min: 0% (A) and 100% (B); 10.1 min: 98% (A) and 2% (B); 11 min: 98% (A) and 2% (B); 12 min: 98% (A) and 2% (B). The scan range was m/z 100–1500, and the ESI source settings were as follows. The spray voltage was 3.2 kV, the sheath gas flow rate was 40 arb, the aux gas flow rate was 10 arb, the capillary temp was 320 °C, the funnel RF level was 40, the aux gas heater temperature was 350 °C, and the polarity was positive, negative; the ESI source MS/MS secondary scan setting was data-dependent scans.

4.5. Asarinin Extraction and Content Determination

Asarinin standard was prepared as a 1 mg.mL−1 solution with methanol. For sample preparation, the sample was passed through a stainless-steel mesh (No. 3 mesh, Xinxiang Zhentai Mechanical Co., Ltd., Xinxiang, China) and placed in a conical flask with a stopper containing 15 mL of methanol and weighed. Ultrasonic treatment (power 500 W, frequency 40 KHz) was then performed for 45 min, then the solution was cooled to room temperature. The loss of weight was made up with methanol. The solution was filtered through a 0.22 μm filter membrane (φ13, Jinteng, Tianjin, China), and the filtrate was injected directly into the UPLC system (Acquity UPLC H-Class, Waters, MA, USA). An Acquity C18 column (50 mm × 2.1 mm, 1.7 μm) (Waters, USA) was used for analysis.
The column temperature and flow rate were set to 30 °C and 0.3 mL.min−1, respectively. The detection wavelength was 287 nm. The injection volume was 2 μL. Gradient elution was performed using acetonitrile (A)–water (B) as the mobile phase: 0 min: 65% (A) and 35% (B); 7 min: 50% (A) and 50% (B); 10 min: 45% (A) and 55% (B); 12 min: 10% (A) and 90% (B); 15 min: 85% (A) and 15% (B); 18 min: 65% (A) and 35% (B). Asarinin standard was added with methanol to prepare a series of standard solutions (1, 5, 10, 50, 100, and 200 μg.mL−1), and the injection volume was 2 uL. The standard curve was plotted with the concentration as the abscissa (x) and the chromatographic peak area as the ordinate (y) in Table S1.

4.6. Study of the Anti-Inflammatory and Analgesic Effect of Asarum

The mice used in the experiments were purchased from Beijing Huafukang Biotechnology Co., Beijing, Ltd., China. The rearing room was designed to maintain environmental control for the animals, with a 12 h light–dark cycle, a temperature of 22 ± 2 °C, and a relative humidity of 50% ± 10%. The animals were allowed to eat and drink ad libitum during feeding. All animals were acclimated for three days prior to the main experiment. The animals were starved the night before the experiment and given unlimited access to water. The animal experiments were approved by the Animal Care and Use Committee of the Shanghai University of Traditional Chinese Medicine (Jilin Agricultural University; ethic number: 2023 02 02 001).
The mice were randomly divided into 14 groups of 10 mice each (Table 5). According to “Fang Pharmacology”, the dose for a 70 kg adult is 3 g, so the equivalent dose was calculated based on the body surface area [41]. The dexamethasone group received dexamethasone solution (0.260 g.kg−1). The extract was administered orally. The ear oedema test was performed as previously reported [42]. The degree of swelling (mg) and the inhibition rate (%) for each group were determined as previously reported [36]. In addition, the acetic acid writhing test was performed according to the method of [42,43].

4.7. Statistical Analyses

LC-MS data were processed and analysed as follows. Pearson’s correlation coefficient and statistical significance were calculated in R using the cor and cor.mtest packages, respectively (https://www.r-project.org/; accessed on 20 December 2023). The correlation was visualized using the corrplot package in R. The obtained total ion current chromatograms were searched and compared with the spectra in the mzCloud (https://www.mzcloud.org/; accessed on 20 December 2023), mzVault [44], and MassList (https://massbank.eu/MassBank/; accessed on 20 December 2023) mass spectrometry databases to determine the chemical components in the EEs. The differentially accumulated metabolites (DAMs) were screened if the variable importance of projection (VIP) > 1, p < 0.05, and fold change ≥2 and ≤0.5. Metabolites were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [45] and lipid maps [46]. The DAMs were enriched in KEGG pathways [47].
Data were analysed using a one-way analysis of variance in SPSS statistical software version 26.0. Tests of significant differences among treatments were analysed using the least significant difference (LSD) test. The significance level was set at p < 0.05 and 0.01.

5. Conclusions

Our results show that the active components in Asarum samples from different producing areas are different, i.e., EO, EE, and asarinin. There was a significant difference in EO components between A. heterotropoides and A. sieboldii samples. In general, wild and cultivated Asarum roots exhibit different EO, EE, and AsCs. Among the four tested methods of drying Asarum roots, washing the roots with water and drying (shade drying or drying at 30 or 40 °C) proved to be better than the method in which the samples were not washed and only the soil was removed prior to drying. Both species differ in terms of contents of six major EOs. Moreover, EEs of A. heterotropoides and A. sieboldii are different, hence can be used to distinguish both species. Our comparative results highlight that EOs have better pharmacodynamic performance compared to EEs. Thus, our results provide an important scientific basis for the cultivation, drying, and clinical application of Asarum.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17101301/s1, Figure S1. Map showing sites of Asarum heterotropoides and Asarum sieboldii samples from China. Table S1. Details of standard curve of each reference substance. Table S2. Biochemical profiles of Asarum roots processed by different methods. Table S3. List of constituents of essential oil detected in Asarum roots by using GC-MS. Table S4. List of components of ethanol extracts in Asarum roots (R) and leaves (L).

Author Contributions

Conceptualization, Z.W. and G.Z.; methodology, X.X. and Y.W. (Yanhong Wang); software, H.L.; validation, G.Z., and Z.Z.; formal analysis, X.X., Y.W. (Yingping Wang); investigation, Z.W.; resources, Z.W. and Y.W. (Yanhong Wang); data curation, G.W.; writing—original draft preparation, G.Z.; writing—review and editing, Z.Z.; visualization, X.X.; supervision, Y.W. (Yingping Wang); project administration, Z.W.; funding acquisition, Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study is funded by the Science and Technology Department Project from Jilin Province, China (20190304017YY), and the National Key Research Special Project of China (2019YFC1710700).

Institutional Review Board Statement

The animal experiments were approved by the Animal Care and Use Committee of the Shanghai University of Traditional Chinese Medicine (Jilin Agricultural University; ethic number: 2023 02 02 001).

Informed Consent Statement

Experimental research and studies on A. heterotropoides comply with relevant institutional, national, and international guidelines and legislation.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Antsyshkina, A.M.; Ars, Y.V.; Bokov, D.O.; Pozdnyakova, N.A.; Prostodusheva, T.V.; Zaichikova, S.G. The Genus Asarum L.: A Phytochemical and Ethnopharmacological Review. Syst. Rev. Pharm. 2020, 11, 472–502. [Google Scholar]
  2. Shaoqing, C.; Xuan, W.; Shu, Z.; Zhicen, L. Herbological study of Chinese traditional drug Xixin (herba asari). Beijing Yi Ke Da Xue Xue Bao J. Beijing Med. Univ. 1997, 29, 233–235. [Google Scholar]
  3. Perumalsamy, H.; Jung, M.Y.; Hong, S.M.; Ahn, Y.-J. Growth-Inhibiting and morphostructural effects of constituents identified in Asarum heterotropoides root on human intestinal bacteria. BMC Complement. Altern. Med. 2013, 13, 245. [Google Scholar] [CrossRef] [PubMed]
  4. Liu, H.; Wang, C. The genus Asarum: A review on phytochemistry, ethnopharmacology, toxicology and pharmacokinetics. J. Ethnopharmacol. 2022, 282, 114642. [Google Scholar] [CrossRef]
  5. Dan, Y.; Liu, H.-Y.; Gao, W.-W.; Chen, S.-L. Activities of essential oils from Asarum heterotropoides var. mandshuricum against five phytopathogens. Crop Prot. 2010, 29, 295–299. [Google Scholar] [CrossRef]
  6. Wang, Y.; Guo, S.; Cao, J.; Pang, X.; Zhang, Z.; Chen, Z.; Zhou, Y.; Geng, Z.; Sang, Y.; Du, S. Toxic and Repellent Effects of Volatile Phenylpropenes from Asarum heterotropoides on Lasioderma serricorne and Liposcelis bostrychophila. Molecules 2018, 23, 2131. [Google Scholar] [CrossRef]
  7. Pang, X.; Almaz, B.; Qi, X.-J.; Wang, Y.; Feng, Y.-X.; Geng, Z.-F.; Xi, C.; Du, S.-S. Bioactivity of essential oil from Atalantia buxifolia leaves and its major sesquiterpenes against three stored-product insects. J. Essent. Oil Bear. Plants 2020, 23, 38–50. [Google Scholar] [CrossRef]
  8. Lee, J.; Lee, Y.J.; Oh, S.-M.; Yi, J.-M.; Kim, N.S.; Bang, O.-S. Bioactive compounds from the roots of Asiasarum heterotropoides. Molecules 2013, 19, 122–138. [Google Scholar] [CrossRef]
  9. Jeong, M.; Kim, H.M.; Lee, J.S.; Choi, J.-H.; Jang, D.S. (−)-Asarinin from the roots of Asarum sieboldii induces apoptotic cell death via caspase activation in human ovarian cancer cells. Molecules 2018, 23, 1849. [Google Scholar] [CrossRef] [PubMed]
  10. Jeong, S.H.; Jang, J.H.; Cho, H.Y.; Lee, Y.B. Simultaneous determination of asarinin, β-eudesmol, and wogonin in rats using ultraperformance liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic studies following administration of standards and Gumiganghwal-tang. Biomed. Chromatogr. 2021, 35, e5021. [Google Scholar] [CrossRef] [PubMed]
  11. Haque, A.T.; Moon, J.N.; Saravana, P.; Tilahun, A.; Chun, B.-S. Composition of Asarum heterotropoides var. mandshuricum radix oil from different extraction methods and activities against human body odor-producing bacteria. J. Food Drug Anal. 2016, 24, 813–821. [Google Scholar] [CrossRef] [PubMed]
  12. Chen, F.; Zhang, Q.; Gu, H.; Yang, L. A modified approach for separating essential oil from the roots and rhizomes of Asarum heterotropoides var. mandshuricum. J. Clean. Prod. 2018, 172, 2075–2089. [Google Scholar] [CrossRef]
  13. Li, J.-H.; Cui, E.-J.; Zheng, C.-J. Chemical constituents from the roots of Asarum sieboldii Miq. var. Seoulense Nakai. Biochem. Syst. Ecol. 2021, 94, 104220. [Google Scholar] [CrossRef]
  14. Wang, Z.-Q. Analysis and evaluation on main active ingredients of Asarum heterotropoides var. mandshuricum from different habitats. Chin. Tradit. Herb. Drugs 2020, 24, 748–756. [Google Scholar]
  15. Liu, D.; Liu, C. Chemical Components of Volatile Oil Chemical of Cultivated Asarum Produced in Different Places. Chin. J. Exp. Tradit. Med. Formulae 2010, 16, 79–82. [Google Scholar]
  16. Ahmed, A.; Ayoub, K.; Chaima, A.J.; Hanaa, L.; Abdelaziz, C. Effect of drying methods on yield, chemical composition and bioactivities of essential oil obtained from Moroccan Mentha pulegium L. Biocatal. Agric. Biotechnol. 2018, 16, 638–643. [Google Scholar] [CrossRef]
  17. Zhu, L.; Li, M.; Yang, W.; Zhang, J.; Yang, X.; Zhang, Q.; Wang, H. Effects of different drying methods on drying characteristics and quality of Glycyrrhiza uralensis (licorice). Foods 2023, 12, 1652. [Google Scholar] [CrossRef] [PubMed]
  18. Jing, Y.; Zhang, Y.-F.; Shang, M.-Y.; Yu, J.; Tang, J.-W.; Liu, G.-X.; Li, Y.-L.; Li, X.-M.; Wang, X.; Cai, S.-Q. Phenanthrene derivatives from roots and rhizomes of Asarum heterotropoides var. mandshuricum. Fitoterapia 2017, 117, 101–108. [Google Scholar] [CrossRef] [PubMed]
  19. Pan, L.; Yang, N.; Sui, Y.; Li, Y.; Zhao, W.; Zhang, L.; Mu, L.; Tang, Z. Altitudinal Variation on Metabolites, Elements, and Antioxidant Activities of Medicinal Plant Asarum. Metabolites 2023, 13, 1193. [Google Scholar] [CrossRef] [PubMed]
  20. Shahat, A.A.; Hammouda, F.M.; Shams, K.A.; Saleh, M.A. Comparative chemical analysis of the essential oil of wild and cultivated fennel (Foeniculum vulgare Mill). J. Essent. Oil Bear. Plants 2012, 15, 314–319. [Google Scholar] [CrossRef]
  21. Arsenijević, J.; Drobac, M.; Šoštarić, I.; Jevđović, R.; Živković, J.; Ražić, S.; Moravčević, Đ.; Maksimović, Z. Comparison of essential oils and hydromethanol extracts of cultivated and wild growing Thymus pannonicus All. Ind. Crops Prod. 2019, 130, 162–169. [Google Scholar] [CrossRef]
  22. Ilić, Z.S.; Kevrešan, Ž.; Šunić, L.; Stanojević, L.; Milenković, L.; Stanojević, J.; Milenković, A.; Cvetković, D. Chemical profiling and antioxidant activity of wild and cultivated sage (Salvia officinalis L.) essential oil. Horticulturae 2023, 9, 624. [Google Scholar] [CrossRef]
  23. Hassiotis, C.; Ntana, F.; Lazari, D.; Poulios, S.; Vlachonasios, K. Environmental and developmental factors affect essential oil production and quality of Lavandula angustifolia during flowering period. Ind. Crops Prod. 2014, 62, 359–366. [Google Scholar] [CrossRef]
  24. Lal, R.; Gupta, P.; Chanotiya, C.; Mishra, A.; Kumar, A. Eminent high essential oil yielding and photosynthesis efficient genotype selection across multi-environments in the palmarosa {Cymbopogon martinii (Roxb.) Wats.}. Ecol. Genet. Genom. 2023, 27, 100167. [Google Scholar] [CrossRef]
  25. Das, S.; Prakash, B. Effect of Environmental Factors on Essential Oil Biosynthesis, Chemical Stability, and Yields. In Plant Essential Oils: From Traditional to Modern-Day Application; Springer: Berlin/Heidelberg, Germany, 2023; pp. 225–247. [Google Scholar]
  26. Elshamy, A.I.; Abd-ElGawad, A.M.; El-Amier, Y.A.; El Gendy, A.E.N.G.; Al-Rowaily, S.L. Interspecific variation, antioxidant and allelopathic activity of the essential oil from three Launaea species growing naturally in heterogeneous habitats in Egypt. Flavour Fragr. J. 2019, 34, 316–328. [Google Scholar] [CrossRef]
  27. Maurya, S.; Chandra, M.; Yadav, R.K.; Narnoliya, L.K.; Sangwan, R.S.; Bansal, S.; Sandhu, P.; Singh, U.; Kumar, D.; Sangwan, N.S. Interspecies comparative features of trichomes in Ocimum reveal insights for biosynthesis of specialized essential oil metabolites. Protoplasma 2019, 256, 893–907. [Google Scholar] [CrossRef]
  28. Xiao, Y.; Liu, Z.; Gu, H.; Yang, F.; Zhang, L.; Yang, L. Improved method to obtain essential oil, asarinin and sesamin from Asarum heterotropoides var. mandshuricum using microwave-assisted steam distillation followed by solvent extraction and antifungal activity of essential oil against Fusarium spp. Ind. Crops Prod. 2021, 162, 113295. [Google Scholar] [CrossRef]
  29. Şanli, A.; Karadoğan, T. Geographical impact on essential oil composition of endemic Kundmannia anatolica Hub.-Mor.(Apiaceae). Afr. J. Tradit. Complement. Altern. Med. 2017, 14, 131–137. [Google Scholar] [CrossRef]
  30. Wu, H.; Li, J.; Zhang, F.; Li, L.; Liu, Z.; He, Z. Essential oil components from Asarum sieboldii Miquel are toxic to the house dust mite Dermatophagoides farinae. Parasitol. Res. 2012, 111, 1895–1899. [Google Scholar] [CrossRef]
  31. Jing, Y.; Zhang, Y.-F.; Shang, M.-Y.; Liu, G.-X.; Li, Y.-L.; Wang, X.; Cai, S.-Q. Chemical constituents from the roots and rhizomes of Asarum heterotropoides var. mandshuricum and the in vitro anti-inflammatory activity. Molecules 2017, 22, 125. [Google Scholar] [CrossRef]
  32. Xie, B.; Shang, M.; Wang, X.; Cai, S.; Lee, K. A new aristolochic acid derivative from Asarum himalaicum. Yao Xue Xue Bao Acta Pharm. Sin. 2011, 46, 188–192. [Google Scholar]
  33. Xie, B.B.; Shang, M.Y.; Lee, K.-H.; Wang, X.; Komatsu, K.; Cai, S.Q. 5–Methoxyaristololactam I, the First Natural 5–Substituted Aristololactam from Asarum ichangense. Nat. Prod. Commun. 2011, 6, 11–14. [Google Scholar] [CrossRef] [PubMed]
  34. Wu, T.-S.; Ou, L.-F.; Teng, C.-M. Aristolochic acids, aristolactam alkaloids and amides from Aristolochia kankauensis. Phytochemistry 1994, 36, 1063–1068. [Google Scholar] [PubMed]
  35. Zhang, Z.; Kang, H. Protective effect of Asarum sieboldii essential oil on ovalbumin induced allergic rhinitis in rat. Biosci. Rep. 2020, 40, BSR20191370. [Google Scholar] [CrossRef] [PubMed]
  36. Liu, H.; Li, S.; Huan, X.; Xie, Y.; Xie, Z.; Sun, Y.; Cao, N.; Xie, Q.; Wang, Y.; Wang, H. The antinociceptive and anti-inflammatory potential and pharmacokinetic study of significant alkamides ingredients from Asarum Linn. J. Ethnopharmacol. 2022, 297, 115569. [Google Scholar] [CrossRef]
  37. Chen, M.-T.; Yang, Y.-J.; Li, Y.-S.; Li, X.-J.; Zhang, W.K.; Wang, J.-P.; Wang, X.; Tian, G.-H.; Tang, H.-B. Shengfu oil enhances the healing of full-thickness scalded skin accompanying the differential regulation of β-catenin, Dlk1, and COX-2. Front. Pharmacol. 2017, 8, 801. [Google Scholar] [CrossRef]
  38. Baumgart, D.C.; Carding, S.R. Inflammatory bowel disease: Cause and immunobiology. Lancet 2007, 369, 1627–1640. [Google Scholar] [CrossRef]
  39. Zuo, X.; Gu, Y.; Wang, C.; Zhang, J.; Zhang, J.; Wang, G.; Wang, F. A systematic review of the anti-inflammatory and immunomodulatory properties of 16 essential oils of herbs. Evid.-Based Complement. Altern. Med. 2020, 2020, 8878927. [Google Scholar] [CrossRef]
  40. Pharmacopoeia, C. Commission. In Pharmacopoeia of the People’s Republic of China; China Medical Science and Technology Press: Beijing, China, 2010. [Google Scholar]
  41. Nair, A.B.; Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 2016, 7, 27. [Google Scholar] [CrossRef]
  42. Xiong, Y.; Jing, Y.; Shang, M.; Li, C.; Ye, J.; Wang, X.; Cai, S. Anti-inflammatory and anti-nociceptive effects in mice of water and ethanol extracts of roots and rhizomes of Asarum heterotropoides var. mandshuricum. Zhongguo Zhong Yao Za Zhi Zhongguo Zhongyao Zazhi China J. Chin. Mater. Medica 2009, 34, 2252–2257. [Google Scholar]
  43. Kim, S.-J.; Zhang, C.G.; Lim, J.T. Mechanism of anti-nociceptive effects of Asarum sieboldii Miq. Radix: Potential role of bradykinin, histamine and opioid receptor-mediated pathways. J. Ethnopharmacol. 2003, 88, 5–9. [Google Scholar] [CrossRef] [PubMed]
  44. Li, Y.; Zhu, W.; Xiang, Q.; Kim, J.; Dufresne, C.; Liu, Y.; Li, T.; Chen, S. Creation of a Plant Metabolite Spectral Library for Untargeted and Targeted Metabolomics. Int. J. Mol. Sci. 2023, 24, 2249. [Google Scholar] [CrossRef] [PubMed]
  45. Kanehisa, M. KEGG bioinformatics resource for plant genomics and metabolomics. Plant Bioinform. Methods Protoc. 2016, 1374, 55–70. [Google Scholar]
  46. Liebisch, G.; Fahy, E.; Aoki, J.; Dennis, E.A.; Durand, T.; Ejsing, C.S.; Fedorova, M.; Feussner, I.; Griffiths, W.J.; Köfeler, H. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J. Lipid Res. 2020, 61, 1539–1555. [Google Scholar] [CrossRef]
  47. Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27–30. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. (A) Essential oil, ethanol extract, and asarinin content in Asarum roots. (B) Number of metabolites accumulated in Assarum roots according to class. (C) Content distribution of six compounds in sample essential oil in 36 Asarum samples (roots). Samples 1–31 are Asarum heterotropoides Fr. var. mandshuricum (Maxim.) Kitag (cultivated), and Samples 32–36 are Asarum sieboldii Miq var. seoulense Nakai (wild).
Figure 1. (A) Essential oil, ethanol extract, and asarinin content in Asarum roots. (B) Number of metabolites accumulated in Assarum roots according to class. (C) Content distribution of six compounds in sample essential oil in 36 Asarum samples (roots). Samples 1–31 are Asarum heterotropoides Fr. var. mandshuricum (Maxim.) Kitag (cultivated), and Samples 32–36 are Asarum sieboldii Miq var. seoulense Nakai (wild).
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Figure 2. Total ion current chromatograms of ethanol extract samples. CN1 indicates L24-3, CN2 indicates L32-3, CN3 indicates R24-3, and CN4 indicates R32-3.
Figure 2. Total ion current chromatograms of ethanol extract samples. CN1 indicates L24-3, CN2 indicates L32-3, CN3 indicates R24-3, and CN4 indicates R32-3.
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Figure 3. Comparative metabolome analysis of A. heterotropoides and A. sieboldii roots and leaves. (A) Venn diagram of common and specific metabolites in roots and leaves of the two species. (Bi) Volcano map of leaf vs. root differential metabolites in negative ion mode and (Bii) Volcano map of leaf vs. root differential metabolites in positive ion mode. The abscissa represents log2 (fold change); the ordinate represents −log10 (p-value); each point represents a compound; the size of each point represents the VIP value, i.e., the larger the point, the larger the VIP value; up (red) represents the compounds with significant difference and upregulation; DW (green) represents the compounds with significant difference and downregulation; no Diff (grey) represents the compounds with no significant difference; the greater the absolute value of the abscissa, the greater the difference between samples; the larger the ordinate value, the more significant the difference between the samples, and the more reliable the result. (Ci) Heatmap displaying the variation in differential metabolite accumulation in roots and leaves of samples, with negative and (Cii) with positive ion modes. The ruler indicates the level of metabolite expression. The redder the colour, the higher the metabolite expression. The bluer the colour, the lower the metabolite expression. Each row represents a metabolite. (Di) KEGG pathway enrichment of the differentially accumulated metabolites in leaf vs. roots, with negative and (Dii) with positive ion modes. (Ei) Annotation of the lipid compounds detected, with negative and (Eii) with positive ion modes. HXXL indicates L32, BXXL indicates L24-3, HXXR indicates R32, and BXXR indicates R24-3. L = leaf and R = root.
Figure 3. Comparative metabolome analysis of A. heterotropoides and A. sieboldii roots and leaves. (A) Venn diagram of common and specific metabolites in roots and leaves of the two species. (Bi) Volcano map of leaf vs. root differential metabolites in negative ion mode and (Bii) Volcano map of leaf vs. root differential metabolites in positive ion mode. The abscissa represents log2 (fold change); the ordinate represents −log10 (p-value); each point represents a compound; the size of each point represents the VIP value, i.e., the larger the point, the larger the VIP value; up (red) represents the compounds with significant difference and upregulation; DW (green) represents the compounds with significant difference and downregulation; no Diff (grey) represents the compounds with no significant difference; the greater the absolute value of the abscissa, the greater the difference between samples; the larger the ordinate value, the more significant the difference between the samples, and the more reliable the result. (Ci) Heatmap displaying the variation in differential metabolite accumulation in roots and leaves of samples, with negative and (Cii) with positive ion modes. The ruler indicates the level of metabolite expression. The redder the colour, the higher the metabolite expression. The bluer the colour, the lower the metabolite expression. Each row represents a metabolite. (Di) KEGG pathway enrichment of the differentially accumulated metabolites in leaf vs. roots, with negative and (Dii) with positive ion modes. (Ei) Annotation of the lipid compounds detected, with negative and (Eii) with positive ion modes. HXXL indicates L32, BXXL indicates L24-3, HXXR indicates R32, and BXXR indicates R24-3. L = leaf and R = root.
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Table 1. Relative content differences in DAMs between leaves and roots of A. heterotropoides.
Table 1. Relative content differences in DAMs between leaves and roots of A. heterotropoides.
CompoundR24L24
(3,4,5-Trihydroxy-6-methyloxan-2-yl)2-(methylamino)benzoate0.17143.7265
3,4-Dimethoxycinnamic acid4.77180.3927
2-(hydroxymethyl)-5-[6-(methylamino)purin-9-yl]oxolan-3-ol4.21070.3638
Saxitoxin0.15016.4897
4-Methoxycinnamaldehyde2.40544.9084
[(2S)-2,3-dihydroxypropyl] (6Z,9Z,12Z)-octadeca-6,9,12-trienoate0.14745.3676
Salsolinol0.28272.7246
Isorhamnetin0.01340.1739
Indoline-2-carboxylic acid0.12480.6896
Choline3.90464.8109
DL-Arginine3.19280.577
6-Methoxyflavanone0.15190.3365
Normorphine0.14691.3902
(S)-Glutamic acid2.99121.0074
(2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide4.44231.4944
Muramic acid0.96210.0958
N-Desmethylvenlafaxine1.04440.0757
gamma-Glutamylglutamic acid2.32826.4897
Asarinin1.51484.9084
O-Desmethylvenlafaxine1.50974.8109
Asparagine1.93773.7265
Sauchinone2.1033.6052
(9Z,11E,15Z)-13-hydroxyoctadeca-9,11,15-trienoic acid1.06822.7246
9-Oxo-10(E),12(E)-octadecadienoic acid1.20092.4944
Adenosine1.15781.6364
Acetyl-trans-resveratrol3.7221.4944
L-Pyroglutamic acid1.60791.3886
(2E,4E)-N-(2-methylpropyl)dodeca-2,4-dienamide5.67641.2843
Methyl caffeate1.06021.1626
5-Methyluridine1.20541.0929
Menadione1.18931.078
Kaempferol0.00981.0608
Stiripentol0.08891.0074
Aristolochic acids0.00060.0062
Table 2. Effect of volatile oil and EEs of Asarum roots pretreatment on oedema degree and inhibition rate.
Table 2. Effect of volatile oil and EEs of Asarum roots pretreatment on oedema degree and inhibition rate.
GroupDegree of Swelling (mg)Inhibition Rate (%)GroupDegree of Swelling (mg)Inhibition Rate (%)
NS11.40 ± 1.220Ibuprofen6.90 ± 0.43 **39.47
V2low8.60 ± 0.94 **29.82E2low9.38 ± 0.53 *17.72
V2mild7.62 ± 0.99 **33.04E2mild8.68 ± 0.92 **23.86
V2high6.52 ± 0.86 **42.81E2high8.38 ± 0.88 **26.49
V8low6.42 ± 0.50 **43.68E8low8.68 ± 0.76 **23.86
V8mild5.46 ± 0.78 **52.11E8medium8.04 ± 0.61 **29.47
V8high4.98 ± 0.61 **56.32E8high7.01 ± 0.80 **38.6
NS = normal saline; V = essential oil extract; E = ethanol extract. The values are mean of n = 10 ± standard deviation. * = significant (p ≤ 0.05) and ** = significant (p ≤ 0.01).
Table 3. Effect of volatile oil and EEs of Asarum roots pretreatment on acetic acid writhing in mice.
Table 3. Effect of volatile oil and EEs of Asarum roots pretreatment on acetic acid writhing in mice.
GroupNumber of Twists (Second Rate)Inhibition Rate (%)GroupNumber of Twists (Second Rate)Inhibition Rate (%)
NS41.2 ± 2.940DEX19.6 ± 2.70 **52.43
V2low28.4 ± 3.20 **31.07E2low31.2 ± 4.54 **24.27
V2mild21.8 ± 3.96 **47.09E2mild26.6 ± 4.80 **35.44
V2high17.6 ± 1.81 **57.28E2high19.2 ± 3.26 **53.4
V8low20.8 ± 3.27 **49.51E8low25.8 ± 4.86 **37.38
V8mild14.4 ± 1.14 **65.05E8medium18.6 ± 3.04 **54.85
V8high6.20 ± 2.58 **84.95E8high11.4 ± 3.02 **72.33
NS = normal saline; DEX = dexamethasone; V = essential oil extract; E = ethanol extract. The values are mean of n = 10 ± standard deviation. ** = significant (p ≤ 0.01).
Table 4. Details of the Asarum samples used in this study.
Table 4. Details of the Asarum samples used in this study.
No.OriginLatitude and LongitudeGrowth YearsCollection DateNo.OriginLatitude and LongitudeGrowth YearsCollection Date
1Fusong, JilinN 41°55′ E 128°01′Four2020.0719Liuhe, JilinN 42°07′ E 125°35′Six2020.08
2Fusong, JilinN 41°55′ E 128°01′Six2020.0720Liuhe, JilinN 42°07′ E 125°35′Eight2020.08
3Jian, JilinN 40°58′ E 126°18′Seven2020.0621Tonghua, JilinN 42°47′ E 126°23′Four2020.06
4Jian, JilinN 40°58′ E 126°18′Seven2020.0722Tonghua, JilinN 42°47′ E 126°23′Four2020.07
5Jian, JilinN 40°58′ E 126°18′Seven2020.0823Tonghua, JilinN 42°47′ E 126°23′Four2020.08
6Jian, JilinN 40°58′ E 126°18′Seven2020.0924Tonghua, JilinN 42°47′ E 126°23′Five2020.07
7Jian, JilinN 40°58′ E 126°18′Wild2020.0625Tonghua, JilinN 42°47′ E 126°23′Five2020.08
8Jian, JilinN 40°58′ E 126°18′Wild2020.0726Tonghua, JilinN 42°47′ E 126°23′Six2020.06
9Jian, JilinN 40°58′ E 126°18′Wild2020.0827Tonghua, JilinN 42°47′ E 126°23′Six2020.07
10Jian, JilinN 40°58′ E 126°18′Wild2020.0928Tonghua, JilinN 42°47′ E 126°23′Six2020.08
11Liuhe, JilinN 42°07′ E 125°35′Four2020.0629Wangqing, JilinN 43°36′ E 129°44′Ten2020.07
12Liuhe, JilinN 42°07′ E 125°35′Four2020.0730Xinbin, LiaoningN 41°22′ E 124°49′Ten2020.06
13Liuhe, JilinN 42°07′ E 125°35′Four2020.0831Xinbin, LiaoningN 41°22′ E 124°49′Wild2020.06
14Liuhe, JilinN 42°07′ E 125°35′Five2020.0632Ningqiang, ShanxiN 33°48′ E 105°31′Five2020.07
15Liuhe, JilinN 42°07′ E 125°35′Five2020.0733Ningqiang, ShanxiN 33°48′ E 105°31′Six2020.07
16Liuhe, JilinN 42°07′ E 125°35′Five2020.0834Ningqiang, ShanxiN 33°48′ E 105°31′Wild2020.07
17Liuhe, JilinN 42°07′ E 125°35′Six2020.0635Lveyang, ShanxiN 33°33′ E 106°16′Six2020.07
18Liuhe, JilinN 42°07′ E 125°35′Six2020.0736Lveyang, ShanxiN 33°33′ E 106°16′Wild2020.07
Note: Samples 1–31 are Asarum heterotropoides Fr. var. mandshuricum (Maxim.) Kitag (cultivated), and Samples 32–36 are Asarum sieboldii Miq var. seoulense Nakai (wild).
Table 5. Doses of volatile oil and ethanol extracts of Asarum roots used for pharmacodynamic study of mice.
Table 5. Doses of volatile oil and ethanol extracts of Asarum roots used for pharmacodynamic study of mice.
Pretreatment GroupDose (μL.kg−1)Dose (μL.kg−1)
Normal SalineNS00
Volatile oilV2 Low 1.651.65
Volatile oilV2 mild 3.33.3
Volatile oilV2 high 6.66.6
Volatile oilV8 Low 1.71.7
Volatile oilV8 mild 3.43.4
Volatile oilV8 high 6.86.8
Dexamethasone/ibuprofenDEX/IBP(DEX) 0.26(IBP) 0.104
Ethanol extractE2 Low 8.858.85
Ethanol extractE2 mild 17.717.7
Ethanol extractE2 high 35.435.4
Ethanol extractE8 Low 13.913.9
Ethanol extractE8 mild 27.827.8
Ethanol extractE8 high 55.655.6
DEX = dexamethasone; IBP = ibuprofen.
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MDPI and ACS Style

Li, H.; Wang, Z.; Zhao, G.; Wang, Y.; Xu, X.; Wang, Y.; Zhang, Z.; Wang, G. Comparative Biochemical and Pharmacodynamic Analyses of Asarum heterotropoides Fr. Schmidt var. Mandshuricum (Maxim) Kitag and Asarum sieboldii Miq var. Seoulense Nakai Roots. Pharmaceuticals 2024, 17, 1301. https://doi.org/10.3390/ph17101301

AMA Style

Li H, Wang Z, Zhao G, Wang Y, Xu X, Wang Y, Zhang Z, Wang G. Comparative Biochemical and Pharmacodynamic Analyses of Asarum heterotropoides Fr. Schmidt var. Mandshuricum (Maxim) Kitag and Asarum sieboldii Miq var. Seoulense Nakai Roots. Pharmaceuticals. 2024; 17(10):1301. https://doi.org/10.3390/ph17101301

Chicago/Turabian Style

Li, Huiling, Zhiqing Wang, Guangyuan Zhao, Yanhong Wang, Xuanwei Xu, Yingping Wang, Ze Zhang, and Guanghui Wang. 2024. "Comparative Biochemical and Pharmacodynamic Analyses of Asarum heterotropoides Fr. Schmidt var. Mandshuricum (Maxim) Kitag and Asarum sieboldii Miq var. Seoulense Nakai Roots" Pharmaceuticals 17, no. 10: 1301. https://doi.org/10.3390/ph17101301

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