A Network Pharmacology-Based Study on Pharmacological Activities and Mechanisms of Essential Oil in Leaves of C. Grandis ‘Tomentosa’

Background and Objective: Citrus grandis ‘Tomentosa’, as the fruit epicarp of C. grandis ‘Tomentosa’ or C. grandis (L.) Osbeck, is widely used in health food and medicine. Actually, based on our survey results, there are also rich essential oils with bioativities in leaves, but the chemical compounds in this part and relevant pharmacological activities have never been studied systematically yet. Therefore, this study was to preliminarily decipher the pharmacological activities and mechanisms of the essential oil in leaves of C. grandis ‘Tomentosa’ by an integrated network pharmacology approach. Methods: Essential oil compositions from leaves of C. grandis ‘Tomentosa’ were identied using GC-MS/MS. And then the targets of these oil compositions were predicted and screened from TCMSP, SwissTargetPrediction, STITCH and SEA databases. STRING database was used to construct the protein-protein interaction networks, and the eligible protein targets were input into WebGestalt 2019 to carry out GO enrichment and KEGG pathway enrichment analysis. Based on the potential targets, disease enrichment information was obtained by TTD databases. Cytoscape software was used to construct the component-target-disease network diagrams. Results: Finally, 61 essential oil chemical components were identied by GC-MS/MS, which correspond to 679 potential targets. Biological function analysis showed that there were 12, 19, and 12 GO entries related to biological processes, cell components and molecular functions, respectively. 43 KEGG pathways were identied, of which the most signicant categories were terpenoid backbone biosynthesis, TNF signaling pathway and leishmaniasis. The component-target-disease network diagram revealed that the essential oil compositions in leaves of C. grandis ‘Tomentosa’ could treat tumors, immune diseases, neurodegenerative diseases and respiratory diseases, which were highly related to CHRM1, PTGS2, CASP3, MAP2K1 and CDC25B. Conclusion: This study may provide a new insight into C. grandis ‘Tomentosa’ or C. grandis (L.) Osbeck and may provide useful information for future utilization and development.


Background
Citri Grandis Exocarpium (Huajuhong), recorded o cially in the current Chinese Pharmacopoeia (2020 edition), is the fruit epicarp of C. grandis 'Tomentosa' or C. grandis (L.) Osbeck particularly originated from Huazhou town in Guangdong province, southern China. Citri Grandis Exocarpium is a medicinal and edible food and is well known as nutritional bene ts and pharmaceutical effects. It is a rich source of avonoids, essential oil, polysaccharides, coumarins and limonoids [1][2][3]. Its therapeutic effects in traditional Chinese medicine include regulating qi-owing and eliminating dampness and phlegm. Modern researches further demonstrated that Citri Grandis Exocarpium has anti-tussive, anti-oxidant, antiin ammatory, anti-microbial, anti-proliferative and anti-atherosclerotic activities [4,5].
Fruit epicarp and whole fruit are the most commonly used parts of C. grandis 'Tomentosa' or C. grandis (L.) Osbeck, in which essential oils play an important in pharmacological effects. Essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds usually extracted from plants.
Essential oils are often used for aromatherapy to induce relaxation and proper application can effectively treat diseases [6]. Actually, based on our survey results, there are also rich essential oil in the leaves of C. grandis 'Tomentosa', but the chemical compounds in this part and relevant pharmacological activities have never been studied systematically yet. Due to the limited availability of reference substances, gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was applied to characterize components in this study, which have been widely accepted to be the predominant tool for the analysis of essential oil contents. It provides signi cant advantages for unequivocal identi cation and quanti cation of very low limits of ingredients [7]. Network pharmacology, proposed by Andrew L Hopkins [8], can build a "compound-target-disease" multilevel network to analyze the active ingredients, relevant pharmacological activities and possible molecular network mechanisms, which is highly in accordance with the connotation of holistic theory, multi-components and multi-targets of Chinese medicine [9][10][11].
In this study, essential oil contents in the leaves of C. grandis 'Tomentosa' were identi ed by GC-MS/MS and network pharmacology was employed to establish the compound-target-disease network to explore the potential pharmacological activities and mechanism. The results obtained will provide direct and reliable evidence for the broader research and application of C. grandis 'Tomentosa', which will contribute to reducing the waste of resource and bringing economic bene ts.

Methods
Identi cation of essential oil composition Leaves of C. grandis 'Tomentosa' (Fig. 7) were obtained from the genuine producing area Huazhou city and were authenticated by Professor Huan-lan Liu from Guangdong University of Chinese Medicine. 50 g leave pieces were extracted by Soxhlet extractor with 150 mL anhydrous ether for 18 h. Then the extract was dried by anhydrous sodium sulfate, and concentrated to dryness with a termovap sample concentrator. The ether was evaporated and the volume was adjusted to 1 mL with n-hexane. The sample was ltered through a 0.22 µm PTFE syringe lter. The chemical composition of essential oil was determined by GC-MS/MS. GC analysis was performed on an Agilent 5977B GC-MS/MS system, equipped with an Agilent Multimode injector. The column used was a HP-5ms, 30 m × 0.25 mm i.d., 0.25µm lm thickness. The carrier gas was helium at a constant ow rate of 1.0 mL/min. The injection was conducted in splitless mode at 250°C for 3 min and the injected volume was 1 µL. The oven temperature program consisted in the following steps: an initial temperature of 60°C maintained for 3 min, heating from 60°C to 110°C at a rate of 5°C/min, and then raised to 150°C at a rate of 4°C/min, where the nal temperature of 240°C was held for 5 min. The temperature of the transfer line was 240°C. The mass spectrometer was operated in electron ionization mode at 70 eV and the ions were detected by a triple quadrupole mass spectrometer. Data acquisition and analyses were performed using the MassHunter Workstation software.
Targets screening of essential oil composition The protein targets of the essential oil composition in leaves of C. grandis 'Tomentosa' were searched via the TCMSP (http://tcmspw.com/tcmsp.php), SwissTargetPrediction (http://www.swisstargetprediction.ch/), STITCH (http://stitch.embl.de/), and SEA [26] (http://sea.bkslab.org/) for each chemical component. Homo sapiens were the only species for the targets and the repetitive targets collected were removed. Then the component-target network of essential oil composition was constructed using Cytoscape software (Version 3.2.1) [27]. The network was analyzed using Cytoscape plugin CentiScaPe to calculate topological parameters, mainly including the degree, betweenness centrality, closeness centrality and average shortest path length [28]. The major ingredients and targets were represented by signi cant node, and the interactions were encoded by edges.

Construction and analysis of PPI
The PPI analysis was constructed by the open STRING database (https://string-db.org/cgi/input.pl), which contains information on protein/gene interactions, including veri ed experimental data, computational predicted data, and public text collections [29]. The targets of the essential oil composition were imported into the STRING database and the species was de ned as Homo sapiens. Then only the data on PPIs with high con dence scores (scores ≥ 0.9) were adopted for further analysis.

Results
Determination of essential oil composition in leaves of C. grandis 'Tomentosa' A total ion chromatogram obtained by GC-MS/MS for essential oil composition in leaves of C. grandis 'Tomentosa' was shown in Fig. 1. In total, 61 essential oil chemical components were identi ed and listed in Table 1. The families of detected essential oils in leaves mainly included terpenes (48.41%) and the oxygen containing derivatives of the terpenes-alcohols (34.73), which were consistent with the previous reports, but the contents was higher than those extracted in the same method from the Citri Grandis Exocarpium (fruit) [12]. The major compound was β-caryophyllene (15.75%), followed by (3R,6E)-nerolidol (12.66%), bicyclogermacrene (10.74%), β-citronellol (5.21%), 1-Methyl-4-(1-methylethylidene)-2-(1methylvinyl)-1-vinylcyclohexane (4.92%), geraniol (4.12%) and phytol (4.03%).  Table S1). Figure 2 showed the component-target network of essential oil composition in leaves of C. grandis 'Tomentosa', which contained 679 targets. The circular nodes represent the targets of essential oil composition, and the diamond nodes represent the chemical composition of essential oil. Each edge represents the interaction between the active component and the target. Only the targets with higher values of "degree" (above two-fold of the median value), "betweenness centrality" and "closeness centrality" (above the median value), and "average shortest path length" (below the median value) were identi ed as the candidate targets of the essential oil composition in leaves of C. grandis 'Tomentosa'. Ultimately, 4 direct targets were found to be highly correlated with the essential oil composition, among which PTGS2, CHRM1, GGPS1 and MAPK14 were associated with 34, 32, 20 and 9 chemical components, respectively ( Table 2). Construction and analysis of the PPI (protein-protein interaction) network The PPI network was showed in Fig. 3. The network contained 54 nodes (representing the action target) and 161 edges (representing association between a pair of action targets). Based on the calculation results from the Search Tool for the Retrieval of Interacting Genes (STRING) database, JUN and FOS were found to have the strongest combination ability and the combined score reached 0.999. According to the PPI network diagram, MAPK14 was in the center of the targets, which could be associated with 38 proteins, followed by JUN and TNF, associating with 16 and 15 respectively.

Functional enrichment analysis of target protein
For biological process, the target proteins were mainly enriched in metabolic process, biological regulation and response to stimulus (Fig. 4A). In terms of cellular component, it was revealed that these target proteins were mainly enriched in nucleus, cytosol and membrane-enclosed lumen (Fig. 4B). For molecular function, it was uncovered that the most target protein were enriched in protein binding, ion binding and transferase activity (Fig. 4C). The enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that 43 enriched categories were identi ed, in which 40 most signi cant categories such as terpenoid backbone biosynthesis, TNF signaling pathway and leishmaniasis were shown in Fig. 5.

Discussion
Traditional Chinese medicine is an important resource bank for the development of innovative new drugs.
However, compound Chinese medicine has the characteristics of multiple components, multiple targets, and multiple levels, and its mechanism is wide and di cult to be elucidated, so all of these greatly limits the use and development of Chinese medicine. In recent years, combination of Chinese medicine and network pharmacology has become a research hotspot, which contributes to systematically exploring the target and synergistic effects of the components of Chinese medicine, further realizing the development and modernization of Chinese medicine.
This study was to excavate the potential targets of the essential oil in leaves of C. grandis 'Tomentosa', explore its pharmacological mechanism and predict its treatable diseases based on the method of network pharmacology. The results showed that a total of 61 chemical components in the essential oil had their corresponding target proteins in the TCMS, Swiss Target Prediction, STITCH and SEA database, and a total of 679 potential targets were obtained. After lter by condition, 4 direct targets were found to be highly correlated with the essential oil composition, of which were PTGS2, CHRM1, GGPS1 and MAPK14. The PPI network was successfully constructed, which contained 54 nodes and 161 edges.
Functional enrichment analysis showed that there were 12, 19, and 12 Gene Ontology (GO) entries related to biological processes, cell components and molecular functions, respectively. A total of 43 KEGG pathways were obtained, of which the most signi cant was terpenoid backbone biosynthesis. 22 diseases were achieved from TTD and they were mainly classi ed as tumors, immune diseases, neurodegenerative diseases and respiratory diseases.
In the KEGG pathways, several experimental and clinical evidences reveal that TNF signaling pathway, infection and apoptosis were demonstrated to have a correlation with immune diseases. In addition, the potential target proteins of the essential oil in leaves of C. grandis 'Tomentosa' were enriched in TNF signaling pathway, toll-like receptor signaling pathway, amyotrophic lateral sclerosis, neurotrophin signaling pathway, human cytomegalovirus infection, apoptosis, which were involved in neurodegenerative diseases. Pertussis, Th17 cell differentiation, NF-kappa B signaling pathway, in uenza A, T cell receptor signaling pathway, herpes simplex infection and apoptosis were involved in the signal transduction of respiratory diseases, which may also be the main mechanism of the essential oil in leaves of C. grandis 'Tomentosa' in treating respiratory diseases.
In these pathways, apoptosis was involved in the regulation of four diseases. Th17 cell differentiation and NF-kappa B signaling pathway were connected with 3 diseases, including tumors, immune diseases and respiratory diseases. TNF signaling pathway, toll-like receptor signaling pathway and human cytomegalovirus infection also have connection with 3 diseases, including tumors, immune diseases and neurodegenerative diseases. Cell apoptosis, sometimes called programmed cell death, is an active process of commanding mutated cells to commit suicide. Apoptosis plays an important role in the occurrence of several diseases, such as tumor/cancer, autoimmune diseases, AIDS, ischemia, and neurodegenerative diseases. It is generally believed that malignant tumors are caused by the uncontrolled growth of cells and excessive proliferation. From the perspective of cell apoptosis, it is believed that the occurrence of malignant tumors is the result of the inhibition of tumor apoptosis [13]. Autoreactive T lymphocytes and antibody-producing B lymphocytes are the main immunopathological mechanisms that cause autoimmune diseases. Under the stimulation of self-antigens, immune cells that recognize selfantigens are activated and eliminated by apoptosis [14]. However, if this mechanism is disturbed, the clearance of immune-competent cells that recognize self-antigens will be obstructed. Cohen observed that Lpr and gld mice developed lymphadenopathy and splenomegaly in an age-dependent manner, by accumulating activated T lymphocytes and B lymphocytes [15]. Alzheimer's disease is an irreversible degenerative neurological disease, which is caused by the acceleration of nerve cell apoptosis. Studies found that presenilin-1 (PS1) and presenilin-2 (PS2) mutations lead to familial Alzheimer's disease [16]. Meanwhile, presenilin was demonstrated to be involved in the regulation of neuronal apoptosis [17].
Apoptosis is also relevant to the pathogenesis of different respiratory diseases. Asthma has been demonstrated to be associated with a defective activation of T-cell apoptosis through FAS death receptor [18]. Reactive oxygen species induced cell apoptosis have also been known to play a factor in the pathogenesis of acute respiratory distress syndrome [19].
In the network of component-target-disease, component-target-tumor network, component-target-immune disease network, component-target-neurodegenerative disease network and component-target-respiratory disease network were established. In which, CHRM1 was regarded as the main target becasue 32 essential oil compositions could act on CHRM1 and further be highly associated with cognitive impairment and chronic obstructive pulmonary disease. Pharmacological evidence suggests that cholinergic receptors are vital members of the cholinergic system, in which CHRM1 plays an important role in cognitive processes, hippocampal synaptic plasticity and neuronal excitability [20]. Some studies also found that CHRM1 was associated with bronchoconstriction of the airways, asthma, nicotine dependence and chronic obstructive pulmonary disease [21][22][23]. Farnesol was regarded as the most active essential oil composition in leaves of C. grandis 'Tomentosa', which was involved in solid tumor/cancer, arthritis, cognitive impairment, asthma and chronic obstructive pulmonary disease.
Farnesol, a natural terpene, is frequently found in essential oils [24]. A systematic review summarized that farnesol possessed broad pharmacological activities, including antimicrobial effect, preventing and treating cancer, promoting neuroprotective and behavioral effects, cardioprotective and hypotensive effects, antioxidant and anti-in ammatory properties [25].

Conclusion
In summary, this study was to study the pharmacological activity and mechanism of the essential oil in leaves of C. grandis 'Tomentosa' based on network pharmacology. The speci c chemical components of the essential oil were identi ed by GC-MS/MS, so the prediction accuracy is relatively high. The results revealed that the essential oil in leaves of C. grandis 'Tomentosa' had the potential to treat tumors, immune diseases, neurodegenerative diseases and respiratory diseases by multi-pathways and multitargets. In which, the most promising evidence was that farnesol may treat cognitive impairment and chronic obstructive pulmonary disease by regulating apoptosis via targeting CHRM1. However, further pharmacological experimental veri cation is needed.
The essential oils of leaves in C. grandis 'Tomentosa' have not been studied before. This study may provide a new insight into C. grandis 'Tomentosa' or C. grandis (L.) Osbeck and may provide useful information for future utilization and development.
Abbreviations GC-MS/MS: Gas chromatography coupled with tandem mass spectrometry;  Figure 1 A total ion chromatogram of essential oil from leaves of C. grandis 'Tomentosa'.

Figure 2
The component-target network of essential oil composition. The diamond nodes represent ingredients, and the circular nodes represent targets. The colors of the nodes are illustrated from red to yellow in descending order of degree values.

Figure 5
Enriched KEGG pathway of potential targets.