This research was supported by the Product Development and Innovation Team of TCM Health Preservation and Rehabilitation (2022C005) and Research on New Business Cross-border Integration of &#34;Health Preservation and Rehabilitation+&#34;.

All procedures involving the handling of animals were conducted in accordance with the Chengdu University of Traditional Chinese Medicine Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of the Chengdu University of Traditional Chinese Medicine (Protocol number 2020-36). Male C57BL/6 mice (20 &#177; 2 g) were used for the present study. The mice were obtained from a commercial source (see Table of Materials).
1. Net...

In recent years, the incidence rate of hyperlipidemia has been increasing, mainly due to long-term unhealthy eating habits. TCM and its chemical ingredients have various pharmacological activities, which have been widely studied in recent years37,38. FP is a kind of fruit resource, used both as medicine and food, and has an important potential for treating hyperlipidemia. However, the potential therapeutic mechanism of FP against hyperlipidemia needs further stud...

Hyperlipidemia is a common metabolic disease with serious impacts on human health, and is also the primary risk factor for cardiovascular diseases1. Recently, there has been a downward age-related trend for this disease, and younger people have become more susceptible because of long-term irregular lifestyles and unhealthy eating habits2. In the clinic, various drugs have been used to treat hyperlipidemia. For example, one of the most commonly used drugs for patients with hyperlipidemia and related atherosclerotic disorders is statins. However, long-term use of statins has side effects that can&#39;t be neglected, which lead to a poor prognosis, such as intolerance, treatment resistance, and adverse events3,4. These shortcomings have become additional pains for hyperlipidemia patients. Therefore, novel treatments for stable lipid-lowering efficacy and fewer side effects should be proposed.
Traditional Chinese Medicine (TCM) has been widely used to treat diseases because of its good efficacy and few side effects5. Fructus Phyllanthi (FP), the dried fruit of Phyllanthus emblica Linn. (popularly known as amla berry or Indian gooseberry), is a famous medicine and food homologous material of traditional Chinese and India medicines6,7. This medicine has been used for clearing heat, cooling blood, and promoting digestion, as per TCM theories8. Modern pharmacological studies have shown that FP is rich in bioactive compounds such as gallic acids, ellagic acids, and quercetin9, which are responsible for a range of multifaceted biological properties, by acting as an antioxidant, an anti-inflammatory, liver protection, an anti-hypolipidaemic, and so on10. Recent research has also showed that FP could effectively regulate the blood lipids of patients with hyperlipidemia. For example, Variya et al.11 have demonstrated that FP fruit juice and its main chemical ingredient of gallic acid can decrease plasma cholesterol and reduce oil infiltration in the liver and aorta. The therapeutic efficacy was related to FP&#39;s regulation in increasing the expression of peroxisome proliferator-activated receptor-alpha and decreasing hepatic lipogenic activity. However, the underlying mechanism of FP in improving hyperlipidemia should be further investigated, because its bioactive ingredients are quite extensive. We sought to explore the potential mechanism of FP&#39;s therapeutic efficacy, which may be beneficial for the further development and utilization of this medicine.
Currently, network pharmacology is regarded as a holistic and efficient technique to study the therapeutic mechanism of TCM. Instead of looking for single disease-causing genes and drugs treating solely an individual target, a complete drug-ingredients-genes-diseases network is constructed to find the multi-target mechanism of the multi-ingredient drug regarding their comprehensive treatment12. This technique is especially suitable for TCM, as their chemical compositions are massive. Unfortunately, network pharmacology can only be used to forecast targets affected by chemical ingredients in theory. The endogenous metabolites in the disease model should be observed to validate the effectiveness of network pharmacology. The metabolomics method, which emerges with the development of systems biology, is an important tool for monitoring the changes in endogenous metabolites13. The changes in metabolites reflect the steady state changes of the host, which is also an important indicator for studying the internal mechanism. Some researchers have successfully integrated network pharmacology and metabolomics to explore the interaction mechanism between drugs and diseases14,15.
This article explores the mechanistic basis of FP against hyperlipidemia by integrating network pharmacology and metabolomics techniques. Network pharmacology was applied to analyze the relationship between the main active ingredients in FP and molecular targets for hyperlipidemia. Subsequently, metabolomics was performed to observe the change of endogenous metabolites in the animal model, which can explain the medicine actions at the metabolic level. Compared with the application of network pharmacology or metabonomics alone, this integrated analysis provided a more specific and comprehensive research mechanism. Additionally, the molecular docking strategy was used to analyze the interaction between active ingredients and key proteins. In general, this integrated approach could compensate for the lack of experimental evidence for network pharmacology and the lack of an endogenous mechanism for the metabolomics method, and can be used for the therapeutic mechanism analysis of natural medicine. The main schematic flowchart of the protocol is shown in Figure 1.

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			<td>101-3B Oven</td>
			<td>Luyue Instrument and Equipment Factory</td>
			<td>\</td>
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			<td>80312/80302 Glass Slide</td>
			<td>Jiangsu Sitai Experimental Equipment Co., LTD</td>
			<td>\</td>
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			<td>80340-1630 Cover Slip</td>
			<td>Jiangsu Sitai Experimental Equipment Co., LTD</td>
			<td>\</td>
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			<td>AccucoreTM C18 (3 mm &#215; 100 mm, 2. 6 &#956;m)</td>
			<td>Thermo Fisher Scientific</td>
			<td>\</td>
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			<td>Acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A998</td>
			<td>Version 1.5.6</td>
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			<td>ACQUITY UPLC HSS T3 Column (2.1 mm &#215; 100 mm, 1.8 &#956;m)</td>
			<td>Thermo Fisher Scientific</td>
			<td>\</td>
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			<td>Aethanol</td>
			<td>Fisher Chemical</td>
			<td>A995</td>
			<td>Version 3.0</td>
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			<td>Ammonia Solution</td>
			<td>Chengdu Cologne Chemicals Co., LTD</td>
			<td>1336-21-6</td>
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			<td>AutoDockTools</td>
			<td>Scripps Institution of Oceanography</td>
			<td>\</td>
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			<td>BS-240VT Full-automatic Animal Biochemical Detection System</td>
			<td>Shenzhen Mindray Bio-Medical Electronics Co., Ltd.</td>
			<td>\</td>
			<td></td>
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		<tr>
			<td>Compound Discoverer</td>
			<td>Thermo Fisher Scientific</td>
			<td>\</td>
			<td></td>
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			<td>Cytoscape</td>
			<td>Cytoscape Consortium</td>
			<td>\</td>
			<td></td>
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			<td>DM500 Optical Microscope</td>
			<td>Leica</td>
			<td>\</td>
			<td></td>
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			<td>DV215CD Electronic Balance</td>
			<td>Ohaus Corporation ., Ltd</td>
			<td>T15A63</td>
			<td></td>
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			<td>Ethyl Alcohol</td>
			<td>Chengdu Cologne Chemicals Co., LTD</td>
			<td>64-17-5</td>
			<td></td>
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			<td>Formic Acid</td>
			<td>Fisher Chemical</td>
			<td>A118</td>
			<td></td>
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			<td>HDL-C Assay Kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A112-1-1</td>
			<td></td>
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			<td>Hematoxylin Staining Solution</td>
			<td>Biosharp</td>
			<td>BL700B</td>
			<td></td>
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			<td>High Fat Diet</td>
			<td>ENSIWEIER</td>
			<td>202211091031</td>
			<td></td>
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			<td>Hitachi CT15E/CT15RE Centrifuge</td>
			<td>Hitachi., Ltd.</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Oulaibo Technology Co., Ltd</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Chengdu Cologne Chemicals Co., LTD</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-forming System</td>
			<td>LIOO</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>JB-L5 Freezer</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>\</td>
			<td></td>
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		<tr>
			<td>JB-L5 Tissue Embedder</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>JK-5/6 Microtome</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>JT-12S Hydroextractor</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>KQ3200E Ultrasonic Cleaner</td>
			<td>Kun Shan Ultrasonic Instruments Co., Ltd</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>LDL-C Assay Kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A113-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Male C57BL/6 Mice</td>
			<td>&#160;SBF Biotechnology Co., Ltd.</td>
			<td>\</td>
			<td>Version 2.3.2</td>
		</tr>
		<tr>
			<td>Neutral Balsam</td>
			<td>Shanghai Yiyang Instrument Co., Ltd</td>
			<td>10021190865934</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Water</td>
			<td>Guangzhou Watson&#39;s Food &#38; Beverage Co., Ltd</td>
			<td>GB19298</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>DeLano Scientific LLC</td>
			<td>\</td>
			<td>Version 14.1</td>
		</tr>
		<tr>
			<td>RE-3000 Rotary Evaporator</td>
			<td>Yarong Biochemical Instrument Factory ., Ltd</td>
			<td>\</td>
			<td></td>
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			<td>RM2016 Pathological Microtome</td>
			<td>Shanghai Leica Instruments Co., Ltd</td>
			<td>\</td>
			<td>Version 26.0</td>
		</tr>
		<tr>
			<td>SIMCA-P</td>
			<td>Umetrics AB</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Simvastatin</td>
			<td>Merck Sharp &#38; Dohme., Ltd</td>
			<td>14202220051</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS</td>
			<td>International Business Machines Corporation</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Assay Kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A111-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TG Assay Kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A110-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>UPLC-Q-Exactive Quadrupole Electrostatic Field Orbital Hydrazine High Resolution Mass Spectrometry</td>
			<td>Thermo Fisher Scientific</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Vibrator</td>
			<td>Beijing PowerStar Technology Co., Ltd.</td>
			<td>LC-Vortex-P1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Chengdu Cologne Chemicals Co., LTD</td>
			<td>1330-20-7</td>
			<td></td>
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network pharmacology prediction and metabolomics validation of the mechanism of fructus phyllanthi against hyperlipidemia

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

Network pharmacology 
A total of 18 potential ingredients in FP were screened according to their pharmacokinetic and pharmacodynamic properties from the database and LC-MS analysis (the total ion chromatograms are shown in Supplementary Figure 1). Through relevant literature, the content of gallic acid is much higher than other ingredients and is effective in lowering lipids9,11. Therefore, this ingredient was considered a p...

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Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

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1.7K visualizações.  Chengdu University of Traditional Chinese Medicine. Nossa pesquisa inclui técnica experimental seriada e o protocolo tem esses detalhes que são necessários para que os leitores entendam nossos métodos. Nosso método integrado compensa a desvantagem da farmacologia de rede e metabolômica, e pode ser usado para a meta-análise terapêutica da medicina natural. Este método é usado para selecionar o composto ativo do ingrediente da empresa e o traje para corredores com composição química maciça, como uma medicina tradicional chinesa.