BACKGROUND: Reproducibility is a major concern in biomedical studies, and existing publication guidelines do not solve the problem. Batch effects and quality imbalances between groups of biological samples are major factors hampering reproducibility. Yet, the latter is rarely considered in the scientific literature.
RESULTS: Our analysis uses 40 clinically relevant RNA-seq datasets to quantify the impact of quality imbalance between groups of samples on the reproducibility of gene expression studies. High-quality imbalance is frequent (14 datasets; 35%), and hundreds of quality markers are present in more than 50% of the datasets. Enrichment analysis suggests common stress-driven effects among the low-quality samples and highlights a complementary role of transcription factors and miRNAs to regulate stress response. Preliminary ChIP-seq results show similar trends. Quality imbalance has an impact on the number of differential genes derived by comparing control to disease samples (the higher the imbalance, the higher the number of genes), on the proportion of quality markers in top differential genes (the higher the imbalance, the higher the proportion; up to 22%) and on the proportion of known disease genes in top differential genes (the higher the imbalance, the lower the proportion). We show that removing outliers based on their quality score improves the resulting downstream analysis.
CONCLUSIONS: Thanks to a stringent selection of well-designed datasets, we demonstrate that quality imbalance between groups of samples can significantly reduce the relevance of differential genes, consequently reducing reproducibility between studies. Appropriate experimental design and analysis methods can substantially reduce the problem.

Cinnamomum cassia Presl is a major food spice as well as traditional herbal medicine with anti-inflammatory, analgesic, and stomachic properties, which must be dried to preserve its quality, but mostly by using traditional, ineffective drying method. In order to find a scientific drying method by evaluating different drying methods that could influence the quality of C. cassia, ten indices were employed to evaluate different drying methods in C. cassia using the Analytic Hierarchy Process (AHP) method though calculating the total scores and ranking the priority. Four quality markers (Q-Markers) (coumarin, cinnamyl alcohol, cinnamaldehyde and o-methoxycinnamaldehyde) were isolated from the samples and analyzed by high performance liquid chromatography (HPLC) method under different drying methods. The results showed that various drying methods had multiple effects on the physicochemical qualities, essential oil content, and Q-Marker contents. Compared with other drying methods, oven-drying of 45 °C (45OD) maintained optimal levels of color and aroma, it also significantly shortened the drying time by 225 h than traditionally shade-drying (SHD) method with the drying rate (48.35 %), and obtained the highest essential oil content (3.05 %) and Q-Marker contents (30.23 mg g-1). Furthermore, the ash content (4.22 %) were satisfied with the stipulation of Chinese pharmacopoeia in 45OD samples. Applying AHP allowed us to identify 45OD as the optimal drying method with the highest total score (9.00), followed by the traditional shade-drying (SHD) method (7.88). The present study is the first report to apply the AHP method for quality evaluation of drying processing in C. cassia. It can provide the theoretical basis for evaluating an excellent method for C. cassia drying processing, as well as the rational use of different drying methods to furtherly develop the high quality C. cassia industry.

Qing-fei-da-yuan granules (QFDYGs) had been proved to be an effective TCM prescription for treating coronavirus disease 2019 (COVID-19), which are composed of a variety of TCMs, and characterized by multiple components, multiple targets and overall regulation. It is meaningful to further study the chemical composition and pharmacology of QFDYGs for quality evaluation. However, due to the complexity of the components of QFDYGs, there are no reliable and simple analytical methods for current quality evaluation. In this work, antipyretic activity assessment of QFDYGs in the LPS-induced New Zealand rabbit model was carried out to verify the efficacy firstly. It was proved that QFDYGs can be used to relieve fever to help preventing or controlling the prevalence of influenza and pneumonia. Subsequently, UHPLC-ESI-QTOF-MS/MS combined with network pharmacology, quality markers and fingerprint analysis were used to establish the quality control condition. The chemical compositions were analyzed by UHPLC-ESI-QTOF-MS/MS, and 79 of them were identified, such as arecoline, mangiferin, paeoniflorin, etc. Then, the network pharmacology strategy based on 45 candidate components (CCs) in conjunction with influenza and pneumonia diseases was employed to screen the potential active ingredients. According to the drug-CCs-genes-diseases (D-CCs-G-D) networks, baicalein, honokiol, baicalin, paeoniflorin, saikosaponin A, glycyrrhizic acid and hesperidin were selected as quality markers. And a method for content determination of the 7 quality markers was established by optimizing extraction methods, chromatographic conditions and methodological verification. Finally, the quality of 15 batches of QFDYGs was evaluated by using the 7 quality markers combined with fingerprints and principal component analysis (PCA). The analyzed results showed that baicalin, paeoniflorin, glycyrrhizic acid and hesperidin were the high content and stable quality markers. QFDYGs were characterized by overall consistency and individual ingredient differences among the 15 batches. Our quality evaluation study will provide reference for the further development and research of QFDYGs.

Phyllanthus emblica (P. emblica) is a vital medicinal plant with both medical and edible values. In the quality standard of P. emblica listed by the Chinese Pharmacopoeia, gallic acid is used as the index component for the content determination. However, a large number of tannin components can be decomposed into gallic acid during its refluxing extraction process, thus affecting the accuracy and specificity of the content determination. Thus, the index component used for the quality control needs to be further determined. In this study, the quality markers of P. emblica was specified by integrating chromatographic fingerprint, serum pharmacochemistry and network pharmacology. The chromatographic fingerprint of 18 batches of P. emblica samples were established by ultra-high-performance liquid chromatography (UPLC), and 8 differential components causing quality fluctuation were identified by chemometric analysis and UPLC-Q-TOF/MS analysis. Afterwards, 14 prototype migration components absorbed into the blood after gavage administration to rats were identified by UPLC-Q-TOF/MS analysis. Subsequently, a network pharmacology approach was used to construct the component-target-disease-pathway network, resulting in the identification of 22 components responsible for efficacy of P. emblica. Finally, by integrating the above results, ellagic acid was screened out as one of the Q-markers and could be employed as a quantitative component of P. emblica to improve the quality standard. The strategy is also informative for discovering Q-markers of other TCMs.

Du Zhong is a valuable Chinese medicinal herb unique to China. It is a national second- class precious protected tree, known as \"plant gold\", which has been used to treat various diseases since ancient times. The main active ingredients are lignans, phenylprophetons, flavonoids, iridoids and steroids and terpenoids, which have pharmacological effects such as lowering blood pressure, enhancing immunity, regulating bone metabolism, protecting nerve cells, protecting liver and gallbladder and regulating blood lipids. In this paper, a comprehensive review of Eucommia ulmoides Oliv. was summarized from the processing and its compositional changes, applications, chemical components, pharmacological effects, and pharmacokinetics, and the Q-marker of Eucommia ulmoides Oliv. is preliminarily predicted from the aspects of traditional efficacy, medicinal properties and measurability of chemical composition, and the pharmacodynamic substance basis and potential Q-marker of Eucommia ulmoides Oliv. are further analyzed through network pharmacology. It is speculated that quercetin, kaempferol, β-sitosterol, chlorogenic acid and pinoresinol diglucoside components are selected as quality markers of Eucommia ulmoides Oliv., which provide a basis for the quality control evaluation and follow-up research and development of Eucommia ulmoides Oliv.

Hua-ju-hong (HJH) is a Chinese medicinal material obtained from Citrus grandis \'Tomentosa\' (CGT) and Citrus grandis (L.) Osbeck (CG) with various commercial specifications. It is known for relieving cough and dispelling phlegm. To reveal the quality marker for distinguishing the various HJH, 215 batches of commercial HJH were studied systematically using multidimensional chemical analysis. Ten major components were identified by high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and quantified via high-performance liquid chromatography coupled with diode array detection. In this study, a rapid, efficient, and low-cost chromatographic method was established. Total coumarin-hemiterpene and total coumarin-monoterpene were first classified and analyzed in HJH. The result indicated that the main component, naringin, was not the quality marker for differentiating CGT from CG. For reflecting the unique medicinal and food value of HJH, coumarins should be the more potential quality markers. Flavonoids were the possible quality markers for distinguishing two growth stages of fruit-exocarp and young fruit. For the first time, two chemotypes of HJH were identified in CG. This study provides a convenient yet reliant chromatographic method and novel yet systematic strategies for overall quality control of commercial HJH.

Chinese medicinal preparations play an equally important role in reducing toxicity and treating tumors. Few studies discriminate the quality markers(Q-markers) conferring different therapeutic effects of traditional Chinese medicine preparations. Therefore, we take Aidi Injection(AD) as an example to comprehensively identify the Q-markers of anti-tumor and cardioprotective effects based on the "spider web" mode. Firstly, based on the principle of measurability, the chemical components in the prescription were qualitatively analyzed, and then the components with high content and capable to be measured were quantitatively analyzed as measurable evaluation indexes. Based on the principle of stability, the effects of light and temperature on the content of each component of AD were investigated as indicators of stability. Based on the principle of compatibility, the compounds were classified according to the law of compatibility of sovereign, minister, assistant, and guide medicinal materials in the prescription. Based on the principle of efficacy, the anti-tumor and antiangiogenic activities of the Q-markers were evaluated, and their synergistic effects with doxorubicin(DOX) in inhibiting tumorigenesis and angiogenesis and lowering cardiotoxicity were evaluated as the evaluation indexes of effectiveness. The seven-dimensional spider web of "compatibility-content-stability-antitumor activity-synergistic anti-tumor activity with DOX-antiangiogenic activity-synergistic anti-angiogenic activity with DOX" and the four-dimensional spider web of "compatibility-content-stability-protective effects against DOX-induced myocardial toxicity" were established, on the basis of which the Q-markers of anti-tumor and cardioprotective effects of AD were comprehensively analyzed. The results showed that 12 components were selected as the Q-markers of AD, among which cantharidin, ginsenoside Re, ginsenoside Rb_1, astragaloside Ⅱ, cryptochlorogenic acid, and ginsenoside Rg_2 were the anti-tumor Q-markers of AD. Ginsenoside Rd, isofraxidin, syringin, eleutheroside E, calycosin-7-O-β-D-glucoside, and azelaic acid were the cardioprotective Q-markers of AD. Taking into account both the anti-tumor and cardioprotective effects, these Q-markers could cover the four herbs constituting the prescription. The findings provides a scientific basis for the quality control of AD and an effective method for identifying comprehensive and reasonable Q-markers for the two effects of Chinese medicinal preparations.

Raphani Semen, with both edible and medicinal values, is a typical Chinese herbal medicine with different effects before and after processing. The raw helps ascending and the cooked helps descending. This paper comprehensively summarizes the differences in chemical constituents and pharmacological effects between raw and processed Raphani Semen that are reported in recent years. Based on the principle of quality markers(Q-markers) of traditional Chinese medicines, the chemical constituent sources, chemical constituent detection techniques, and correlation between bidirectional regulatory efficacy and chemical constituents are compared between raw and processed Raphani Semen. The results suggest that sulforaphene and glucoraphanin could be used as candidate Q-markers of raw and processed Raphani Semen, respectively. This review is expected to provide a reference for further research on the processing, new drug development, and improvement of safety and effectiveness of Raphani Semen in clinical application.

Studies have reported that the hemostatic effect of Sanguisorbae Radix(SR) is significantly enhanced after processing with charcoal. However, the standard components(tannins and gallic acid) specified in the Chinese Pharmacopeia decrease in charcoal-fried Sanguisorbae Radix(CSR), which is contrast to the enhancement of the hemostatic effect. Therefore, this study aimed to optimize the charcoal-frying process of SR based on its hemostatic efficacy and comprehensively analyze the components of SR and its processed products, thus exploring the material basis for the hemostatic effect. The results indicated that SR processed at 250 ℃ for 14 min(14-min CSR) not only complied with the description in the Chinese Pharmacopeia but also demonstrated improved blood-coagulating and blood-adsorbing effects compared with raw SR(P<0.05). Moroever, 14-min CSR reduced the bleeding time in the rat models of tail snipping, liver bleeding, and muscle injury, surpassing both raw and excessively fried SR(16 min processed) as well as tranexamic acid(P<0.05). Ellagitannin, ellagic acid, methyl gallate, pyrogallic acid, protocatechuic acid, Mg, Ca, Mn, Cu, and Zn contributed to the hemostatic effect of CSR over SR. Among these substances, ellagitannin, ellagic acid, Mg, and Ca had high content in the 14 min CSR, reaching(106.73±14.87),(34.86±4.43),(2.81±0.23), and(1.21±0.23) mg·g~(-1), respectively. Additionally, the color difference value(ΔE~*ab) of SR processed to different extents was correlated with the content of the aforementioned hemostatic substances. In summary, this study optimized the charcoal-frying process as 250 ℃ for 14 min for SR based on its hemostatic effect. Furthermore, ellagic acid and/or the powder chromaticity are proposed as indicators for the processing and quality control of CSR.

BACKGROUND: Appropriate conditions for storage of Artemisia argyi leaves reduce irritation during treatment and increase the active ingredient content. Naturally aged A. argyi leaves (≥1 year) are optimal for moxibustion; however, this process is time-consuming and costly. A comprehensive understanding of the conditions for artificial aging of A. argyi leaves and the mechanism of quality-marker conversion are required to guarantee A. argyi quality and moxibustion efficacy.
OBJECTIVE: To identify the optimal conditions for artificial aging of A. argyi leaves and clarify the mechanism of quality-marker conversion.
METHODS: Gas chromatography (GC), high-performance liquid chromatography (HPLC), colorimeter (CD), and near-infrared spectroscopy (NIRS) were used to determine the chemical composition of A. argyi leaves before and after artificial and natural (1 year) aging and to determine the optimal artificial aging conditions. The effects of both artificially and naturally aged A. argyi leaves were then evaluated in a mouse model of ulcerative colitis (UC). The main chemical components of aged A. argyi leaves were then analyzed to determine quality-markers and the transformation mechanism.
RESULTS: Comprehensive analysis of volatile and non-volatile components, color values, and characteristic near-infrared spectra revealed that the quality of artificially aged A. argyi leaves was similar to that of naturally aged A. argyi leaves. In the mouse model, artificially and naturally aged A. argyi leaves not only improved the symptoms of UC with the same therapeutic effects, but also safeguarded the barrier of the colonic mucosa and prevented the release of colitis-related substances. In addition, the content of caffeic acid converted from L-phenylalanine in A. argyi leaves increased during the aging process.
CONCLUSIONS: Conditions for artificial aging of A. argyi leaves were identified for the first time, and the equivalent efficacy of artificially aged A. argyi leaves and naturally aged A. argyi leaves for improving UC was confirmed. This method for artificial aging of A. argyi leaves not only reduces the time and cost associated with this process, but also provides technical support to ensure the quality and stability of artificially aged A. argyi leaves. In addition, caffeic acid was identified as a potential quality-marker for establishing standards and specifications for aging A. argyi leaves for the first time, and its possible transformation mechanism was preliminarily elucidated.