Research Progress of Yihuang Decoction and Prediction Analysis on Quality Markers

Background Yihuang Decoction, a classical prescription found in Fu Qingzhu's Obstetrics and Gynecology, is formulated with fried Dioscoreae Rhizoma, fried Euryales Semen, wine-fried Plantaginis Semen, salt-fried Phellodendri Chinensis Cortex and Ginkgo Semen. Quality marker (Q-marker) plays a crucial role in promoting the standardization of the quality of traditional Chinese medicine (TCM). Hence, this study aimed to predict and analyze the Q-marker of Yihuang Decoction based on TCM Q-marker theory. Methods A comprehensive review of relevant literature and the TCMSP database was

Plantaginis Semen, with a bitter taste and cold properties, clears heat and dry dampnesses by acting on the kidney [3].Phellodendri Chinensis Cortex, with a sweet taste and cold properties, can clear heat and promote diuresis [4].Yihuang Decoction is primarily employed to treat kidney deficiency, damp-heat and leukorrheal diseases, which has the functions of reinforcing the kidney for stopping leucorrhea, clearing heat and expelling damp.
Clinically, Yihuang Decoction is mainly used to treat female reproductive system inflammation as well as other diseases, including human papillomavirus (HPV) infection and urinary tract infection [5,6].

Chinese medicine proposed by Academician Liu
Changxiao and other scholars in recent years [7].
Zhang Tiejun et al. have expounded the core theory and research method of Q-marker from five perspectives, including effectiveness, specificity, measurability, transmissibility and traceability, and compatibility [8].This study aims to summarize the chemical constituents, pharmacological effects and clinical application of Yihuang Decoction in recent years, and predict potential Q-markers for the formulation.The findings will provide a foundation for the quality evaluation and rational application of Yihuang Decoction.
Polysaccharides are the main active constituent known for its complex composition and structure.Xu Chunping et al. [10] employed GC/MS and fourier transform infrared spectrometer to characterize the structure of polysaccharides in Dioscoreae Rhizoma, and found that the main monosaccharides are glucose (84.5%), xylose (11.4%), galactose (2.3%) and arabinose (1.4%).Liu Lijie et al. [11]  Dioscoreae Rhizoma also contains polyphenols including catechin, quercetin and kaempferol, as well as essential unsaturated fatty acids such as linoleic acid and linolenic acid [9].Allantoin, as a derivative of uric acid, is another quality control indexes for Dioscoreae Rhizoma [13].
Euryales Semen refers to the dried ripe kernel of Euryale ferox Salisb., containing virous medicinal components, such as polysaccharides, polyphenols, lignans, tocopherols and cerebrosides, as well as Exploration and Verfication Publishing 4 factors to decrease the number of inflammatory cells and thus modulate the immunity system.Yihuang Decoction can reduce IL-6, IL-17 and IL-12 levels, while elevating the levels of IL-2, IL-4 and INF-γ in vaginal secretions of patients.These changes help to ameliorate the vaginal environment of patients infected with high-risk cervical human papillomavirus [25,26].Moreover, the clysis of Yihuang Decoction has been verified to shorten the time for leukocytes, neutrophils and C-reactive protein to return to normal levels, thereby alleviating damp-heat-toxin-type pelvic inflammation [27,28].

Anti-bacteria activities
Vaginal lactobacillus maintains the acidic vaginal environment and inhibits or kills foreign bacteria by producing hydrogen peroxide.Disruption of the local vaginal environment leads to a reduction or complete disappearance of lactobacillus, allowing excessive growth of other bacteria such as anaerobic bacteria, Gardnerella, and mycoplasma, resulting in vaginal dysbacteriosis [29].The pH value in vagina can impact vaginal bacteria, and Yihuang Decoction can improve vaginal pH value to mitigate vaginal dysbacteriosis in senile vaginitis [30][31][32][33].

Anti-virus effects
Hypersensitivity C-reactive protein (hs-CRP) reflects the body's immunity and inflammatory response, while TNF-α is implicated in the immune response and inflammatory factor expressions in patients infected with cervical virus.HPV virus can decrease immunity, thereby increasing levels of inflammatory factors such as TNF-α and hs-CRP.The combination of Yihuang Decoction and self-made Qingdu prescription can reduce the viral load of HPV patients and decrease the levels of TNF-α and hs-CRP [34].
Duan Qianni et al. [35] proved that modified Yihuang Decoction downregulates the expressions of viral proteins gB, gD, VP16, ICP5 and ICP4, improves host cell morphology, enhances host cell viability, and notably affects the adhesion and penetration stages of herpes simplex virus type 2 (HSV-2).In addition, Li Shi et al. [36] validated that Yihuang Decoction, combined with chitosan anti-bacteria membrane, interferes with HPV infection through modulating expressions of HPV encoded proteins HPV E6/E7.

Other functions
Yihuang Decoction also possesses the functions of regulating the spleen and stomach, alleviating diarrhea, promoting diuresis and providing neuroprotection.
Chronic colitis and infantile diarrhea are caused by damp evil, weakness of the spleen and the stomach, rise and fall disorder of Qi and blood, and dysfunction in separating the clear Qi from the turbid Qi, with diarrhea as the main manifestation.Yihuang Decoction can warm kidney and strengthen spleen, elevate clear Qi, remove dampness, as well as astringe intestine to relieve diarrhea [37].

Inflammation of the female reproductive system
Inflammation of the female reproductive system is a prevalent gynecological condition, encompassing vaginitis, cervicitis and pelvic inflammation.These diseases belong to "leukorrheal diseases" in TCM, manifested by the apparent increase of leucorrhea, chromatic aberration, and pruritus vulvae or ulceration.
However, Yihuang Decoction has shown significant efficacy in alleviating these symptoms.
Cervicitis: Modified Yihuang Decoction can be utilized in the late stage of chronic cervicitis treatment for cervical erosion [44].Liu Shimei et al. [44] conducted a study in which 104 patients received microwave treatment as the control group and 102 patients received modified Yihuang Decoction as the treatment group.No statistically significant differences were found in the treatment efficacy between the two approaches for damp-heat-type cervical erosion; however, the vaginal fluid rates were increased in control group, while the treatment group has no complications.
In addition to the aforementioned diseases, Yihuang Decoction combined with or without other prescription can also be used to treat ovulatory bleeding, vulvovaginal candidiasis, female mycoplasma genitalium infection, etc. [45][46][47].

HPV infection
Clinically, the common symptoms of HPV infection include increased leucorrhea and vaginal pruritus, which are closely resemble the symptoms of the "leukorrheal diseases" in TCM.Several studies have confirmed that Yihuang Decoction can assist in the treatment of damp-heat type of cervical HPV infection [6].Numerous studies have revealed that Yihuang Decoction can reduce HPV viral load and increase the infection negative conversion ratio, thereby potentiating the ability of recombinant human interferon to clear HPV virus and improving the body's antiviral capability [25,48,49].Lin Lan et al.
[6] found that Yihuang Decoction reduced the viral load of patients infected with HR-HPV.

Prediction and analysis of Q-marker
The Exploration and Verfication Publishing 6 transmissibility and traceability, component specificity, component effectiveness, compound compatibility, and component measurability (Figure 2).These findings lay a foundation for quality control of Yihuang Decoction.When being orally administered, gallic acid has relatively low absolute bioavailability [56], and its distribution is rapid and extensive, mainly in the small intestine, kidney, and liver tissues [57].Ginkgolide A/B and bilobalide are primary components in Ginkgo Semen.YARO PETER [58]  and has a fastest distribution rate in the spleen and kidneys [62], which is also rapidly metabolized and eliminated [63].Verbascoside is rapidly absorbed, and quickly and widely distributed after oral administration, with small amounts excreted from urine, bile, and feces, and its absolute bioavailability is as low as 1% [64].

Predictive analysis on the Q-marker of Yihuang
Decoction based on component specificity

Dioscoreae Rhizoma
Dioscoreae Rhizoma contains abundant functional components such as polysaccharide, saponin, allantoin, polyphenol, fatty acid and protein.Among these, allantoin and diosgenin are important active substances of Dioscoreae Rhizoma.However, the content of these substances can vary significantly in medicinal materials sourced from different regions and can be influenced by factors such as harvesting time and processing methods [65][66][67][68].Therefore, allantoin and diosgenin can serve as reliable Q-markers for assessing the quality of Dioscoreae Rhizoma medicinal materials.

Euryales Semen
Euryales Semen contains nutrients such as starch, protein, amino acids, etc., and medicinal ingredients including polysaccharides, polyphenols, lignans, tocopherols, cerebrosides, etc. Euryales Semen from different places of origin greatly vary in quality.
Among Semen [69].Polyphenols have complex components with various activities, and the combination of different polyphenols directly affects their efficacy.
Ginkgo terpene lactones are specific components in gingko with profound pharmacological activities [72].

Bilobalide and ginkgolide have different contents in
Ginkgo Semen from various places of origin or batches [17], making them reliable specific components for evaluating the quality of Ginkgo Semen.

Plantaginis Semen
Plantaginis Semen is composed of alkaloids, flavonoids, phenolic acids, triterpenoids, phenylpropanoids, amides, etc.Among these, alkaloids occupy the most content, primarily including berberine, phellodendrine, palmatine, magnoflorine, and jatrorrhizine.Berberine and phellodendrine have been identified as Q-markers of Plantaginis Semen in Pharmacopoeia of the People's Republic of China (2020 edition).Berberine occupies a large content in Plantaginis Semen, and phellodendrine is a specific component of Plantaginis Semen.Notably, the places of origin, harvesting time and stir-frying with salt-water process make significantly influence the levels of these two components [73][74][75].The content of magnoflorine varies depending on the source of Plantaginis Semen [22].Accordingly, berberine, phellodendrine, and magnoflorine can be used as the exclusive characteristic markers of Plantaginis Semen.

Predictive analysis on the Q-marker of Yihuang
Decoction with constituents related to medicinal effectiveness

Anti-inflammation and immune modulation
The immunity comprises cellular immunity, humoral Exploration and Verfication Publishing 10 parmatine and magnoflorine can effectively suppress E6 expression [95].Plantaginis Semen has been evidenced to inhibit cellular viral replication and upregulate the expressions of antiviral genes [96].
Verbascoside may exert antiviral effects through dwindling HBx protein level, reducing antigen secretion, and blocking the transcription of the viral genome [97].

Decoction based on compound compatibility
The therapeutic effect of drugs in traditional Chinese Hualu et al. [8] found that the contents of allantoin, gallic acid, geniposidic acid and berberine in 15 batches of Yihuang Decoction ranged from 0.0801% to 1.087%, 0.047% to 0.057%, 0.037% to 0.081% and 0.0250% to 0.387%, respectively.Luo Jing et al. [99] determined the content of diosgenin in 14 different places of origin with HPLC, and confirmed that its content from different places of origin is between  1 and Figure 3).
described in Fu Qingzhu's Obstetrics and Gynecology, a monograph of gynecology written by Fu Shan in Qing Dynasty, and is composed of 50 g Dioscoreae Rhizoma, 50 g Euryales Semen, 10 g Plantaginis Semen, 5 g Phellodendri Chinensis Cortex, and 10 g Ginkgo Semen.This herbal combination is conventionally prepared by decocting in water.Yihuang Decoction has been included in the 100 list of Catalogue of Ancient Classical Formulas (The First Batch) issued by the State Administration of Traditional Chinese Medicine in 2018.In the decoction, Dioscoreae Rhizoma and Euryales Semen serve as principal herbs, addressing deficiency of Ren meridian and promoting urination.Ginkgo Semen serve as the ministerial herb, primarily promoting astringent and mitigating leukorrheal diseases, and concurrently eliminating dampness and heat [1,2].Plantaginis Semen and Phellodendri Chinensis Cortex are adjuvants.
adopted HPLC and spectrophotometry to determine the contents of allantoin, dioscin and total polyphenols in the long Dioscoreae Rhizoma with fine hairs from different regions of Northeast China, and detected the content of active polysaccharides after water extraction and alcohol precipitation.The study found higher levels of these active components in Dioscoreae Rhizoma from Shenyang city compared to Qiqihar city and Changchun city.Based on the interactive application of various chromatographs as well as UV, IR, HRESIMS, NMR, and chemical analysis, Zhao Weixue et al. [12] isolated and analyzed 9 new and 11 known steroidal saponins from Dioscoreae Rhizoma, with diosgenin often used as a quality control index for Dioscoreae Rhizoma medicinal materials.

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distributed 102 elderly patients with vaginitis into control and study groups, where patients in control group were treated with Baofukang suppository and those in study group were treated with Baofukang suppository and modified Yihuang Decoction.The results showed that the cure rate, total effective rate, vaginal pH value, bacterial culture score, vaginal cleanliness and symptom score in study group are superior to those in control group.Pelvic inflammation: In a study by Hao Furong [42], divided into control (treated with conventional Western medicine) and treatment (treated with conventional Western medicine and Yihuang Decoction) groups.The data revealed higher cure rates and overall effectiveness in the treatment group compared to the control group.Kuang Xunhong et al.
compound Yihuang Decoction is composed of 5 TCM materials (Figure 1), which contains complex chemical components and can be influenced by place of origin, climate, processing methods, etc., resulting in varied quality of TCM.At present, the research of Q-marker focus on single medicinal material, and there is a lack of studies elucidating the Q-markers of Yihuang Decoction as a compound formulation.This study predicted and summarized the Q-marker of Yihuang Decoction from the perspectives of quality Journal of Experimental and Clinical Application of Chinese Medicine 2024; 5(1): 2-18.

acetonitrile- 0 . 8 representative
1% phosphoric acid aqueous solution as the mobile phase to determine the HPLC fingerprint of 15 batches of Yihuang Decoction at a wavelength of 230 nm.Later, 15 common peaks were detected, and 8 characteristic peaks information were confirmed, of which 6 were from Plantaginis Semen (chlorogenic acid, phellodendrine, magnoflorine, 5-O-feruloylquinic acids, 4-feruloylquinic acids, and berberine), 1 from Phellodendri Chinensis Cortex marinated with liquor (geniposidic acid), and 1 from stir-baked rhizoma dioscorea (allantoin).Zhou Jinxin et al. [52] confirmed 90 constituents of Yihuang Decoction through HPLC-Q-TOF-MS, including 23 alkaloids (berberine, palmatine, phellodendrine, jatrorrhizine, and tetrahydropalmatine), 14 organic acids, 3 phenylethanoid glycosides, 4 iridoid glycosides, 5 terpene lactones, 10 flavonoids, 8 nucleobases and nucleosides, 12 amino acids and 11 other chemical compounds.Besides, they tested -feruloylquinic acid, gallic acid, geniposidic acid and phellodendrine) in the 10 batches of Yihuang Decoction samples using HPLC-DAD, and the results indicated that this method is fast, accurate, reliable, and easy to operate, which provides strong evidence for the quality control of Yihuang Decoction.Compound formulation of TCM have complicated constituents that exert pharmacologic effects through Journal of Experimental and Clinical Application of Chinese Medicine 2024; 5(1): 2-18.Exploration and Verfication Publishing 7 absorption, distribution and metabolism in the body.Hence, selecting appropriate absorbed constituents and metabolites is pivotal to the research on quality control of compound formulation of TCM.Allantoin and diosgenin are the main constituents of Dioscoreae Rhizoma.By means of HPLC analysis has demonstrated that allantoin act as the absorbed constituents in Psammosilene tunicoides medicinal materials, and as the index [53].Chang Jinhua et al. [54] determined the concentration of diosgenin in rat plasma with UPLC-MS/MS and found diosgenin blood concentration is relatively reduced after oral administration and diosgenin is widely distributed and metabolized slowly after entering the blood.It is worthwhile to explore the metabolism of α-tocopherol and gallic acid, two main active components in Euryales Semen, in the body.α-tocopherol undergoes ω-oxidation, β-oxidation, and oxidation with free radicals in the body, and metabolites are excreted into urine mainly through conjugating with sulphate [55].
. Iridoids (including geniposidic acid) and phenylethanoid glycosides (including verbascoside) are the main active constituents of Phellodendri Chinensis Cortex, with several pharmacologic action [24].Geniposidic acid and verbascoside have been listed as Q-markers of Phellodendri Chinensis Cortex in Pharmacopoeia of the People's Republic of China (2020 edition) and are indicators during processing of Phellodendri Chinensis Cortex [76].Collectively, geniposidic acid and verbascoside can act as specific components of Phellodendri Chinensis Cortex.

3 . 3 . 2 3 . 3 . 3
TLR signal cascade reaction, and activate NF-кB, resulting in expressions of inflammatory factors, including TNF-α and IL-1β.Dioscoreae Rhizoma and Euryales Semen can elevate thymus index, improve T cell proliferation, increase serum IL-2 and IL-4 levels, and decrease IFN-γ level, so as to ameliorate the immunity[77].Moreover, diosgenin in Dioscoreae Rhizoma has been identified to downregulate expressions of inflammatory factors (IL-1β, IL-6 and TNF-α), protein levels of COX-2 and iNOS, and augment levels of genes related to antioxidant (haem oxygenase-1, superoxide dismutase 3, etc.)[78].Bilobalide can increase expressions of antioxidant genes (Nrf and Keap), dwindle levels of inflammatory factors (TNF-α, IL-1β and VEGF) and NF-кB, restore Th1/Th2, and thus promote the immunity[79,80].Reportedly, Ginkgolide attenuates ), and downregulates TNF-α and IL-1β levels[81].It has been documented that total polysaccharide from Phellodendri Amurensis Cortex evidently suppress proliferation of T lymphocytes and spleen cells, and exert immunosuppressive effects through cellular immunosuppression [82].Phellodendrine plays an anti-inflammatory role through blocking the activation of TLR-4/MyD88/NF-κB (p65) pathway and inhibiting expressions of iNOS, COX-2 and TNF-α [83].Additionally, verbascoside has been corroborated to promote the proliferation of mouse dendritic cells, stimulate the proliferation of naïve T cells, and inhibit transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1)/JNK/AP-1 pathway and its downstream COX-2 and iNOS gene expressions, so as to generate anti-inflammatory and immunomodulatory effects [84, 85].Polysaccharides of Phellodendri Chinensis Cortex repress the overproduction of TNF-α, IL-2, IL-6, IL-10 and IL-1β, elevate glutathione peroxidase and improve total antioxidant capacity in mouse liver [86].Also, polysaccharides of Phellodendri Chinensis Cortex can enhance the phagocytic activity of peritoneal macrophages in immunosuppressed mice, potentiate the transformation function of splenic lymphocytes, increase hemolysin secretion, and promote the secretion of IL-2 by splenic lymphocytes, thereby strengthening the immunity [87].Based on the above information, it can be concluded that Yihuang Decoction can exert anti-inflammatory and immunomodulatory effects via mediating inflammatory factors and immune cells.Anti-bacteria Several studies have indicated that TCM exerts antibacterial effects by disrupting the integrity of bacterial cell walls and membranes, and dampening the synthesis of proteins and nucleic acids.Polysaccharides in TCM have good antibacterial effects.For example, ] can suppress the growth of staphylococcus aureus, escherichia coli and bacillus subtilis, among which Ginkgo Semen polysaccharides can play a bacteriostatic role through inhibiting the formation of staphylococcus aureus biofilm.Moreover, gallic acid curbs the growth of staphylococcus aureus via disrupting bacterial membrane structure and suppressing bacterial protein synthesis [91].Berberine, the main active component of Plantaginis Semen, regulates colitis-triggered intestinal flora imbalance and restores the content of tryptophan, a metabolite of intestinal bacteria [92].Berberine hydrochloride concentration-dependently produces bacteriostatic effects by limiting the number of staphylococcus aureus and suppressing the formation of biofilm [93].Anti-virus HPV encodes two key oncoproteins E6 and E7.The dimer formed by E6 and E6AP can specifically binds to the tumor suppressor protein p53, and promotes the degradation of p53, thereby blocking cell apoptosis.E7 binds to the tumor suppressor protein pRB to dissociate E2F and pRB complexes and to make the cell cycle out of control.E6/E7 expression is regulated by URR which contains transcription factors that can bind to viral regulatory protein E2 and host-derived transcription factors (AP-1, STAT3).Further, berberine can inhibit AP-1 and the expressions of viral oncoproteins E6 and E7 [94].Berberis aquifolium ethanolic plant extract alters STAT3 and AP-1 expressions to control E6 and E7 expressions, and its chemical components berberine, Journal of Experimental and Clinical Application of Chinese Medicine 2024; 5(1): 2-18.
medicine (TCM) is influenced by the compatibility of compound prescriptions.Drug compatibility based on drug property can enhance curative effect and alleviate partial effect.In Yihuang Decoction, Dioscoreae Rhizoma and Euryales Semen are principle drugs with mild property, which are neither dry nor hard to digest, and can tonify Qi deficiency, strengthen astringent, invigorate spleen for diuresis, and arrest spontaneous emission and leukorrhagia.Ginkgo Semen, as the ministerial drug, has a sweet, bitter and astringent flavor and mild nature, which can reinforce Qi deficiency and reduce leucorrhea.Plantaginis Semen has a bitter flavor and cold nature, and Phellodendri Chinensis Cortex has a sweet flavor and cold nature, which together function as adjuvants for clearing dampness-heat in lower energizer.Fu Yuanyuan et al. [98] investigated the main chemical components of salt-fried Phellodendri Chinensis Cortex and Yihuang Decoction and found that the mass fraction of 4-O-feruloylquinic acid calculated based on salt-fried Phellodendri Chinensis Cortex in Yihuang Decoction is increased, while those of phellodendrine chloride, 5-O-feruloylquinic acid and berberine hydrochloride are decreased, indicating that the components of Yihuang Decoction may interact during the decoction process.Medicinal materials in Yihuang Decoction are matched with each other, and jointly clear heat, induce diuresis and tonify Chongand Ren channels, so that all diseases can be cured.3.5 Predictive analysis on the Q-marker ofYihuang Decoction based on content detection Q-marker of Yihuang Decoction is set based on its specificity and activity as well as components that can be easily detected.As per the Pharmacopoeia of the People's Republic of China (2020 edition), the indicators for content detection of Plantaginis Semen and Phellodendri Chinensis Cortex are berberine, phellodendrine, geniposidic acid and verbascoside.The indicators of Dioscoreae Rhizoma, Euryales Semen and Ginkgo Semen are not listed in the Pharmacopoeia of the People's Republic of China (2020 edition).Through detection using HPLC, Li medicine and lay a crucial role in the discovery of novel drugs.In recent years, China has laid greater emphasis on the research and development of classical prescriptions.The quality of Chinese medicine is of utmost importance for ensuring clinical efficacy and drug safety, and it serves as the lifeline for the development of the Chinese medicine industry.The research on the quality of Chinese medicine has always been the difficulty and hot spot, which, together with standard setting, is the national strategic issue related to Chinese medicine science and industrial development.However, owing to the multi-families genera, complex compositions, unclear pharmacodynamic substances and undefined mechanism of action of Chinese medicine, coupled with the long industrial chain, the quality research and the quality standard setting for Chinese medicine face a great challenge.Yihuang Decoction, included in the first batch of the 100 ancient classical prescriptions released in 2018, has been clinically used for the treatment of vaginitis, pelvic inflammation and other gynecological diseases for hundreds of years.It exhibits a wide range of pharmacological effects, such as anti-inflammation, anti-bacteria, immunoregulation, and antioxidation.This report summarizes the main chemical components, pharmacological effects and clinical application of Yihuang Decoction, and analyzes Yihuang Decoction from the perspectives of quality transmissibility and traceability, component specificity, component effectiveness, compound compatibility, and component measurability based on Chinese medicine Q-marker theory.The results revealed that allantoin, diosgenin, gallic acid, a-tocopherol, bilobalide, ginkgolide A, ginkgolide B, berberine, phellodendrine, magnoflorine, geniposidic acid and verbascoside can serve as Q-markers of Yihuang Decoction, which lays a basis for the research and development of Yihuang Decoction and provides a reference for its quality evaluation system.
[18] Qiutong et al.[16]summarized the previous processing methods and modern research of Euryales Semen, confirming that α-tocopherol and gallic acid may serve as the quality control indexes for Euryales Semen.Ginkgo Semen refers to the dried ripe kernel of Ginkgo biloba L., which mainly contains bilobol, terpene lactones, flavonoids, polysaccharides, alkaloids and other active components.Zhang Qunqun et al.[17]measured the contents of 4 terpene trilactones (bilobalide and ginkgolide A/B/C) and unveiled that the highest content of the four is 4 times that of the lowest.They accordingly suggested to use the content limit of these four terpene trilactones as quality standard, laying a foundation for research on Ginkgo Semen quality control.Shan Shujun[18]

Table 1
Information of Q-marker of Yihunag Decoction