Next Article in Journal
Enhancing the Photocatalytic Performance of Antibiotics Using a Z-Scheme Heterojunction of 0D ZnIn2S4 Quantum Dots and 3D Hierarchical Inverse Opal TiO2
Next Article in Special Issue
Challenges and Advances in the Bioproduction of L-Cysteine
Previous Article in Journal
2-Acetyl-5,8-dihydro-6-(4-methyl-3-pentenyl)-1,4-naphthohydroquinone-Derived Chalcones as Potential Anticancer Agents
Previous Article in Special Issue
Effect of Nano-Selenium on Nutritional Quality of Cowpea and Response of ABCC Transporter Family
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 20
10.3390/molecules28207173
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium

by
Xiaoling Zhang
1,2,3,
Bingling Tang
1,2,
Sijie Wen
1,2,
Yitong Wang
1,2,
Chengxue Pan
1,*,
Lingbo Qu
4,
Yulong Yin
5 and
Yongjun Wei
2,*
1
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
2
Laboratory of Synthetic Biology, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
3
Key Laboratory of Food Safety Quick Testing and Smart Supervision Technology for State Market Regulation, Zhengzhou 450003, China
4
College of Chemistry, Zhengzhou University, Zhengzhou 450001, China
5
Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410081, China
*
Authors to whom correspondence should be addressed.
Molecules 2023, 28(20), 7173; https://doi.org/10.3390/molecules28207173
Submission received: 21 September 2023 / Revised: 12 October 2023 / Accepted: 17 October 2023 / Published: 19 October 2023
(This article belongs to the Special Issue Synthesis of Natural Products Using Engineered Plants and Microorganisms)
Browse Figures
Versions Notes

Abstract

:
Epimedium is a classical Chinese herbal medicine, which has been used extensively to treat various diseases, such as sexual dysfunction, osteoporosis, cancer, rheumatoid arthritis, and brain diseases. Flavonoids, such as icariin, baohuoside I, icaritin, and epimedin C, are the main active ingredients with diverse pharmacological activities. Currently, most Epimedium flavonoids are extracted from Epimedium plants, but this method cannot meet the increasing market demand. Biotransformation strategies promised huge potential for increasing the contents of high-value Epimedium flavonoids, which would promote the full use of the Epimedium herb. Complete biosynthesis of major Epimedium flavonoids by microbial cell factories would enable industrial-scale production of Epimedium flavonoids. This review summarizes the structures, pharmacological activities, and biosynthesis pathways in the Epimedium plant, as well as the extraction methods of major Epimedium flavonoids, and advancements in the biotransformation and complete microbial synthesis of Epimedium flavonoids, which would provide valuable insights for future studies on Epimedium herb usage and the production of Epimedium flavonoids.

1. Introduction

The Epimedium genus, belonging to the Berberidaceae family, contains 68 species worldwide, with 58 of them (85.3%) distributed in China [1]. China is the center of geographical distribution and varieties of Epimedium. Over 15 Epimedium species have a long history of use in Traditional Chinese Medicine (TCM) and are believed to have kidney-nourishing and Yang-reinforcing properties [2]. Tao Hongjing, a renowned medical scientist, learned from shepherds that male sheep consuming a certain plant experienced significantly increased penile erections and mating frequency. Tao believed that this plant could enhance “Yang” energy, and named it “Yin-Yang-Huo” in Chinese [3]. It was later discovered that this was an Epimedium plant.
Epimedium was first mentioned over 2000 years ago in the “Shen Nong Ben Cao Jing”. It was later listed as a medium-grade herb in the “Ben Cao Gang Mu” by Li Shizhen during the Ming Dynasty [4]. In the Chinese Pharmacopoeia (2020 edition), Epimedii Folium (EF) refers to the dried leaves of four Epimedium plants, namely E. brevicornum Maxim, E. sagittatum (Sieb. et Zucc.) Maxim, E. pubescens Maxim, and E. koreanum Nakai [5]. EF is a classical herbal medicine. Alone, or combined within diverse prescriptions, it has been used to treat various diseases, including sexual dysfunction [6,7], osteoporosis [8], cancer [9], rheumatoid arthritis [10], and brain diseases [11]. Additionally, EF has been used in functional food production, and is available in alcoholic health beverages, health tea, and medicated gruel and noodle diets [2,12]. Published reviews have primarily concentrated on the compositions and molecular structures of the active components of EF, as well as the pharmacological activities of EF flavonoids [3,8,9,10,11]. In this review, we aim to emphasize the recent advancements in the biotransformation and biosynthesis of EF’s active flavonoids.

2. The Pharmacological Activities of Major Epimedium Flavonoids

More than 379 compounds have been detected in EF, including flavonoids, lignans, organic acids, terpenoids, dihydrophenanthrene derivatives, alkaloids, and other constituents [13]. Flavonoids, such as epimedin A, epimedin B, epimedin C, icariin, baohuoside I (also known as icariside II), icariside I, and icaritin have been recognized as major phytochemical and pharmacological active ingredients (Figure 1) [13,14]. These compounds differ in varying degrees of glycosylation at the C-3 and C-7 of icaritin [2,14]. There were great variations among the flavonoid contents in Epimedium from different species, collection and/or storage times and/or locations [15,16]. Icariin was the most abundant component in E. brevicornum Maxim and E. koreanum Nakai, followed by epimedin B, epimedin C, and epimedin A. However, epimedin C was the most abundant component in E. sagittatum (Sieb. et Zucc.) Maxim, E. pubescens Maxim, and E. wushanense T.S. Ying, followed by icariin, epimedin B, and epimedin A [15,16]. The average proportions of the total contents of epimedin A, B, C, and icariin to the 15 investigated flavonoid contents were 85.6%, 82.3%, 68.8%, 74.9%, and 69.8% in E. brevicornum Maxim, E. koreanum Nakai, E. sagittatum (Sieb. et Zucc.) Maxim, E. pubescens Maxim, and E. wushanense T.S. Ying, respectively [15,16]. In another study, epimedin A, B, C, and icariin accounted for over 52% of the total flavonoid contents in E. brevicornum Maxim [17]. In the Chinese Pharmacopoeia (2020 edition), the total amount of epimedin A, B, C and icariin was identified as the quality control indicator for the EF herb [5]. The major flavonoids in EF exhibit significant and diverse pharmacological activities (Figure 1).

2.1. Icariin and Its Pharmaceutical Effects

Icariin, the major bioactive component in EF (about 1%) [17], has been found to possess various pharmacological effects. These include improved reproductive system function, a neuroprotective effect, an anti-osteoporosis effect, protective effects from cardiovascular disease, an anti-inflammation effect, an anti-oxidative stress effect, an anti-depressive effect, and an anti-tumor effect [18,19,20,21]. In ancient China, EF was commonly used to treat sexual dysfunction [3]. Icariin can enhance erectile function in spontaneously hypertensive rats by reducing endothelial microparticle levels in the blood and inhibiting platelet activation [22]. In male mice, icariin can improve sexual function through the PI3K/AKT/eNOS/NO signaling pathway [23]. In the female reproductive system, icariin promotes estrogen biosynthesis in human ovarian granulosa-like KGN cells, and upregulates the expression of aromatase, which is responsible for the conversion of androgens to estrogens in vertebrates [24]. Additionally, icariin has exhibited protective effects in various nervous system disorders, including Alzheimer’s disease, Parkinson’s disease, and depressive disorder [20,25,26,27]. Moreover, icariin is regarded as a potential drug for osteoporosis treatment. Recent studies have demonstrated that icariin could prevent bone loss in ovariectomized rat models by modulating gut microbiota and regulating metabolite alterations [28] or by activating autophagy [29], as well as protect against iron overload-induced bone loss via suppressing oxidative stress [30].

2.2. Baohuoside I and Its Pharmaceutical Effects

Baohuoside I, although presents in low contents (<0.15%) in the raw material of EF compared to icariin, exhibits a wider range of pharmacological activities [31,32]. Baohuoside I has better bioavailability in vivo than icariin, as it is more easily absorbed by the capillaries of intestinal epithelial cells because of its lower polarity [33]. Cheng et al. found that 91.2% of icariin was converted to baohuoside I after oral administration in rats [34]. Similarly, human intestinal microflora metabolized most icariin to baohuoside I in a short time before absorption in the human intestine [35]. Baohuoside I has been proved to have a significant therapeutic effect on various diseases, such as sexual dysfunction, osteoporosis, and cancers [9,36,37,38]. For improving erectile dysfunction, baohuoside I could facilitate the differentiation of adipose-derived stem cells into Schwann cells and preserve the erectile function of bilateral cavernous nerve injury (BCNI) in rats [36,39]. The anti-osteoporotic activity of baohuoside I was suggested to be associated with its ability to induce bone marrow stromal cell differentiation into osteoblasts while inhibiting adipocyte formation, regulating immune functions, and providing antioxidant activity [40]. Baohuoside I could inhibit osteoclastogenesis and protect against ovariectomy-induced bone loss in mice, surpassing the effects of icariin [38]. Current studies have shown that baohuoside I exhibits promising anti-tumor effects on lung cancer cells [41], melanoma cells [42], breast cancer cells [43], prostate cancer cells [44], and osteosarcoma cells [45]. Furthermore, baohuoside I has shown its potential application in type 2 diabetes treatment [46], neuroprotection [47], and asthma inhibition [48].

2.3. Icaritin and Its Pharmaceutical Effects

Icaritin is a flavonoid aglycone in EF [14], which can be generated by hydrolytic reactions that remove the glycone parts of icariin, baohuoside I, and icariside I [49,50]. Icaritin possesses diverse pharmacological activities [51], including protection of neurons against amyloid-induced neurotoxicity [52], promotion of differentiation from embryonic stem cells into cardiomyocytes [53], anti-osteoporosis effects and osteogenesis promotion [54,55], immunomodulation [56,57], and recovery of UVB-induced photoaging of human keratinocytes [58]. Moreover, icaritin is considered as a promising candidate for the treatment of various cancers [59,60], including hepatocellular carcinoma [61,62,63], breast cancer [64], lung cancer [65], ovarian cancer [66], endometrial cancer [67], human oral squamous cell carcinoma [68], and multiple hematological malignancies [59,69,70,71]. In the treatment of hepatocellular carcinoma, icaritin can suppress cell growth and promote cell apoptosis via down-regulating alpha-fetoprotein gene expression in hepatocellular carcinoma [62] and inducing anti-tumor immune responses [61,63]. In 2022, an icaritin soft capsule was marketed as a small molecule immunomodulatory drug, providing a solution for patients with advanced hepatocellular carcinoma with poor prognosis, and significantly improving the life quality of patients with hepatocellular carcinoma [72,73].

2.4. Epimedin C and Its Pharmaceutical Effects

Epimedin C is a trioglycoside ingredient in EF, with the highest content among all flavonol glycosides in certain Epimedium species, such as E. brevicornu Maxim [74], E. wushanense T.S. Ying, and E. sagittatum Maxim [75]. Epimedin C is considered as the quality control standard for evaluating the quality of E. wushanense T.S. Ying in the Chinese Pharmacopoeia (2020 edition) [5]. The pharmacological activities of epimedin C mainly include treatment of bone loss, anti-oxidant effects, and anti-inflammation. Epimedin C has shown significant anti-inflammatory and chondroprotective effects by increasing the expression of extracellular matrix components in osteoarthritis chondrocytes [76]. Epimedin C could alleviate the suppressive impact of dexamethasone on the osteogenesis of larval zebrafish and MC3T3-E1 cells via triggering the PI3K/AKT/RUNX2 signaling pathway [77]. Notably, epimedin C has a stronger anti-osteoporosis effect than icariin at the same dose on dexamethasone-induced osteoporosis in a mouse model [78]. Furthermore, epimedin C has been found to protect against H2O2-induced peroxidation injury by enhancing the function of endothelial progenitor human umbilical vein endothelial cells, which plays an important role in repairing endothelial cell vascular injury [79]. In an ovalbumin-induced murine asthma model, epimedin C was demonstrated to dose-dependently decrease the protein levels of p52 and RelB, and the phosphorylation of ERK1/2, and p38 MAPK, which are pivotal in the development of Th9 cells and Treg cells, thereby inhibiting airway inflammation [80].

2.5. Other Flavonoids and Their Pharmaceutical Effects

Other flavonoids presented in EF include epimedin A and B, icariside I, and sagittatoside A, B, and C [2,13]. Their contents in EF are very low, and limited pharmacological research is available on them. However, similar to the major flavonoids described above, icariside I, epimedin A, and epimedin B also exhibit anti-osteoporosis, neuroprotective, and anti-cancer effects. Epimedin A has shown excellent efficacy against senile osteoporosis [8], and in vitro and in vivo experiments demonstrated that a complex epimedin A drug significantly enhances bone regeneration [81]. In addition, epimedin A could ameliorate 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis in mice, due to its ability to suppress the NF-κB/NLRP3 pathway, enhance the Nrf2 pathway, and modulate local inflammation [82]. Diao et al. provided evidence that epimedin B ameliorates osteoporosis in male mice via regulating PI3K-Akt, MAPK, and PPAR signaling pathways [83]. Additionally, epimedin B can exert a neuroprotective effect against Parkinson’s disease in an MPTP-induced mouse model [84]. Chen et al. suggested that icariside I performed tumor immunotherapy activity by blocking the kynurenine-AhR pathway and tumor immune escape [85]. Icariside I could significantly inhibit B16F10 melanoma growth in vivo through regulation of gut microbiota and host immunity [85]. Moreover, icariside I also effectively ameliorated estrogen deficiency-induced osteoporosis in an ovariectomy mouse model [86].
In addition to the various beneficial effects of EF flavonoids, it is important to note that EF can potentially cause drug-induced liver injury (Table 1). In clinical applications, there are increasing evidences indicate that Zhuangguguanjie pills and Xianlinggubao capsules have toxic effects, leading to liver injury in humans [87,88]. Both medicines contain EF as their major components, and are used to treat rheumatism, bone pain, arthritis, osteoporosis, and other diseases. Recently, animal studies have indicated that EF extracts can cause liver toxicity in mice and rats, with the severity of hepatotoxic effects increasing with higher dosages and prolonged exposure [89,90]. However, the exact compound(s) and the underlying mechanisms contributing to the observed liver toxicity remain unclear. Zhang et al. suggested that icariside I and sagittatoside A are the most relevant compounds related to the hepatotoxicity of EF extracts [91]. Epimedin C has been reported to have potential hepatotoxicity. Song et al. revealed that mRNA methylation might be associated with epimedin C-induced liver injury by the UPLC-MS/MS method [92]. When treated with the normal human liver cell line (HL-7702) and human hepatocellular carcinoma cell line (HepG2), 2″-O-Rhamnosyl icariside II, baohuoside I, and baohuoside II showed significant dose-toxic effects, and baohuoside I was more likely to be involved in the hepatotoxicity of EF [93]. Therefore, the hepatotoxicity of EF, like other TCMs, is probably due to the combined effects of multiple components. Further investigations are needed to fully understand the hepatotoxicity mechanism in order to avoid EF-induced liver injury.

3. The Biosynthetic Pathway of Prenylated Flavonoids in EF

The biosynthetic pathway of Epimedium flavonoids has been explored in E. sagittatum Maxim and E. pubescens Maxim [95,96,97]. It can be divided into three phases (Figure 2): phase 1 involves the phenylpropanoid pathway, phase 2 refers to the core pathway, and phase 3 involves further enzymatic modification pathways [97]. The starting precursors for the phenylpropanoid pathway are phenylalanine and tyrosine, which are produced via the shikimate pathway [98]. In plant cells, glucose metabolism produces phosphoenolpyruvate (PEP) and erythrose-4-phosphate, and they are catalyzed by seven enzymes to convert to chorismite, which is the common precursor for the synthesis of phenylalanine and tyrosine [98,99].
In phase 1, phenylalanine is converted to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL); cinnamic acid is then catalyzed by cinnamate-4-hydroxylase (C4H) to produce p-coumaric acid, and tyrosine is converted to p-coumaric acid by tyrosine ammonia lyase (TAL) [100]. Then, 4-coumarate CoA ligase (4CL) converts p-coumaric acid to 4-coumaroyl-CoA. Another precursor, malonyl-CoA, is mainly derived from acetyl-CoA by acetyl-CoA carboxylase (ACC), and acetyl-CoA is often produced from the classic glucose metabolic pathway. In phase 2, one molecule of 4-coumaryl-CoA and three molecules of malonyl-CoA are condensed by chalcone synthase (CHS) to form naringenin chalcone (a C6-C3-C6 backbone unit for all flavonoids) [97,100]. Subsequently, naringenin chalcone is converted to naringenin by chalcone isomerase (CHI) to complete ring closing. Naringenin is transformed to dihydroflavonol by flavanone 3-hydroxylase (F3H). Flavonol synthase (FLS) further converts dihydroflavonol to flavonols. In phase 3, isopentenyl is added to flavonols by prenyltransferase (PT), and further modifications are carried out by various post-modification enzymes, such as UDP-glycosyltransferase (UGT) and O-methyltransferase (OMT), to produce a series of icaritin flavonoids, including epimedin A, epimedin B, epimedin C, icariin, baohuoside I, icariside I, and icaritin [96,97].

4. Extraction Methods of Epimedium Flavonoids

Currently, commercially available Epimedium flavonoids are extracted from Epimedium plants. Several techniques have been developed for isolating flavonoids from Epimedium (Figure 3), including hot water extraction, alcohol extraction, ultrasonic extraction, microwave-assisted extraction, and ultra-high-pressure extraction. Among these techniques, hot water extraction and alcohol extraction have been implemented in industrial production, while others are at the lab-scale stage.

4.1. Hot Water Extraction

Hot water extraction is a traditional method used for decocting Chinese herbs. In this method, the crushed herbs are immersed in water in a container for an appropriate amount of time, then heated and gently boiled for a certain period of time. The liquid is subsequently filtered, and the process of decoction is repeated 2–3 times. The decocted liquids from each iteration are mixed and concentrated to achieve the desired flavonoid concentration. Wang et al. optimized the hot water extraction procedure with an orthogonal test [101]. The results showed that the optimized extraction procedure was 2% Na2CO3, 15 times the water volume of the weight of dried material, with three 1.5 h extractions. The final extracting ratio of the total flavonoids was 97.92%. Other new technologies, such as microwave technology, have been used to enhance hot water extraction. Compared to the conventional hot water extraction method, microwave-assisted extraction offers higher extraction efficiency and is time-saving [102]. The hot water extraction process is simple and cost-effective, and utilizes water as a safe solvent. The whole process generates minimal pollution. Therefore, hot water extraction is suitable for the large-scale production of flavonoids. However, the efficiency of hot water extraction for flavonoid extraction is low, and it lacks selectivity in capturing specific flavonoids.

4.2. Alcohol Extraction

The alcohol extraction method is the most commonly used technique for extracting flavonoids, adopted by the Chinese Pharmacopoeia (2020 edition) [5]. In this method, ethanol is generally employed as the extraction solvent. The process of the alcohol extraction method is relatively simple, and well-suited for industrial applications. However, a large amount of ethanol is added to the extraction reactor, which subsequently needs to be removed using extra instruments. As a result, the overall cost of this method is higher compared to hot water extraction. Zhang et al. demonstrated that the extraction rate of icariin using the alcohol extraction method was significantly higher than that of the water extraction method [75]. The optimal extraction parameters were determined to be 50% ethanol, 1:10 solid–liquid ratio, 60 °C extraction temperature, 2 h extraction time, and two extraction cycles. In addition, an ultrasonic-assisted ethanol extraction procedure has shown to increase the extraction yield of epimedin A, epimedin B, epimedin C, and icariin from Herba Epimedii, when compared to the conventional ethanol boiling extraction method [75].

4.3. Other Extraction Methods

Ultrasonic extraction utilizes the effects of strong vibrations, cavitation, and thermal energy generated by ultrasound to extract the active components of plants into solvents. Ultrasonic extraction is regarded as a powerful tool for extracting flavonoids from plant biomass, offering several advantages, such as increased extraction yield, shorter extraction time, and lower extraction temperature [103]. Microwave technology utilizes the ability to generate heat within cells and vaporize water to break down the cell walls, allowing for better release of active ingredients in plant cells. The microwave technique presents numerous benefits, including high efficiency, low energy consumption, short processing time, low cost, cleanliness, easy controllability, and low solvent requirement [104]. Both ultrasonic extraction and the microwave technique are often used to assist common extraction methods, such as water extraction and alcohol extraction, to improve the efficiency of extracting Epimedium flavonoids [75,102,105,106]. Furthermore, ultra-high-pressure extraction has also been utilized for extracting flavonoids from E. sagittatum. Compared to heating extraction and ultrasonic-assisted extraction, ultra-high-pressure extraction presents distinctive advantages in superior extraction yield and a higher percentage of marker compounds [107].

5. Biotransformation and Biosynthesis of Epimedium Flavonoids

The natural resources of medicinal Epimedium species have experienced a significant decline over the past several decades, primarily due to over-harvesting and curtailment of habitats [108]. Epimedium is a low-growing perennial plant that is commonly propagated by rhizome division because of its low seed viability [109]. Epimedium needs specific growth conditions and thrives in acidic soil with shade and relatively high humidity [2]. The growth conditions directly affect the contents of the major components present in Epimedium plants, such as Epimedium flavonoids. The cultivation of large-scale Epimedium plants is challenging [110], as selecting high-quality and stable Epimedium species, advancing seedling propagation technology, and enhancing farmers’ planting income are difficult [111]. Thus, improving the use efficiency of the existing wild resources of Epimedium and exploring new alternative methods for producing Epimedium flavonoids to meet the huge market demand are necessary and of great interest.

5.1. Biotransformation of Epimedium Flavonoids by Enzymes

Epimedium flavonoids mainly consist of epimedin A, epimedin B, epimedin C, icariin, baohuoside I, and icaritin. These compounds share a similar structure with the common aglycone skeleton icaritin, while differing in the type and number of lined sugar groups at the C-3 or C-7 positions [2,14]. Notably, icariin and baohuoside I are considered as the most effective components [18,33,112]. However, the contents of icariin and baohuoside I in Epimedium plants are extremely rare [17,32]. Therefore, converting other flavonoid components (epimedin A, epimedin B, and epimedin C) into icariin and baohuoside I through enzymatic hydrolysis of the terminal extra sugar groups, provides a feasible strategy to increase their contents in Epimedium extracts (Figure 4). Additionally, enzymatic hydrolysis shows enormous potential in the preparation of icariin and baohuoside I due to its notable selectivity, mild reaction conditions, high efficiency, and environmental friendliness [113,114].
The transformation of Epimedium flavonoids includes the hydrolysis of three types of sugar moieties: the glucose group, rhamnose group, and xylose group (Figure 1 and Figure 4). Three types of enzymes, namely glucosyl hydrolase, rhamnosyl hydrolase, and xylosyl hydrolase, are required to hydrolyze the corresponding sugar moieties (Table 2). High glycosylation flavonoids gradually hydrolyze sugar groups to form low glycosylation flavonoids (Figure 4). For example, epimedin C can be converted to icariin by removing its terminal rhamnose using α-l-rhamnosidase. To produce baohuoside I from icariin, β-glucosidase is employed to hydrolyze the glucose attached to the 7-O position of icariin. The aglycone icaritin can be generated by releasing the rhamnose moiety from the 3-O position of baohuoside I by α-l-rhamnosidase.
Previous studies have identified multiple enzymes capable of facilitating the conversion of Epimedium flavonoids (Table 2). Xie et al. discovered a novel thermostable GH78 family α-l-rhamnosidase (PodoRha) from Paenibacillus odorifer, which exhibited high specific activity in cleaving the outer α-1,2-rhamnopyranosyl moieties of epimedin C to produce icariin [114]. Another thermostable glucose-tolerant GH1 β-glucosidase (IagBgl1) derived from the hyperthermophile Ignisphaera aggregans was found to efficiently produce baohuoside I from icariin [32]. Based on these two enzymes, Xie et al. built a two-step conversion system consisting of PodoRha and IagBgl1 to transform epimedin C into baohuoside I with a conversion rate of 98% under optimized conditions [114]. A fungal α-l-rhamnosidase, AmRha, could hydrolyze the α-1,2-rhamnoside bond between two rhamnoses in epimedin C, achieving the production of icariin with a molar conversion rate of 92.3% in vitro [112]. When AmRha cooperated with β-glucosidase/β-xylosidase Dth3 [115], epimedin A, epimedin B, epimedin C, and icariin in the total Epimedium flavonoids could be converted to baohuoside I efficiently and simultaneously [112]. In addition, snailase, a kind of β-D-glucose hydrolase, was reported to contain more than 20 kinds of enzymes, including cellulase, pectinase, hemicellulose, and β-glucuronidase [116]. Snailase was applied to hydrolyze the total Epimedium flavonoids to the hydrolysate with enhanced anti-tumor activity [117].
Table 2. Three types of functional enzymes were used to transform Epimedium flavonoids.
Table 2. Three types of functional enzymes were used to transform Epimedium flavonoids.
Enzyme TypeEnzyme NameEnzyme SourceEnzyme CharacterizationEnzyme FunctionsReferences
Rhamnosyl hydrolaseα-l-rhamnosidase (AmRha)Aspergillus
mulundensis		107.27 kDa, glycoside hydrolase (GH) 78 family; the optimal activity was achieved at 65 °C and pH 5.5, wide application temperature range, high level of enzyme catalytic ability and stability in the range of pH 5.0–7.5, acting on α-1,2-rhamnoside and α-1,6-rhamnoside bonds directly connected with glucose.Catalyzes the bioconversion of epimedin C to icariin[112]
α-l-rhamnosidasePapiliotrema laurentii ZJU-L07100 kDa; the optimal activity was achieved at 55° C and pH 7.0, sensitive to temperature, stable at a pH range of 5.5–9.0 with an activity of over 80%, higher selectivity to cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside and the α-1,2 glycosidic linkage between rhamnoside and rhamnoside.Produces icariin from epimedin C[118]
α-l-rhamnosidase (PodoRha)Paenibacillus odoriferThe molecular weight of the monomer was 100.12 kDa, the native recombinant PodoRha was a trimer, GH78 family; the optimal activity was achieved at 45 °C and pH 6.5, a broad range of activity within a pH range of 5.0–8.5, excellent thermostability at 40 °C and 35 °C, high specificity on α-1,2-glycoside in epimedin C.Converts epimedin C into icariin[114]
α-l-rhamnosidase (DthRha)Dictyoglomus thermophilum DSM3960106.96 kDa, GH78 family; the optimal activity was achieved at 95 °C and pH 6.5, stable within the pH range of 4.5–7.5, residual activities exceeded 50% after incubation at 85 °C for 3 h and exceeded 90% after incubation at 75 °C for 3 h. Efficient hydrolyzation of the α-l-rhamnosidic bond of Epimedium flavonoids.Converts epimedin C into icariin, converts icariin into icariside I, and converts baohuoside I into icaritin[50]
α-l-rhamnosidase (synAnRhaE)Aspergillus nidulans95 kDa; the optimal activity was achieved at 55 °C and pH 4.5, stable in an acidulous pH range below 55 °C, high specificity on α-1,2 rhamnoside glycosidic bond in epimedin C.Converts epimedin C into icariin[119]
α-l-rhamnosidase (SPRHA2)Novosphingobium sp. GX9120 kDa, GH106 family, when combined with PBGL, the optimal temperature for the reaction was 55 °C, and the highest activity was observed in 200 mM borate saline buffer at pH 8.5.Catalyzes icariin into icariside I, converts baohuoside I into icaritin[14]
α-l-rhamnosidase (BtRha)Bacteroides thetaiotaomicron VPI-548283.3 kDa, GH78 family; the optimal activity was achieved at 55 °C and pH 6.5, high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively.Transforms epimedin C to icariin[120]
α-l-rhamnosidase (Rhase-I)Talaromyces stollii CLY-6140 kDa, GH106 family; the optimal activity was achieved at 45 °C and pH 4.5, high thermal stability at a temperature lower than 50 °C and superior stability in an acidic environment (pH 2.0–5.0), be activated by Ca2+ and Mg2+, efficiently cleaving both the outer and inner rhamnosidic bonds of epimedin C.Converts epimedin C into icariin, and converts icariin into icariside I[121]
α-l-rhamnosidase (AtRha)Aspergillus terreus CCF305996.9 kDa, GH78 family; the optimal activity was achieved at 60 °C and pH 6.5, excellent thermal stability and pH stability, hydrolyzed icariin containing the α-1 rhamnoside linkage.Hydrolyzes icariin to icariside I[122]
Glucosyl hydrolaseβ-glucosidase (Tpebgl3)Thermomotoga petrophila DSM 13,99581.24 kDa, GH3 family; the optimal activity was achieved at 90 °C and pH 5.0, the thermostability of the enzyme was improved by Ca2+, good stability at high temperatures and organic solvents.Produces baohuoside I from icariin[31,123]
β-glucosidase (IagBgl1)Ignisphaera aggregansThe molecular weight of the monomer was 56.36 kDa, the native recombinant IagBgl1 was a trimer, GH1 family; the optimal activity was achieved at 95 °C and pH 6.5, thermostable and glucose-tolerant, retained more than 70% after incubation at 90 °C for 4 h, high catalytic activity towards icariin.Produces baohuoside I from icariin[32]
β-glucosidase (PBGL)Paenibacillus cookii GX-484 kDa, GH3 family, when combined with SPRHA2, the optimal temperature for the reaction was 55 °C, and the highest activity was observed in 200 mM borate saline buffer at pH 8.5.Converts icariin into baohuoside I, converts icariside I to icaritin[14]
β-1,3-glucanase (CtLam55)Chaetomium thermophilum82.7 kDa, GH55 family; the optimal activity was achieved at 60 °C and pH 5.0, thermostable at 50 °C, exo-β-1,3-glucanase activity.Hydrolyzes icariin to baohuoside I[113,124]
β-glucosidase (Dth3)Dictyoglomus thermophilum DSM396088.7 kDa, GH3 family; the optimal activity was achieved at 90 °C and pH 5.5, highly tolerant to glucose.Converts epimedin A into baohuoside I, converts icariin into baohuoside I[112,115]
β-glucosidaseTrichoderma viride60 kDa; the optimal activity was achieved at 41 °C and pH 4.0.Prepares baohuoside I from icariin[125]
dextranase-The optimal activity was achieved at 40 °C and pH 5.4, sensitive to pH.Hydrolyzes icariin to baohuoside I[126]
cellulase-The optimum conditions for the cellulase were 50 °C and pH 5.0.Transforms icariin to baohuoside I[127]
Xylosyl hydrolaseβ-xylosidase
(BbXyl)		Bifidobacterium breve K-11070 kDa, GH43 family; the optimal activity was achieved at 45 °C and pH 5.5, the residual activity was more than 80% after being incubated at 45 °C for 4 h, showed over 70% of the maximum activity at a pH from 4.5 to 7.0, higher catalytic efficiency and selection specificity.Converts epimedin B into icariin[128]
β-xylosidase (Dt-2286)Dictyoglomus turgidum85.1 kDa, GH3 family; the optimal activity was achieved at 98 °C and pH 5.0, excellent thermostable/haloduric/organic solvent-tolerance, a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities.Converts epimedin B into sagittatoside B, and converts sagittatoside B into icariside I[129]
β-xylosidase (Dth3)Dictyoglomus thermophilum DSM396088.7 kDa, GH3 family; the optimal activity was achieved at 90 °C and pH 5.5, activity was not affected by xylose in high concentration.Converts epimedin B into baohuoside I[112,115]
-: Not determined.

5.2. Biotransformation of Epimedium Flavonoids by Whole-Cell Catalysis

Icariin, baohuoside I, and icaritin are the most effective components in Epimedium [31,118,119,122], and considered as high-value Epimedium flavonoids. Enzyme transformation is the preferred method for the preparation of icariin, baohuoside I, and icaritin, due to its high selectivity and catalytic efficiency, fewer by-products, mild reaction conditions, and convenient purification of the products [114,130]. However, enzymes, as a kind of biological macromolecule catalyst, have limitations in the large-scale use in industry, such as easily reduced activity and the high cost associated with enzyme expression and purification [131]. To address these challenges, enzymes are usually combined with carriers to improve their operational stability and reusability [131]. For example, Lu et al. successfully prepared an immobilized enzyme (4LP-Tpebgl3@Na-Y) by fusing the 4LP linker to thermostable β-glucosidase (Tpebgl3) on Na-Y zeolite. This immobilized enzyme exhibited enhanced tolerance of organic solvent and glucose, higher activity, and more reuse cycles, as compared to free enzymes during the production of baohuoside I from icariin [31]. Whole-cell catalysis is an alternative strategy that eliminates enzyme isolation and purification processes, thereby reducing costs (Figure 5). In this strategy, the cells provide a natural and protective environment for the enzymes [132]. Therefore, hydrolysis of flavonoid glycosides by whole-cell catalysis has the potential to be a low-cost strategy while retaining high efficiency.
Several studies have used whole microbial cells to transform Epimedium flavonoids. The α-l-rhamnosidase synAnRhaE from A. nidulans was expressed in Escherichia coli, and the recombinant E. coli could efficiently transform epimedin C to icariin [119]. Another α-l-rhamnosidase SPRHA2 and β-glucosidase PBGL were expressed in E. coli, respectively. The mixture of SPRHA2 cells and PBGL cells was used to transform icariin into icaritin, and the yield rate was 95.23%. Moreover, crude icariin extracts were also efficiently hydrolyzed by the recombinant E. coli strain that co-expressed SPRHA2 and PBGL [14]. Additionally, the recombinant E. coli strain transformed with α-l-rhamnosidase BtRha and β-xylosidase BbXyl could convert the total flavonoids of Epimedium into icariin [128]. Furthermore, the engineered Komagataella phaffii GS115 strain expressed with α-l-rhamnosidase AmRha could transform epimedin C to icariin efficiently. This strain also largely removed the feedback inhibition of l-rhamnose and enabled a five-fold increase in the concentration of raw Epimedium flavonoids [112]. Whole-cell catalysis, as an intermediate approach between fermentation and in vitro enzyme catalysis, offers several advantages over the use of purified or immobilized enzymes. These include significantly reduced catalyst costs, increased stability due to residual cell wall compounds, and no need for external cofactor addition [133]. Overall, these findings highlight the potential of whole-cell catalysis as a promising approach for future industrial production of high-value Epimedium flavonoids.

5.3. Complete Biosynthesis of Epimedium Flavonoids

Biotransformation processes have demonstrated the ability to convert low-value flavonoids into high-value flavonoids. However, the production of natural products from plant biomass requires large amounts of arable land and labor-intensive cultivation. Metabolic engineering and synthetic biology methods have emerged as alternative, simple, and eco-friendly strategies for the production of high-value natural herb products [134,135]. Currently, microbial cell factories have been built to produce diverse natural products, such as artemisinic acid [136], opioids [137], and glycyrrhetinic acid [135].
The biosynthetic pathways of specific Epimedium flavonoids have been elucidated, and successful production of icaritin has been achieved. Wang et al. utilized a metabolic engineering strategy to build yeast strains by introducing 11 heterologous genes and modifying 12 native yeast genes. After relocating the methyltransferase GmOMT2 into yeast mitochondria or co-culturing the engineered yeast with an E. coli strain expressing GmOMT2, they realized the biosynthesis of icaritin from glucose, and the yields of icaritin were 7.2 and 19.7 mg/L [110].
The biosynthesis of other Epimedium flavonoids, such as monoglycoside flavonoids of baohuoside I and icariside I, diglycoside flavonoid of icariin, and trioglycoside flavonoids of epimedin A, B, and C, requires specific glycosyltransferase, rhamnosyltransferase, xylosyltransferase, and other modifying enzymes [97]. Some of these enzymes are not well characterized, and their compatibility with eukaryotic chasses and the sequential reactions involved in Epimedium flavonoid biosynthesis have not yet been determined. Despite these challenges, the application of metabolic engineering and synthetic biology holds great potential for advancing the biosynthesis of Epimedium flavonoids (Figure 6). Future research efforts focused on characterizing enzymes, improving compatibility with yeasts and other microbial hosts, and optimizing biosynthetic flavonoid pathways will contribute to the de novo production of Epimedium flavonoids in microbial cell factories.

6. Perspective

EF, an important TCM, has been safely used for more than 2000 years to treat various diseases, such as sexual dysfunction, osteoporosis, rheumatoid arthritis, cardiovascular disease, nervous system diseases, and tumors [138,139,140,141,142,143]. Modern evidences have confirmed that the major active pharmacological ingredients of EF, including epimedin A, epimedin B, epimedin C, icariin, baohuoside I, and icaritin, have corresponding pharmacological activities.
To enhance the production of bioactive high-value Epimedium flavonoids, multiple methods have been developed, including biotransformation by enzymes or whole cells [113,115], chemical synthesis [144], and biological synthesis by microbial cell factories [107]. Among these methods, whole-cell catalysis is regarded as a low-cost, stable, highly efficient, and environmentally friendly strategy. Actually, the treatment of Chinese herbs with microorganisms is a traditional technique; TCM fermentation was recorded thousands of years ago in China [145]. Fermenting Chinese herbs with microorganisms, especially probiotics, could promote the release of effective ingredients, reduce toxicities, generate new bioactive substances, enhance bioavailability, and improve pharmacological activities [146]. Fermenting EF with Lactobacillus plantarum has shown to increase the relative contents of flavonoid aglycones, monoglycosides, and phenolic acids, while the relative contents of multilevel and secondary flavonoid glycosides decreased, and the antioxidation of fermentation products was significantly increased [147].
Currently, synthetic biology is a powerful tool for the production of complex and high-value natural products using microbial cell factories [136]. However, the recovery of the key enzymes and building of the desirable engineered microorganisms remain challenging tasks. Omics technologies have been widely used to identify new functional genes, while metabolic engineering and metabolic flux analyses help to build complete and efficient natural-product synthetic pathways in microbial cell factories. These advanced technologies would greatly promote the efficient production of Chinese herb-derived bioactive ingredients in the near future, including Epimedium flavonoids.

Author Contributions

Y.W. (Yongjun Wei), C.P. and L.Q. conceived the study. X.Z., B.T. and S.W. drafted the manuscript, and X.Z., B.T., S.W. and Y.W. (Yitong Wang) prepared the figures and tables. Y.W. (Yongjun Wei), L.Q., C.P. and Y.Y. revised and polished the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32101003, 32371485, and 32111530179) and Clinical Laboratories, Shenyou Bio.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Xu, Y.Q.; Jiang, Y.; Huang, H.; Li, R.Q.; Li, F.Q.; Liu, Y.; Huang, X.F. Taxonomic study of Epimedium L.: Status, issues and prospect. Guihaia 2020, 40, 601–617. [Google Scholar]
  2. Ma, H.; He, X.; Yang, Y.; Li, M.; Hao, D.; Jia, Z. The genus Epimedium: An ethnopharmacological and phytochemical review. J. Ethnopharmacol. 2011, 134, 519–541. [Google Scholar] [CrossRef] [PubMed]
  3. Niu, Y.; Lin, G.; Pan, J.; Liu, J.; Xu, Y.; Cai, Q.; Wang, T.; Luan, Y.; Chen, Y.; Feng, Y.; et al. Deciphering the myth of icariin and synthetic derivatives in improving erectile function from a molecular biology perspective: A narrative review. Transl. Androl. Urol. 2022, 11, 1007–1022. [Google Scholar] [CrossRef] [PubMed]
  4. Seyedi, Z.; Amiri, M.S.; Mohammadzadeh, V.; Hashemzadeh, A.; Haddad-Mashadrizeh, A.; Mashreghi, M.; Qayoomian, M.; Hashemzadeh, M.R.; Simal-Gandara, J.; Taghavizadeh Yazdi, M.E. Icariin: A promising natural product in biomedicine and tissue engineering. J. Funct. Biomater. 2023, 14, 44. [Google Scholar] [CrossRef] [PubMed]
  5. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China; China Medical Science Press: Beijing, China, 2020; Volume 1. [Google Scholar]
  6. Punyawudho, B.; Puttilerpong, C.; Wirotsaengthong, S.; Aramwit, P. A randomized, double-blind, placebo-controlled crossover study of Cappra® for the treatment of mild or mild to moderate erectile dysfunction in Thai male. Afr. J. Tradit. Complement. Altern. Med. 2012, 10, 310–315. [Google Scholar] [CrossRef]
  7. Zhang, H.; Sun, Z.X.; Men, B.; Fu, X.J.; Chen, J.S. Clinical and mechanism research on functional erectile dysfunction treated with moxibustion and qiangshen shugan qiwei decoction. Zhongguo Zhen Jiu 2021, 41, 1325–1330. [Google Scholar]
  8. Wang, L.; Li, Y.; Guo, Y.; Ma, R.; Fu, M.; Niu, J.; Gao, S.; Zhang, D. Herba Epimedii: An ancient Chinese herbal medicine in the prevention and treatment of osteoporosis. Curr. Pharm. Des. 2016, 22, 328–349. [Google Scholar] [CrossRef]
  9. Chen, M.; Wu, J.; Luo, Q.; Mo, S.; Lyu, Y.; Wei, Y.; Dong, J. The anticancer properties of Herba Epimedii and its main bioactive components icariin and icariside II. Nutrients 2016, 8, 563. [Google Scholar] [CrossRef]
  10. Zhang, L.B.; Yan, Y.; He, J.; Wang, P.P.; Chen, X.; Lan, T.Y.; Guo, Y.X.; Wang, J.P.; Luo, J.; Yan, Z.R.; et al. Epimedii Herba: An ancient Chinese herbal medicine in the prevention and treatment of rheumatoid arthritis. Front. Chem. 2022, 10, 1023779. [Google Scholar] [CrossRef]
  11. Cho, J.H.; Jung, J.Y.; Lee, B.J.; Lee, K.; Park, J.W.; Bu, Y. Epimedii Herba: A promising herbal medicine for neuroplasticity. Phytother. Res. 2017, 31, 838–848. [Google Scholar] [CrossRef]
  12. Zhang, H.F.; Yang, X.H. Application of Herba Epimedii in food industry: Current status and prospect. Sci. Technol. Food Ind. 2010, 31, 390–393. [Google Scholar]
  13. Qian, H.Q.; Wu, D.C.; Li, C.Y.; Liu, X.R.; Han, X.K.; Peng, Y.; Zhang, H.; Zhao, B.Y.; Zhao, Y. A systematic review of traditional uses, phytochemistry, pharmacology and toxicity of Epimedium koreanum Nakai. J. Ethnopharmacol. 2024, 318, 116957. [Google Scholar] [CrossRef] [PubMed]
  14. Lin, Y.; Chen, W.W.; Ding, B.; Guo, M.; Liang, M.; Pang, H.; Wei, Y.T.; Huang, R.B.; Du, L.Q. Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis. Microb. Cell Fact. 2023, 22, 64. [Google Scholar] [CrossRef] [PubMed]
  15. Chen, X.J.; Ji, H.; Zhang, Q.W.; Tu, P.F.; Wang, Y.T.; Guo, B.L.; Li, S.P. A rapid method for simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and ultra-performance liquid chromatography. J. Pharm. Biomed. Anal. 2008, 46, 226–235. [Google Scholar] [CrossRef]
  16. Chen, X.J.; Guo, B.L.; Li, S.P.; Zhang, Q.W.; Tu, P.F.; Wang, Y.T. Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography. J. Chromatogr. A 2007, 1163, 96–104. [Google Scholar] [CrossRef]
  17. Gao, Y.; Shi, W.; Tu, C.; Li, P.; Zhao, G.; Xiao, X.; Wang, J.; Bai, Z. Immunostimulatory activity and structure-activity relationship of epimedin B from Epimedium brevicornu Maxim. Front. Pharmacol. 2022, 13, 1015846. [Google Scholar] [CrossRef] [PubMed]
  18. He, C.; Wang, Z.; Shi, J. Pharmacological effects of icariin. Adv. Pharmacol. 2020, 87, 179–203. [Google Scholar]
  19. Li, Z.; Li, D.; Chen, R.; Gao, S.; Xu, Z.; Li, N. Cell death regulation: A new way for natural products to treat osteoporosis. Pharmacol. Res. 2023, 187, 106635. [Google Scholar] [CrossRef]
  20. Wang, S.; Ma, J.; Zeng, Y.; Zhou, G.; Wang, Y.; Zhou, W.; Sun, X.; Wu, M. Icariin, an up-and-coming bioactive compound against neurological diseases: Network pharmacology-based study and literature review. Drug Des. Devel. Ther. 2021, 15, 3619–3641. [Google Scholar] [CrossRef]
  21. Zeng, Y.; Xiong, Y.; Yang, T.; Wang, Y.; Zeng, J.; Zhou, S.; Luo, Y.; Li, L. Icariin and its metabolites as potential protective phytochemicals against cardiovascular disease: From effects to molecular mechanisms. Biomed. Pharmacother. 2022, 147, 112642. [Google Scholar] [CrossRef]
  22. Li, X.; Yang, H.F.; Chen, Y.; Pei, L.J.; Jiang, R. Effect of the icariin on endothelial microparticles, endothelial progenitor cells, platelets, and erectile function in spontaneously hypertensive rats. Andrology 2022, 10, 576–584. [Google Scholar] [CrossRef]
  23. Ding, J.; Tang, Y.; Tang, Z.; Zu, X.; Qi, L.; Zhang, X.; Wang, G. Icariin improves the sexual function of male mice through the PI3K/AKT/eNOS/NO signalling pathway. Andrologia 2018, 50, e12802. [Google Scholar] [CrossRef] [PubMed]
  24. Yang, L.; Lu, D.; Guo, J.; Meng, X.; Zhang, G.; Wang, F. Icariin from Epimedium brevicornum Maxim promotes the biosynthesis of estrogen by aromatase (CYP19). J. Ethnopharmacol. 2013, 145, 715–721. [Google Scholar] [CrossRef] [PubMed]
  25. Angeloni, C.; Barbalace, M.C.; Hrelia, S. Icariin and its metabolites as potential protective phytochemicals against Alzheimer’s Disease. Front. Pharmacol. 2019, 10, 271. [Google Scholar] [CrossRef]
  26. Wang, G.Q.; Li, D.D.; Huang, C.; Lu, D.S.; Zhang, C.; Zhou, S.Y.; Liu, J.; Zhang, F. Icariin reduces dopaminergic neuronal loss and microglia-mediated inflammation in vivo and in vitro. Front. Mol. Neurosci. 2017, 10, 441. [Google Scholar] [CrossRef] [PubMed]
  27. Wei, K.; Xu, Y.; Zhao, Z.; Wu, X.; Du, Y.; Sun, J.; Yi, T.; Dong, J.; Liu, B. Icariin alters the expression of glucocorticoid receptor, FKBP5 and SGK1 in rat brains following exposure to chronic mild stress. Int. J. Mol. Med. 2016, 38, 337–344. [Google Scholar] [CrossRef] [PubMed]
  28. Wang, S.; Wang, S.; Wang, X.; Xu, Y.; Zhang, X.; Han, Y.; Yan, H.; Liu, L.; Wang, L.; Ye, H.; et al. Effects of icariin on modulating gut microbiota and regulating metabolite alterations to prevent bone loss in ovariectomized rat model. Front. Endocrinol. 2022, 13, 874849. [Google Scholar] [CrossRef]
  29. Liang, X.; Hou, Z.; Xie, Y.; Yan, F.; Li, S.; Zhu, X.; Cai, L. Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy. J. Cell Biochem. 2019, 120, 13121–13132. [Google Scholar] [CrossRef] [PubMed]
  30. Jing, X.; Du, T.; Chen, K.; Guo, J.; Xiang, W.; Yao, X.; Sun, K.; Ye, Y.; Guo, F. Icariin protects against iron overload-induced bone loss via suppressing oxidative stress. J. Cell Physiol. 2019, 234, 10123–10137. [Google Scholar] [CrossRef]
  31. Lu, S.; Zou, K.; Guo, B.; Pei, J.; Wang, Z.; Xiao, W.; Zhao, L. One-step purification and immobilization of thermostable β-glucosidase on Na-Y zeolite based on the linker and its application in the efficient production of baohuoside I from icariin. Bioorg. Chem. 2022, 121, 105690. [Google Scholar] [CrossRef]
  32. Xie, J.; Xu, H.; Jiang, J.; Zhang, N.; Yang, J.; Zhao, J.; Wei, M. Characterization of a novel thermostable glucose-tolerant GH1 β-glucosidase from the hyperthermophile Ignisphaera aggregans and its application in the efficient production of baohuoside I from icariin and total epimedium flavonoids. Bioorg. Chem. 2020, 104, 104296. [Google Scholar] [CrossRef] [PubMed]
  33. Yan, H.; Song, J.; Jia, X.; Zhang, Z. Hyaluronic acid-modified didecyldimethylammonium bromide/ d-a-tocopheryl polyethylene glycol succinate mixed micelles for delivery of baohuoside I against non-small cell lung cancer: In vitro and in vivo evaluation. Drug Deliv. 2017, 24, 30–39. [Google Scholar] [CrossRef] [PubMed]
  34. Cheng, T.; Zhang, Y.; Zhang, T.; Lu, L.; Ding, Y.; Zhao, Y. Comparative pharmacokinetics study of icariin and icariside II in rats. Molecules 2015, 20, 21274–21286. [Google Scholar] [CrossRef] [PubMed]
  35. Wu, H.; Kim, M.; Han, J. Icariin metabolism by human intestinal microflora. Molecules 2016, 21, 1158. [Google Scholar] [CrossRef]
  36. Ge, P.; Guo, Y.; Shen, J. Icariside II facilitates the differentiation of ADSCs to SCs via let-7i/STAT3 axis to preserve erectile function. Biol. Res. 2019, 52, 54. [Google Scholar] [CrossRef]
  37. Khan, M.; Maryam, A.; Qazi, J.I.; Ma, T. Targeting apoptosis and multiple signaling pathways with icariside II in cancer cells. Int. J. Biol. Sci. 2015, 11, 1100–1112. [Google Scholar] [CrossRef]
  38. Ma, M.; Fan, A.Y.; Liu, Z.; Yang, L.Q.; Huang, J.M.; Pang, Z.Y.; Yin, F. Baohuoside I inhibits osteoclastogenesis and protects against ovariectomy-induced bone loss. Front. Pharmacol. 2022, 13, 874952. [Google Scholar] [CrossRef]
  39. Zheng, T.; Zhang, T.; Zhang, W.; Lv, K.; Jia, D.; Yang, F.; Sun, Y.; Lian, J.; Wang, R. Icariside II facilitates the differentiation of ADSCs to schwann cells and restores erectile dysfunction through regulation of miR-33/GDNF axis. Biomed. Pharmacother. 2020, 125, 109888. [Google Scholar] [CrossRef]
  40. Xi, Y.; Jiang, T.; Yu, J.; Xue, M.; Xu, N.; Wen, J.; Wang, W.; He, H.; Ye, X. Preliminary studies on the anti-osteoporosis activity of Baohuoside I. Biomed. Pharmacother. 2019, 115, 108850. [Google Scholar] [CrossRef]
  41. Kong, Q.; Ma, M.; Zhang, L.; Liu, S.; He, S.; Wu, J.; Liu, B.; Dong, J. Icariside II potentiates the anti-PD-1 antitumor effect by reducing chemotactic infiltration of myeloid-derived suppressor cells into the tumor microenvironment via ROS-mediated inactivation of the SRC/ERK/STAT3 signaling pathways. Phytomedicine 2023, 110, 154638. [Google Scholar] [CrossRef]
  42. Peng, Y.G.; Zhang, L. Baohuoside-I suppresses cell proliferation and migration by up-regulating miR-144 in melanoma. Pharm. Biol. 2018, 56, 43–50. [Google Scholar] [CrossRef] [PubMed]
  43. Wang, S.; Wang, N.; Huang, X.; Yang, B.; Zheng, Y.; Zhang, J.; Wang, X.; Lin, Y.; Wang, Z. Baohuoside i suppresses breast cancer metastasis by downregulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 pathway. Phytomedicine 2020, 78, 153331. [Google Scholar] [CrossRef] [PubMed]
  44. Li, S.; Zhan, Y.; Xie, Y.; Wang, Y.; Liu, Y. The impact of icariside II on human prostate cancer cell proliferation, mobility, and autophagy via PI3K-AKT-mTOR signaling pathway. Drug Des. Devel. Ther. 2020, 14, 4169–4178. [Google Scholar] [CrossRef] [PubMed]
  45. Choi, H.J.; Eun, J.S.; Kim, D.K.; Li, R.H.; Shin, T.Y.; Park, H.; Cho, N.P.; Soh, Y. Icariside II from Epimedium koreanum inhibits hypoxia-inducible factor-1alpha in human osteosarcoma cells. Eur. J. Pharmacol. 2008, 579, 58–65. [Google Scholar] [CrossRef] [PubMed]
  46. Kim, D.H.; Jung, H.A.; Sohn, H.S.; Kim, J.W.; Choi, J.S. Potential of icariin metabolites from Epimedium koreanum Nakai as antidiabetic therapeutic agents. Molecules 2017, 22, 986. [Google Scholar] [CrossRef]
  47. Gao, J.; Ma, C.; Xia, D.; Chen, N.; Zhang, J.; Xu, F.; Li, F.; He, Y.; Gong, Q. Icariside II preconditioning evokes robust neuroprotection against ischaemic stroke, by targeting Nrf2 and the OXPHOS/NF-κB/ferroptosis pathway. Br. J. Pharmacol. 2023, 180, 308–329. [Google Scholar] [CrossRef]
  48. Zhou, Y.; Huang, X.; Yu, H.; Shi, H.; Chen, M.; Song, J.; Tang, W.; Teng, F.; Li, C.; Yi, L.; et al. TMT-based quantitative proteomics revealed protective efficacy of Icariside II against airway inflammation and remodeling via inhibiting LAMP2, CTSD and CTSS expression in OVA-induced chronic asthma mice. Phytomedicine 2023, 118, 154941. [Google Scholar] [CrossRef]
  49. Wang, Z.; Liu, C.; Yu, H.; Wu, B.; Huai, B.; Zhuang, Z.; Sun, C.; Xu, L.; Jin, F. Icaritin preparation from icariin by a special Epimedium flavonoid-glycosidase from Aspergillus sp. y848 strain. J. Microbiol. Biotechnol. 2022, 32, 437–446. [Google Scholar] [CrossRef]
  50. Zhang, S.; Luo, J.; Dong, Y.; Wang, Z.; Xiao, W.; Zhao, L. Biotransformation of the total flavonoid extract of epimedium into icaritin by two thermostable glycosidases from Dictyoglomus thermophilum DSM3960. Process Biochem. 2021, 105, 8–18. [Google Scholar] [CrossRef]
  51. Huong, N.T.; Son, N.T. Icaritin: A phytomolecule with enormous pharmacological values. Phytochemistry 2023, 213, 113772. [Google Scholar] [CrossRef]
  52. Wang, Z.; Zhang, X.; Wang, H.; Qi, L.; Lou, Y. Neuroprotective effects of icaritin against beta amyloid-induced neurotoxicity in primary cultured rat neuronal cells via estrogen-dependent pathway. Neuroscience 2007, 145, 911–922. [Google Scholar] [CrossRef] [PubMed]
  53. Zhu, D.Y.; Lou, Y.J. Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitro. Acta Pharmacol. Sin. 2005, 26, 477–485. [Google Scholar] [CrossRef]
  54. Gao, L.; Zhang, S.Q. Antiosteoporosis effects, pharmacokinetics, and drug delivery systems of icaritin: Advances and prospects. Pharmaceuticals 2022, 15, 397. [Google Scholar] [CrossRef] [PubMed]
  55. Wei, Q.; Wang, B.; Hu, H.; Xie, C.; Ling, L.; Gao, J.; Cao, Y. Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression. Int. J. Mol. Med. 2020, 45, 816–824. [Google Scholar] [CrossRef] [PubMed]
  56. Bi, Z.; Zhang, W.; Yan, X. Anti-inflammatory and immunoregulatory effects of icariin and icaritin. Biomed. Pharmacother. 2022, 151, 113180. [Google Scholar] [CrossRef]
  57. Liao, J.; Liu, Y.; Wu, H.; Zhao, M.; Tan, Y.; Li, D.; Long, H.; Dai, Y.; Yung, S.; Chan, T.M.; et al. The role of icaritin in regulating Foxp3/IL17a balance in systemic lupus erythematosus and its effects on the treatment of MRL/lpr mice. Clin. Immunol. 2016, 162, 74–83. [Google Scholar] [CrossRef]
  58. Hwang, E.; Lin, P.; Ngo, H.T.T.; Gao, W.; Wang, Y.S.; Yu, H.S.; Yi, T.H. Icariin and icaritin recover UVB-induced photoaging by stimulating Nrf2/ARE and reducing AP-1 and NF-κB signaling pathways: A comparative study on UVB-irradiated human keratinocytes. Photochem. Photobiol. Sci. 2018, 17, 1396–1408. [Google Scholar] [CrossRef]
  59. Yang, X.J.; Xi, Y.M.; Li, Z.J. Icaritin: A novel natural candidate for hematological malignancies therapy. Biomed. Res. Int. 2019, 2019, 4860268. [Google Scholar] [CrossRef]
  60. Zhang, C.; Sui, X.; Jiang, Y.; Wang, X.; Wang, S. Antitumor effects of icaritin and the molecular mechanisms. Discov. Med. 2020, 29, 5–16. [Google Scholar]
  61. Fan, Y.; Li, S.; Ding, X.; Yue, J.; Jiang, J.; Zhao, H.; Hao, R.; Qiu, W.; Liu, K.; Li, Y.; et al. First-in-class immune-modulating small molecule Icaritin in advanced hepatocellular carcinoma: Preliminary results of safety, durable survival and immune biomarkers. BMC Cancer 2019, 19, 279. [Google Scholar] [CrossRef]
  62. Li, H.; Liu, Y.; Jiang, W.; Xue, J.; Cheng, Y.; Wang, J.; Yang, R.; Zhang, X. Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma. BMC Cancer 2021, 21, 318. [Google Scholar] [CrossRef]
  63. Tao, H.; Liu, M.; Wang, Y.; Luo, S.; Xu, Y.; Ye, B.; Zheng, L.; Meng, K.; Li, L. Icaritin induces anti-tumor immune responses in hepatocellular carcinoma by inhibiting splenic myeloid-derived suppressor cell generation. Front. Immunol. 2021, 12, 609295. [Google Scholar] [CrossRef]
  64. Wang, X.; Zheng, N.; Dong, J.; Wang, X.; Liu, L.; Huang, J. Estrogen receptor-α36 is involved in icaritin induced growth inhibition of triple-negative breast cancer cells. J. Steroid Biochem. Mol. Biol. 2017, 171, 318–327. [Google Scholar] [CrossRef] [PubMed]
  65. Zheng, Q.; Liu, W.W.; Li, B.; Chen, H.J.; Zhu, W.S.; Yang, G.X.; Chen, M.J.; He, G.Y. Anticancer effect of icaritin on human lung cancer cells through inducing S phase cell cycle arrest and apoptosis. J. Huazhong Univ. Sci. Technolog. Med. Sci. 2014, 34, 497–503. [Google Scholar] [CrossRef] [PubMed]
  66. Gao, L.; Chen, M.; Ouyang, Y.; Li, R.; Zhang, X.; Gao, X.; Lin, S.; Wang, X. Icaritin induces ovarian cancer cell apoptosis through activation of p53 and inhibition of Akt/mTOR pathway. Life Sci. 2018, 202, 188–194. [Google Scholar] [CrossRef]
  67. Jin, Y.B.; Liang, X.C.; Cai, J.H.; Wang, K.; Wang, C.Y.; Wang, W.H.; Chen, X.L.; Bao, S. Mechanism of action of icaritin on uterine corpus endometrial carcinoma based on network pharmacology and experimental evaluation. Front. Oncol. 2023, 13, 1205604. [Google Scholar] [CrossRef] [PubMed]
  68. Yang, J.G.; Lu, R.; Ye, X.J.; Zhang, J.; Tan, Y.Q.; Zhou, G. Icaritin reduces oral squamous cell carcinoma progression via the inhibition of STAT3 signaling. Int. J. Mol. Sci. 2017, 18, 132. [Google Scholar] [CrossRef]
  69. Wu, T.; Wang, S.; Wu, J.; Lin, Z.; Sui, X.; Xu, X.; Shimizu, N.; Chen, B.; Wang, X. Icaritin induces lytic cytotoxicity in extranodal NK/T-cell lymphoma. J. Exp. Clin. Cancer Res. 2015, 34, 17. [Google Scholar] [CrossRef]
  70. Zhu, J.; Li, Z.; Zhang, G.; Meng, K.; Kuang, W.; Li, J.; Zhou, X.; Li, R.; Peng, H.; Dai, C.; et al. Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3/AKT signalings. PLoS ONE 2011, 6, e23720. [Google Scholar] [CrossRef]
  71. Zhu, S.; Wang, Z.; Li, Z.; Peng, H.; Luo, Y.; Deng, M.; Li, R.; Dai, C.; Xu, Y.; Liu, S.; et al. Icaritin suppresses multiple myeloma, by inhibiting IL-6/JAK2/STAT3. Oncotarget 2015, 6, 10460–10472. [Google Scholar] [CrossRef]
  72. Liu, X.; Yang, F.; Jia, D.; Dong, X.; Zhang, Y.; Wu, Z. Case report: A case study on the treatment using icaritin soft capsules in combination with lenvatinib achieving impressive PR and stage reduction in unresectable locally progressive pancreatic cancer and a literature review. Front. Genet. 2023, 14, 1167470. [Google Scholar] [CrossRef]
  73. Tang, X.; Zhang, Y.; Dong, X.; Jiang, G.; Hong, D.; Liu, X. The synergy of gene targeting drug icaritin soft capsule with immunomodulator and TACE brings new hope for drug combination in patients with advanced liver cancer: A case report and literature review. Cancer Manag. Res. 2023, 15, 707–717. [Google Scholar] [CrossRef] [PubMed]
  74. Guo, Y.; Wang, X.; Gao, J. Simultaneous preparation and comparison of the osteogenic effects of epimedins A–C and icariin from Epimedium brevicornu. Chem. Biodivers. 2018, 15, e1700578. [Google Scholar] [CrossRef] [PubMed]
  75. Zhang, H.F.; Yang, T.S.; Li, Z.Z.; Wang, Y. Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique. Ultrason. Sonochem. 2008, 15, 376–385. [Google Scholar] [CrossRef] [PubMed]
  76. Ziadlou, R.; Barbero, A.; Martin, I.; Wang, X.; Qin, L.; Alini, M.; Grad, S. Anti-inflammatory and chondroprotective effects of vanillic acid and epimedin C in human osteoarthritic chondrocytes. Biomolecules 2020, 10, 932. [Google Scholar] [CrossRef]
  77. Xu, Y.; Chen, S.; Huang, L.; Han, W.; Shao, Y.; Chen, M.; Zhang, Y.; He, R.; Xie, B. Epimedin C alleviates glucocorticoid-induced suppression of osteogenic differentiation by modulating PI3K/AKT/RUNX2 signaling pathway. Front. Pharmacol. 2022, 13, 894832. [Google Scholar] [CrossRef] [PubMed]
  78. Liu, Y.L.; Huang, M.; Feng, J.; Xia, P.; Wang, Y.; Wei, X.; Qiu, L. Effects of icariin and epimedium C on microstructure of bone tissue in glucocorticoid osteoporosis model mice based on Micro-CT technique. Drug Eval. Res. 2020, 43, 1733–1739. [Google Scholar]
  79. Wei, D.H.; Deng, J.L.; Shi, R.Z.; Ma, L.; Shen, J.M.; Hoffman, R.; Hu, Y.H.; Wang, H.; Gao, J.L. Epimedin C protects H2O2-induced peroxidation injury by enhancing the function of endothelial progenitor HUVEC populations. Biol. Pharm. Bull. 2019, 42, 1491–1499. [Google Scholar] [CrossRef]
  80. Huang, M.; Wei, Y.; Dong, J. Epimedin C modulates the balance between Th9 cells and Treg cells through negative regulation of noncanonical NF-κB pathway and MAPKs activation to inhibit airway inflammation in the ovalbumin-induced murine asthma model. Pulm. Pharmacol. Ther. 2020, 65, 102005. [Google Scholar] [CrossRef]
  81. Liu, Y.; Bi, Y.; Chai, L.; Song, L.; Huang, J.; Wang, Q.; Li, Y.; Zhou, K. Development of epimedin A complex drugs for treating the osteoporosis. J. Mater. Sci. Mater. Med. 2021, 32, 17. [Google Scholar] [CrossRef]
  82. Balaha, M.F.; Ahmed, N.J.; Almalki, Z.S.; Alahmari, A.K.; Alshehri, A.M.; Soliman, G.A.; Hamad, A.M. Epimedin A ameliorates DNFB-induced allergic contact dermatitis in mice: Role of NF-κ B/NLRP3-driven pyroptosis, Nrf2/HO-1 pathway, and inflammation modulation. Life Sci. 2022, 302, 120653. [Google Scholar] [CrossRef] [PubMed]
  83. Diao, X.; Wang, L.; Zhou, Y.; Bi, Y.; Zhou, K.; Song, L. The mechanism of Epimedin B in treating osteoporosis as revealed by RNA sequencing-based analysis. Basic Clin. Pharmacol. Toxicol. 2021, 129, 450–461. [Google Scholar] [CrossRef]
  84. Zhang, M.; Hu, Z.F.; Dong, X.L.; Chen, W.F. Epimedin B exerts neuroprotective effect against MPTP-induced mouse model of Parkinson’s disease: GPER as a potential target. Biomed. Pharmacother. 2022, 156, 113955. [Google Scholar] [CrossRef]
  85. Chen, G.; Huang, J.; Lei, H.; Wu, F.; Chen, C.; Song, Y.; Cao, Z.; Zhang, C.; Zhang, C.; Ma, Y.; et al. Icariside I—A novel inhibitor of the kynurenine-AhR pathway with potential for cancer therapy by blocking tumor immune escape. Biomed. Pharmacother. 2022, 153, 113387. [Google Scholar] [CrossRef]
  86. Chen, C.; Wu, M.; Lei, H.; Cao, Z.; Wu, F.; Song, Y.; Zhang, C.; Qin, M.; Zhang, C.; Du, R.; et al. A novel prenylflavonoid icariside I ameliorates estrogen deficiency-induced osteoporosis via simultaneous regulation of osteoblast and osteoclast differentiation. ACS Pharmacol. Transl. Sci. 2023, 6, 270–280. [Google Scholar] [CrossRef] [PubMed]
  87. Cheng, J.; Cai, H. Adverse reactions to Zhuangguguanjie Wan and cause analysis. Advers. Drug React. J. 2000, 2, 15–19. [Google Scholar]
  88. Du, Q.; Wang, Z.; Yun, N.R.; Huang, Y.H.; Xu, Q.; Wang, B.H. Literature analysis of 185 cases of ADR induced by Xianling Gubao capsule. China Pharm. 2017, 28, 3785–3787. [Google Scholar]
  89. Wang, Q.; Zhang, P.Y.; Yuan, X.M.; Bi, Y.N.; Zhou, K.; Zhang, Y. Long-term toxicity of different extracts of Epimedium brevicornu maxim in mice. Chin. J. Pharmacovigil. 2018, 15, 65–69. [Google Scholar]
  90. Zhang, L.; Zhang, J.X.; Fan, Q.Y.; Su, Z.Q.; Chen, C.; Peng, L.; Wang, T. Hepatoxicity of Epimedii folium in rat model based on uniform design and regression analysis. Chin. J. Exp. Tradit. Med. Formulae. 2018, 24, 189–197. [Google Scholar]
  91. Zhang, L.; Xu, A.L.; Yang, S.; Zhao, B.S.; Wang, T. In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of Herba Epimedii extracts. Toxicol. In Vitro 2020, 62, 104660. [Google Scholar] [CrossRef]
  92. Song, Z.; Li, Z.; Wen, X.; Liu, R.; Tian, X. UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model. Chin. Med. 2021, 16, 91. [Google Scholar] [CrossRef] [PubMed]
  93. Zhang, L.; Wang, T.; Zhao, B.S.; Zhang, J.X.; Yang, S.; Fan, C.L.; Li, P. Effect of 2″-O-rhamnosyl icariside II, baohuoside I and baohuoside II in Herba Epimedii on cytotoxicity indices in HL-7702 and HepG2 Cells. Molecules 2019, 24, 1263. [Google Scholar] [CrossRef] [PubMed]
  94. Li, P.; Zhang, L.; Guo, Z.; Kang, Q.; Chen, C.; Liu, X.; Ma, Q.; Zhang, J.; Hu, Y.; Wang, T. Epimedium koreanum Nakai-induced liver injury-A mechanistic study using untargeted metabolomics. Front. Pharmacol. 2022, 13, 934057. [Google Scholar] [CrossRef] [PubMed]
  95. Huang, W.; Zeng, S.; Xiao, G.; Wei, G.; Liao, S.; Chen, J.; Sun, W.; Lv, H.; Wang, Y. Elucidating the biosynthetic and regulatory mechanisms of flavonoid-derived bioactive components in Epimedium sagittatum. Front. Plant. Sci. 2015, 6, 689. [Google Scholar] [CrossRef] [PubMed]
  96. Shen, G.; Luo, Y.; Yao, Y.; Meng, G.; Zhang, Y.; Wang, Y.; Xu, C.; Liu, X.; Zhang, C.; Ding, G.; et al. The discovery of a key prenyltransferase gene assisted by a chromosome-level Epimedium pubescens genome. Front. Plant Sci. 2022, 13, 1034943. [Google Scholar] [CrossRef] [PubMed]
  97. Xu, C.; Liu, X.; Shen, G.; Fan, X.; Zhang, Y.; Sun, C.; Suo, F.; Guo, B. Time-series transcriptome provides insights into the gene regulation network involved in the icariin-flavonoid metabolism during the leaf development of Epimedium pubescens. Front. Plant Sci. 2023, 14, 1183481. [Google Scholar] [CrossRef]
  98. Mir, R.; Jallu, S.; Singh, T.P. The shikimate pathway: Review of amino acid sequence, function and three-dimensional structures of the enzymes. Crit. Rev. Microbiol. 2015, 41, 172–189. [Google Scholar] [CrossRef]
  99. Maeda, H.; Dudareva, N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 2012, 63, 73–105. [Google Scholar] [CrossRef]
  100. Pandey, R.P.; Parajuli, P.; Koffas, M.A.G.; Sohng, J.K. Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. Biotechnol. Adv. 2016, 34, 634–662. [Google Scholar] [CrossRef]
  101. Wang, Q.; Li, Z.; Ren, X.X. Optimization of the water-extraction method of total flavonoids from Epimedium sagittatum Maxin. Tianjin Agric. Sci. 2012, 18, 3. [Google Scholar]
  102. Huang, R.H.; Zhou, Y.C.; Han, W.; Deng, X. Study on water extraction process of Herba epimedii with microwave technology. Zhongguo Zhong Yao Za Zhi. 2005, 30, 107–110. [Google Scholar]
  103. Kazemi, M.; Khodaiyan, F.; Hosseini, S.S. Eggplant peel as a high potential source of high methylated pectin: Ultrasonic extraction optimization and characterization. LWT 2019, 105, 182–189. [Google Scholar] [CrossRef]
  104. Spinei, M.; Oroian, M. Microwave-assisted extraction of pectin from grape pomace. Sci. Rep. 2022, 12, 12722. [Google Scholar] [CrossRef] [PubMed]
  105. Yang, X.H.; Li, L.; Xue, Y.B.; Zhou, X.X.; Tang, J.H. Flavonoids from Epimedium pubescens: Extraction and mechanism, antioxidant capacity and effects on CAT and GSH-Px of Drosophila melanogaster. PeerJ 2020, 8, e8361. [Google Scholar] [CrossRef] [PubMed]
  106. Karbuz, P.; Tugrul, N. Microwave and ultrasound assisted extraction of pectin from various fruits peel. J. Food Sci. Technol. 2021, 58, 641–650. [Google Scholar] [CrossRef] [PubMed]
  107. Zhang, R.; Su, D.; Hou, F.; Liu, L.; Huang, F.; Dong, L.; Deng, Y.; Zhang, Y.; Wei, Z.; Zhang, M. Optimized ultra-high-pressure-assisted extraction of procyanidins from lychee pericarp improves the antioxidant activity of extracts. Biosci. Biotechnol. Biochem. 2017, 81, 1576–1585. [Google Scholar] [CrossRef]
  108. Xu, Y.; Li, Z.; Wang, Y. Fourteen microsatellite loci for the Chinese medicinal plant Epimedium sagittatum and cross-species application in other medicinal species. Mol. Ecol. Resour. 2008, 8, 640–642. [Google Scholar] [CrossRef]
  109. Mihaljević, S.; Vršek, I. In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU. Sci. Hortic. 2009, 120, 406–410. [Google Scholar] [CrossRef]
  110. Wang, P.; Li, C.; Li, X.; Huang, W.; Wang, Y.; Wang, J.; Zhang, Y.; Yang, X.; Yan, X.; Wang, Y.; et al. Complete biosynthesis of the potential medicine icaritin by engineered Saccharomyces cerevisiae and Escherichia coli. Sci. Bull. 2021, 66, 1906–1916. [Google Scholar] [CrossRef]
  111. Yin, T.; Li, Y.F.; Xu, N.; Meng, F.Y. Current situation and thinking of artificial cultivation of Epimedium. Lishizhen Med. Mater. Medica Res. 2020, 31, 1468–1471. [Google Scholar]
  112. Zhang, S.; Lu, C.; Cao, S.; Li, Q.; Wu, G.; Zhao, L. Efficient production of icariin and baohuoside I from Epimedium Folium flavonoids by fungal α-l-rhamnosidase hydrolysing regioselectively the terminal rhamnose of epimedin C. Biotechnol. Biofuels Bioprod. 2023, 16, 107. [Google Scholar] [CrossRef]
  113. Shen, Y.; Wang, M.; Zhou, J.; Chen, Y.; Xu, L.; Wu, M.; Xia, G.; Tam, J.P.; Yu, J.; Teng, X.; et al. Eco-efficient biphasic enzymatic hydrolysis for the green production of rare baohuoside I. Enzyme Microb. Technol. 2019, 131, 109431. [Google Scholar] [CrossRef]
  114. Xie, J.; Zhao, J.; Zhang, N.; Xu, H.; Yang, J.; Ye, J.; Jiang, J. Efficient production of isoquercitin, icariin and icariside II by a novel thermostable α-l-rhamnosidase PodoRha from Paenibacillus odorifer with high α-1,6-/α-1,2-glycoside specificity. Enzyme Microb. Technol. 2022, 158, 110039. [Google Scholar] [CrossRef]
  115. Li, Q.; Wu, T.; Zhao, L.; Pei, J.; Wang, Z.; Xiao, W. Highly efficient biotransformation of astragaloside IV to cycloastragenol by sugar-stimulated β-glucosidase and β-xylosidase from Dictyoglomus thermophilum. J. Microbiol. Biotechnol. 2019, 29, 1882–1893. [Google Scholar] [CrossRef]
  116. Jin, X.; Zhang, Z.; Sun, E.; Li, S.; Jia, X. Statistically designed enzymatic hydrolysis of an icariin/β-cyclodextrin inclusion complex optimized for production of icaritin. Acta Pharm. Sin. B 2012, 2, 83–89. [Google Scholar] [CrossRef]
  117. Liu, C.; Li, R.; Peng, J.; Qu, D.; Huang, M.; Chen, Y. Enhanced hydrolysis and antitumor efficacy of Epimedium flavonoids mediated by immobilized snailase on silica. Process Biochem. 2019, 86, 80–88. [Google Scholar] [CrossRef]
  118. Lou, H.; Liu, X.; Liu, S.; Chen, Q. Purification and characterization of a novel α-l-rhamnosidase from Papiliotrema laurentii ZJU-L07 and its application in production of icariin from epimedin C. J. Fungi 2022, 8, 644. [Google Scholar] [CrossRef]
  119. Lyu, Y.; Zeng, W.; Du, G.; Chen, J.; Zhou, J. Efficient bioconversion of epimedin C to icariin by a glycosidase from Aspergillus nidulans. Bioresour. Technol. 2019, 289, 121612. [Google Scholar] [CrossRef]
  120. Wu, T.; Pei, J.; Ge, L.; Wang, Z.; Ding, G.; Xiao, W.; Zhao, L. Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C. Bioorg. Chem. 2018, 81, 461–467. [Google Scholar] [CrossRef]
  121. Cheng, L.; Zhang, H.; Cui, H.; Cheng, J.; Wang, W.; Wei, B.; Liu, F.; Liang, H.; Shen, X.; Yuan, Q. A novel α-l-Rhamnosidase renders efficient and clean production of icaritin. J. Clean. Prod. 2022, 341, 130903. [Google Scholar] [CrossRef]
  122. Li, Q.; Ge, L.; Zheng, D.; Zhang, X.; Zhao, L. Screening and characterization of a GH78 α-l-rhamnosidase from Aspergillus terreus and its application in the bioconversion of icariin to icaritin with recombinant β-glucosidase. Enzyme Microb. Technol. 2022, 153, 109940. [Google Scholar] [CrossRef] [PubMed]
  123. Xie, J.; Zhao, D.; Zhao, L.; Pei, J.; Xiao, W.; Ding, G.; Wang, Z. Overexpression and characterization of a Ca(2+) activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(S)-Rg3 bioconversion productivity. J. Ind. Microbiol. Biotechnol. 2015, 42, 839–850. [Google Scholar] [CrossRef] [PubMed]
  124. Papageorgiou, A.C.; Chen, J.; Li, D. Crystal structure and biological implications of a glycoside hydrolase family 55 β-1,3-glucanase from Chaetomium thermophilum. Biochim. Biophys. Acta Proteins Proteom. 2017, 1865, 1030–1038. [Google Scholar] [CrossRef] [PubMed]
  125. Cheng, T.; Yang, J.; Zhang, T.; Yang, Y.S.; Ding, Y. Optimized biotransformation of icariin into icariside II by β-glucosidase from Trichoderma viride using central composite design method. Biomed. Res. Int. 2016, 2016, 5936947. [Google Scholar] [CrossRef] [PubMed]
  126. Yang, Q.; Wang, L.; Zhang, L.; Xiao, H. Baohuoside I production through enzyme hydrolysis and parameter optimization by using response surface and subset selection. J. Mol. Catal. B. Enzym. 2013, 90, 132–138. [Google Scholar] [CrossRef]
  127. Cui, L.; Zhang, Z.; Sun, E.; Jia, X.; Qian, Q. Effect of β-cyclodextrin complexation on solubility and enzymatic hydrolysis rate of icariin. J. Nat. Sci. Biol. Med. 2013, 4, 201–206. [Google Scholar] [PubMed]
  128. Su, J.; Wu, T.; Cao, S.; Pei, J.; Zhao, L. Screening and characterization of a β-xylosidase from Bifidobacterium breve K-110 and its application in the biotransformation of the total flavonoids of epimedium to icariin with α-l-rhamnosidase. Bioorg. Chem. 2023, 132, 106364. [Google Scholar] [CrossRef]
  129. Tong, X.; Qi, Z.; Zheng, D.; Pei, J.; Li, Q.; Zhao, L. High-level expression of a novel multifunctional GH3 family β-xylosidase/α-arabinosidase/β-glucosidase from Dictyoglomus turgidum in Escherichia coli. Bioorg. Chem. 2021, 111, 104906. [Google Scholar] [CrossRef]
  130. Lu, Y.; Luo, Q.; Jia, X.; Tam, J.P.; Yang, H.; Shen, Y.; Li, X. Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedii Folium: Integration of herbal medicine, enzyme engineering, and nanotechnology. J. Pharm. Anal. 2023, 13, 239–254. [Google Scholar] [CrossRef]
  131. Mohamad, N.R.; Marzuki, N.H.; Buang, N.A.; Huyop, F.; Wahab, R.A. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol. Biotechnol. Equip. 2015, 29, 205–220. [Google Scholar] [CrossRef]
  132. De Carvalho, C.C. Enzymatic and whole cell catalysis: Finding new strategies for old processes. Biotechnol. Adv. 2011, 29, 75–83. [Google Scholar] [CrossRef] [PubMed]
  133. Wachtmeister, J.; Rother, D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Curr. Opin. Biotechnol. 2016, 42, 169–177. [Google Scholar] [CrossRef] [PubMed]
  134. Gao, Q.; Wang, L.; Zhang, M.; Wei, Y.; Lin, W. Recent advances on feasible strategies for monoterpenoid production in Saccharomyces cerevisiae. Front. Bioeng. Biotechnol. 2020, 8, 609800. [Google Scholar] [CrossRef] [PubMed]
  135. Guan, R.; Wang, M.; Guan, Z.; Jin, C.Y.; Lin, W.; Ji, X.J.; Wei, Y. Metabolic engineering for glycyrrhetinic acid production in Saccharomyces cerevisiae. Front. Bioeng. Biotechnol. 2020, 8, 588255. [Google Scholar] [CrossRef]
  136. Ro, D.K.; Paradise, E.M.; Ouellet, M.; Fisher, K.J.; Newman, K.L.; Ndungu, J.M.; Ho, K.A.; Eachus, R.A.; Ham, T.S.; Kirby, J.; et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006, 440, 940–943. [Google Scholar] [CrossRef]
  137. Galanie, S.; Thodey, K.; Trenchard, I.J.; Filsinger Interrante, M.; Smolke, C.D. Complete biosynthesis of opioids in yeast. Science 2015, 349, 1095–1100. [Google Scholar] [CrossRef]
  138. Chen, X.J.; Tang, Z.H.; Li, X.W.; Xie, C.X.; Lu, J.J.; Wang, Y.T. Chemical constituents, quality control, and bioactivity of Epimedii folium (Yinyanghuo). Am. J. Chin. Med. 2015, 43, 783–834. [Google Scholar] [CrossRef]
  139. Szabó, R.; Rácz, C.P.; Dulf, F.V. Bioavailability improvement strategies for icariin and its derivates: A review. Int. J. Mol. Sci. 2022, 23, 7519. [Google Scholar] [CrossRef]
  140. Jiang, W.; Ding, K.; Yue, R.; Lei, M. Therapeutic effects of icariin and icariside II on diabetes mellitus and its complications. Crit. Rev. Food Sci. Nutr. 2023, 1–26. [Google Scholar] [CrossRef]
  141. Tan, H.L.; Chan, K.G.; Pusparajah, P.; Saokaew, S.; Duangjai, A.; Lee, L.-H.; Goh, B.-H. Anti-cancer properties of the naturally occurring aphrodisiacs: Icariin and its derivatives. Front. Pharmacol. 2016, 7, 191. [Google Scholar] [CrossRef]
  142. Yang, X.; Lang, S.; Li, S.; Jiang, C.; Han, J. Preparation of icariside I and icariside II, an exploration of their protective mechanism against cyclophosphamide-induced bone marrow suppression in mice, and their regulatory effects on immune function. Pharmazie 2022, 77, 32–37. [Google Scholar] [PubMed]
  143. Zhang, D.; Zhao, N.; Wan, C.; Du, J.; Lin, J.; Wang, H. Icariin and icariside II reciprocally stimulate osteogenesis and inhibit adipogenesis of multipotential stromal cells through ERK signaling. Evid. Based Complement. Altern. Med. 2021, 2021, 8069930. [Google Scholar] [CrossRef] [PubMed]
  144. Son, N.; Shi, L.; Li, Y.; Wang, Q.A. Total synthesis of icaritin via microwave-assistance Claisen rearrangement. Lett. Org. Chem. 2014, 11, 677–681. [Google Scholar]
  145. Li, L.; Wang, L.; Fan, W.; Jiang, Y.; Zhang, C.; Li, J.; Peng, W.; Wu, C. The application of fermentation technology in traditional Chinese medicine: A review. Am. J. Chin. Med. 2020, 48, 899–921. [Google Scholar] [CrossRef] [PubMed]
  146. Zhang, X.; Miao, Q.; Pan, C.; Yin, J.; Wang, L.; Qu, L.; Yin, Y.; Wei, Y. Research advances in probiotic fermentation of Chinese herbal medicines. iMeta 2023, 2, e93. [Google Scholar] [CrossRef]
  147. Xie, Y.; Ya, H.; Zhang, X.; Fan, Y.; Zhao, B.; Wang, R.; Yang, Z. Biotransformation of flavonoid glycosides in Epimedium by Lactobacillus plantarum. China Brew. 2022, 41, 103–109. [Google Scholar]
Molecules 28 07173 g001
Figure 1. Chemical structures and pharmaceutical properties of the major flavonoids from Epimedii Folium.
Figure 1. Chemical structures and pharmaceutical properties of the major flavonoids from Epimedii Folium.
Molecules 28 07173 g001
Molecules 28 07173 g002
Figure 2. The biosynthetic pathway of prenylated flavonoids recovered from EF. Phase 1: the phenylpropanoid pathway; phase 2: the core pathway; phase 3: further enzymatic modification pathways. PAL: phenylalanine ammonia lyase, C4H: cinnamate-4-hydroxylase, TAL: tyrosine ammonia lyase, 4CL: 4-coumarate CoA ligase, ACC: acetyl-CoA carboxylase, CHS: chalcone synthase, CHI: chalcone isomerase, F3H: flavanone 3-hydroxylase, FLS: flavonol synthase, PT: prenyltransferase, UGT: UDP-glycosyltransferase, OMT: O-methyltransferase, Glu: Glucose, Rha: Rhamnose, Xyl: Xylose.
Figure 2. The biosynthetic pathway of prenylated flavonoids recovered from EF. Phase 1: the phenylpropanoid pathway; phase 2: the core pathway; phase 3: further enzymatic modification pathways. PAL: phenylalanine ammonia lyase, C4H: cinnamate-4-hydroxylase, TAL: tyrosine ammonia lyase, 4CL: 4-coumarate CoA ligase, ACC: acetyl-CoA carboxylase, CHS: chalcone synthase, CHI: chalcone isomerase, F3H: flavanone 3-hydroxylase, FLS: flavonol synthase, PT: prenyltransferase, UGT: UDP-glycosyltransferase, OMT: O-methyltransferase, Glu: Glucose, Rha: Rhamnose, Xyl: Xylose.
Molecules 28 07173 g002
Molecules 28 07173 g003
Figure 3. The advantages and disadvantages of different extraction methods of Epimedium flavonoids.
Figure 3. The advantages and disadvantages of different extraction methods of Epimedium flavonoids.
Molecules 28 07173 g003
Molecules 28 07173 g004
Figure 4. Schematic diagram of the transformation of high glycosylation flavonoids (epimedin A, epimedin B, and epimedin C) into low glycosylation flavonoids (icariin, baohuoside I, icariside I, and icaritin) by different enzymes. This conversion involves the enzymatic hydrolysis of the terminal sugar groups attached to the flavonoid molecules, resulting in the formation of the desired low glycosylation flavonoids.
Figure 4. Schematic diagram of the transformation of high glycosylation flavonoids (epimedin A, epimedin B, and epimedin C) into low glycosylation flavonoids (icariin, baohuoside I, icariside I, and icaritin) by different enzymes. This conversion involves the enzymatic hydrolysis of the terminal sugar groups attached to the flavonoid molecules, resulting in the formation of the desired low glycosylation flavonoids.
Molecules 28 07173 g004
Molecules 28 07173 g005
Figure 5. The work flow for biotransformation of Epimedium flavonoids by whole-cell catalysis, and their industrial applications.
Figure 5. The work flow for biotransformation of Epimedium flavonoids by whole-cell catalysis, and their industrial applications.
Molecules 28 07173 g005
Molecules 28 07173 g006
Figure 6. Complete biosynthesis of Epimedium flavonoids in a microbial cell factory.
Figure 6. Complete biosynthesis of Epimedium flavonoids in a microbial cell factory.
Molecules 28 07173 g006
Table 1. The published mechanisms of the hepatotoxicity effects of Epimedium flavonoids.
Table 1. The published mechanisms of the hepatotoxicity effects of Epimedium flavonoids.
Epimedium FlavonoidsResearch SystemsMechanismsReference
Alcohol extracts of E. koreanum Nakai and E. wushanense T.S. YingSD ratsCompared with the normal group, animal groups treated with EF extracts showed severer hepatotoxicity, which was positively correlated with the dose and course. Additionally, the females experienced more significant damage compared to the males.[90]
Icariside I and sagittatoside AHL-7702 and HepG2 cellsIcariside I could destroy the cell structure and cause oxidative stress. Sagittatoside A could cause oxidative stress and damage to mitochondria.[91]
Epimedin CMale Balb/c miceEpigenetic modification changed in mouse liver after epimedin C treatment with a test dose, and the m6A and m5C may be associated with epimedin C-induced liver injury.[92]
Baohuoside I HL-7702 and HepG2 cellsThe toxicity mechanism(s) of baohuoside I may be involved in increasing oxidative stress and inducing apoptosis.[93]
E. koreanum Nakai ethanol extractMale Sprague Dawley ratsThe mechanism of hepatotoxicity of E. koreanum Nakai was probably related to the induction of ferroptosis in hepatocytes.[94]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhang, X.; Tang, B.; Wen, S.; Wang, Y.; Pan, C.; Qu, L.; Yin, Y.; Wei, Y. Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium. Molecules 2023, 28, 7173. https://doi.org/10.3390/molecules28207173

AMA Style

Zhang X, Tang B, Wen S, Wang Y, Pan C, Qu L, Yin Y, Wei Y. Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium. Molecules. 2023; 28(20):7173. https://doi.org/10.3390/molecules28207173

Chicago/Turabian Style

Zhang, Xiaoling, Bingling Tang, Sijie Wen, Yitong Wang, Chengxue Pan, Lingbo Qu, Yulong Yin, and Yongjun Wei. 2023. "Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium" Molecules 28, no. 20: 7173. https://doi.org/10.3390/molecules28207173

Article Metrics

Back to TopTop