Synonym name: Calendoflavoside
Catalogue No.: BP0795
Cas No.: 55033-90-4
Formula: C₂₈H₃₂O₁₆
Mol Weight: 624.548
Botanical Source: Nerisyrenia sp., Parietaria officinalis, Typha latifolia and Zea mays (sweet corn)

Purity: 95%~99%
Analysis Method: HPLC-DAD or/and HPLC-ELSD
Identification Method: Mass, NMR
Packing: Brown vial or HDPE plastic bottle
Can be supplied from milligrams to grams.

For Reference Standard and R&D, Not for Human Use Directly.
Inquire for bulk scale.

Storage conditions：Short-term storage at 2~8℃, long-term storage at -20 ~ -80℃

Introduction/Overview

Natural products, as an important source of drug discovery, play an irreplaceable role in the long struggle between humans and diseases. Flavonoids, as the largest class of polyphenolic compounds in plant secondary metabolites, have attracted much attention due to their widespread biological activity. They not only give plants color, resist UV radiation and pathogen invasion, but also demonstrate multiple effects such as antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protection in human health maintenance. Among numerous flavonoids, Isorhamnetin-3-O-neohesperoside (I3ON), as a flavonol glycoside with unique structural characteristics, has gradually become a research hotspot in the field of natural product pharmacology in recent years.

The discovery of I3ON and seabuckthorn（Hippophae rhamnoides L. The medicinal value is closely related. Sea buckthorn, an ancient medicinal and edible plant, is known as the "longevity fruit" in the traditional medical system. Its fruit, leaves, and seeds are rich in various bioactive components, including vitamins, fatty acids, organic acids, and a wide variety of flavonoids. I3ON is one of the key active ingredients isolated and identified from seabuckthorn. Early research mainly focused on its antioxidant activity as a component of total flavonoids in seabuckthorn. With the advancement of separation and purification techniques and pharmacological evaluation methods, the unique pharmacological spectrum of I3ON, especially its potential in anti skin cancer, has gradually been revealed.

Skin cancer, including basal cell carcinoma, squamous cell carcinoma and malignant melanoma, is one of the malignant tumors with the highest incidence rate in the world. Its occurrence is closely related to ultraviolet radiation, genetic factors and immunosuppression. Although surgical resection is the main treatment method, for late stage, metastatic, or recurrent cases, targeted therapy and immunotherapy have made breakthroughs, but still face challenges such as drug resistance, toxic side effects, and high costs. Therefore, it is of great scientific significance and clinical value to search for efficient and low toxicity new anti skin cancer drugs or lead compounds from natural products. I3ON inhibits the proliferation of skin cancer cells by directly suppressing mitogen activated protein kinase 1 (MEK1) and phosphatidylinositol 3-kinase (PI3K). This discovery not only elucidates the molecular basis of its anti-tumor effect, but also provides a solid theoretical basis for its development as a potential therapeutic drug or functional food ingredient. In addition, I3ON also exhibits significant activity in antioxidant damage by regulating the nuclear factor E2 related factor 2 (NFE2L2/NRF2) signaling pathway, upregulating the expression of a series of antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase 1 (GPX1), heme oxygenase 1 (HMOX1), and superoxide dismutase 2 (SOD2), thereby protecting cells from oxidative stress damage. The dual effects of antioxidant and anti-tumor activity make I3ON a highly promising multifunctional natural product for development.

This article aims to systematically review the research progress of isorhamnetin-3-O-neohesperidoside, covering its chemical structure and physicochemical properties, plant sources and extraction methods, pharmacological activity research, mechanism of action and molecular targets, pharmacological evaluation and pharmacokinetic characteristics. It also looks forward to its clinical application prospects, in order to provide comprehensive references for the in-depth research and development of this compound.

Chemical structure and physicochemical properties

The chemical structure of isorhamnetin-3-O-neohesperidoside determines its unique physicochemical properties and biological activity. From the naming, it can be seen that the compound is composed of the glycoside Isorhamnetin and the glycosyl part Neohesperidose connected by glycosidic bonds.

Glycogen structure Isorhamnetin (3,5,7-trihydroxy-4 '- methoxyflavonol) is a 3' - methoxy derivative of quercetin and belongs to the class of flavonol compounds. Its core structure is 2-phenylchromenone, with hydroxyl and methoxy groups attached to the A and B rings, respectively. Specifically, the C-5 and C-7 positions of the A ring each have a hydroxyl group (- OH), the C-3 position of the C ring has a hydroxyl group, and the C-3 'position of the B ring has a methoxy group (- OCH ∝) and a hydroxyl group at the C-4' position. This polyphenolic hydroxyl structure is a key pharmacophore for I3ON to exert antioxidant activity, effectively scavenging free radicals and chelating metal ions. The presence of methoxy groups may affect their lipid solubility and metabolic stability.

Glycosyl structure New orange peel sugar is a disaccharide composed of rhamnose and glucose linked by alpha-1,2-glycoside bonds. In I3ON, the disaccharide is connected to the C-3 hydroxyl group of the glycoside isorhamnetin through a β - glycosidic bond. The introduction of the sugar moiety greatly alters the physicochemical properties of the aglycone, particularly increasing its polarity and water solubility, while also affecting its interaction, absorption, distribution, metabolism, and excretion (ADME) processes with biological targets.

Physical and chemical property parameters According to computational chemistry and experimental data, the key physicochemical parameters of I3ON are as follows:
- molecular weight 624.5480 Da. This molecular weight is moderate to large and may pose a certain challenge for the absorption of oral drugs, as the classic "Lipinski Five Rules" typically require a molecular weight of less than 500 Da.
- Lipid water partition coefficient (LogP)-0.2313. A negative LogP value indicates that I3ON has strong hydrophilicity and good water solubility. This is consistent with the presence of multiple hydroxyl and sugar groups in the molecule. High water solubility is beneficial for its dissolution in the gastrointestinal tract, but may limit its passive diffusion through cell membranes.
- Topological Polarity Surface Area (TPSA): 258.4300 Å ². TPSA is an important indicator for measuring molecular polarity and hydrogen bonding ability, closely related to oral absorption and blood-brain barrier penetration. It is generally believed that molecules with TPSA greater than 140 Å ² have poor oral absorption and are difficult to penetrate the blood-brain barrier. The TPSA value of I3ON is as high as 258.43 Å ², indicating that its oral bioavailability may be low and difficult to enter the central nervous system.
- Water solubility 3.4409 (possibly logS value, indicating molar solubility). This value further confirms its good water solubility.
- Blood-brain barrier penetrability: Low. This is consistent with high TPSA and low LogP values, indicating that I3ON mainly acts on peripheral tissues and has a relatively small impact on the central nervous system, which to some extent reduces the risk of neurotoxicity.
- HERG inhibition: No. The hERG (human ether - à - go related gene) potassium ion channel is an important target for evaluating drug cardiac toxicity. I3ON has no inhibitory effect on hERG channels, indicating a low risk of causing cardiac QT interval prolongation and arrhythmia, which is a good safety signal.
- Ames test: 0.6. The Ames test is used to detect the mutagenicity of compounds. A result of 0.6 usually indicates a negative or weakly positive result under testing conditions, suggesting a low risk of genetic toxicity for I3ON.

In summary, the chemical structure of I3ON determines its high polarity, high water solubility, and low fat solubility. These properties make it mainly distributed in the blood and extracellular fluid in the body, making it difficult to penetrate cell membranes and the blood-brain barrier. Its good water solubility, low cardiac toxicity, and low mutagenicity provide a favorable safety basis for it as a candidate drug, but low oral bioavailability may be a key obstacle to overcome in its development process.

Plant sources and extraction methods

Discovery of isorhamnetin-3-O-neohesperidin and seabuckthorn（Hippophae rhamnoides L. The research is inseparable. Sea buckthorn is a deciduous shrub or small tree of the genus Sea buckthorn in the family Elapidae, widely distributed in temperate and cold regions of Eurasia, and mainly distributed in northwest, north, and southwest China. The fruit, leaves, seeds, and bark of seabuckthorn are rich in various bioactive compounds, among which flavonoids are one of its most important active ingredient groups. In addition to seabuckthorn, I3ON is also present in some other plants, such as certain types of bayberries（Myrica Spp. and sage（Salvia spp.）， But seabuckthorn is currently recognized as the most important and abundant source.

The content of I3ON in seabuckthorn varies significantly depending on the variety, origin, harvesting season, location, and extraction method. Usually, seabuckthorn fruit (especially skin and flesh) and leaves are the main raw materials for extracting I3ON. Research has shown that the total flavonoid content in seabuckthorn fruit is relatively high, and I3ON is one of its representative components. In order to efficiently and high-purity obtain I3ON, researchers have developed various extraction and purification methods.

extraction method：
1. Solvent extraction method This is the most classic and commonly used method. Based on the high polarity of I3ON, polar solvents are usually used for extraction. Common solvents include methanol, ethanol, acetone, or their aqueous solutions. For example, using a 70% -80% ethanol aqueous solution for extraction under heating reflux or ultrasound assisted conditions can achieve higher extraction rates. Extraction temperature, time, solid-liquid ratio, and solvent concentration are key parameters that affect extraction efficiency.
2. Ultrasound assisted extraction (UAE)By utilizing the cavitation effect and mechanical vibration of ultrasound, it is possible to accelerate the breakdown of plant cell walls and promote the dissolution of target components. UAE has the advantages of short extraction time, relatively low temperature, and low solvent usage, making it particularly suitable for the extraction of thermosensitive components.
3. Microwave assisted extraction (MAE)By utilizing the penetrability and selective heating of microwaves, the internal temperature and pressure of plant cells rapidly increase, leading to cell rupture and release of contents. MAE also has the characteristics of high efficiency and speed.
4. Enzyme Assisted Extraction (EAE)By adding cellulases, pectinases, etc., the structure of plant cell walls is disrupted, mass transfer resistance is reduced, and the extraction rate of flavonoids is improved. This method has mild conditions and is beneficial for maintaining the stability of the active ingredients.

Purification Method：
Due to the high content of impurities such as sugars, proteins, pigments, lipids, etc. in the crude extract, further purification is required to obtain high-purity I3ON.
1. Liquid-liquid extraction Preliminary separation of I3ON and impurity solubility differences using different solvents. For example, first degreasing with petroleum ether or n-hexane, and then extracting flavonoids with ethyl acetate or n-butanol.
2. Column chromatography method This is the core technology for purifying I3ON. Common fixed phases include:
- Macroporous adsorption resin Such as HPD-100, D101, AB-8, etc. By selecting appropriate resins and elution conditions (such as ethanol aqueous solutions of different concentrations), total flavonoids can be effectively enriched and separated, and I3ON can be preliminarily purified.
- Polyamide resin Polyamide has a special adsorption effect on flavonoids, which is based on the formation of hydrogen bonds between amide groups and phenolic hydroxyl groups of flavonoids. By gradient elution, high-purity I3ON can be obtained.
- Silica gel column chromatography Commonly used for further separation and purification. Use solvent systems such as chloroform methanol water for elution.
- Sephadex LH-20 column chromatography Separation based on molecular size and polarity is commonly used in the final refining step to remove pigments and trace impurities.
3. Preparation type high performance liquid chromatography (Prep HPLC)For research or production requiring high purity (>98%) I3ON, Prep HPLC is the most effective method. By optimizing chromatographic conditions such as C18 reverse phase column, acetonitrile water or methanol water mobile phase, baseline separation of I3ON from other structurally similar flavonoid glycosides can be achieved, and high-purity target compounds can be obtained.

In summary, the extraction and purification of I3ON from seabuckthorn usually adopts a combination process of "solvent extraction macroporous resin enrichment polyamide/silica gel column chromatography separation preparation HPLC purification". With the promotion of green chemistry concepts, efficient and environmentally friendly extraction technologies such as ultrasound, microwave, and enzyme assisted are gradually being applied. However, achieving large-scale, low-cost, and high-purity preparation of I3ON remains a key link in its subsequent research and industrialization.

Pharmacological activity research

The pharmacological activity research of isorhamnetin-3-O-neohesperidin mainly focuses on its antioxidant and anti-tumor aspects, and gradually expands to fields such as anti-inflammatory and cardiovascular protection.

1. Antioxidant damage activity

Oxidative stress is a common pathological basis for various diseases, including cancer, cardiovascular disease, neurodegenerative diseases, and aging. I3ON, as a polyphenolic compound, exhibits strong antioxidant capacity. Its antioxidant mechanism is multifaceted:
- Directly eliminate free radicals Multiple phenolic hydroxyl groups in I3ON molecules can serve as hydrogen atom donors, directly neutralizing reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydroxyl radicals (· OH), superoxide anions (O ₂⁻·), peroxynitrite (ONOO ⁻), etc., thereby blocking free radical chain reactions.
- Chelate transition metal ions The ortho dihydroxy structure of I3ON (3 ′ - OCH ∝ and 4 ′ - OH of the B ring) can chelate iron ions (Fe ² ⁺/Fe ³ ⁺) and copper ions (Cu ² ⁺), inhibit Fenton reaction and Haber Weiss reaction, thereby reducing the generation of highly active hydroxyl radicals.
- Activate endogenous antioxidant defense system This is the key to I3ON exerting long-term and systematic antioxidant effects. Research has shown that I3ON can activate the nuclear factor E2 related factor 2 (NFE2L2/NRF2) signaling pathway. NRF2 is the main transcriptional regulator that cells use to respond to oxidative stress. Under normal physiological conditions, NRF2 binds to Kelch like ECH related protein 1 (KEAP1) and is degraded by ubiquitination. When subjected to oxidative stimulation or inducers such as I3ON, NRF2 dissociates from KEAP1, translocates into the nucleus, binds to antioxidant response elements (ARE), and initiates the transcription of downstream antioxidant and detoxifying enzyme genes, including:
- Superoxide dismutase 1 and 2 (SOD1, SOD2)Catalytic dismutation of superoxide anions into hydrogen peroxide and oxygen.
- Catalase (CAT)Decompose hydrogen peroxide into water and oxygen.
- Glutathione peroxidase 1 (GPX1)Reduce hydrogen peroxide and organic peroxides to water or alcohol using reduced glutathione (GSH).
- Heme oxygenase 1 (HMOX1)Catalytic degradation of hemoglobin into biliverdin, carbon monoxide, and iron ions, biliverdin and its product bilirubin are potent antioxidants.
By upregulating the expression of these key enzymes, I3ON can significantly enhance the overall antioxidant capacity of cells and protect them from subsequent oxidative damage. The mechanism of arming cells by activating the NRF2 signaling pathway is considered to be the core of I3ON's antioxidant activity.

2. Anti skin cancer activity

The most notable pharmacological activity of I3ON is its inhibitory effect on skin cancer. Research has confirmed that I3ON can effectively inhibit the proliferation of various skin cancer cells, induce their apoptosis, and suppress their migration and invasion abilities. Its anti skin cancer effect is mainly achieved through the following two key signaling pathways:
- Inhibition of MEK1/ERK signaling pathway The RAS-RAF-MEK1/2-ERK1/2 (MAPK) signaling pathway is one of the core pathways regulating cell proliferation, differentiation, and survival, and is often abnormally activated in various cancers. I3ON can directly bind to and inhibit the activity of MEK1, thereby blocking the phosphorylation of downstream ERK1/2 and inhibiting the activity of downstream transcription factors such as c-Fos and c-Jun, ultimately inhibiting the proliferation of skin cancer cells and inducing G0/G1 phase cell cycle arrest.
- Inhibiting the PI3K/AKT signaling pathway The PI3K AKT mTOR signaling pathway is another critical pathway for cell survival and growth, and its abnormal activation is closely related to the occurrence, development, drug resistance, and metastasis of tumors. I3ON can also directly bind and inhibit the activity of PI3K, leading to a decrease in the phosphorylation level of downstream AKT, thereby inhibiting the activity of downstream effector molecules such as mTOR, GSK-3 β, and Bad. This can not only inhibit cell proliferation, but also induce cell apoptosis by activating pro apoptotic proteins (such as Bax) and inhibiting anti apoptotic proteins (such as Bcl-2).

Importantly, the dual inhibitory effect of I3ON on MEK1 and PI3K means that it can simultaneously block two important oncogenic signaling pathways, which may have stronger anti-tumor effects than single target inhibitors and may delay or overcome drug resistance caused by compensatory activation of a single pathway. In addition, as I3ON directly acts on these two kinases, its mechanism of action is clear, providing a new lead compound for the development of targeted anti skin cancer drugs.

3. Other pharmacological activities

In addition to antioxidant and anti skin cancer activities, preliminary studies also suggest that I3ON may have other beneficial pharmacological effects:
- anti-inflammatory activity By inhibiting the NF - κ B signaling pathway, the expression of pro-inflammatory cytokines (such as TNF - α, IL-6, IL-1 β) and inflammatory mediators (such as COX-2, iNOS) is reduced.
- Cardiovascular protection: It may play a protective role in cardiovascular diseases such as atherosclerosis and myocardial ischemia reperfusion injury through mechanisms such as antioxidant, anti-inflammatory, improving vascular endothelial function, inhibiting platelet aggregation, etc.
- neuroprotection Although its blood-brain barrier penetration is low, it may provide protection to nerve cells through antioxidant and anti-inflammatory effects in certain pathological states, such as when the blood-brain barrier is damaged.

Mechanism of action and molecular targets

Based on existing research, the pharmacological mechanism of isorhamnetin-3-O-neohesperidoside can be summarized as a multi-target and multi pathway regulatory mode, with its core being the direct or indirect regulation of key signaling molecules and transcription factors.

1. Direct target inhibition: MEK1 and PI3K

This is the most direct and critical molecular mechanism of I3ON's anti skin cancer effect. Research has shown that I3ON can directly bind to MEK1 and PI3K proteins in a non ATP competitive manner, thereby inhibiting their kinase activity.
- Inhibition of MEK1 MEK1 is a key kinase in the MAPK signaling pathway, responsible for phosphorylation and activation of ERK. The binding of I3ON to MEK1 may induce conformational changes, hindering its binding to ATP or substrate ERK, thereby blocking signal transmission downstream. This direct inhibition method has a similar mechanism of action to many clinically used MEK inhibitors (such as trametinib), but I3ON, as a natural product, may have different binding sites and modes, providing new ideas for designing novel MEK inhibitors.
- Inhibition of PI3K PI3K is the initiating kinase of the PI3K/AKT signaling pathway. I3ON can directly bind to the p110 catalytic subunit of PI3K, inhibiting its ability to convert phosphatidylinositol-4,5-diphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). The reduction of PIP3 prevents AKT from being recruited to the cell membrane and activated by PDK1 phosphorylation, thereby inhibiting the entire PI3K/AKT signaling pathway.

2. Regulation of key signaling pathways

By inhibiting the upstream kinases MEK1 and PI3K, I3ON can effectively regulate multiple downstream signaling cascades:
- MAPK/ERK pathway Inhibition of MEK1 → Inhibition of ERK1/2 phosphorylation → Inhibition of downstream transcription factors (such as Elk-1, c-Myc) activity → Downregulation of Cyclin D1 and CDK4/6 expression → Induction of G0/G1 phase cell cycle arrest, inhibition of cell proliferation.
- PI3K/AKT/mTOR pathway Inhibition of PI3K → inhibition of AKT phosphorylation → activation of downstream pro apoptotic proteins (such as Bad, Caspase-9), inhibition of anti apoptotic proteins (such as Bcl-2, Bcl xL) and mTOR activity → induction of cell apoptosis, inhibition of protein synthesis and cell growth.
- NF - κ B pathway Inhibition of AKT can weaken the activity of I κ B kinase (IKK), thereby reducing the phosphorylation and degradation of I κ B α, causing NF - κ B to be retained in the cytoplasm and unable to enter the nucleus to initiate transcription of pro-inflammatory and pro survival genes. This explains the anti-inflammatory activity of I3ON.

3. Transcription factor regulation: activation of NRF2

The antioxidant effect of I3ON mainly depends on the activation of NRF2 transcription factors. The mechanism may involve:
- Modify the thiol group of KEAP1 I3ON or its metabolites may covalently modify key cysteine residues on KEAP1 protein (such as Cys151, Cys273, Cys288), altering the conformation of KEAP1 and reducing its affinity for NRF2, thereby releasing NRF2.
- Inhibition of KEAP1 mediated NRF2 ubiquitination By interfering with the interaction between KEAP1 and Cullin3 ubiquitin ligase complex, the ubiquitination degradation of NRF2 is prevented, leading to its accumulation in cells.
- Activate upstream kinase I3ON may also phosphorylate NRF2 by activating certain protein kinases (such as PKC, PI3K, MAPK, etc.), promoting its dissociation and nuclear translocation from KEAP1.

Once NRF2 enters the nucleus, it forms a heterodimer with Maf protein and binds to the ARE sequence of the target gene promoter region, initiating the transcription of over 200 antioxidant and detoxifying genes including SOD1/2, CAT, GPX1, HMOX1, NQO1, GCLC, etc., thus constructing a powerful cellular defense system.

4. Molecular target network

In summary, the molecular targets of I3ON are not singular, but form a complex network. Its core targets include MEK1, PI3K, and KEAP1/NRF2 systems. By acting on these key nodes, I3ON can simultaneously regulate multiple biological processes such as cell proliferation, apoptosis, oxidative stress, and inflammatory response. This multi-target mode of action is a significant feature that distinguishes natural products from many highly selective synthetic drugs, and also makes them potentially more effective and lower in the treatment of complex diseases such as cancer.

Evaluation of drug properties and pharmacokinetics

The development of natural products with good pharmacological activity into clinical drugs must undergo strict pharmacological evaluation, among which pharmacokinetic (ADME) properties are one of the key factors determining whether they can become successful drugs. For isorhamnetin-3-O-neohesperidin, its medicinal properties present both opportunities and challenges.

1. Analysis of pharmacological parameters

Based on the aforementioned physicochemical property parameters, we can conduct a preliminary evaluation of the pharmacological properties of I3ON:
- Advantages：
- High water solubility LogP is negative, with good water solubility, which is beneficial for drug dissolution in the gastrointestinal tract and formulation development.
- Low risk of cardiac toxicity Lack of hERG inhibitory activity reduces the risk of causing arrhythmia.
- Low genetic toxicity risk The Ames test result is negative, indicating a low risk of mutagenicity.
- Low blood-brain barrier penetration Although it limits its application in central nervous system diseases, it also means that its peripheral effects may be safer and the risk of neurotoxicity may be lower.
- challenge：
- Low oral bioavailability This is the biggest challenge faced by I3ON. Its molecular weight (624.5 Da) exceeds 500 Da, and its TPSA (258.4 Å ²) is much higher than 140 Å ², both of which strongly indicate poor oral absorption. The high polarity and multiple hydrogen bond donors/acceptors make it difficult for them to passively diffuse through the lipid bilayer of intestinal epithelial cells. In addition, as a glycoside, it may be hydrolyzed by gut microbiota or glycosidase on the intestinal wall, leading to its absorption in the form of aglycones (isorhamnetin) or smaller metabolites, thereby altering the exposure level of the original drug.
- Metabolic instability Flavonoids are prone to extensive phase II metabolism in the body, such as glucuronidation, sulfation, and methylation. The multiple phenolic hydroxyl groups on I3ON are ideal substrates for phase II metabolic enzymes, which may lead to their rapid metabolism and clearance in the liver and intestine, further reducing their systemic exposure.

2. Pharmacokinetic characteristics (speculation and preliminary study)

At present, there are relatively limited detailed research reports on the pharmacokinetics of I3ON in vivo. However, based on its structural characteristics and studies on similar flavonoid glycosides (such as rutin and quercitrin), it can be inferred that its general characteristics are:
- absorb After oral administration, the absorption of I3ON in the stomach and small intestine may be poor. Most of them may reach the colon and be hydrolyzed by the rhamnosidase and glucosidase produced by the gut microbiota, releasing the glycoside isorhamnetin. As a glycoside element, isorhamnetin has a small molecular weight (316.3 Da), a LogP of about 1.4, significantly improved lipid solubility, and is more easily absorbed by colonic epithelial cells. Therefore, the systemic exposure of I3ON after oral administration may mainly exist in the form of isorhamnetin and its phase II metabolites.
- distribution Due to its high polarity, the I3ON prototype drug is mainly distributed in plasma and extracellular fluid, and its tissue distribution may be limited. Its metabolite isorhamnetin may have a wider distribution.
- Metabolism As mentioned earlier, I3ON undergoes hydrolysis and II binding reactions in vivo. The main metabolic pathways include: 1) hydrolysis into isorhamnetin and glycosides in the intestine or liver; 2) Isorhamnetin undergoes further glucuronidation, sulfation, and methylation.
- excretion Metabolites are mainly excreted through bile and urine.

3. Strategies for improving drug efficacy

Given the inherent challenge of low oral bioavailability of I3ON, future drug development may require the following strategies:
- Structural modification Design prodrugs for the sugar moiety or phenolic hydroxyl group of I3ON. For example, it can be prepared into ester or phosphate prodrugs to improve their lipid solubility and intestinal permeability, and the original drug can be released after enzymatic hydrolysis in vivo.
- Optimization of administration route: Develop non oral drug delivery routes, such as transdermal drug delivery systems (transdermal patches, gel), which can directly act on the skin for the treatment of skin cancer or skin oxidative damage. This can bypass the first pass effect, increase local drug concentration, and reduce systemic side effects.
- Nanoformulation technology Using nanocarriers such as liposomes, nanoparticles, and micelles to encapsulate I3ON can increase its solubility, protect it from metabolism, and promote its transmembrane transport, thereby improving oral bioavailability or achieving targeted delivery.
- Combined with absorption enhancers Combined use with P-glycoprotein (P-gp) inhibitors or intestinal permeability enhancers may enhance their oral absorption.

Clinical application prospects and prospects

Based on the unique pharmacological activity and mechanism of action of isorhamnetin-3-O-neohesperidin, it has shown broad application prospects in multiple therapeutic fields, especially in skin health and tumor prevention and treatment.

1. Treatment and Prevention of Skin Cancer

This is the most promising application direction for I3ON conversion. Its mechanism of inhibiting skin cancer cell proliferation and inducing apoptosis by directly inhibiting MEK1 and PI3K is clear and efficient. Compared with traditional chemotherapy drugs, I3ON, as a natural product, theoretically has lower toxic side effects. Its application prospects include:
- Local drug preparation: developed into cream, gel or lotion for the treatment of actinic keratosis (precancerous lesions), basal cell carcinoma and squamous cell carcinoma. Local administration can maximize the concentration of drugs at the lesion site while minimizing systemic exposure, thereby reducing systemic toxic side effects. This is particularly important for patients who require long-term treatment or prevention of recurrence.
- adjuvant therapy As an adjuvant therapy after surgical resection, it is used to remove residual small lesions and reduce the risk of recurrence.
- combination therapy Combined use with existing targeted therapy drugs (such as BRAF inhibitors, MEK inhibitors) or immune checkpoint inhibitors (such as PD-1/PD-L1 antibodies) may enhance efficacy through synergistic effects and delay or overcome the development of drug resistance. For example, the inhibitory effect of I3ON on PI3K may help overcome the adaptive activation of certain MEK inhibitor resistant tumors.
- Chemical prevention For high-risk populations such as long-term outdoor workers and immunocompromised patients after organ transplantation, develop active ingredients for sunscreen or skincare products to prevent skin cancer induced by ultraviolet radiation.

2. Antioxidant and anti-aging

The strong antioxidant activity of I3ON, especially by activating the NRF2 pathway to enhance the ability of endogenous defense systems, makes it potential for anti-aging and prevention of oxidative stress-related diseases.
- Functional food/health products As a standardized ingredient of sea buckthorn extract, it is added to functional foods or dietary supplements for daily health care, combating oxidative damage, and delaying aging.
- cosmetic ingredient Added to skincare products as an antioxidant and photoprotective agent, used to reduce UV induced skin photoaging, pigmentation, and wrinkles.

3. Other potential applications

• cardiovascular disease: It may have an auxiliary therapeutic effect on atherosclerosis, hypertension and other diseases through antioxidant, anti-inflammatory, vascular function improvement and other mechanisms.
• Metabolic diseases The preliminary study suggests that it may improve insulin resistance and lipid metabolism disorder, and has exploratory value in the prevention and treatment of diabetes and its complications.
• Inflammatory diseases Its anti-inflammatory activity makes it potentially applicable in the treatment of chronic inflammatory diseases such as arthritis, dermatitis, and enteritis.

Future research directions：

Despite the promising prospects, the clinical translation of I3ON still faces many challenges, and future research should focus on the following aspects:
1. In depth pharmacokinetic research Systematically elucidate the absorption, distribution, metabolism, and excretion processes of I3ON in animals and humans, particularly its metabolic fate and systemic exposure forms after oral administration. Clearly determine whether the prototype drug or its metabolites (such as isorhamnetin) exert the main in vivo efficacy.
2. Pharmacodynamic validation in vivo Systematic evaluation of the anti-tumor efficacy and safety of local or systemic administration of I3ON in animal models, such as UV induced skin cancer mouse models and xenograft tumor models. Evaluate its synergistic effect with existing treatment plans.
3. Formulation development: Focus on the development of local drug delivery agents (such as nano emulsion, liposome gel) suitable for the treatment of skin cancer, as well as new agents to improve oral bioavailability (such as phospholipid complex, nano crystal).
4. toxicological evaluation Conduct comprehensive acute, subchronic, and chronic toxicity studies, particularly evaluating the skin irritation and sensitization of long-term topical use, as well as assessing gastrointestinal and liver toxicity of oral administration.
5. In depth exploration of the mechanism of action Using structural biology, chemical biology, and other methods, elucidate the specific sites and patterns of I3ON binding to MEK1, PI3K, and KEAP1 proteins, providing a basis for structure based drug design. Meanwhile, explore whether it also acts on other targets, such as affecting the tumor microenvironment and regulating immune responses.

Conclusion

Isorhamnetin-3-O-neohesperidoside, a natural flavonol glycoside derived from seabuckthorn, has become a remarkable new star in the field of natural product pharmacology due to its unique chemical structure and various pharmacological activities, especially its anti skin cancer potential demonstrated by directly inhibiting MEK1 and PI3K, as well as its strong antioxidant capacity achieved by activating the NRF2 pathway. Its clear molecular targets and mechanisms of action provide a solid scientific foundation for the development of new anti-tumor drugs or functional health products.

However, the road from laboratory discovery to clinical application is still long and challenging. The inherent high polarity and high molecular weight of I3ON result in low oral bioavailability, which is the biggest bottleneck for its drug development. Future research must focus on addressing this core issue, fully unleashing its therapeutic potential through innovative formulation techniques, rational structural modifications, or optimized delivery routes. Meanwhile, in-depth pharmacokinetic, in vivo pharmacodynamic, and toxicological studies are necessary prerequisites for promoting its clinical translation.

In summary, isorhamnetin-3-O-neohesperidoside is a highly valuable natural product lead compound for development. With the continuous deepening of research and the advancement of technology, we have reason to believe that this "treasure molecule" from seabuckthorn is expected to play an important role in the prevention and treatment of skin cancer, antioxidant health care, and even broader health fields, contributing to the cause of human health. The study of it is not only an exploration of a single compound, but also a vivid practice of combining traditional herbal wisdom with modern pharmaceutical science.