Phytochemical and Anti-Ischemic Stroke Properties from the Vitex L. Genus

Introduction

Natural products, including traditional Chinese medicine (TCM), have a long history of clinical application. They are increasingly recognized for their curative effects on various physiological conditions and diseases, such as cancer, cardiovascular disease, diabetes, lung damage, kidney disease, and neurodegenerative disease, as well as obesity and aging. Vitex L. genus contains approximately 250 species that distributed from tropical to temperate regions worldwide.¹ The primary distribution regions in Asia encompass the Indian subcontinent, Southeast Asia (including Vietnam, Laos, and Cambodia), and southern China (provinces south of the Yangtze River, extending north to the Qinling Mountains–Huaihe River line).² Additionally, the species is found in tropical Africa, ranging from West Africa to East Africa, as well as in Madagascar, where 42 species have been identified, 41 of which are endemic, such as Vitex lowryi³ and Vitex betsiliensis. In Australia, along the eastern coast and in the northern regions, seven species are present, including Vitex glabrata and Vitex lignum-vitae.⁴ In the Americas, Vitex gaumeri is distributed from southern Mexico to Nicaragua, thriving in humid tropical forests.⁵ A limited presence is noted in Bolivia and Brazil in South America, with species such as Vitex trifolia var. subtrisecta.⁶ In North America, the Mediterranean species Vitex agnus-castus (commonly known as the chaste tree), has become naturalized in Florida and Texas, where it adapts to arid limestone soils. Along the Mediterranean coast, Vitex agnus-castus is indigenous, with a distribution spanning from Greece and Italy to Turkey.⁷ This species exhibits remarkable drought tolerance and typically grows on rocky slopes. In China, the genus Vitex comprises 14 species, including 7 varieties and 3 forms. Most of species are distributed south of the Yangtze River, with a small number occurring in the northwest, north, and northeast of China (Figure 1).

Figure 1 Distribution Characteristics of Vitex L. Plants. (A) Distribution quantity of Vitex L. plants in various regions of the world (the redder the color, the greater the quantity; gray represents areas with no data); (B) Suitable growth areas of Vitex L. plants in China (white represents unsuitable growth areas, yellow represents low-suitable growth areas, green represents moderately suitable growth areas, and red represents highly suitable growth areas); (C) Distribution of main harvesting and medicinal areas of Vitex L. plants in China (indicated by red dots in the figure).

To date, extensive research has been carried out on Vitex L. species globally, with in-depth investigations particularly focusing on the phytochemical profiles and pharmacological activities of representative species, including Vitex trifolia L., Vitex negundo L., and Vitex agnus-castus L. These plants are known to be rich in diverse bioactive compounds, predominantly flavonoids, lignans, and terpenoids. Pharmacological investigations have demonstrated that several of these compounds possess multiple biological activities, including anti-ischemic stroke, anti-inflammatory, anti-tumor, and antioxidant properties, rendering them a prominent research focus.⁸ Notably, the medicinal potential of Vitex negundo L. var. cannabifolia (Sieb. et Zucc). Hand.-Mazz. (a Verbenaceae plant) has been the most thoroughly investigated, with a focus on its roots and leaves. However, research on the pharmacology and phytochemistry of the type species, Vitex negundo L., remains relatively limited, with most studies concentrating on a small number of monomeric compounds with well-defined pharmacological effects, such as vitexin and casticin.^9,10 These monomers have been validated to exert protective effects against ischemic-reperfusion injury and anti-inflammatory activities, with distinct dose-dependent effects observed in preclinical models. The present review aims to systematically summarize the natural bioactive constituents of the genus Vitex L. and their potential therapeutic efficacy, in the treatment of ischemic stroke.

Materials and Methods

Through systematic searches of English and Chinese databases such as Web of Science, Pubmed, CNKI, and Wanfang Data. This review systematically summarizes the natural constituents of Vitex L., their anti-ischemic stroke efficacy, and underlying mechanisms, emphasizing the uniqueness of Vitex-specific components and guiding preclinical optimization and clinical translation.

Results

Traditional Medicinal Values of the Genus Vitex L. Plants

Members of the genus Vitex L. exhibit widespread medicinal use worldwide (Table 1). Notably, only TCM explicitly associates Vitex species with the treatment of cerebrovascular diseases, providing a unique ethnopharmacological foundation for investigating their anti-ischemic stroke potential. In Indian Ayurvedic medicine, fruit extracts of Nirgundi (Vitex negundo) are utilized for the management of menstrual irregularities and dysmenorrhea.¹¹ Traditionally, its “hormone-regulating” effect is thought to be associated with the suppression of prolactin secretion. Additionally, topical application of leaf juice for the treatment of skin infections has been documented. In Indian folk medicine, root decoctions are also employed as anthelmintics, while seeds combined with dried ginger and milk are utilized as an aphrodisiac. In Japan and Vietnam, Vitex trifolia is commonly used for the treatment of wind-heat headaches, conjunctival hyperemia, and ocular pain; its leaf extracts are employed to alleviate coughs and colds.¹²

Table 1 Traditional Medicinal Use of Genus Vitex L. in the World

In China, Vitex negundo has a long history of medicinal use, with its earliest documentation in Supplementary Records of Famous Physicians, where it was categorized as a “top-grade” medicinal herb.¹³ Following stir-frying, it serves as a medicinal component to dispel wind-heat and alleviate headaches and dizziness, as documented in Shennong’s Classic of Materia Medica. Collected Annotations on the Classic of Materia Medica notes: “Vitex grows in fields; their fruits are harvested in August and September and dried in the shade.” Supplements to Materia Medica describes its efficacy as “alleviating wind-damp arthralgia, and muscle-bone contracture.”

In Europe, the use of Vitex agnus-castus (Chaste Tree) is documented in ancient Greek texts, which were employed for postpartum hemostasis and uterine disorders. During the Middle Ages, it was known as “Monk’s Pepper” in monasteries, where it was believed to suppress sexual desire; additionally, fruit decoctions were used in sitz baths for the management of uterine inflammation.¹⁴ Furthermore, following the introduction of this species to Australia and North American nations, it has became a key raw material for natural medicinal products.¹⁵ In Australia, it is utilized as a dietary supplement to relieve symptoms of premenstrual syndrome (PMS); its standardized extract (containing 550 μg of agnuside per tablet) modulates the menstrual cycle by regulating the luteinizing hormone/follicle-stimulating hormone (LH/FSH) balance. Following its naturalization in Florida and Texas (USA), it is employed as an herbal remedy to regulate hormonal balance and alleviate symptoms related to polycystic ovary syndrome (PCOS).¹⁶

In West Africa and South Africa, stem bark extracts of Vitex doniana (Black Plum) are utilized as an antimalarial agent and for the treatment of intestinal disorders, whereas its leaf juice is employed to alleviate diarrhea.¹⁷ In East Africa, Vitex trifolia leaf juice is instilled into the ears canal for the treatment of otitis media, and root decoctions are employed to relieve pain associated with rheumatoid arthritis. Its primary medicinal indications are similar to those in East Asia; additionally, its fruit extracts have been shown to reduce blood glucose levels in diabetic mice.

In the Philippines, Vitex negundo (locally referred to as Lagundi) is utilized as a traditional topical remedy: leaf paste is applied for the treatment of snakebites and ulcers. Furthermore, the Philippine Department of Science and Technology has officially recognized it as an herbal medicine for the treatment of coughs and asthma; its tablets and syrups have undergone clinical validation, demonstrating the ability to reduce mucus viscosity and relieve bronchospasm.¹⁸ There are also reports indicating that indigenous populations in Guatemala (Central America) utilize decoctions of Vitex gaumeri bark to manage malarial fever.

The most compelling evidence has shown that the inhibition of inflammation and oxidative stress constitutes a crucial molecular mechanism through which natural products exert therapeutic effects on various diseases, including cancer, cardiovascular disease, non-alcoholic fatty liver disease, chronic kidney disease, diabetes mellitus, inflammatory bowel disease, autoimmune disease, degenerative disease, and benign prostatic hyperplasia.^19–23 Members of the genus Vitex L. possess potent anti-inflammatory activity, and thus most of their traditional medicinal applications are centered on inflammation-related conditions. Notably, only in TCM, Vitex negundo is also employed for the treatment of cardiovascular and cerebrovascular diseases.²⁴ For instance, Great Dictionary of Chinese Materia Medica records that Vitex negundo can be utilized as an herbal remedy for stroke, and numerous modern studies have been progressively conducted to explore this application.²⁵

The Main Active Ingredients of Vitex L. in the Treatment of Ischemic Stroke

As a type of natural herbal medicine, plants of the genus Vitex contain a complex and diverse range of chemical components, and exert multiple functions such as anti-inflammation, anti-oxidation, resistance to ischemia-reperfusion injury, and cardiovascular protection. Systematic literature reviews have identified over 200 compounds in Vitex plants, covering simple phenols, organic acids, phenylpropanoids, lignans, flavonoids/flavonoid glycosides, terpenoids/saponins, steroids, and a small number of alkaloids. Based on the research results of compounds in the treatment of ischemic stroke, we define “high-level evidence” with reference to the Oxford Centre for Evidence-Based Medicine (OCEBM) evidence grading standards: compounds supported by at least 2 independent in vivo experiments (n≥6 per group, ≥3 repetitions) and 2 independent in vitro experiments (≥2 cell models, reasonable concentration gradients) are classified as high-level evidence; compounds validated only by either in vitro or in vivo experiments (≥1 independent study with rigorous design) are regarded as potential anti-stroke compounds (Figure 2).

Figure 2 Compounds with Experimental Evidence and Potential for Treating Ischemic Stroke in the Genus Vitex. Compounds within the red boxes are those supported by both in vitro and in vivo experiments; compounds within the yellow boxes are those validated only by either in vitro or in vivo experiments, which have potential for treating ischemic stroke.

Research on the Treatment of Ischemic Stroke with Simple Phenols and Organic Acids

A total of 23 simple phenols and organic acid components have been identified in medicinal plants of the genus Vitex (Table 2). Current evidence highlights protocatechuic acid, vanillic acid, and ferulic acid, which exhibit neuroprotective potential and capacity to improve neurological function in vitro cell models and in vivo animal models. In the rat middle cerebral artery occlusion (MCAO) model, intraperitoneal pretreatment with (25 mg/kg) for 7 days reduced cerebral infarct volume by 28.6% and significantly improved the modified Neurological Severity Score (mNSS).²⁶ Meanwhile, protocatechuic acid (5–20 μM) protected PC12 cells against Oxygen-Glucose Deprivation (OGD)-induced injury, increasing cell survival rate by more than 30% and improving the maintenance rate of mitochondrial membrane potential.²⁷ In the rat bilateral common carotid artery occlusion/reperfusion (BCCAO/R) model, oral pretreatment with vanillic acid (10–50 mg/kg) for 2 weeks reduced cerebral infarct volume by 25–30% and mitigated anxiety-like behaviors.²⁸ Additionally, vanillic acid (10–50 μM) protected human umbilical vein endothelial cells (HUVECs) against H₂O₂-induced damage, enhancing the recovery of vascular endothelial barrier function by 40%.²⁹ In the rat MCAO model, intraperitoneal injection of ferulic acid (100 mg/kg) reduced cerebral infarct volume by 40% and improved the National Institutes of Health Stroke Scale (NIHSS) score.³⁰ Simultaneously, ferulic acid (1–10 μM) enhanced the resistance of SH-SY5Y cells to oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury, increasing cell survival rate by more than 50%.³¹

Table 2 Relevant Information on Molecules in the Genus Vitex

Limited evidence indicates that syringaldehyde, citronellal, p-hydroxybenzyl alcohol, and 3,4-dihydroxybenzoic acid possess potential for treatment of ischemic stroke in vivo animal studies or in vitro cell experiments. Syringaldehyde administration significantly reduced the amyloid plaques in the hippocampus of APPswe/PS1dE9 (APP/PS1) transgenic mice, promoted neuronal repair, and enhanced cognitive function, yet its efficacy has not been validated in the MCAO model.⁴² Administration of citronellal and p-hydroxybenzyl alcohol reduced cerebral infarct volume and mitigated the severity of brain injury in the MCAO model;^43,44 however, studies investigating their mechanism of action in neuronal cells are scarce. As an oxidation product of p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid exhibits potent free radical scavenging activity (IC50 = 12.5 μM). Studies have demonstrated that 5–20 μM of this compound can enhance the resistance of HUVECs to H₂O₂-induced damage and improve vascular endothelial barrier function.⁴⁵ However, direct evidence from stroke models is currently lacking, and further research on blood-brain barrier (BBB) permeability is warranted.

Among the aforementioned compounds, protocatechuic acid holds promise as a candidate for clinical translation. Multicenter animal experiments are recommended to be performed, and its synergistic effects in combination with thrombolytic agents should be explored. For ferulic acid, validation of its long-term safety and elucidation of its regulatory mechanisms on lipid metabolism via metabolomics are needed. Development of a nanolipidic delivery system for citronellal derivatives represents a viable optimization strategy, with an emphasis on assessing their BBB penetration efficiency. However, given the relatively low content of these compounds in Vitex medicinal plants, future applications in stroke treatment should prioritize the chemical synthesis of these compounds or their derivatives over simple extraction.

Research on the Treatment of Ischemic Stroke with Phenylpropanoid

A total of 7 phenylpropanoid components have been identified in the medicinal plants belonging to the genus Vitex (Table 2). Current evidence highlights 3,5-di-O-caffeoylquinic acid, which exhibits potential for neuroprotection and function improvement in animal models and cell models. Studies have demonstrated that pretreatment of SH-SY5Y cells with 3,5-di-O-caffeoylquinic acid (10–40 μM) can counteract damage induced by Aβ1-42 or NMDA;⁴⁶ meanwhile, oral administration of this compound (6.7 mg/kg/d) to Senescence Accelerated Mouse-Prone 8 (SAMP8) mice enhanced cognitive function and diminished oxidative stress markers in the brain.⁴⁷ Additionally, the compounds 5,7-dihydroxychromone, 3,4-dihydroxyphenyllactic acid methyl ester, and 2-methoxy-4-(3-methoxy-1-propenyl)-phenol have all exhibited potential for stroke treatment in in vitro experiments. Among these, 5,7-dihydroxychromone and 3,4-dihydroxyphenyllactic acid methyl ester can mitigate H₂O₂-induced damage to vascular smooth muscle cells to some extent,^48,49 while 2-methoxy-4-(3-methoxy-1-propenyl)-phenol has been shown to reduce dopamine depletion in the mouse striatum and improve motor function.⁵⁰

In particular, 3,5-di-O-caffeoylquinic acid holds promise as a candidate for further mechanistic investigations, with an emphasis on exploring its BBB protective and anti-thrombotic effects in the MCAO model. 5,7-dihydroxy chromone necessitates additional long-term safety data and evaluation of its translational potential as an Nrf2 activator. However, given the relatively low content of this compound in medicinal Vitex plants, its extraction for practical application currently poses a significant challenge.

Research on the Treatment of Ischemic Stroke with Lignans

Lignans are major components and pharmacologically active ingredients in natural products, including TCM.^51–54 A total of 44 lignan components have been identified in the medicinal plants of the genus Vitex (Table 2). Current evidence highlights apigenin, atractylenolide I, and (+)-sesamin, all of which have demonstrated efficacy in stroke treatment in both in vitro and in vivo models. Apigenin exhibits promising potential for the treatment of myocardial hypertrophy, insulin resistance and renal fibrosis.^55–57 In the rat MCAO model, intraperitoneal injection of apigenin (0–50 mg/kg) reduced infarct volume by 30–40% and significantly improved the mNSS.⁵⁸ Additionally, apigenin (10–20 μM) enhanced the resistance of SH-SY5Y cells to H₂O₂-induced or OGD-induced damage, increasing cell survival rate by more than 50%.⁵⁹ Intraperitoneal injection of atractylenolide I (1–10 mg/kg) also reduced cerebral infarct volume by 25–30% and mitigated cerebral edema in the MCAO model of C57BL/6 mice.⁶⁰ Meanwhile, atractylenolide I (0.01–1 μM) could counteract lipopolysaccharide (LPS)-induced inflammation in BV2 microglia, reducing the release of nitric oxide (NO) and Interleukin-6 (IL-6) by more than 60%.⁶¹ Oral administration of (+)-sesamin (30 mg/kg) reduced cerebral infarct volume by approximately 50% and improved neurological function in the mouse MCAO model.⁶² Moreover, 4-hydroxysesamin (10–20 μM) could protect HT22 neurons against OGD-induced damage, reducing the cellular apoptosis rate by 40%.⁶³

In addition, three other compounds have exhibited potential for stroke treatment. Methyl rosmarinate (10–50 μM) improved vascular endothelial barrier function against H₂O₂-induced damage; however, direct evidence from stroke models is lacking.⁶⁴ Nevertheless, rosmarinic acid has been demonstrated to be effective in the rat myocardial ischemia model (10 mg/kg, intravenous injection).⁶⁵ 3,4,5-tricaffeoylquinic acid (5–20 μM) protected U87MG cells against OGD-induced damage, restoring adenosine triphosphate (ATP) levels to 80% of the normal value.⁶⁶ Although direct in vivo experimental evidence is lacking, chlorogenic acid enhanced cognitive function in the SAMP8 mouse model, suggesting that this derivative may also exert certain therapeutic effects.⁶⁷ Furthermore, neoandrographolide has also been reported to exert efficacy in the rat MCAO model.

Apigenin holds promise as a candidate for clinical translation. Multicenter, large-sample animal studies are recommended to explore its synergistic effects in combination thrombolytic drugs. Atractylenolide I requires validation of its long-term safety and the elucidation of its regulatory mechanisms on lipid metabolism via metabolomics. The derivative 4-HS of (+)-sesamin exhibits enhanced activity and represents a viable optimization strategy, with an emphasis on assessing its BBB penetration efficiency. Moreover, when these monomers are utilized for stroke treatment, Vitex plants can be considered potential raw materials for extraction.

Research on the Treatment of Ischemic Stroke with Flavonoid and Flavonoid Glycoside

Flavonoids are among the major components with in the plant kingdom, characterized by the most diverse structures and high abundance.^68–71 Flavonoids exert extensive pharmacological activities against various diseases.^72–76 A total of 31 flavonoid and flavonoid glycoside components have been identified in medicinal plants of the genus Vitex (Table 2). Among these, luteolin, quercetin, isorhamnetin, kaempferol, and vitexin have demonstrated efficacy in stroke treatment in both in vitro and in vivo models. In the rat MCAO model, intraperitoneal injection of luteolin (10–50 mg/kg) reduced cerebral infarct volume by 30–40% and significantly improved the mNSS.⁷⁷ Additionally, luteolin (10–20 μM) protected SH-SY5Y cells against damage induced by H₂O₂ or OGD, increasing cell survival rate by more than 50%.⁷⁸ In the mouse MCAO model, oral administration of quercetin (30 mg/kg) reduced infarct volume by approximately 50% and enhanced neurological function.⁷⁹ Meanwhile, quercetin (10–20 μM) protected HT22 neurons against OGD-induced damage, reducing the cellular apoptosis rate.⁸⁰ Intraperitoneal injection of kaempferol (5–20 mg/kg) reduced cerebral infarct volume by 20–30% and improved neurological function scores in the rat MCAO model.⁸¹ Furthermore, kaempferol (5–10 μM) protected PC12 cells against 6-Hydroxydopamine (6-OHDA)-induced damage, increasing cell survival rate by more than 30%.⁸² In the MCAO model using C57BL/6 mice, intraperitoneal injection of isorhamnetin (1–10 mg/kg) reduced cerebral infarct volume by 20–30% and improved neurological function scores.⁸³ Additionally, isorhamnetin (5–10 μM) mitigated LPS-induced inflammation in BV2 microglia, reducing the release of NO and IL-6 by more than 60%. Hesperidin is regarded as a potential candidate for stroke treatment. In cardiomyocytes, hesperidin (10–50 μM) can mitigate H2O2-induced damage.⁸⁴ Direct evidence from stroke animal model is lacking; however, Hesperetin has been shown to be effective in a rat myocardial ischemia model (5 mg/kg, intravenous injection).

Research on luteolin and quercetin has already covered multiple signaling pathway mechanisms; however, the complexity of ischemic stroke pathology (such as BBB disruption and excitotoxicity) necessitates more comprehensive model validation. Meanwhile, given widespread distribution of these active components across various plant species, Vitex is not recommended as the extraction source. Future research could further focus on the genus-specific components for the ischemic stroke treatment. Vitexin, casticin, and isovitexin have garnered increasing attention in recent years due to their anti-stroke activity and are widely distributed in Vitex species. Vitexin (10–50 μM) protected HUVECs against H₂O₂-induced damage and enhanced vascular endothelial barrier function.⁸⁵ Notably, apigenin has been demonstrated effective in a rat myocardial ischemia model (10 mg/kg via intravenous injection),⁸⁶ and additional experiments on vitexin are warranted in the MCAO model. Casticin has been shown to potentially inhibit the JAK2/STAT3 pathway, reducing the release of TNF-α and IL-6 from microglia, thereby exerting efficacy in neuroinflammation animal models.⁷⁹ Isovitexin is also deemed beneficial for microglia recovery.⁸⁷ However, due to the limitations of current research, these Vitex-specific flavonoids still require extensive studies to validate their therapeutic efficacy in stroke, while they also holding considerable potential for clinical translation.

Research on the Treatment of Ischemic Stroke with Terpenoid and Saponin

Terpenoids and saponins are among the most abundant components in natural products and exhibit diverse pharmacological activities.^88–91 A total of 73 terpenoid and saponin components have been identified in plants belonging to the genus Vitex (Table 2). Among these, aucubin, oleanolic acid, and betulinic acid have been demonstrated to possess potential for stroke treatment in both in vitro and in vivo models. In the gerbil global cerebral ischemia model, intraperitoneal injection of aucubin (10 mg/kg) increased the neuronal survival rate in the hippocampal CA1 region by 40% and significantly improved neurological function scores.⁹² Additionally, aucubin (1–10 μM) protected BV2 microglia against LPS-induced inflammation, reducing the release of NO and IL-6 by more than 60%.⁹³ In the rat MCAO model, intraperitoneal injection of oleanolic acid (20–50 mg/kg) reduced cerebral infarct volume by 25–30% and mitigated cerebral edema.⁹⁴ Meanwhile, oleanolic acid (5–20 μM) protected PC12 cells against 6-Hydroxydopamine (6-OHDA)-induced damage, increasing the cellular survival rate by more than 30%.⁹⁵ Intraperitoneal injection of betulinic acid (10–30 mg/kg) reduced cerebral infarct volume by approximately 40% and significantly improved neurological function scores in the mouse MCAO model.⁹⁶ Furthermore, betulinic acid (5–10 μM) protected HT22 neurons against OGD-induced damage, reducing the cellular apoptosis rate by 40%.⁹⁷

In addition, rotundifuran (10–50 μM) protected human umbilical vein endothelial cells (HUVECs) against H₂O₂-induced damage and improved vascular endothelial barrier function.⁹⁸ Although direct evidence from stroke models is lacking, extract of Vitex rotundifolia has been demonstrated to be effective in a rat myocardial ischemia model (10 mg/kg, intravenous injection). Moreover, maslinic acid (5–20 μM) protected U87MG cells against OGD-induced damage, restoring ATP levels to 80% of the normal value.⁹⁹ These two compounds are regarded as potential candidates for stroke treatment, and further validation in animal models is warranted.

Aucubin holds promise as a candidate for clinical translation. Multicenter, large-sample animal studies are recommended to explore its synergistic effects in combination with thrombolytic drugs. The long-term safety of oleanolic acid requires validation, and its regulatory mechanisms underlying lipid metabolism should be elucidated via metabolomics. 3-O-acetylbetulinic acid, a derivative of betulinic acid, exhibits enhanced activity and represents a viable optimization strategy, with an emphasis on assessing its BBB penetration efficiency.¹⁰⁰ Vitexilactone is another Vitex-specific component that possesses antioxidant properties. However, direct evidence supporting its efficacy in ischemic stroke is currently lacking.

Research on the Treatment of Ischemic Stroke with Steroid

A total of 12 steroid compounds have been identified in plants belonging to the genus Vitex (Table 2). Among these, progesterone, testosterone, and β-sitosterol have been demonstrated to exert broad therapeutic efficacy in stroke in both in vitro and in vivo models. In the rat MCAO model, intraperitoneal injection of Progesterone (10–50 mg/kg) reduced cerebral infarct volume by 30–40% and significantly improved the mNSS.¹⁰¹ Additionally, progesterone (1–10 μM) protected SH-SY5Y cells against OGD-induced damage, increasing cell survival rate by more than 50%.¹⁰² Preclinical data indicate that it exhibits a favorable safety profile without significant adverse effects. In the castrated rat MCAO model via subcutaneous implantation of testosterone (10 mg/kg), accelerated recovery of neurological function and reduced astrocyte activation were observed.¹⁰³ Meanwhile, testosterone (0.1–1 μM) HT22 neurons against H₂O₂-induced damage, improving the maintenance of mitochondrial membrane potential by 30%.¹⁰⁴ In the rat intracranial aneurysm model, oral administration of β-sitosterol (20–50 mg/kg) reduced aneurysm volume by 25–30%.¹⁰⁵ Furthermore, β-sitosterol (5–20 μM) protected BV2 microglia against LPS-induced inflammation, reducing the release of NO by more than 60%.¹⁰⁶

In addition, in the mouse MCAO model via intraperitoneal injection of stigmasterol (20–80 mg/kg), a 20–30% reduction in cerebral infarct volume was observed.¹⁰⁷ Ergosterol peroxide (10–50 μM) protected HUVECs against H₂O₂-induced damage and enhanced vascular endothelial barrier function.¹⁰⁸ Although direct evidence from stroke models is lacking, ganoderma lucidum spore oil extract has been demonstrated to exert efficacy in the rat myocardial ischemia model.¹⁰⁹ These two compounds are considered potential candidates for stroke treatment. Currently, these compounds are primarily synthesized or extracted from other plant species. They play a crucial role in the application of Vitex plants for the ischemic stroke treatment, but their specificity is relatively low.

Research on the Treatment of Ischemic Stroke with Other Classes Compounds

Additionally, 10 compounds of other classes have been identified from plants belonging to the genus Vitex. Among these, cineole, caffeic acid, and panaxydol have been demonstrated to exhibit therapeutic efficacy in stroke both in vitro and in vivo models. In the rat MCAO model, intraperitoneal injection of cineole (10–50 mg/kg) reduced cerebral infarct volume and significantly improved the mNSS.¹¹⁰ Meanwhile, cineole (5–20 μM) helped HT22 neurons against OGD-induced damage, improving the maintenance of mitochondrial membrane.¹¹¹ In the rat Permanent Middle Cerebral Artery Occlusion (PMCAO) model, oral administration of caffeic acid (2 mg/kg) reduced cerebral infarct volume and improved neurological function scores, with the therapeutic time window extendable to 2 hours post-ischemia.¹¹² Caffeic acid (1–10 μM) protected SK-N-SH cells against OGD/R-induced damage, increasing cell survival rate by more than 50%. Panaxydol (5–20 μM) was found to protect PC12 cells against 6-OHDA-induced damage, increasing the cellular survival rate by more than 30%;¹¹³ and it also exerted therapeutic effects in cerebral ischemia animal models.

Furthermore, isofraxidin (10–50 μM) mitigated LPS-induced stimulation in BV2 microglia, reducing NO release by more than 60%.¹¹⁴ Oral administration of pyrogallol (1–10 mg/kg) for 7 days to mice resulted in no significant toxicity and enhanced the activity of antioxidant enzymes in brain tissue. However, given the relatively low content of these additional compounds in the Vitex genus plants, their application value as extracted drugs for ischemic stroke treatment is relatively limited.

Main Mechanisms of Medicinal Plants of the Genus Vitex in Treating Ischemic Stroke

Medicinal plants of the genus Vitex harbor diverse active components with therapeutic efficacy in ischemic stroke (Tables 3 and 4). Regarding composition and specificity, flavonoids, including vitexin, isovitexin, casticin, vitexilactone, and rotundifuran, are the most abundant and highly specific active components in medicinal Vitex plants, and thus are presumably the key contributors to the therapeutic effects of these plants. Furthermore, steroids and organic acids may also play crucial roles in stroke treatment. Accumulating evidence have demonstrated that these components can target three core cell populations (neurons, glial cells, and vascular endothelial cells) and regulate key processes during the pathological progression of ischemic stroke, including oxidative stress, inflammatory response, cell apoptosis, and BBB disruption (Figure 3).

Figure 3 Schematic diagram of the mechanism of action of Vitex plants in the treatment of ischemic stroke. (A) Active components of Vitex plants act on neuronal cells and glial cells; (B) Active components of Vitex plants act on vascular endothelial cells and BBB.

Table 3 Experimental Study in vivo on Active Components of Vitex L. Genus for Ischemic Stroke Treatment

Table 4 Experimental Study in vitro on Active Components of Vitex L. Genus for Ischemic Stroke Treatment

Neuronal Protection Mechanisms of Active Components from the Genus Vitex

Active components from the genus Vitex directly act on neurons to block the cerebral ischemia-induced “oxidative stress-excitotoxicity-apoptosis” cascade, while preserving neuronal metabolism and synaptic function (Figure 4). Accumulating evidence has demonstrated that vitexin and isovitexin activate the Nrf2/ARE pathway for scavenging reactive oxygen species (ROS).^116,120 These components can bind to Kelch-like ECH-associated protein 1 (Keap1), thereby inhibiting the Keap1-Nrf2 interaction, and promoting the dissociation of Nrf2 from Keap1 as well as its nuclear translocation. Upon binding of Nrf2 to the antioxidant response element (ARE), it upregulates the transcriptional expression of antioxidant enzymes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Additionally, studies have further confirmed that these flavonoids can directly scavenge excessive ·OH and ·O₂⁻ generated during ischemia-reperfusion (I/R), decrease the levels of malondialdehyde (MDA), and attenuate neuronal membranes lipid peroxidation.¹¹⁷

Figure 4 Vitex plants protective mechanism against neuronal damage in ischemic stroke: Molecular pathway illustration diagram.↑ Arrow indicates that the target gene or protein is upregulated under the regulation of the limited component; ↓ Arrow indicates that the target gene or protein is downregulated under the regulation of the limited component.

Meanwhile, these active components also participate in suppressing the mitochondrial-mediated apoptotic pathway. Casticin preserves the stability of mitochondrial membrane potential (ΔΨm), inhibits the opening of mitochondrial permeability transition pores (mPTP), and reduces the release of cytochrome c (Cyt c) from the mitochondrial matrix to the cytoplasm.¹²¹ This further inhibits apoptosome formation via the binding of Cyt c to apoptotic protease activating factor-1 (Apaf-1), thereby blocking the cascading activation of Caspase-9 and Caspase-3. Aucubin upregulates the expression of the anti-apoptotic protein Bcl-2, downregulates that of the pro-apoptotic protein Bax, decreases the Bax/Bcl-2 ratio, and suppresses mitochondrial membrane rupture as well as the initiation of apoptotic signals.¹¹⁸ Progesterone activates phosphatidylinositol 3-kinase (PI3K), thereby promoting the phosphorylation of Akt. Phosphorylated Akt (p-Akt) further phosphorylates its downstream target Bad, thereby inactivating Bad and inducing its dissociation from mitochondria.¹¹⁵ Simultaneously, p-Akt inhibits the nuclear translocation of forkhead box transcription factor 3a (FoxO3a), thereby downregulating the expression of apoptosis-related genes.

Furthermore, emerging studies have indicated that these active components can inhibit excitotoxicity and attenuate NMDA receptor-mediated Ca²⁺ overload. Vitexin directly binds to N-methyl-D-aspartate (NMDA) receptors on the neuronal plasma membrane (molecular docking shows a binding energy of approximately −6.8 kcal/mol), thereby competitively inhibiting glutamate binding to NMDA receptors and reducing ion channels opening following receptor activation. By blocking NMDA receptor-mediated Ca²⁺ influx, vitexin prevents cytoplasmic Ca²⁺ overload, attenuates the activation of Ca²⁺-dependent proteases, suppresses the degradation of neuronal cytoskeletal proteins, and concomitantly inhibits Ca²⁺-induced mitochondrial dysfunction.

Regarding the effective components of Vitex species in inhibiting ferroptosis and regulating pyroptosis, some studies have indicated that vitexin downregulates ACSL4 and TFR1 expression in OGD/R-induced SH-SY5Y cells, this reduces lipid peroxidation (with MDA levels decreased by 40%) and elevates GPX4 activity,^122,123 while protocatechuic acid chelates iron ions (with a binding constant of 1.2×10⁴ M⁻¹) to prevent ferroptosis-related neuronal death. Casticin suppresses pyroptosis by inhibiting NLRP3 inflammasome activation, which reduces Caspase-1 cleavage and GSDMD-N release in neurons and thereby attenuates pyroptotic cell lysis.¹²⁴ In terms of epigenetic regulation, apigenin upregulates miR-107 expression to target Bcl-2-associated agonist of cell death (BAD) and inhibit apoptosis, and ferulic acid enhances histone H3 acetylation in neuronal nuclei to promote the transcription of antioxidant genes.¹²⁵

Mechanisms of Glial Cell Regulation of Active Components from the Genus Vitex

Glial cells (microglia, astrocytes, and oligodendrocytes) are critical mediators of the inflammatory response and neural repair during ischemic stroke. Active components from the genus Vitex exert precise regulation on the functional states of these glial cells, attenuating detrimental responses and potentiating protective effects (Figure 5). Specifically, these active components can promote the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

Figure 5 Vitex plants protective mechanism against ischemic stroke in neural glial cells: Molecular mechanism diagram.↑ Arrow indicates that the target gene or protein is upregulated under the regulation of the limited component; ↓ Arrow indicates that the target gene or protein is downregulated under the regulation of the limited component.

Inhibition of M1 Phenotype Activation and Pro-Inflammatory Cytokine Release

Casticin inhibits the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway, attenuating the phosphorylation of JAK2 (p-JAK2) and nuclear translocation of STAT3.¹²⁶ This further downregulates the transcriptional and expression of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS), thereby mitigating the toxicity of NO (nitric oxide); as excessive NO reacts with ·O₂⁻ to form ONOO⁻, a highly toxic reactive nitrogen species. Meanwhile, vitexin inhibits the TLR4/NF-κB pathway, abrogating the LPS-induced inflammatory cascade triggered by Toll-like receptor 4 (TLR4) activation.¹²⁷ This not only reduces the nuclear translocation of the NF-κB p65 subunit but also suppresses the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E₂ (PGE₂). Vitexin activates peroxisome proliferator-activated receptor γ (PPAR-γ), promoting microglia expression of M2 phenotype markers (CD206, Arg-1) along with the secretion of anti-inflammatory cytokines, including interleukin-10 (IL-10) and transforming growth factor-β (TGF-β).^126,128 This restricts the propagation of local inflammation and enhances the survival of injured neurons.

Regulation of Astrocytes: Inhibition of Glial Scar Formation and Enhancement of Nutritional Support

First, active components derived from Vitex species inhibit excessive astrocyte activation and glial scar formation. Vitexin suppresses the Notch1 signaling pathway, attenuating the interaction between the Notch1 receptor and its ligand Jagged1, as well as the nuclear translocation of the Notch intracellular domain (NICD).¹²⁹ This further inhibits astrocyte proliferation and the expression of glial fibrillary acidic protein (GFAP), precluding overactivated astrocytes from secreting collagen fibers to form glial scars and alleviating the physical barrier that impairs axon regeneration. Second, these components augment neurotrophic factor secretion and metabolic support. Under ischemic stress, vitexilactone induces astrocytes to upregulate the expression and secretion of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF).¹³⁰ Upon binding to TrkB and Ret receptors on the neuronal plasma membrane, BDNF and GDNF activate the PI3K/Akt pathway, thereby suppressing neuronal apoptosis. Additionally, astrocytes upregulate glutamate transporter 1 (GLT-1), facilitating the clearance of excessive extracellular glutamate and mitigating excitotoxicity.¹³¹ Active components from Vitex species also exert beneficial effects on oligodendrocytes by preserving myelin integrity and promoting remyelination. Vitexin activates the Nrf2/HO-1 signaling pathway, mitigating ischemia-induced oxidative stress in oligodendrocytes and sustaining their viability.¹²² Moreover, Vitex-derived components upregulate the expression of myelin-associated proteins, including myelin basic protein (MBP) and proteolipid protein (PLP).¹³² They also activate the Kir4.1 potassium channel in oligodendrocyte precursor cells (NG2 glia), facilitating the differentiation of NG2 glia into mature oligodendrocytes, restoring ischemia-impaired myelin structures, and preserving axonal conduction integrity.

Regarding the frontier mechanisms of active ingredients in Vitex plants inhibiting ferroptosis and regulating astrocyte function, some studies have indicated that isovitexin reduces LPS-induced NLRP3/caspase-1/GSDMD pathway activation in BV2 cells, decreasing the release of pro-inflammatory cytokines (TNF-α, IL-1) by more than 50%.⁸⁷

Repair Mechanisms of Vascular Endothelial Cells and BBB of Active Components from the Genus Vitex

Ischemic stroke induces vascular endothelial cell injury, BBB disruption, and cerebral microcirculation dysfunction. Active components from the genus Vitex enhance cerebral blood supply and tissue perfusion by preserving the function of vascular endothelial cells, repairing the BBB, and facilitating angiogenesis (Figure 6). Vitexin activates the PI3K/Akt signaling pathway to induce vascular endothelial cells to upregulate expression of tight junction proteins,¹³³ including Occludin, zonula occludens-1 (ZO-1), and Claudin-5, thereby augmenting intercellular junction integrity.¹³⁴ Meanwhile, these components suppress the expression and enzymatic activity of matrix metalloproteinase-9 (MMP-9), attenuate the MMP-9-mediated degradation of tight junction proteins, reduce BBB permeability, and mitigate the leakage of macromolecular substances (Evans Blue, EB) into the brain parenchyma.¹³⁵ Casticin maintains the structural integrity of the BBB by upregulating Bcl-2 and downregulating Bax,¹³⁶ thereby suppressing vascular endothelial cell apoptosis.

Figure 6 Vitex plants protective mechanism against vascular endothelial damage in ischemic stroke: Molecular mechanism diagram.↑ Arrow indicates that the target gene or protein is upregulated under the regulation of the limited component; ↓ Arrow indicates that the target gene or protein is downregulated under the regulation of the limited component.

Vitex-derived active components suppress the NF-κB pathway to downregulate the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelial cells.¹³⁷ This abrogates the adhesion of peripheral leukocytes (eg, neutrophils, monocytes) to vascular endothelial cells and their transmigration cross the BBB, thereby attenuating inflammatory cell infiltration and cerebral parenchymal inflammatory injury. Vitexin activates endothelial nitric oxide synthase (eNOS) to promote the NO production,¹³⁸ which dilates blood vessels, inhibits platelet aggregation, scavenges ROSe, and mitigates oxidative injury to vascular endothelial cells.

Vitexin upregulates the expression of vascular endothelial growth factor (VEGF). Upon binding of VEGF to VEGF receptor 2 (VEGFR2) on the vascular endothelial cell surface, it activates the PI3K/Akt/eNOS signaling pathway, facilitating the proliferation, migration, and tube formation of endothelial cells.¹³⁹ Additionally, vitexin upregulates the expression of angiopoietin-1 (Ang-1) to augment the stability of newly formed blood vessels and inhibit vascular leakage.¹⁴⁰ By promoting angiogenesis and vasodilation, vitexin increases blood perfusion in the ischemic region, shortens the duration of cerebral ischemia-hypoxia, supplies sufficient oxygen and nutrients to neurons and glial cells, and suppresses the progression of ischemic injury.

Regarding the effective components of Vitex species in inhibiting ferroptosis and regulating pyroptosis, some studies have pointed out that vanillic acid confers ferroptosis resistance in endothelial cells by upregulating SLC7A11 expression,¹⁴¹ this maintains GSH levels and inhibits H2O2-induced endothelial ferroptosis, while vitexin mediates epigenetic regulation of angiogenesis, it promotes histone acetylation in the VEGF promoter region by inhibiting HDAC1, thereby enhancing VEGF transcription and angiogenesis.^142,143

Research Value and Development Prospects of Unique Components in Vitex L

Vitex L. harbors specific components with structural uniqueness and targeted anti-ischemic stroke activity, which are the core basis for its therapeutic advantages and research value.

Vitexilactone

As a diterpenoid lactone unique to Vitex (eg, Vitex trifolia), vitexilactone exhibits potent antioxidant activity (IC50 = 15.3 μM for DPPH scavenging) and neuroprotective effects. Preclinical studies show it promotes astrocyte secretion of BDNF/GDNF via activating the PI3K/Akt pathway, may inhibiting neuronal apoptosis.¹⁴⁴ Its unique structure (C22H34O5) enables specific binding to Keap1 (molecular docking binding energy = −7.2 kcal/mol), enhancing Nrf2 nuclear translocation more efficiently than common flavonoids. Future directions include verifying its inhibition of ferroptosis via regulating GPX4 expression and optimizing synthesis to improve yield.

Rotundifuran

This diterpenoid (C22H34O4) is enriched in Vitex rotundifolia, with demonstrated vascular protective effects. Rotundifuran (10–50 μM) improves HUVEC barrier function by 35% via upregulating Occludin/ZO-1.^119,145 Unlike non-specific vascular protectants, it specifically targets VEGFR2 to promote angiogenesis without inducing abnormal vascular proliferation.¹¹⁹ Its mechanism involves inhibiting MMP-9 activity and reducing BBB permeability, making it a potential candidate for BBB repair.⁹⁸

Casticin

A flavonoid unique to Vitex (Vitex negundo, Vitex agnus-castus), casticin inhibits microglial M1 polarization via targeting JAK2/STAT3 (p-JAK2 reduction by 60%) and NLRP3 inflammasome activation (IL-1β release reduction by 55%).¹⁰ It also blocks neuronal excitotoxicity by binding to NMDA receptors (binding energy = −6.9 kcal/mol), synergizing with vitexin to enhance neuroprotection.⁷⁹ Clinical translation potential is supported by its good safety profile (LD50 = 150 mg/kg in mice) and potential for oral formulation.¹²¹

These unique components, together with other common active ingredients, form a “multi-component, multi-target” therapeutic network, highlighting the irreplaceable value of Vitex L. in anti-ischemic stroke research.

Discussion

Scientific Evidence and Mechanistic Prospects for Vitex in Ischemic Stroke Treatment

Accumulating preclinical studies provide compelling evidence for the efficacy of Vitex L. species against ischemic stroke. In vitro models (SH-SY5Y, BV2, HUVEC cells) and in vivo models (rat/mouse MCAO, BCCAO/R) have confirmed that Vitex-derived components reduce cerebral infarct volume, enhance neurological function (mNSS/NIHSS), and attenuate key pathological insults (oxidative stress, inflammation, BBB disruption). Mechanistically, the “multi-cellular target, multi-pathway regulation” characteristic is well-documented: these components target neurons to block the “oxidative stress-excitotoxicity-apoptosis” cascade (via Nrf2/ARE, PI3K/Akt, and NMDA receptor inhibition), regulate glial cells to balance inflammation and tissue repair (via PPAR-γ, Notch1, and Kir4.1 pathways), and protect vascular endothelial cells to restore BBB integrity and cerebral perfusion (via PI3K/Akt-tight junctions signaling and VEGF/Ang-1-mediated angiogenesis).

Current preclinical evidence confirms Vitex L.’s anti-ischemic stroke efficacy, with a mechanistic network spanning both classic and frontier pathways. Future efforts should include validating ferroptosis and pyroptosis regulation, this could involve focusing on vitexin’s role in the ACSL4/TFR1 axis and casticin’s impact on the NLRP3 inflammasome via molecular dynamics simulation, decoding intercellular communication by exploring how astrocyte-derived lncRNA MALAT1 coordinates with neuronal miR-107 to modulate synaptic remodeling, and clarifying epigenetic mechanisms through investigating component-mediated histone modifications (eg, acetylation) and non-coding RNA interactions within ischemic brain tissue.

However, mechanistic understanding of these effects remains incomplete. Future research directions should focus on: (1) Exploring underinvestigated pathological pathways, such as ferroptosis (ie, whether vitexin regulates ACSL4/TFR1) and autophagy (the role of betulinic acid in SIRT1/FoxO1-mediated autophagy balance);¹⁴⁶ (2) Investigating epigenetic regulation, including the crosstalk between Vitex-derived components and non-coding RNAs (miR-107, lncRNA MALAT1) or histone modifications (acetylation) in ischemic neurons;¹⁴⁷ (3) Elucidating intercellular communication mechanisms, such as how astrocyte-derived BDNF/GDNF crosstalk with neuronal TrkB/Ret receptors to promote synaptic remodeling, and how microglial M2 polarization modulates oligodendrocyte differentiation.¹⁴⁸ These investigations will further expand the mechanistic network underlying the anti-ischemic stroke effects of Vitex L. species.

Active Components of Vitex and Progress in Extraction/Application

Vitex L. species harbor diverse active components with distinct anti-ischemic stroke potential, which can be classified based on evidence strength and application potential: High-evidence components, flavonoids (vitexin, isovitexin, casticin, luteolin), are the most specific and potent, with vitexin and isovitexin exhibiting potent BBB-protective effects and NMDA receptor inhibitory activity; terpenoids (oleanolic acid, betulinic acid, aucubin) exert robust anti-apoptotic and anti-inflammatory effects; simple phenols (protocatechuic acid, ferulic acid) possess prominent antioxidant activity but low abundance in Vitex species. Potential components, phenylpropanoids (3,5-di-O-caffeoylquinic acid) and steroids (progesterone, β-sitosterol), demonstrate efficacy in single experimental models but necessitate further validation in stroke-specific models.

Although some key active components (eg, protocatechuic acid) have low abundance in Vitex plants, optimized extraction techniques (ultrasonic-assisted extraction), chemical synthesis, and nanodelivery systems (eg, vitexin microcapsules) have effectively overcome this limitation. Moreover, the unique components of Vitex (eg, vitexilactone) and the synergistic effects of multi-component mixtures highlight the irreplaceable value of Vitex-centered research.

Limitations of This Review and Future Improvements

This review comprehensively collates existing research on Vitex in ischemic stroke treatment, but several limitations should be noted: First, most studies rely on single-cell lines or single animal models (Sprague-Dawley rats, C57BL/6 mice), lacking multi-center, large-sample animal experiments to verify reproducibility. For example, although syringaldehyde reduces amyloid plaques in APP/PS1 mice, its efficacy in MCAO models (the gold standard for ischemic stroke) has not been validated, leading to uncertain translational value.¹⁴⁹ Second, while core pathways (Nrf2, NF-κB) are well-studied, molecular dynamics verification of component-target binding (the interaction between vitexin and NMDA receptors at the atomic level) is scarce, and the cross-talk between pathways (how PI3K/Akt interacts with NF-κB to regulate endothelial cell apoptosis) is not clarified. Third, many components (progesterone, β-sitosterol) are widely distributed in other plants, and Vitex-specific components (vitexilactone, rotundifuran) have limited research. Additionally, only progesterone has entered early clinical trials, while other high-evidence components (apigenin, oleanolic acid) lack human safety and efficacy data, hindering clinical translation. Fourth, Vitex extracts contain multiple active components, but current studies focus on monomers rather than exploring how flavonoids, terpenoids, and phenols synergistically regulate pathology. This limits the development of holistic herbal therapies based on Vitex plants.

Single-cell sequencing technology holds great promise for deciphering the cell-type-specific regulatory effects of diverse active components from Vitex L. on ischemic brain tissue, enabling precise identification of subtype-specific responses of core cell populations (eg, microglia, astrocytes, and vascular endothelial cells) and clarification of their distinct functional alterations under the intervention of Vitex-derived compounds. This technology can further uncover the synergistic regulatory mechanisms of multi-component mixtures from Vitex L., such as the crosstalk between flavonoids and terpenoids in modulating inflammatory cascades, ferroptosis, and metabolic pathways across different cell subtypes, while identifying hub genes and key signaling axes analogous to the Spp1-mediated regulation revealed in KBA-Z-GS studies.¹⁵⁰ Future research should address these limitations by conducting multi-model validation, integrating multi-omics techniques (proteomics, metabolomics) to decode mechanisms, developing Vitex-specific component screening platforms, and designing Phase I/II clinical trials for promising candidates. These efforts will promote the transformation of Vitex plants from traditional medicine to evidence-based anti-ischemic stroke therapies.

Conclusion

Vitex L. exhibits significant anti-ischemic stroke potential, with unique components and multi-pathway regulation as core advantages. Future research should focus on multi-center validation, synergistic mechanism exploration, and clinical trials of high-evidence components to advance translation.