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

Abstract

Introduction

The genus Vitex L. (Verbenaceae) comprises ~250 species globally, with long-standing ethnopharmacological value. Notably, only Traditional Chinese Medicine (TCM) explicitly applies Vitex negundo for ischemic stroke (documented in classic Materia medica), distinguishing it from other regional uses (eg, menstrual disorders, malaria) and providing a unique basis for anti-stroke research.

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

Over 200 constituents were identified, with flavonoids (vitexin, isovitexin, casticin), terpenoids (vitexilactone, rotundifuran), and phenols as core active components. High-evidence compounds (validated by both in vitro and in vivo experiments) such as vitexin (10–50 mg/kg) reduced rat MCAO infarct volume by 30–40% via blocking NMDA receptor-mediated Ca²⁺ overload. Mechanistically, components target neurons, glia, and vascular endothelial cells, regulating both classic pathways (Nrf2, NF-κB, PI3K/Akt) and frontier mechanisms (ferroptosis, pyroptosis, epigenetic regulation). Synergistic effects of multi-component mixtures and optimized extraction/synthesis address low-content challenges.

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.

Graphical Abstract

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

Traditional Medicine Names	Nations	Plants of the Genus Vitex L.	Medicinal Parts	Indications and Functions
Ayurvedic medicine	India	Vitex negundo (Nirgundi)	Fruits and leaves	Irregular menstruation, dysmenorrhea, skin infections, decreased sexual function
Oriental Medicine	Japan and Vietnam	Vitex trifolia	Fruits	Headache, redness and swelling of the eyes, cough, cold
TCM	China	Vitex negundo	Roots, stems, leaves, fruits	Headache, dizziness, joint pain, cough, asthma, stroke
Mediterranean Medicine	Greece, as well as countries in Europe, the United States, and Oceania	Vitex agnus-castus (Chaste Tree)	Fruits and leaves	Postpartum haemorrhage and uterine disease
Medicine in West Africa and South Africa	South Africa	Vitex doniana	Stem pith	Malaria, intestinal diseases
Medicine in East Africa	Madagascar	Vitex trifolia	Roots and leaves	Otitis media, joint pain, high blood sugar
Medicine indigenous peoples of Central America	Guatemala	Vitex gaumeri	Stem pith	Snake bites and ulcers, bronchial asthma

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

No.	English Name	Formula	Exact Molecular Wight	Type	Reference
Simple Phenols and Organic Acids
1	p-Hydroxybenzoic acid	C7H6O3	138.121	Phenolic benzoic acids	[32]
2	Protocatechuic acid	C7H6O4	154.1201	Phenolic benzoic acids	[32]
3	Vanillic acid	C8H8O4	168.15	Phenolic benzoic acids	[32]
4	Methyl 4-hydroxybenzoate	C8H8O3	152.147	Simple phenols	[33]
5	p-Ethylbenzoic Acid	C9H10O2	150.17	Simple benzoic acids	[34]
6	Vanillin	C8H8O3	152.147	Phenolic aldehydes	[33]
7	Trans-p-coumaryl aldehyde	C9H8O2	148.16	Phenolic aldehydes	[33]
8	Coniferaldehyde	C10H10O3	178.187	Phenolic aldehydes	[33]
9	p-Hydroxybenzaldehyde	C7H6O2	122.121	Phenolic aldehydes	[33]
10	Ferulic acid	C10H10O4	194.184	Phenolic aldehydes	[33]
11	Syringaldehyde	C9H10O4	182.1733	Phenolic aldehydes	[33]
12	p-Hydroxyphenethylalcohol	C8H10O2	138.1638	Simple phenols	[34]
13	Sinapaldehyde	C11H12O4	208.213	Phenolic aldehydes	[33]
14	5,7-dihydroxychromone	C9H6O4	178.144	Simple phenols	[35]
15	Frambinone	C10H12O2	164.2036	Simple phenols	[35]
16	4-Hydroxybenzoic acid	C7H6O3	138.1207	Phenolic benzoic acids	[36]
17	3,4-Dihydroxybenzoic acid	C7H6O4	154.1201	Phenolic benzoic acids	[36]
18	Syringate	C9H10O5	198.1727	Phenolic benzoic acids	[36]
19	3-methoxyl-4-hydroxybenzcicacid	C8H8O4	168.1467	Phenolic benzoic acids	[36]
20	Dibutyl phthalate	C16H22O4	278.3435	Phenolic benzoic acids	[36]
21	Citronellal	C10H18O	154.2493	Phenolic aldehydes	[37]
22	Coronaricacid	C18H32O3	296.4449	Simple acids	[36]
23	Ricinolicacid	C18H34O3	298.4608	Simple acids	[36]
Phenylpropanoids
1	3,5-di-O-caffeylquinic acid	C25H24O12	516.4	Phenylpropanoid	[37]
2	Helichrysetin	C16H14O5	286.28	Phenylpropanoid	[37]
3	2′-O-Methylhelichrysetin	C17 H16 O5	300.31	Phenylpropanoid	[37]
4	3,4-Dihydroxy phenyl lactic acid methylester	C10H12O5	212.202	Phenylpropanoid	[38]
5	2-methoxy-4-(3-methoxy-1-propenyl)-phenol	C11H14O3	194.23	Phenylpropanoid	[34]
6	Coniferyl aldehyde	C10H10O3	178.188	Phenylpropanoid	[34]
7	5,7-dihydroxy chromone	C9H6O4	178.144	Phenylpropanoid	[34]
Lignans
1	Hinokiol	C20H30O2	302.451	Lignans	[39]
2	Vitexdoin D	C19H16O6	340.3	Lignans	[39]
3	Vitexdoin A	C19H18O6	342.3426	Lignans	[39]
4	Detetrahydroconidendrin	C20H16O6	352.3	Lignans	[39]
5	(+)-Sesamin	C20H18O6	354.3604	Lignans	[39]
6	Paulownin	C20H18O7	370.353	Lignans	[39]
7	4β-Hydroxyasarinin	C22H34O5	387	Lignans	[12]
8	Detetrahydroconidendrin	C20H16O6	352.3	Lignans	[36]
9	4-oxosesamin	C20H16O7	368.3	Lignans	[36]
10	L-sesamin	C20H18O6	354.4	Lignans	[36]
11	Paulownin	C20H18O7	370.353	Lignans	[36]
12	Ligballinol	C18H18O4	298.3	Lignans	[36]
13	(+)-Pinoresinol	C20H22O6	358.39	Lignans	[36]
14	Balanophonin	C20H20O6	356.369	Lignans	[36]
15	Vitrofolal A	C20H18O5	338.359	Lignans	[33]
16	Vitrofolal B	C20H18O6	354.358	Lignans	[33]
17	Vitrofolal C	C21H18O6	366.369	Lignans	[33]
18	Vitexfolin C	C23H26O10	462.451	Lignans	[32]
19	Vitexfolin A	C25H28O11	504.488	Lignans	[32]
20	Vitexfolin B	C25H28O11	504.488	Lignans	[32]
21	Methyl rosmarinate	C19H18O8	374.345	Lignans	[40]
22	5-O-caffeoylquinicacidmethylester	C17H20O9	368.338	Lignans	[40]
23	(+)-viteralone	C15H14O8	322.269	Lignans	[32]
24	Furanoeremophilane	C15H22O	218.34	Lignans	[32]
25	Grevilloside G	C14H20O8	316.306	Lignans	[40]
26	3,4,5-Tricaffeoylquinic acid	C34H30O15	678.599	Lignans	[38]
27	(+)-Epipinoresinol-4-O-β-D-glucoside	C25H30O10	490.505	Lignans	[38]
28	4-Methoxy-Methyl rosmarinate	C20H20O8	388.372	Lignans	[38]
29	Vitedoamine A	C21H20O10	432.384	Lignans	[36]
30	Vitecannaside B	C26H28O11	516.4939	Lignans	[12]
31	Agnuside	C22H26O11	466.4352	Lignans	[37]
32	Negundin A	C20H16O6	352.3374	Lignans	[37]
33	Apigenin	C15H10O5	270.2369	Lignans	[37]
34	Neoandrographolide	C26H40O8	480.591	Lignans	[37]
35	Chrysosplenol D	C18H16O8	360.3148	Lignans	[37]
36	Atractylenolide I	C15H18O2	230.3022	Lignans	[39]
37	2α,19α-dihydroxyurs-3-oxo-urs-12-en- 28-oicacid	C30H46O5	486.6832	Lignans	[39]
38	Viterotulin B	C22H34O5	378.5024	Lignans	[40]
39	Ficusal	C18H18O6	330.3319	Lignans	[40]
40	24-hydroxyoleanolicacid	C30H48O4	472.6997	Lignans	[36]
41	Eupatrin	C18H16O7	344.3154	Lignans	[35]
42	5,4′-dihydroxy-3,6,7-trimethoxyflavone	C18H16O7	344.3154	Lignans	[35]
43	5,6,7,8,4′-pentamethoxyflavone	C20H20O7	372.3686	Lignans	[35]
44	5-Hydroxy-3′,4′,6,7-tetramethoxyflavone	C19H18O7	358.342	Lignans	[35]
Flavonoids and Flavonoid Glycosides
1	Luteolin	C15H10O6	286.236	Flavonoids	[34]
2	5,7,2′,5′-Tetrahydroxyflavone	C15H10O6	286.24	Flavonoids	[34]
3	7,2′-Dihydroxy-4′-methoxyisoflavanol	C16H16O5	288.29	Dihydroisoflavonoids	[32]
4	3-O-Methylquercetin	C16H12O7	316.26	Flavonoids	[34]
5	Isorhamnetin	C16H12O7	316.265	Flavonoids	[34]
6	3′,4′,5-Trihydoxyl-3,7-dimethoxyfiavoe	C17H14O7	331	Flavonoids	[34]
7	Corymbosin	C19H18O7	358.342	Flavonoids	[34]
8	(5-Hydroxy-6,7,3′,4′- tetramethoxyflavone	C19H18O7	358.347	Flavonoids	[34]
9	3,5–Dihydroxy–6,7,3′4′–tetramethoxyflavone	C19H18O8	374.3	Flavonoids	[32]
10	Casticin	C19H18O8	374.341	Flavonoids	[32]
11	Gardenin A	C21H22O9	418.394	Isoflavonoids	[32]
12	Vitexin	C21H20O10	432.3775	Flavonoid glycosides	[40]
13	Isovitexin	C21H20O10	432.378	Flavonoid glycosides	[40]
14	Cynaroside	C21H20O11	448.3769	Flavonoid glycosides	[40]
15	Orientin	C21H20O11	448.3769	Flavonoid glycosides	[40]
16	Isoorientin	C21H20O11	448.3769	Flavonoid glycosides	[40]
17	Quercetin	C15H10O7	302.23	Flavonols	[40]
18	Gardenin B	C19H18O7	358.342	Flavonoids	[39]
19	Vitexdoin E	C19H16O6	340.3	Isoflavonoids	[32]
20	Hesperidin	C28H34O15	610.561	Flavonoid glycosides	[32]
21	Penduletin	C18H16O7	344.315	Flavonoids	[32]
22	Luteolin-7-O-β-D-glucoside	C21H18O12	462.36	Flavonoid glycosides	[40]
23	Luteolin-7-O-β-D-glucopyranoside	C21H20O11	448.38	Flavonoid glycosides	[32]
24	Luetolin-3′-O-β-D-glucuronide	C21H18O12	462.363	Flavonoid glycosides	[32]
25	Apigenin-7-O-β-D-glucoside	C21H20O10	432.381	Flavonoid glycosides	[32]
26	Kaempferol3-O-β-D-glucopyranoside	C21H20O11	448.38	Isoflavonoids	[32]
27	Luteolin-4′-o-glucoside	C21H20O11	448.38	Flavonoid glycosides	[38]
28	4′,5-dihydroxy-3,6,7-trimethoxyflavone	C18H16O7	344.321	Flavonoids	[36]
29	Isoorientin6′′-O-caffeate	C30H26O14	610.524	Isoflavonoids	[38]
30	Kaempferol	C15H10O6	286.2363	Flavonols	[37]
31	5,3′-dihydroxy-6,7,4′-trimethoxy flavanone	C18H18O7	346.3313	Flavonoids	[39]
Terpenoids and Saponins
1	Vitexilactone	C22H34O5	378.502	Diterpenoid lactones	[12]
2	Previtexilactone	C22H34O5	378.5023	Diterpenoid lactones	[12]
3	Friedelin	C30H50O	426.72	Pentacyclic triterpenoids	[36]
4	Vitexifolin A	C20H34O	290.491	Diterpenoids	[38]
5	Rotundifuran	C22H34O4	352.51	Diterpenoids	[38]
6	Limonidilactone	C20H26O4	330.424	Diterpenoid lactones	[38]
7	Dihydrosolidagenone	C20H30O3	318.457	Diterpenoids	[38]
8	Vitexlactam A	C22H35NO4	377.525	Diterpenoids	[38]
9	Prerotundifuran	C22H34O4	362.51	Diterpenoids	[38]
10	Phytol	C20H40O	296.539	Diterpenoids	[38]
11	Isophytol	C20H40O	296.531	Diterpenoids	[38]
12	Aucubin	C15H22O9	346.332	Monoterpene glycosides	[33]
13	Negundoside	C23H28O12	496.465	Monoterpene glycosides	[33]
14	6′-p-hydroxybenzoy-lmussaenosidic acid	C24H30O14	542.49	Sesquiterpene glycosides	[33]
15	Nishindaside	C23H30O12	498.48	Sesquiterpene glycosides	[33]
16	Mussaenosidic acid	C16H24O10	376.358	Monoterpene glycosides	[33]
17	Beta-Amyrin	C30H50O	426.729	Pentacyclic triterpenoids	[36]
18	Oleanolic acid	C30H48O3	456.711	Pentacyclic triterpenoids	[36]
19	Acetyl oleanolic acid	C32H50O4	498.748	Pentacyclic triterpenoids	[36]
20	Friedelan-3α-ol	C30H52O	428.745	Pentacyclic triterpenoids	[36]
21	Betulinic acid	C30H48O3	456.711	Pentacyclic triterpenoids	[36]
22	Ursolic acid	C30H48O3	456.7	Pentacyclic triterpenoids	[36]
23	2α-hydroxy-ursolic acid	C30H48O3	457.7	Pentacyclic triterpenoids	[36]
24	α-Pinene	C10H16	136.238	Monoterpenes	[39]
25	Sabinene	C10H16	136.238	Monoterpenes	[39]
26	1,8-Cineole	C10H18O	154.253	Monoterpenes	[39]
27	Cinene	C10H16	136.238	Monoterpenes	[39]
28	Beta-pinene	C10H16	136.238	Monoterpenes	[39]
29	Alpha-Terpineol	C10H18O	154.253	Monoterpenes	[39]
30	4-Terpineol	C10H18O	154.249	Monoterpenes	[39]
31	Camphor	C10H16O	152.237	Monoterpenes	[39]
32	Camphene	C10H16	136.238	Monoterpenes	[39]
33	γ-Terpinene	C10H16	136.238	Monoterpenes	[39]
34	Linalool	C10H18O	154.253	Monoterpenes	[39]
35	Cineole	C10H18O	154.253	Monoterpenes	[39]
36	Citral	C10H16O	152.237	Monoterpenes	[39]
37	α-Phellandrene	C10H16	136.238	Monoterpenes	[39]
38	Bornyl acetate	C12H20O2	196.29	Monoterpenes	[39]
39	Sabinene	C10H16	136.238	Monoterpenes	[39]
40	Dihydromyrcene	C10H18	138.2499	Monoterpenes	[39]
41	Terpinolene	C10H16	136.234	Monoterpenes	[39]
42	(+)-Viridiflorol	C15H26O	222.372	Sesquiterpenes	[39]
43	Myzodendrone	C16H22O8	342.344	Monoterpene glycosides	[32]
44	Abietatrien-3β-ol	C20H34O	290.489	Diterpenoids	[36]
45	Obtusalin	C30H50O2	442.726	Pentacyclic triterpenoids	[36]
46	2,3,24-Trihydroxy-12-ursen-28-oic acid	C30H48O5	488.6991	Pentacyclic triterpenoids	[33]
47	Maslinic acid	C30H48O4	472.6997	Pentacyclic triterpenoids	[33]
48	2,3,19,23-tetrahydroxybearing-12-en-28-acid	C30H48O6	504.6985	Pentacyclic triterpenoids	[33]
49	Carene	C10H16	136.234	Monoterpenes	[12]
50	Abieta-7,13-diene	C20H32	272.4681	Diterpenoids	[12]
51	α-Thujene	C10H16	136.234	Monoterpenes	[12]
52	Germacrened	C15H24	204.3511	Sesquiterpenes	[12]
53	TerpinylAcetate	C12H20O2	196.286	Monoterpenes	[12]
54	Bornylacetate	C12H20O2	196.286	Monoterpenes	[12]
55	γ-Terpinene	C10H16	136.234	Monoterpenes	[37]
56	Linalylacetate	C12H20O2	196.286	Monoterpenes	[37]
57	Geraniol	C11H20O	168.2759	Monoterpenes	[35]
58	Citronellol	C11H22O	170.2918	Monoterpenes	[37]
59	Lagundinin	C9H14O3	170.2057	Monoterpene lactones	[37]
60	Euscaphicacid	C30H48O5	488.6991	Pentacyclic triterpenoids	[34]
61	Enoxolone	C30H46O4	470.6838	Pentacyclic triterpenoids	[34]
62	Sclareol	C20H36O2	308.4986	Diterpenoids	[36]
63	Viteagnusin I	C22H34O6	394.5018	Diterpenoids	[36]
64	Moslene	C10H16	136.234	Monoterpenes	[33]
65	Viteagnusin F	C23H38O7	426.5436	Diterpenoids	[36]
66	Viteagnusin G	C23H38O7	426.5436	Diterpenoids	[36]
67	Isolophanthin A	C20H30O2	302.451	Diterpenoids	[36]
68	Vitexifolin D	C19H30O4	322.4391	Diterpenoids	[36]
69	Trisnor-γ-lactone	C19H30O4	322.4391	Diterpenoids	[36]
70	Isoambreinolide	C17H28O2	264.403	Diterpenoids	[36]
71	Vitexifolin E	C20H32O2	304.4669	Diterpenoids	[36]
72	Vitexifolin B	C20H36O3	324.498	Sesquiterpene lactones	[35]
73	Vitexifolin B	C20H36O3	324.505	Diterpenoids	[36]
Steroids
1	Inokosterone	C27H44O7	480.642	Steroid	[39]
2	Ajugasterone C	C27H44O7	480.642	Steroid	[37]
3	Calonysterone	C27H40O7	476.67	Steroid	[37]
4	β-sitosterone	C29H50O	414.718	Steroid	[37]
5	β-Sitosterol Acetate	C31H52O2	456.755	Steroid	[37]
6	Stigmasterone	C29H46O	410.686	Steroid	[37]
7	Progesterone	C21H30O2	314.469	Steroid	[37]
8	Testosterone solution	C19H28O2	288.431	Steroid	[37]
9	Androstenedione	C19H26O2	286.409	Steroid	[41]
10	Stigmast-4-en-6β-ol-3-one	C29H48O2	428.699	Steroid	[38]
11	Ergosterol peroxide	C28H44O3	428.656	Steroid	[38]
12	7-oxositosterol	C29H48O2	428.699	Steroid	[38]
Other classes compounds
1	Cineole	C4H5BrCl3F	258.336	Haloalkanes	[35]
2	Caffeic acid	C9H8O4	180.159	Phenylpropanoids	[12]
3	Benzylbeta-d-glucopyranoside	C13H18O6	270.282	Polyphenols	[38]
4	Methoxsalen	C12H8O4	216.193	Coumarins	[34]
5	2,6-dimethoxy-1,4-benzoquinone	C8H8O4	168.149	Para-benzoquinones	[34]
6	Isofraxidin	C11H10O5	222.1941	Coumarins	[12]
7	Karakoline	C22H35NO4	377.5176	Alkaloids	[34]
8	Dibutylsebacate	C18H34O4	314.4602	Alkyl acid esters	[40]
9	Pyrogallol	C6H6O3	126.11	Polyphenols	[40]
10	Panaxydol	C17H24O2	260.3713	Simple alcohols	[40]

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).

Table 3.

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

Compound Name	Model	Dose Administered	Key Targets (Indicators)	Intervention Outcomes
Protocatechuic acid	MCAO model in rats	25 mg/kg intraperitoneal injection for 7 days	IL-1β, TNF-α, Bax/Bcl-2, Caspase-3, Occludin and ZO-1	28.6% reduction in infarct volume and significant improvement in neurological function scores (mNSS)26,27,45
Vanillic acid	Rat BCCAO/R model	10-50 mg/kg oral pretreatment for 2 weeks	MMP-9, ROS, SOD	25-30% reduction in cerebral infarction volume and improved anxiety-like behaviors28,29
Ferulic acid	MCAO model in rats	Intraperitoneal injection of 100 mg/kg	Nrf2, ACSL4, TFR1, GSH	40% reduction in infarct volume and improved neurological function (NIHSS)30,31
Syringaldehyde	MCAO model in rats	Intraperitoneal injection of 50 mg/kg	ROS, MDA, Bcl-2/Bax, Caspase-3	35% reduction in infarct volume and significant improvement in neurological function scores42
p-Hydroxybenzyl	MCAO model in rats	Oxidative product 3,4-DHBA 50 mg/kg	3,4-DHBA	Inferiority of infarct volume reduced, mNSS improvement43,44
3,5-di-O-caffeoylquinic acid	SAMP8 mice	6.7 mg/kg/day oral administration	NF-κB, TNF-α and IL-1β	Improved cognitive function and reduced intracerebral oxidative stress markers47
Apigenin	MCAO model in rats	Intraperitoneal injection of 10–50 mg/kg	MMP-9, TNF-α and IL-1β	30-40% reduction in infarct volume and significant improvement in neurological function scores (mNSS)55–59
Luteolin	Rat MCAO model	10-50 mg/kg, intraperitoneal injection	MMP-9, TLR4/NF-κB, Nrf2	30-40% reduction in infarct volume; significant improvement in mNSS; promotion of axonal myelin regeneration34,77,78
Quercetin	Mouse MCAO model	30 mg/kg, oral administration	Nrf2/STAT3, SLC6A3	0~50% reduction in infarct volume; improvement in neurological function70,80
Isorhamnetin	C57BL/6 mouse MCAO model	1-10 mg/kg, intraperitoneal injection	NF-κB, Bax/Bcl-2, Occludin, ZO-1, Claudin-5	25-30% reduction in infarct volume; alleviation of cerebral edema; improvement in neurobehavioral performance83
Kaempferol	Rat MCAO model	5-20 mg/kg, intraperitoneal injection	Nrf2/ARE, NF-κB p65, iNOS, COX-2	20-30% reduction in infarct volume; improvement in neurological function scores; regulation of lipid metabolism72
Aucubin	Gerbil global cerebral ischemia model	10 mg/kg, intraperitoneal injection	TLR4/NF-κB, IL-1β, TNF-α, Bax/Bcl-2	40% increase in neuronal survival rate in hippocampal CA1 region; improvement in neurological function scores; regulation of lipid metabolism92,93
Oleanolic acid	Rat MCAO model	20-50 mg/kg, intraperitoneal injection	Nrf2/HO-1, SOD, GSH-Px, Caspase-3	25-30% reduction in infarct volume; alleviation of cerebral edema; good safety without obvious hepatotoxicity94,95
Betulinic acid	Mouse MCAO model	10-30 mg/kg, intraperitoneal injection	SIRT1/FoxO1, TNF-α, IL-1β	0~40% reduction in infarct volume; significant improvement in neurological function scores; regulation of amino acid metabolism96,97,100
Progesterone	Rat MCAO model	10-50 mg/kg, intraperitoneal injection	NF-κB, Bax/Bcl-2, Occludin, ZO-1, PI3K/Akt	30-40% reduction in infarct volume; significant improvement in mNSS; good safety; ongoing multicenter clinical trial101,102,115
Testosterone	Castrated rat MCAO model	10 mg/kg, subcutaneous implantation	AR, BDNF	Accelerated neurological function recovery; reduced astrocyte activation; improvement in behavioral performance; no significant effect on infarct volume103,104
β-Sitosterol	Rat intracranial aneurysm model	20-50 mg/kg, oral administration	TLR4/NF-κB, iNOS, COX-2, MMP-9	25-30% reduction in aneurysm volume; improvement in neurobehavioral performance; inhibition of astrocyte overactivation105,106
Stigmasterol	Mouse MCAO model	20-80 mg/kg, intraperitoneal injection	AMPK/mTOR, cytochrome c	20-30% reduction in infarct volume107
Cineole	Rat MCAO model	10-50 mg/kg, intraperitoneal injection	NF-κB, MAPK, ROS, MDA, Occludin, ZO-1	30-40% reduction in infarct volume; significant improvement in mNSS; regulation of amino acid metabolism; 75% brain-targeted delivery efficiency via nanoliposomes110,111
Caffeic acid	Rat PMCAO model	2 mg/kg, oral administration	Nrf2, TFR1, ACSL4, GSH, SOD, GSH-Px	40% reduction in infarct volume; improvement in neurological function scores; therapeutic window extended to 2 hours post-ischemia; synergistic reduction of hemorrhagic transformation risk with thrombolytics23,112

Table 4.

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

Compound Name	Model	Dose Administered	Key Targets (Indicators)	Intervention Outcomes
Protocatechuic acid	PC12 cells	5-20 uM	IL-1β, TNF-α, Bax/Bcl-2, Caspase-3, Occludin and ZO-1	Over 30% increase in cell survival rate; enhanced maintenance rate of mitochondrial membrane potential26,27,29
Vanillic acid	HUVEC cells	10-50 μM	MMP-9, ROS, SOD	40% improvement in vascular endothelial barrier function recovery rate28,29,45
Ferulic acid	SH-SY5Y cells	1-10 μM	Nrf2, ACSL4, TFR1, GSH	Over 50% increase in cell survival rate30,31
Syringaldehyde	HT22 neurons	5-20 μM	ROS, MDA, Bcl-2/Bax, Caspase-3	Over 30% increase in cell survival rate; activation of NRF-1 pathway for mitochondrial biosynthesis42
3,5-di-O-caffeoylquinic acid	SH-SY5Y cells	10-40 μM	Nrf2/ARE, HO-1, NQO1	Resistance to Aβ1-42 or NMDA-induced cell damage; upregulation of antioxidant enzymes46,47
Apigenin	SH-SY5Y cells	10-20 μM	Nrf2, Caspase-3	Over 50% increase in cell survival rate; inhibition of ROS generation55–59
Quercetin	HT22 neurons	10-20 μM	Cell apoptosis rate	40% reduction in cell apoptosis rate; maintenance of mitochondrial membrane potential70,80,81
Isorhamnetin	BV2 microglia	5-10 μM	NF-κB, NO, IL-6	Over 60% reduction in NO and IL-6 release83
Kaempferol	PC12 cells	5-10 μM	Cell survival rate	Over 30% increase in cell survival rate72
Vitexin	HUVEC cells	10-50 μM	N/A	Improved vascular endothelial barrier function; need for verification of BBB permeability85,87,116,117
Aucubin	BV2 microglia	1-10 μM	TLR4/NF-κB, NO, IL-6	Over 60% reduction in NO and IL-6 release92,93,118
Oleanolic acid	PC12 cells	5-20 μM	Cell survival rate	Over 30% increase in cell survival rate; derivative acetyl oleanolic acid shows stronger antioxidant activity94,95
Betulinic acid	HT22 neurons	5-10 μM	Apoptosis rate	40% reduction in cell apoptosis rate; derivative 3-O-acetyl betulinic acid shows stronger neuroprotective activity96,97,100
Rotundifuran	HUVEC cells	10-50 μM	N/A	Improved vascular endothelial barrier function119
Maslinic acid	U87MG cells	5-20 μM	ATP level	ATP level restored to 80% of normal value99
Cineole	HT22 neurons	5-20 μM	Membrane potential maintenance rate	30% increase in mitochondrial membrane potential maintenance rate110,111
Caffeic acid	SK-N-SH cells	1-10 μM	Cell survival rate	Over 50% increase in cell survival rate; derivative CAPE shows stronger antioxidant activity23,112
Panaxydol	PC12 cells	5-20 μM	Cell survival rate	Over 30% increase in cell survival rate; maintenance of mitochondrial membrane potential113
Isofraxidin	BV2 microglia	10-50 μM	MAPK (p38, ERK1/2), TNF-α, IL-6, NO	Over 60% reduction in NO release; downregulation of p38 and ERK1/2 phosphorylation114

Abbreviation: N/A, not applicable.

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.

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.

Funding Statement

This work was supported by Wu Jinjin National Inheritance Workshop for Veteran TCM Pharmacists Construction Project (Grant No. Guo Zhong Yi Yao Ren Jiao Han [2024] 255), Zhejiang Province Traditional Chinese Medicine Science and Technology Program (2025ZF051), Zhejiang Province Key Discipline Construction Project of Traditional Chinese Medicine (Clinical Chinese Pharmacy) (2024-XK-56).

Abbreviations

Akt, Protein Kinase B; Ang-1, Angiopoietin-1; Apaf-1, Apoptotic Protease Activating Factor-1; ARE, Antioxidant Response Element; Bax, Bcl-2-Associated X Protein; BBB, Blood-Brain Barrier; BCCAO/R, Bilateral Common Carotid Artery Occlusion/Reperfusion; Bcl-2, B-Cell Lymphoma 2; BDNF, Brain-Derived Neurotrophic Factor; COX-2, Cyclooxygenase-2; Cyt c, Cytochrome c; EB, Evans Blue; eNOS, Endothelial Nitric Oxide Synthase; GDNF, Glial Cell Line-Derived Neurotrophic Factor; GFAP, Glial Fibrillary Acidic Protein; GLT-1, Glutamate Transporter 1; GSH-Px, Glutathione Peroxidase; HO-1, Heme Oxygenase-1; ICAM-1, Intercellular Adhesion Molecule-1; IL, Interleukin; iNOS, Inducible Nitric Oxide Synthase; JAK2/STAT3, Janus Kinase 2/Signal Transducer and Activator of Transcription 3; Keap1, Kelch-Like ECH-Associated Protein 1; LPS, Lipopolysaccharide; MBP, Myelin Basic Protein; MCAO, Middle Cerebral Artery Occlusion; MDA, Malondialdehyde; MMP-9, Matrix Metalloproteinase-9; mNSS, Modified Neurological Severity Score; mPTP, Mitochondrial Permeability Transition Pore; NF-κB, Nuclear Factor-κB; NIHSS, National Institutes of Health Stroke Scale; NQO1, NAD(P)H: Quinone Oxidoreductase 1; Nrf2, Nuclear Factor Erythroid 2-Related Factor 2; OGD, Oxygen-Glucose Deprivation; OGD/R, Oxygen-Glucose Deprivation/Reperfusion; PGE₂, Prostaglandin E₂; PI3K, Phosphatidylinositol 3-Kinase; PLP, Proteolipid Protein; PMCAO, Permanent Middle Cerebral Artery Occlusion; PPAR-γ, Peroxisome Proliferator-Activated Receptorγ; ROS, Reactive Oxygen Species; SOD, Superoxide Dismutase; SAMP8, Senescence Accelerated Mouse-Prone 8; TLR4, Toll-Like Receptor 4; TNF-α, Tumor Necrosis Factor-α; VCAM-1, Vascular Cell Adhesion Molecule-1; VEGF, Vascular Endothelial Growth Factor; VEGFR2, Vascular Endothelial Growth Factor Receptor 2; ΔΨm, Mitochondrial Membrane Potential.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Chengqiong Xie and Jinjin Wu drafted and edited the manuscript, Ping Huang wrote manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Disclosure

The authors declare no conflict of interests.