Bio-effectiveness of the main flavonoids of Achillea millefolium in the pathophysiology of neurodegenerative disorders- a review.

The Achillea millefolium L. (Yarrow) is a common herb which is widely being used, worldwide. Achillea is being used for treatment of many disorders since centuries. It is considered safe for supplemental use and flavonoids such as kaempferol, luteolin and apigenin are of main constituents present in Achillea. Most of both antioxidant and anti-inflammatory properties of this herb have been attributed to its flavonoid content. Oxidative and inflammatory processes play important roles in pathogenesis of neurodegenerative diseases. Present review was aimed to review the latest literature evidences regarding application of Achillea and/or its three main flavonoid constituents on epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease and stroke.

Introduction

Achillea millefolium L. (yarrow), as a medicinal plant which grows worldwide, has been used for treatment of diseases and injuries since ancient historical era. Most often is called yarrow. Achillea by people and belongs to the family of Asteraceae and contains more than 100 species (1). Achillea has erect stems, with 30–60 cm height, herbaceous, perennial herb, simple or branched from the base, fern-like feathery leaves and pink-white as well as dense flowers (Figure 1) (2).

Figure 1

Achillea millefolium L.

For many years, Achillea has popularly been used for treatment of ulcers and gastrointestinal disorders. Regardingly, recent studies have reported some other medicinal properties for this herb, including antioxidant (3), anti-inflammatory (4), antibacterial (5) and anti-hepatotoxic (6) effects.

A wide variety of bioactive components, including amino acids, fatty acids, salicylic and succinic acids (7, 8), ascorbic acid, folic acid (9), caffeic acid (10) and flavonoids (9) are found in Achillea. Among them, flavonoids (kaempferol: 3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one, luteolin: 3’,4’, 5,7-tetrahydroxyflavone and apigenin: 4’,5,7-trihydro-xyflavone) have attracted more attentions in recent years and most of antioxidant and anti-inflammatory properties of this herb attributed to them (Figure 2).

Figure 2

Chemical structure of main flavonoids of Achillea millefolium L.

Regarding the extensive body of literature on the critical role of oxidative stress in neuronal damage induced by neurodegenerative diseases (such as Parkinson’s disease and Alzheimer’s disease) and also considering the rich flavonoid content of Achillea, as aforementioned, in present article, we reviewed the studies which have used Achillea and/or its three main flavonoid constituents on nervous system related diseases (Table 1).

Table 1

Summary of in vivo and in vitro studies of Achillea millefolium L. and its flavonoid contents on neurodegenerative diseases

Herb and its flavonoid contents Disease	Achillea millefolium	Apigenin	Luteolin	Kaempferol
Multiple Sclerosis	Attenuate disease severity, inflammatory responses and demyelinating lesions in EAE (11)	Reduces the progression and relapse in EAE (12)	Suppresses behavioral deficits, prevented relapse and reduced inflammation in EAE (13), improve maturation of OPCs (14), delay the recovery of behavioral deficits in EAE (15)
Alzheimer’s disease		Inhibits activated microglial cells and neuronal death in animal models of AD (16, 17), protect copper-mediated β-amyloid neurotoxicity in AD cell model (18), ameliorate AD-associated learning and memory deficits and relieve Aβ plaque formation (19)	Inhibits Aβ-induced neuronal death in mouse cortical cultures (20, 21), inhibits memory impairment, degeneration of cholinergic neurons and amyloid deposition (22, 23), the protective effect in traumatic brain injury induced AD (24)	Inhibits formation, extension, and destabilization of beta-amyloid fibrils in Escherichia coli (25), inhibits Aβ-induced toxicity in mouse-derived hippocampal neuronal cells (HT22) (26, 27), revers memory impairment in mice model of AD (28)
Parkinson’s disease	Improves motility disorders and muscle tone in a rat (29)	Inhibited the disaggregation of alpha-synuclein oligomers (30), improves the locomotor and muscular activity and protected dopaminergic neurons (31)	Protects neural pheochromocytoma (PC12) and glial C6 cells (32), protect dopaminergic neurons from oxidant and inflammatory insults in PC12 cell line (33), improved the locomotor and muscular activities (31)	Prevents oxidative stress-induced cell death in rat PC12 and primary neuronal cells (34), improves motor coordination, raise striatal dopamine level and prevent the loss of dopaminergic neurons in mice (35)
Stroke	Reduction in infarct volume, neurological disorders and sensorimotor disorder in rat (36)	Inhibition of activated murine microglia cell line (37), neuroprotective effects on ROS-insulted primary neurons (38)	Lowered the risk of stroke (39), reduce serum levels of inflammatory markers (40), neuroprotective effect on ROS-insulted primary neurons (41), decreases infarct volume and suppresses edema in rat (42, 43), improve neurological status, cognitive abilities in human (44)	Lowered the risk of stroke (39), prevents ischemic brain injury and neuroinflammation in rats (45, 46)
Epilepsy	Reduces the seizure severity in rat (47)	Reduces the latency in the onset of picrotoxin-induced convulsions and inhibitory effects on GABA activated Cl(-) currents in cerebellar granule cells (48), antiepileptic effect and inhibition of Kainic acid-induced cytotoxicity in neurons (49)	Suppresses seizure intensity and duration (50)	Anti-epileptic effects (51), anticonvulsant effects (52)

Experimental autoimmune encephalomyelitis (EAE), reactive oxygen species (ROS), oligodendrocyte precursor cells (OPCs), Alzheimer’s disease (AD)

Multiple Sclerosis

Multiple Sclerosis (MS) is a neuroinflammatory disorder with autoimmune base, characterized by a relapsing-remitting demyelinating phenomenon (53, 54).

Inflammatory and oxidant pathways (via modulation of immune system) are thought to be involved in pathogenesis of the disease (55). The experimental autoimmune encephalitis (EAE) is a well-established animal model of MS in which, in addition to demyelinating lesions and inflammatory reactions (56, 57), cognitive disturbances are also evident within EAE albeit (58) not in the early phase of this model (56, 59). Herbal medicine as an alternative or complementary therapy protocol for MS has been shown to enhance the efficacy of current treatments (60-62).

A recent study showed that oral administration of aqueous extract of Achillea (40, 200 and 400 mg/kg) attenuated the disease severity in combination with inflammatory responses, and demyelinating lesions in EAE model of MS in male C57BL/6 mice (11). An ongoing author-designed clinical trial is now assessing the effects of A. millefolium aqueous extract on MS patients by the authors of this manuscript.

In addition, for luteolin; Hendriks and colleagues reported that both oral and intraperitoneal administration of luteolin (100 mg/kg) in Lewis rat model at EAE suppressed behavioral deficits, prevented relapse and reduced inflammation, as well as axonal damage (13). Furthermost, it was recently reported that luteolin (10 μM) could improve maturation of oligodendrocyte precursor cells (OPCs) in tissue culture. These cells serve as myelin sheet producer around neurons in mice EAE model (14). However, in a different study it was reported that oral administration of luteolin (10 mg) delayed the recovery of behavioral defects rather than any reduction in disease severity in EAE female SJL/J mice (15). Further studies are requisite to be undertaken to clarify the effect(s) of luteolin in improvement of MS related damages.

For apigenin; it was reported that oral and intraperitoneal (IP) administration of apigenin (40 mg/kg of body weight) reduces the progression and relapse in two mouse models of MS (female C57/BL/6 and SJL/J mice) by modulating the immune system responses (12).

Anxiety disorders are prevalent and disabling conditions are usually accompanied by other neurological disorders, such as MS (63). There exist some reports in favor of sedative effects of Achillea and its flavonoid components; for example, injection of Achillea extract (100 mg/kg) in Wistar rats by intraperitoneal showed more sedative and anxiolytic effects than diazepam (2 mg/kg) (64). Further, chronic gavage (300-600 mg/kg) (65) or thereby being intraperitonealy injected (8, 10 or 12.0 mg/kg), Achillea extract showed anti-anxiety effects in albino Swiss male mice (66). Apigenin modulates the GABAergic system and show anxiolytic effect in rodents (67). Grundmann et al. demonstrated anxiolytic activity for kaempferol (0.02 to 1.0 mg/kg IP) and they also showed that this effect was partially antagonized by flumazenil (68). Despite the low affinity of luteolin for the GABAA receptors, it has anxiolytic-like effects. Coleta and coworkers showed that luteolin (0.1–50 mg/kg IP) increased exploration in the open areas and increased the number of entries in the open areas of the elevated plus maze, suggesting an anxiolytic-like effect in male adult Swiss mice (69). Also in another study Crupi colleagues investigated the effects of luteolin (1 mg/kg IP) in an animal model of anxiety/depression (129Sv/Ev mice) and reported similar results in elevated plus maze (70).

Alzheimer’s disease

The Alzheimer’s disease (AD) is described as one of the main subtypes of neurodegenerative diseases. The prominent hallmark of AD includes formation of Amyloid β (Aβ) plaques, degeneration of cholinergic neurons and neuronal cell death (71).

The effects of Achillea flavonoids have been previously evaluated in AD. A beneficial effect of luteolin on AD complications has drawn attentions during last two years. Cell culture studies demonstrated that luteolin (30, 27, 2.7 and 0.27 μM) inhibits Aβ-induced neuronal death in cultured mouse cortical cells and it had strong scavenging activities (20, 21). In line with these data, experimental studies using animal models of AD (male Wistar rats) demonstrated that luteolin (10 and 20 mg/kg) is able to inhibit memory impairment, prohibits degeneration of cholinergic neurons and amyloid deposition (22, 23). Brain injuries following trauma are recognized as a major risk factors for AD, and interestingly, the protective effect of luteolin (1.25, 2.5, 5 and 10 mg/kg/day) on AD complications was also found in traumatic brain injury-induced AD in Tg2576 mice (24). Considering beneficial effects of luteolin in animal models and in vitro studies, designing of clinical trial studies by luteolin is highly recommended.

Studies on the effects of apigenin in AD have longer history. In earlier studies it was reported that apigenin (25 μM and 50 μM) can inhibit activated microglial cells, which may contribute for creation of gliosis and neuronal death in AD-induced animal models (16, 17). It was also reported that apigenin (25 μM) could protect copper-mediated β-amyloid neurotoxicity in AD cell model (copper dyshomeostasis

is evident within AD-affected brain) (18). In, a later study, using animal model of AD (APP/PS1 mice) demonstrated that apigenin in a dose of 40 mg/kg could ameliorate AD-associated learning and memory deficits and relieve as well as formation of Aβ plaques (19).

The kaempferol is a polyphenol compound that was reported to inhibit formation, extension, and destabilization of beta-amyloid fibrils at a dose of 30 μl in Escherichia coli (E. coli). As a result, the fibril Aβ plaque was remodeled to non-toxic oligomers and fibrils (25, 72). In cell culture studies, it was demonstrated that kaempferol (25 mM) inhibit Aβ- induced toxicity in mouse-originated hippocampal neuronal cells (HT22) (26, 27). In vivo studies showed that kaempferol (10, 20 and 40 mg/kg) reversed memory impairment in mouse model of AD (28).

Parkinson’s disease

Parkinson’s disease (PD) is a progressive neuro-degenerative disorder characterized by injuries to dopaminergic neurons of the basal ganglia. Accumulating evidences have suggested that both inflammation and oxidative stress are involved in PD pathogenesis (73).

It was reported that gavage of A. millefolium aqueous extract (1.4 and 2.8 mg/kg) can improve motility disorders and muscle tone in rat models of PD (29).

Some studies have investigated the effects of Achillea flavonoids on PD. In a study, Wruck et al. reported that luteolin (3.13, 6.25, 12.5, 25 or 50 μM) was able to protect neural pheochromocytoma (PC12) and glial C6 cells against oxidative-medieted inturies in a rat cell line (32). Later, evidences demonstrated that luteolin (100 μM) could prohibit dopaminergic neurons death, raised by oxidant and inflammatory insults (PC12 cell line) (33, 74, 75). In accordance with in vitro results, in vivo studies also showed that gavage of luteolin (10 and 20 mg/kg) to mice model of PD ameliorated the locomotor and muscular activities and protected dopaminergic neurons (31). Interestingly, co-administration of luteolin with palmitoylethanolamide by a ratio of 1:10 potentiated the protective effect of luteolin on dopaminergic neurons in, in vivo models of PD (76).

In vitro studies indicated that apigenin (100 μM) inhibited the disaggregation of alpha-synuclein oligomers (77) and lipid membrane damage was also prevented by this protein (30). During recent years, studies demonstrated that alpha-synuclein protein plays a fundamental part in pathophysiology of PD. As a matter of fact, increased expression and/or pathologically altered forms of this protein could degenerate nigrostriatal dopaminergic neurons (78). Results of a new in vivo study revealed that gavage of apigenin (5, 10 and 20 mg/kg) to mice model of PD has improved the locomotor and muscular activity along with protection of dopaminergic neurons (31).

There also exist positive reports regarding the effect of kaempferol (20 mM) on PD. It has been shown that this compound was able to prevent oxidative stress-induced cell death in rat PC12 and primary neuronal cells, in in vitro cell culture studies (34) and enhances mitochondrial turnover by autophagy (79). Interestingly, it appears that the antioxidant properties of kaempferol are mediated through DJ-1 dependent pathways (34). DJ-1 is a causative gene product that plays a role in anti-oxidative stress, and its dysfunctional state would result in the onset of PD (80, 81). In line with the in vitro studies, Li and Pu, reported that kaempferol (25, 50 and 100 mg/kg) ameliorated motor coordination, elevated striatal dopamine level and prohibited the loss of dopaminergic neurons in male C57BL/6 mice model of PD (35).

In conclusion, both Achillea and its three flavonoid contents have shown anti-parkinson properties. Altogether this may evolve a new avenue to the treatment of neurodegenerative disorders or at least, partially be considered as a promising target for reduction of harmful clinical symptoms.

Stroke

It was reported that oral administration of the aqueous extract of Achillea (50, 500 mg/kg) in female rats subjected to ovariectomy, caused a significantly reduced the infarct volume, neurological disorders and sensorimotor disorder in middle cerebral artery occlusion in animal models of stroke (36).

Studies about the possible beneficial effects of luteolin in stroke were initially come from two interesting human studies. Marniemi et al. showed that high dietary intake of the two flavonoids such as, luteolin and kaempferol (as supplemental nutrient at least once a week) has attenuated risk of stroke (39). Holt and colleagues also reported that serum levels of inflammatory markers were inversely associated with the dietary input of luteolin (40). Cell culture studies demonstrated that luteolin (there by used as an aqueous solution of 500 ml) has exerted a neuroprotective effect on reactive oxygen species (ROS)-insulted primary neurons (41). Accordingly, in vivo studies showed that luteolin (5 and 20 mg/kg) alleviates neurological deficit, decreases infarct volume and suppresses edema in rat stroke models (41-43). A recently published clinical trial cohort study demonstrated that the stroke patients treated with co-ultramicronized palmitoylethanolamide (PEA)/luteolin (contained co-ultramicronized 700 mg PEA and 70 mg luteolin, in microgranular form) preparation for 60 days, have improved neurological status, cognitive abilities and independence in daily living activities (44).

Studies about the effects of apigenin on stroke emerged to the literature since eight years ago.

Firstly, Ha and colleagues reported that apigenin (1, 5, and 10 mM) had anti-inflammatory effect through inhibition of activated murine microglia cell line (BV-2 cells). They also demonstrated that this flavonoid decreased the infarct volume in an animal model of stroke (37). Interestingly, it is likely that stroke also induces astrocytes impairment and apigenin (10, 30, or 50 μM) could inhibit these pathologic state in astrocytes which were obtained from stroke-prone

spontaneously hypertensive rats (especially the expression of adhesion molecules) (82). Recent evidences, also reported that apigenin (1, 10, 20 and 40 μM) had neuroprotective effects against reactive oxygen species (ROS)-induced insalts in primary neurons (in cultured PC12 Cells) (38). Taken together, these results may suggest that apigenin has protective effects on both glial and neuronal cells in stroke.

Two various in vivo studies demonstrated that further acute ischemia administration of kaempferol (injection, at a dose of 10–15 μM/l of blood) prevented ischemic brain injuries as well as neuroinflammation in rats (45, 46).

Epilepsy

Epilepsy is a frequent, seriously neurological disorder, worldwide. Oxidative processes and free radicals both play pivotal roles in pathogenesis of epilepsy.

It has been established that hydro-alcoholic extract of Achillea (100, 200 or 400 mg/kg) was able to decrease seizure severity in animal models (male Wistar rat epilepsy model) (47).

Reports about the effect of luteolin in epilepsy are controversial. As an instance Zhen et al. studied the effect of luteolin in pentylenetetrazole induced epileptic male Sprague–Dawley rats and that oral administration of luteolin (50 or 100 mg/kg/day) suppressed the seizure intensity and duration in this animal model of epilepsy (50). Interestingly, the anticonvulsant effects of luteolin may be due to its inhibitory effects on neuronal and oxidative stress damages which are evident in chronic seizure activities (50). Alternatively, it has been shown that luteolin (3 and 10 mg/kg) can bind the benzodiaze-pine site on the GABAA receptors on the surface of BV-2 microglia-like cells (83). However, in an earlier study, Shaikh and colleagues examined the effect of luteolin in both acute and chronic animal models of seizure. In this set of discrepant data they failed to find any indications that luteolin (3 and 10 mg/kg) is pro- or anti-convulsant neither in chronic nor in acute mouse models (CD1 mice) of seizure (84). These discrepant findings may be due to various doses of luteolin used by two different studies (50 - 100 mg/kg in first study vs. 3-10 mg/kg in last study).

The effect of luteolin on epilepsy was as similar as apigenin disputing. In an early study, it was reported that apigenin (there by was IP injected in a 25 and 50 mg/kg dose) reduced the latency in the onset of picrotoxin-induced convulsions. This effect was attributed to the inhibitory effects of apigenin on GABA activated Cl(-) currents on cerebellar granule cells (48). Later, Campbell and co-workers demons-trated a novel second order modulatory action of apigenin on GABA recombinant human α1β2γ2L GABAA receptors. Actually, they showed that apigenin (1 μM) could enhance GABA responses to diazepam (85). In accordance to these results, it has been proposed that apigenin might also potentiate the effect of the endogenous benzodiazepine-like agents like endozepines which can be used clinically to control convulsions (85). A recent study demonstrated a protective effect for apigenin (at 25 and 50 mg/kg dose) in Kainic acid-induced seizure in male ICR in mouse model. This antiepileptic effect of apigenin is associated with the inhibition of Kainic acid-induced cytotoxicity in neurons (49).

Juan Song et al. showed the anti-epileptic effects of kaempferol (8 and 30 μM) in a genetic model of epilepsy in Drosophila (51). The anti-epileptic activity of kaempferol may be due to its effects on inhibition of DNA topoisomerase I enzyme. This could be especially beneficial for brain tumor patients who are suffering from epilepsy. Nugroho et al. showed the anticonvul-sant effects of kaempferol (per os (p.o) injection at 100 and 200 mg/kg dose) in extract of Aster glehni on pentylenetetrazole-induced seizure in ICR mouse model (52).

Conclusion

There is an extensive body of evidences on beneficial effects of Achillea and its main flavonoids on nervous system related disorders within the database. However, almost all of performed studies have focused on in vitro and animal preparations. Also, almost all of these in vivo and in vitro reports showed beneficial preventive or therapeutic properties of Achillea and/or its flavonoids on epilepsy, Alzheimer’ diseases, multiple sclerosis, Parkinson’ diseases and stroke (Figure 3). Considering little side effects of Achillea and its derivative flavonoids (86) on main brain functions (87, 88), as well as their availability for supplemental use, one can suggest to use them in neurodegenerative disorders.

Figure 3

Schematic description for protective mechanisms of Achillea millefolium L. in ameliorating neurodegenerative diseases

As tried our best, we could not find any further clinical trial study about the effects of Achillea and/or its three flavonoids in nervous system related disorders. Future studies are required to address a possible preventative/therapeutic role for Achillea and/or its flavonoids on neurodegenerative diseases with focusing on human subjects.

Conflict of interest

The authors have declared that no conflict of interest exists.