Nepeta menthoides Boiss . & Buhse , an endemic species in Iran : A review of traditional uses , phytochemistry and pharmacology

Introduction Plants have long been used for the treatment of various conditions and many researches are being done in pharmaceutical industries because of the belief that these natural remedies might have lesser side effects than current drugs (1). Lamiaceae family is one of the largest and specific flowering plants with about 220 genera and 4000 species all around the world (2,3). The genus Nepeta L. is one of the genera of Nepetoideae subfamilies and consists of about 300 species widely distributed in Europe, Asia and some areas of Africa (3,4). Iran is one of the major sources of this genus with 79 species (5). Various species of the genus Nepeta have been used traditionally for their therapeutic effects including anticonvulsant, antitussive, http://www.herbmedpharmacol.com doi: 10.15171/jhp.2019.xx


Introduction
Plants have long been used for the treatment of various conditions and many researches are being done in pharmaceutical industries because of the belief that these natural remedies might have lesser side effects than current drugs (1).Lamiaceae family is one of the largest and specific flowering plants with about 220 genera and 4000 species all around the world (2,3).The genus Nepeta L. is one of the genera of Nepetoideae subfamilies and consists of about 300 species widely distributed in Europe, Asia and some areas of Africa (3,4).Iran is one of the major sources of this genus with 79 species (5).Various species of the genus Nepeta have been used traditionally for their therapeutic effects including anticonvulsant, antitussive, anti-microbial and anti-asthma properties (6).Nepeta menthoides is one of the endemic species distributed in different areas of Iran including Azarbaijan, Tabriz, Sabalan, and Marand (7,8).Nepeta menthoides is a herbaceous, perennial, climbing plant with the height about 15-45 cm with the purple flowers (8).Archaeological excavation evidence suggests that some species of the Lamiaceae family were widely cultivated in the past locally (9).Many species of this family have been shown to possess similar essential oils (2,3).Many studies have been conducted on the chemical composition of the Nepeta sp.essential oils in Iran and other countries; based on these studies the main components of these species are nepetalactone , 1,8-cineole, β-pinene and geranyl acetate.Phenolic compounds such as flavonoids have also been reported as the main component of this genus (10).Nepeta menthoides is one of the plants named "Ostokhodus" through the time in TPM and widely used for the treatment of neurological disorders including seizure, chronic pain, depression and anxiety disorder (11,12).The original Ostokhodus (Lavandula stoechas) was used in Persia for a long time as an imported drug (13).As an endemic medicinal plant and a common substitute of L. stoechas in Iran, this herb is of importance in traditional Persian medicine (TPM).Some researches on N. menthoides have been carried out to evaluate scientifically the traditional uses of this herb.The aim of this study is to review the pharmacological and phytochemical scientific evidence on N. menthoides.This review presents a comprehensive assessment report on phytochemical aspects, pharmacological activities, and toxicity of Nepeta menthoides by focusing on the data from the year 1980 until the May 30 th 2018 via papers on databases including PubMed, Scopus, Google Scholar, Web of Science, and SID (a Persian scientific database).The keywords "Nepeta menthoides" and "Ostokhodus" were used for the search.

Nepeta menthoides in traditional medicine
Nepeta menthoides is an endemic species of the genus Nepeta in Iran which is distributed in the northwest of the country.Nepeta menthoides is named "ostokhodus" in Persian traditional medicine and herbal stores (10).Ostokhodus or stoechas is one of the oldest herbs mentioned in TPM books and has been represented by several herbs during the time.Today Nepeta menthoides is used as ostokhodus in herbal markets of Iran and traditional medicine because its therapeutic effects are similar to those of ostokhodus in TPM books (13,14).
Ostokhodus has been mentioned in TPM books for reinforcing the major organs including brain, heart and liver, and considered as one of the brain-targeted medications based on TPM concepts.There are TPM indications of ostokhodus including amnesia, sadness and depression, obsession, epilepsy and melancholy.In addition, based on TPM theories some ailments of the other organs such as gastric disorders and respiratory problems including cough and catarrh are related to the brain disorders, and with this viewpoint, ostokhodus has also been recommended for these diseases in TPM (12,15).

Pharmacological profile
Different pharmacological effects of N. menthoides have been reported in both in vitro and in vivo studies.These pharmacological activities have been summarized in Table 1.

Phytochemistry
Various types of chemical constituents such as monoterpene derivatives, sesquiterpenes, diterpenes, triterpenes, phenolic compounds such as flavonoids, phenolic acids, and some other constituents have been identified in Nepeta species (16)(17)(18).Phytochemical screening tests of N. menthoides have been shown to be positive for triterpene, sterol, saponin and flavonoid (10,19,20).The names, chemical structure and sources of identified compounds are collected in Table 2.As can be seen from the Table, terpenoids and flavonoids are dominant constituents within this plant.
Anti-infective properties A number of reports have been published on the antibacterial effects of N. menthoides extracts or its oil.Methanol extract from aerial parts of N. menthoides showed significant antibacterial activity against the gram-positive bacteria: Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus aureus and Bacillus cereus.However, aqueous extracts of the plant had no antibacterial activity (20).A poor inhibitory activity of methanol extracts from N. menthoides root and leaves was observed against Bordetella bronchiseptica and Staphylococcus aureus respectively.(21,22).In addition, N. menthoides essential oil from aerial parts and its crude methanolic extract have been shown to have larvicidal activity against Anopheles stephensi with LC50=234.3ppm and LC50=69.5 ppm, respectively.The authors indicated that nepetalactone isomers and 1,8 cineol must be mentioned as effective compounds related to larvicidal activity (8).Moreover, the antibacterial activity of the essential oil of N. menthoides and its 2 main constituents, 1,8-cineol (57%) and 4aα, 7α, 7aα-nepetalactone, was evaluated against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsiella pneumonia, and Escherichia coli (23).

Antioxidant activity
The antioxidant effect of the essential oil from N. menthoides was investigated by Kahkeshani et al (24).The authors demonstrated its effects by two different methods: DPPH and FRAP assays and concluded that the antioxidant activity of the essential oil may partly be attributed to its components like myrcene, 1,8-cineole, limonene, α-terpinene and α-terpinolene.Moreover, this activity may play a crucial role in protecting cells from oxidative stress and disease prevention.
Anti-Inflammatory and antinociceptive activities A hydroalcoholic extract of N. menthoides was reported to possess anti-nociceptive and anti-inflammatory activity as evidenced by effects in formalin test, Hot water tailimmersion test and xylene induced ear oedema in mice.
The study reported that N. menthoides aerial parts had anti-inflammatory effect at all tested doses, particularly at dose of 2800 mg/kg.Also, it caused a significant reduction of pain in the formalin test (acute and chronic phases) and the dose of 2800 mg/kg had the highest anti-nociceptive effect in chronic phases.Meanwhile, N. menthoides hydroalcoholic extract showed an acceptable analgesic effect in the tail immersion test.These effects might be due to its constituents: Nepetalactone and 1,8 cineole (25).

Antidepressant and anxiolytic activities
Nepeta menthoides aerial parts have shown to possess antidepressant activity in an 8-week triple-blind randomized controlled trial study.In this study, fifteen patients received only N. menthoides capsules (400 mg) and fifteen patients treated with N. menthoides capsules and conventional drugs.Pharmaceutical capsules of N. menthoides could be potentially effective in depressed patients (26).In parallel, Kolouri et al assessed the effectiveness of N. menthoides in the treatment of major depression (MD).The results of this double blind randomized controlled trial study showed that in a short-term therapy, patients with MD receiving capsules containing 400 mg freeze-dried powder of N. menthoides aqueous extract experienced a significant reduction in their Beck Depression Inventory (BDI) scores after four weeks of treatment compared with the

Name of compound Chemical structure Plant part References
Table 2. Continued standard treatment group (sertraline).Furthermore, two weeks follow up after intervention showed a statistically significant lower recurrence rate in the N. menthoides group (27).The anxiolytic activity of N. menthoides was investigated by evaluating its influence on the Beck anxiety inventory (BAI) score in patients with depression.The N. menthoides group who received freeze-dried aqueous extract formulation (400 mg/BID) demonstrated a greater reduction of BAI score and lower symptom recurrence rate as compared to the sertraline group.Moreover, the prevalence of reported side effects was lower in the N. menthoides than the SSRI group (28).These studies demonstrated the effects of N. menthoides on depression and anxiety induced by monoterpenes, sesquiterpenes, polyphenolic compounds and flavonoids.The findings of these studies suggested that N. menthoides might be an effective and safe treatment for depression and anxiety along with conventional medications.

Sedative-hypnotic effects
In an effort to find new effective substances that relieve insomnia while avoiding side effects, numerous studies tending to focus on herbal medicines have been done.In 2012, Bozorgmehr et al reported a sedative effect for the ethanolic extract of N. menthoides (29).Nepeta menthoides (freeze-dried aqueous extract) has been shown to possess hypnotic effect.In this triple-blind randomized placebo controlled study, the subjects received three 500 mg capsules containing 1000 mg Melissa officinalis plus 400 mg of N. menthoides nightly for four weeks.The results showed that the Insomnia Severity Index (ISI) significantly decreased in treatment group and the patients who received herbal treatment experienced a significant improvement in their total sleep time as compared to the placebo group (30).

Effect on cognition and memory
The efficacy of N. menthoides extract in the treatment of patients with Alzheimer's disease was assessed in a clinical trial.The study was performed on two groups of AD patients.While the first group was prescribed the oral capsule (500 mg) of Nepeta extraction, the second group was given the placebo capsules (500 mg).Both groups were prescribed the conventional Alzheimer medications as the primary drugs.The remarkable improvement in the MMSE (Mini-Mental State Examination) scores in patients who received oral capsule of N. menthoides, three times daily, emphasized the potential of this plant accompanied by the conventional medications in decreasing the AD symptoms (31).Furthermore, some pharmacological evidence has been indicated the anticholinesterase activity, and memory enhancing effects of N. menthoides in vivo and in vitro.Essential oil from N. menthoides aerial parts was found to inhibit the acetylcholinestrase enzyme (AChE).
In AchE inhibitory test, the essential oil had desirable anticholinesterase activity with IC50 value of 64.870 μg/ mL.1,8-cineol, the greatest percentage composition of N. menthoides essential oil, is a potent inhibitor of AchE which plays a major role in inhibiting the enzyme (24).Moreover, AchE inhibitory activity of N. menthoides was also measured in a study conducted by Adhami et al.Both the methanol and dichloromethane extract showed significant AchE inhibitory effect (32).More recent studies showed the significant inhibition of AchE activity by essential oil and 1,8-cineole from N. menthoides.1,8-Cineole exhibited higher inhibitory than the essential oil and both of them were more potent than Rivastigmine (33).Taken together, these findings implicate that N. menthoides would be excellent candidate for future Alzheimer's disease therapy.
Behavioral studies have demonstrated that N. menthoides extract affects learning and memory in experimental animals.Hydroalcoholic extract of N. menthoides was reported to improve scopolamine-induced impairments of memory retention and retrieval in Y-maze task in mice (34).In similar work, Sarahroodi et al (35) demonstrated that the aqueous extract of N. menthoides leaves had promising memory enhancing effects at dose of 800 mg/ kg using passive avoidance down step model.The presence of antioxidants such as rosmarinic acid, luteolin, cinnamic acid derivatives, caffeic acid, tocopherols, phenolic acids and coumarins can be a major contributor to the memoryenhancing effects of N. menthoides extract.Moreover, the water extract of N. menthoides was found to reverse the destructive effect of chronic cold-water-induced hypothermia on learning and memory in rats.The results showed that lower dose of N. menthoides neutralized learning and memory impairment, but higher dose had adverse effects on learning.Moreover, the extract could reduce hyperphosphorylation of tau protein that seems to be one of the mechanisms of its anti-dementia activities (36).
Neuroprotective effects Different doses of N. menthoides were investigated for neuroprotective activity.It was observed that intraperitoneal administration of axotomized rats with ethanolic extract of N. menthoides resulted in an increased survival of axotomized motoneurons and a decreased apoptotic rate in a dose-dependent manner which was most prominent with the dose of 500 mg/kg (37).However, the study on prophylactic capacity of N. menthoides revealed that administration of N. menthoides ethanolic extract before neural injury causes no more neuroprotective effect on axotomized motoneurons (38).In another study, alcoholic extract of N. menthoides aerial parts by virtue of its anti-apoptotic effects induced neuroprotective effect on axotomized sensory neurons in dorsal root ganglion (DRG) of neonate rats by attenuating the apoptotic cell death (39).The results confirm the traditional use of this plant for the treatment of neural disorders such as epilepsy and melancholia (12).

Cytotoxic activity
The anti-proliferative effects of essential oil of N. menthoides were investigated in colon carcinoma (HT-29), colorectal adeno-carcinoma (Caco-2), breast ductal carcinoma (T47D) cell lines and the Swiss mouse embryo fibro blast (NIH-3T3) as a normal cell line using the MTT method.As the IC50 value for the normal cell line was low, the MTT test showed that the essential oil exhibited the highest cytotoxic activity against T47D followed by HT-29 and Caco-2 cell lines with the IC50 values of 19.37±4.92,30.7±7.36 and 32.24±5.98μg/mL, respectively.This cytotoxic activity of N. menthoides might be observed due to specific components like α-terpineol, terpinolene, 1,8-cineole and limonene (24).In 2018, Kahkeshani et al studied the inhibitory effects of the essential oil and 1,8-cineole isolated from N. menthoides against three breast cancer cell lines (MCF-7, T47D and MDA-MB-231).Both compounds displayed inhibitory activity, but the essential oil was more cytotoxic than 1,8-cineole to breast cancer cell lines (33).
Effect on opioid dependence and tolerance Chronic pretreatment with N. menthoides hydro-alcoholic extract could dose-dependently attenuate the development of dependence in morphine-treated mice.Furthermore, N. menthoides prevented the acquisition of morphine tolerance and potentiated morphine analgesic effect in a synergistic manner.It appears that inhibition of the nitric oxide (NO) overproduction is the main mechanism responsible for these effects of N. menthoides (40).

Toxicity and safety
Nepeta menthoides has been widely used in Persian traditional medicine.Although previous clinical studies on this herb have reported no significant adverse effects, some clinical and experimental studies have shown that N. menthoides has mild to moderate side effects.In one clinical trial anorexia, bulimia, xerostomia, emesis, impotence, and drowsiness were the most commonly side effects reported by the patients and one patient stopped N. menthoides capsules due to severe dermal irritation [16,28].Moreover, N. menthoides was found to increase susceptibility to seizures (41).

Discussion
Ostokhodus, one of the oldest herbs in Iran, had been widely introduced in TPM textbooks for treating various ailments.This herb has been substituted with other herbs with similar therapeutic effects over the time.Since 20 century the plant N. menthoides has been utilized as ostokhodus in Iran by people for different medicinal effects.This review collected findings about phytochemical and pharmacological properties of N. menthoides and presented comprehensive analysis of researches published since the year 2000.Ethnopharmacological published data about this herb is rare because of limited distribution as an endemic plant in Iran.Despite the lack of comprehensive pharmacological studies on N. menthoides, this plant is widely used in traditional medicine as antinociceptive, anti-Alzheimer's disease, memory enhancer, neuroprotective, antidepressant and anxiolytic agent.It seems that more preclinical studies are needed to assess this herb clinically specially in the field of seizure because one study indicated the increased susceptibility to seizures.Moreover, there are several medicinal properties discussed in Persian medicine such as anti-tussive, gastrotonic and gastrointestinal effects which are not supported by any modern scientific evidence and N. menthoides should also be assessed in these fields.
Despite high consumption of this plant, little phytochemical and quality control studies have been done on it.However, with respect to the limited published phytochemical assays, triterpenes and monoterpenes are the most abundant constituents of the essential oil of N. menthoides.

Conclusion
Considering the medicinal properties of isolated components, it can be concluded that terpenoids might be associated with anti-inflammatory and antimicrobial activities.Also, antioxidant and anticancer activities could be related to its phenols and flavonoids.Many of the phytochemical studies on this herb are based on essential oil analysis, hence there is still a huge need for extensive analysis on its different extracts.