Zataria multiflora Boiss.: A review study on chemical composition, anti-fungal and anti-mycotoxin activities, and ultrastructural changes

Shokri H and Sharifzadeh A Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 2 and is the aborigine of Iran, Afghanistan and Pakistan (5). Zataria multiflora can be recognized by the orbicular, densely gland-dotted, ovate leaves and the dense white, hairy, round buds on the leaf axils. It is an aromatic shrub that reaches 60-90 cm in height. Mature branches are woody and leafless whereas young branches are white with dense glandular, spreading, pilose indumentums. Leaves (5-10 × 5-10 mm2) are orbicular ovate to orbicular. Flowering stems are usually un-branched, sometimes having short lateral branches. Flowers are white, subsessile, very small and often male sterile (6). Zataria multiflora (aerial parts) is not only a popular condimental plant but is also used in traditional folk remedies for its anti-microbial, analgesic, carminative, anthelmintic and anti-diarrheal properties (7). Modern pharmacological studies have shown that Z. multiflora possesses wide ranging biological properties including anti-nociceptive, anti-microbial, spasmolytic and anti-inflammatory effects (5,8-10). Currently, some pharmaceutical forms of this plant, such as syrups, oral drops, soft capsules and vaginal creams are sold as treatment remedies for various diseases. To outline the extensive uses of Z. multiflora in healthcare and medicine and to provide a probable scope for future research, several pharmacological and clinical studies on this plant and its active components are provided in this review article. Methods Google Scholar, PubMed, EBSCO, Directory of open access journals (DOAJ), EMBASE, and Web of Science were searched using the keywords Z. multiflora and pathogenic and toxigenic fungi. Chemical composition of Zataria multiflora Various extraction techniques like distillation, effleurage, CO2 extraction, expression and solvent extraction are applied for essential oil (EO) extraction from various plants. But conventional methods that are usually used to extract EO oil from plants, are the distillation methods like steam-distillation, hydro-distillation and watersteam-distillation method. Quantitatively, the most abundant components in hydrodistilled Z. multiflora EOs are oxygenated monoterpenes (approximately 70%), followed (in order) by monoterpene hydrocarbons, sesquiterpene hydrocarbons and oxygenated sesquiterpenes (10,11). Two studies from Pakistan identified carvacrol as the main constituent of the oil (12,13). To date, a large number of studies have focused on Z. multiflora EO: some reported carvacrol as the main compound, but others reported thymol, the isomer of carvacrol, as the main compound. Saei-Dehkordi et al (14) collected Z. multiflora from five different areas of Iran and analyzed its EOs. According to the GC–MS data, the main EO constituents remained similar between plants from different geographical regions, but their relative quantities differed among plants from different regions. Thymol was the most abundant compound among all constituents in all samples (14). In a study conducted by Shokri et al (15), Z. multiflora main components were carvacrol (61%) and thymol (25%). Additionally, Saleem et al (16) showed that thymol was the main constituent of the fresh plant (73.21%), while carvacrol was the primary constituent in the dried plant (62.87%). It is clear that geographical variation, cultivar differences, stage of plant growth, preparation process and other factors may influence the EO composition both quantitatively and qualitatively. According to previous studies, the EOs of Z. multiflora contain significant amounts of thymol and carvacrol, which are well-known anti-fungal agents (17-19). p-Cymene is the other main component in Z. multiflora EO. Zatariol, zataroside A (glycoside and non-volatile), zataroside B (glycoside and non-volatile), multiflotriol and multiflorol have been reported as new derivatives of p-cymene isolated from Z. multiflora (20,21). Linalool, caryophyllene, γ-terpinene and borneol are some of the other main components in the EOs (22). Zataria multiflora also contains other compounds belonging to different classes of natural products, including alkanes such as n-nonacosane (C29), n-hentriacontane (C31), n-dotriacontane (C32), n-tritriacontane (C33) and n-pentatriacontane (C35); fatty acids such as behenic acid (C22), lignoceric acid (C24), cerotic acid (C26) and montanic acid (C28) (23); phytosterols such as β-sitosterol and stigmasterol; triterpenes such as betulin, betulinic acid and oleanolic acid (24); and hydroxycinnamic acids such as rosmarinic acid (25). Moreover, flavonoids such as apigenin, luteolin and 6-hydroxyluteolin are also among the phytochemicals reported from Z. multiflora (22). This plant also contains small amounts of tannins, resins and saponins while lacking alkaloids (26). Pharmacological and therapeutic activities of Zataria multiflora 1. Anti-yeast activity of Zataria multiflora Various studies have shown that many species of yeasts, especially Candida and Malassezia species, have developed resistance to standard antifungal drugs. Nowadays traditional medicine and the use of herbal medicines to treat yeast infections are important because herbal medicines have fewer side effects and are less likely to develop drug resistance compared with chemical ones (27,28). Medicinal plants have been shown to eliminate the drug resistant yeasts and can be beneficial for their therapeutic effects (29). Zarei-Mahmoudabadi et al evaluated anti-Candida activity of three extracts of Z. multiflora (aqueous, ethanolic and methanolic) against 14 isolates of Candida. Aqueous extract of Z. multiflora showed no activity against Candida species. The ethanolic and methanolic extracts showed remarkable activities against Candida species. The minimum inhibitory concentration (MIC) for both extract was between 50 and 150 mg/L. The lowest MIC was for seven isolates of C. albicans (125 mg/L). Others MICs were respectively C. glabrata (126 mg/L), C. parapsilosis (125 mg/L) and C. tropicalis (131 mg/L). Totally, the MIC of ethanolic extract for 14 isolates of Candida was 127 mg/L. Both C. albicans and A review on Z. multiflora Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 3 C. parapsilosis were more susceptible than other species. The isolates of C. parapsilosis (64 mg/L) were more susceptible to methanolic extract of Z. multiflora, followed by C. glabrata (66 mg/L), C. albicans (76 mg/L) and C. tropicalis (76 mg/L). In addition, the MIC of methanolic extract for tested Candida was 70 mg/L. In that study methanolic extract showed more activities than ethanolic extract against 14 isolates of Candida (30). In Moghim et al (31) study, the obtained MIC of Z. multiflora extract was 0.13 mg/mL for C. albicans, which was lower than the effect on C. albicans obtained for this extract in the study of Zarei Mahmoudabadi et al (30). In another study by Fuladi et al (32), the MIC of Z. multiflora methanol and ethanol extracts were estimated to be 0.079 and 0.125 mg/ mL, respectively. The contrasting results could be due to the different species of Z. multiflora, plant chemical compounds and/or methodologies. Katiraei et al (33) compared the MIC of EOs of Z. multiflora, Geranium and Artemisia with regard to the growth of C. albicans isolates resistant to azole drugs. They showed that the MIC levels of EOs of the plants were statistically significant from those of azoles. In that study, the obtained MIC of Z. multiflora for C. albi cans was 0.18 mg/mL. In a study conducted by Esfandiary et al (34), a set of C. glabrata (29 strains), C. krusei (3 stains) and C. patapsilosis (2 strains) were studied. The results revealed that 33 isolates were resistant (MIC = 64 μg/mL), 4 isolates were susceptible (MIC ≤ 8 μg/mL) and 7 isolates had dose-dependent susceptibility (MIC = 16-32 μg/mL) to fluconazole, respectively. With regard to fluconazole, high resistance rate was observed in C. glabrata and C. krusei. However, ciclopirox olamine was found to inhibit the growth of all non-albicans Candida species (MIC ≤8 μg/mL). Also, favorable anti-fungal activity against non-albicans Candida species was obtained by Z. multiflora despite having a wide range of MICs (34.9-139.5 mg/mL). Mohammadi-Pourfard and Kavoosi (35) showed that EO from Z. multiflora significantly (P < 0.01) inhibited the growth of C. albicans. MIC for C. albicans was 2.8 ± 0.8 mg/mL of EO. At concentration > 5 mg/mL, EO significantly (P < 0.01) reduced the growth of C. albicans by 100%. However, the aqueous extract did not show any such activity at any concentration used. In addition, Zomorodian et al (36) determined the MICs and minimum fungicidal concentrations (MFCs) of the EO from Z. multiflora against Candida and Trichosporon species, showing strong anti-yeast activities with MIC values ranging from 0.007 to 0.5 μg/mL. This finding is similar to that of the study by Mahboubi et al (37) who reported strong anti-Candida activity of Z. multiflora EO with high thymol and carvacrol concentrations. In a randomized clinical trial conducted by Khosravi et al (38), the application of 0.1% Z. multiflora cream in patients with acute vaginal candidiasis decreased vulvar pruritus in 80.9% of patients, vaginal pruritus in 65.5% of patients, vaginal burning in 73.9% of patients, urinary burning in 100% of patients, painful intercourse in 92.6% of patients, and vaginal secretion in 90% of patients. In addition, 0.1% Z. multiflora cream reduced erythema and satellite vulvar lesions in 100% of patients, vulvar edema in 100% of patients, vaginal edema in 83.3% of patients, vulvo-vaginal excoriation and fissures in 92% of patients, and white, sticky vaginal secretions in 86.2% of patients. After treatment with 0.1% Z. multiflora cream a laboratory using standard methods reported negative mycologic results on microscopic evaluation for 90% of patients; negative mycologic cultures in 86.7% of patients; and negative mycologic results on microscopy and culture combined for 93.3% of patients (38). Abou Fazeli et al (39) demonstrated the activity of the EO against C. albicans. They also suggested Z. multiflora oil-containing vaginal suppositories as a successful replacement for current drugs for the treatment of vaginitis caused by C. albicans (39). In a comparative study, a 7-day therapy with Z. multiflora as an intravaginal cream was more effective than clotrimazole vaginal cream in the treatment of Candidal vaginitis (40). An open-label, randomized and controlled study with two parallel treatment groups was conducted to evaluate the efficacy of a miconazole 2% gel compared with a Z. multiflora 0.1% gel applied four times daily for two weeks in the treatment of Candida-associated denture stomatitis. The results indicated that the Z. multiflora gel reduced the surface erythema of the palate more efficiently than miconazole gel but did not reduce the colony count on the denture surface as efficiently as miconazole (41). In another study, the physicochemical properties and stability of creams containing different concentrations (1%–3%) of Z. multiflora were evaluated and suggested as a successful replacement in the treatment of C. albicans induced vaginitis. In a previous study by Khosravi et al (43), intraperitoneally administration of 64 mg/kg of the EO in mice with systemic candidiasis had the highest efficacy in reducing C. albicans and produced 39.5, 21.8, 141.5, 174 and 501-fold reductions in mean colony forming units per 0.1 gram in Candida infections of the liver, spleen, lungs, brain and kidneys, respectively, as compared to itraconazole. Avaei et al (44) demonstrated that Saccharomyces cerevisiae (MIC = 200 μg/mL and MFC = 1600 μg/mL) was more resistant than C. utilis to Z. multiflora EO. Naeini et al (45) showed that Malassezia species were susceptible to Z. multiflora EO ranging from 10 to > 50 mm (mean value: 28.1 mm). The highest inhibitory effect was recorded with Malassezia obtusa, followed by M. furfur and M. globosa. In another study conducted by Naeini et al (46), the EO from Z. multiflora presented anti-Malassezia activity against the tested yeasts. All Malassezia species were very susceptible to Z. multiflora EO, with MICs ranging from 0.015 to 0.06% (v/v), and approximately 52.4% of the strains had a MIC value of 0.015% (v/v). The mean MIC values of the EO against M. sympodialis, M. furfur and M. pachydermatis were 0.03, 0.024 and 0.02%, respectively. Khosravi et al (47) showed that Z. multiflora EO had the best anti-fungal activity against various Malassezia species isolated from dogs with atopic dermatitis, with MIC values ranging from 30 to 80 μg/mL. M. nana isolate was the most susceptible one (30 Shokri H and Sharifzadeh A Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 4 μg/mL), while M. sloofiae isolates showed the highest MIC value (80 μg/mL) (P<0.05). 2. Anti-dermatophyte activity of Zataria multiflora Dermatophytes are the major cause of superficial mycoses, and remain a public health problem. They have the ability to invade keratinized tissues and cause dermatophytosis, the most common human contagious fungal disease (48). In recent years, the incidence of dermatophytosis has increased considerably, especially among individuals with impaired immunity, as well as in pediatric and geriatric populations (49). Some of these infections are still difficult to resolve completely, and remissions and relapses are often observed. The poor availability of antifungals and the increasing number of treatment failures have motivated current searches for therapeutic alternatives which can work effectively as potential anti-dermatophyte agents. Khosravi et al (50) showed that EO obtained from Z. multiflora possessed anti-fungal activity against a wide number of clinical isolates of various dermatophytes, with MIC ranging from 0.25 to 4 mg/mL. However, variation of susceptibility from one species to another was evident. The MIC values were 0.5 mg/mL for Microsporum canis and M. gypseum, 1.5 mg/ mL for Trichophyton mentagrophytes and Epidermophyton floccosum and 2 mg/mL for T. rubrum. The MFCs recorded for plant oil tested ranged from 0.5 to 8 mg/mL. In a study conducted by Effatpanah et al (51), total (100%) fungal growth inhibition was observed from Z. multiflora EO at concentration of > 8 mg/mL for T. mentagrophytes, T. rubrum and E. floccosum (P < 0.01). In another study, the minimum inhibitory activity of Z. multiflora methanolic extract against various dermatophytes was found to be approximately 0.5% (w/v) (42). 3. Anti-filamentous fungi activity of Zataria multiflora Literature studies indicated that there are many reports on non-dermatophytic filamentous fungi causing fungal infections in human and animals and food spoilage (18,52). Gandomi Nasr-Abadi et al (53) found that all concentrations of EO from Z. multiflora had a significant effect on the growth and sporulation of Aspergillus flavus. Also, they reported the levels of MIC and MFC 400 and 1000 ppm, respectively. In a study conducted by Mohammadi-Pourfard and Kavoosi (35), the EO from Z. multiflora at all tested concentrations significantly (P < 0.01) inhibited the growth of A. niger. MIC for A. niger was 2.2 ± 0.5 mg/mL of EO. At concentration > 5 mg/mL, EO significantly (P < 0.01) reduced the growth of A. niger by 100%. In a study by Shokri et al (18), the EO from Z. multiflora showed the inhibitory effects on four tested fungi including A. flavus, A. parasiticus, A. ochraceus and Fusarium verticillioides at all concentrations (500-2000 ppm). It completely inhibited four fungi at 2000 ppm. At 1000 ppm concentration, Z. multiflora EO significantly decreased the growth of Aspergillus species compared with the control, whereas it caused complete growth inhibition of F. verticillioides (P < 0.05). Therefore, F. verticillioides had a higher susceptibility than Aspergillus species against the EO tested. In addition, Nasseri et al (54) showed that the EO from Z. multiflora has anti-fungal activity; the lowest inhibition (75%) was observed in A. niger, while the highest inhibition (95.3%) was observed in Alternaria solani. The MICs for A. solani, Rhizoctonia solani, Rhizopus stolonifer, A. flavus, A. ochraceus and A. niger were 200, 200, 200, 300, 300 and 200 ppm, respectively. In addition, the MFCs for A. solani, R. solani, R .stolonifer, A. niger and A. ochraceus were 600, 400, 300, 900 and 700 ppm, respectively, and none of the tested concentrations were fatal for A. flavus. A. solani and R. solani showed a strong susceptibility to Z. multiflora EO at all concentrations. The fungal pathogens studied were classified according to their susceptibility to the EO in the following order: A. solani > R. stolonifer > R. stolonifer > A. ochraceus > A. niger > A. flavus. Sharif Rohani et al (55) showed that dosage of 800 ppm of Z. multiflora EO for Fusarium solani had obvious inhibitory rate. In a study by Amini et al (56), Z. multiflora EO was very effective on the four studied pathogenic fungi including Pythium aphanidermatum, R. solani, Fusarium graminearum and Sclerotinia sclerotiorum with growth inhibition average of 100% at 200 μL/L concentration. Nevertheless, MIC and MFC of the EO were variable depending to species of fungi. P. aphanidermatum and S. sclerotiorum were the most susceptible and most resistant to the studied EO with average growth inhibition 89.54% and 75.35%, respectively. Khosravi et al (57) determined the anti-fungal assay of EO from Z. multiflora against Saprolegnia parasitica isolated from fish eggs. The infected fish eggs were treated with EO oil at concentrations of 1, 5, 10, 25, 50, and 100 ppm daily. The MIC of Z. multiflora EO against S. parasitica was 0.9 ppm. Z. multiflora at concentrations of 25, 50, and 100 ppm had significant differences in comparison with negative control (P<0.05). The most hatching rates were recorded with Z. multiflora (11%) EO. Z. multiflora was effective for the treatment of S. parasitica-infected rainbow trout eggs in aquaculture environment (57). 4.Inhibition of mycotoxin production by Zataria multiflora Fungi are significant spoilage microorganisms of foodstuffs during the storage, rendering them unfit for human consumption by retarding their nutritive value and sometimes by producing mycotoxins (58). Mycotoxins are polyketide secondary metabolites produced by the important food and feed contaminating species Aspergillus, Penicillium and Fusarium and are known as potent carcinogens for a wide variety of animal species, including humans (59). EOs and their phenolic compounds have been used as natural inhibitors of fungal growth and mycotoxin production to preserve foods and feeds in some countries during recent decades. Many of the spices and herbal EO which have been tested have an antagonistic effect against aflatoxigenic Aspergillus strains (60). Previous studies demonstrated that thymus EO, which mainly consists of thymol, has been shown to inhibit both the growth and aflatoxin production in A. A review on Z. multiflora Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 5 flavus and A. parasiticus (61,62). Yahyaraeyat et al (63) determined the effects of Z. multiflora EO on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of A. parasiticus. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in yeast extract sucrose medium. RT-PCR showed that in the presence of Z. multiflora EO (100 ppm), nor-1, ver-1 and omtA genes expression was reduced. It was suggested that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity (63). Also, Gandomi et al (64) investigated the effect of Z. multiflora EO against growth, spore production and aflatoxin formation by A. flavus ATCC 15546. EO effectively inhibited radial growth and spore production on potato dextrose agar (PDA) in a dose-dependent manner. At 200 ppm, the radial growth and sporulation reduced by 79.4% and 92.5%, respectively. The growth was completely prevented at EO ≥400 ppm on PDA, and MFC of the oil was estimated at 1000 ppm. The EO also significantly suppressed mycelial growth and aflatoxin synthesis in broth medium at all concentrations tested (P < 0.05). In that study, Z. multiflora EO had a significant inhibitory effect on aflatoxin formation, which was reduced by 31% and 99.4% at 50 and 150 ppm, respectively. In a similar study conducted by Farag et al (65), Z. multiflora EO was effective at concentrations ≥100 ppm in reducing aflatoxin formation by 50%. It has been suggested that the regulation of aflatoxin synthesis and conidiogenesis may be interlinked, since the loss of aflatoxigenic capabilities in the nonaflatoxigenic variant strains of A. parasiticus was correlated with alterations in the conidial morphology (66). Furthermore, it has been shown that lysis of the mycelia and spores of the toxigenic fungi are one of the characteristics of aflatoxin deactivation process (67). It was shown by several investigators that chemical compounds of the EOs may cause reduction or stimulation in the toxin production and the anti-toxigenic effects of the EOs are not necessarily related to their anti-fungal activity. Values for growth inhibition were calculated as 0.79 and 0.86mM for carvacrol and thymol, while for AFB1 and AFG1, it was reported as 0.50 and 0.06mM for carvacrol and 0.69 and 0.55mM for thymol (68). It was also shown by Wright et al (69) that aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. Their results indicated that all three volatile compounds reduced radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus. Difference in antifungal and aflatoxin inhibition efficacy of Thymus and Zataria EOs in different studies may be attributable to the EO compositions. Two major components of Thyme and Zataria are thymol and carvacrol. Thyme contains higher thymol than Zataria and Zataria contains more carvacrol. The other factors are also important for causing these differences such as differences in culture media used, culture conditions, temperature, pH and durations of culture. There are little reports on the effect of Z. multiflora in inhibiting the other mycotoxins. In this regard, Vazquez et al (70) reported that phenolic compounds, i.e., thymol and eugenol, inhibited growth and citrinin production by Penicillium citrinum in some Galician cheeses. In another study by Noori et al (71), the EO from Z. multiflora significantly (P < 0.05) suppressed citrinin formation by P. citrinum in mozzarella cheese at all concentrations, as citrinin accumulation was reduced by 30% and 87% at 50 and 1000 ppm, respectively. Ultrastructural changes of fungi treated with Zataria multiflora 1. Scanning electron microscopy The effect of Z. multiflora EO on morphology of A. flavus observed by Scanning electron microscopy (SEM) is illustrated in Figure 1. Hyphae grown on PDA without EO showed swollen and turgid feature with smooth and uniform surface. In presence of 50 ppm EO, hyphae lost their turgidity and uniformity to some extent and these modifications increased at higher concentrations of EO. Furthermore, 100 ppm EO induced the shrinkage of the hyphae and formation of pits along the mycelia. The severity of damage increased with 200 ppm EO and the hyphae were frequently collapsed and their destruction was often evident (72). 2. Light and transmission electron microscopy Semi-thin sections of A. flavus grown in broth medium containing different concentrations of Z. multiflora Figure 1. Scanning electron micrographs ×2000, Aspergillus flavus mycelia: (a) untreated; (b) treated with 50 ppm Zataria multiflora essential oil (EO); (c) treated with 100 ppm Z. multiflora EO; (d) treated with 200 ppm Z. multiflora EO (72). Shokri H and Sharifzadeh A Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 6 EO are illustrated in Figure 2. Longitudinal sections of untreated culture displayed normal mycelia that had a steady arrangement with continuous and homogenous cytoplasm (Figure 2a). Vacuolization of the cytoplasm was the major effect induced in the presence of 50 ppm EO, which led to the cell swelling (Figure 2b). However, mycelia treated with 100 ppm EO revealed greater damage characterized with detachment of the cell membrane from the cell wall that was collapsed and herniated in different intervals along the longitudinal sections resulting in the deformation of mycelia (Figure 2c). In presence of 150 ppm EO, cell membrane was fully disconnected from the cell wall and mycelia obtained an electron-lucent appearance due to cytoplasm shedding from the cell (Figure 2d). Moreover, swelling and deformation of the mycelia were the other changes observed (Figure 2d). The concentration of 200 ppm EO effectively inhibited the germination of the fungal conidia and no mycelia formed at this concentration (Figure 2e). Some of the conidia had a normal shape but the others showed changes ranging from the vacuolization to cell membrane detachment and deformation (Figure 2e). With increasing EO concentration to 400 ppm, more severe damages including depletion of the cytoplasm and frequent degradation of the cell wall were observed (Figure 2f). Furthermore, deformation and destruction of conidia were often evident (Figure 2f) (72). Examination of ultrathin sections under transmission electron microscopy (TEM) resulted in more obvious findings. Untreated hypha was enclosed by the cell wall that was integrated and intact. The plasma membrane was Figure 2. Semi-thin sections × 1000, Aspergillus flavus grown in broth medium: (a) untreated; (b) treated with 50 ppm Zataria multiflora essential oil (EO); (c) treated with 100 ppm Z. multiflora EO; (d) treated with 150 ppm Z. multiflora EO; (e) treated with 200 ppm Z. multiflora EO; (f) treated with 400 ppm Z. multiflora EO. fully attached to the cell wall along the hypha and was smooth and unwrinkled. Cytoplasm was homogeneous and dense and intercellular septum was intact and healthy. At concentration of 50 ppm of Z. multiflora EO, the cell wall lost its integrity and uniformity to some extent. The cell membrane in some areas was detached from the cell wall and invaginated into the cytoplasm. The loss of cytoplasm density and vacuolization were the other changes. At concentration of 100 ppm of Z. multiflora EO, the plasma membrane was completely separated from the cell wall and vacuolization of the cytoplasm was more evident. The presence of 150 ppm of EO increased the severity of injury characterized by the depletion of cytoplasm content, and hypha lost its normal shape and was collapsed. In addition, detachment and fragmentation of the plasma membrane and formation of the lomasome, small membrane-bound vesicles, beneath the cell wall, were seen and membrane fragments were spread over the cytoplasm. At concentration of 200 ppm EO, no evident change was observed in the cell wall, whereas cell membrane was completely destroyed and cytoplasm was released from the cell. With increase in oil concentration to 400 ppm, complete destruction of the plasma membrane and cell wall was evident and hypha was totally free of cytoplasm (73). Conclusion In summary, the microscopic examinations showed that Z. multiflora EO suppressed the size of the colony as well as sporulation of fungi. Mycelia treated with EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. The major change at level as low as 50 ppm of EO was limited to vacuolization of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites of action of EO were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin production. Authors’ contributions All authors contributed to the conception of the study, confirmed the final version of the article and approved all aspects of the study. Conflict of interests The authors declared no competing interests. Ethical considerations Ethical issues (including plagiarism, misconduct, data fabrication, falsification, double publication or submission and redundancy) were completely observed by authors. A review on Z. multiflora Journal of Herbmed Pharmacology, Volume 6, Number 1, January 2017 http://www.herbmedpharmacol.com 7 Funding/Support This work was supported by the Council Research of Amol University of Special Modern Technologies, Amol, Iran (Grant No. 18902).