Investigation of ethnopharmacology and diversity in yield components and phytochemical characteristics of some species of the genus Viola (Viola spp.) in the habitats of northern Iran
Subject Areas : Medicinal PlantsMina Dordi 1 , sarah khorasaninejad 2 , Khalil ghorbani 3 , mohsen farhadpour 4
1 - Graduate Student, Department of Horticultural Siences, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
2 - Gorgan University Agricultural Sciences and Natural re sources
3 - Associate Professor, Department of Water Engineering, Faculty of Water and Soil, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
4 - Assistant Professor, National Research Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Keywords: phenols, Flavonoids, antioxidant compounds, Ethnopharmacology, Habitates, Viola,
Abstract :
In this research, in order to investigate the ethnopharmacological diversity, yield components and phytochemical characteristics of Viola species (Viola spp.) from 16 habitats in the north and northwest of Iran (Golestan, Mazandaran, Ardabil, East Azarbaijan and Kermanshah provinces), a research based on a completely Randomization was done with three replications. Ethnopharmacology information was obtained from local people and plant samples were collected from the mentioned habitats in the spring of 2018 at the time of flowering. At the same time, the samples of each barium were collected and prepared for identification at the species level and receiving the code. Soil samples were also taken from a depth of 0-30 for physical and chemical analysis, and the geographic coordinates and height were also recorded. The yield components and phytochemical characteristics (including chlorophyll a, b and total, carotenoid by spectrophotometer, total phenol by Folin-Siocatio colorimetric method, total flavonoid by aluminum-chloride colorimetric method, antioxidant compounds by ABTS and DPPH methods) of the collected samples were measured. As a result of the botanical investigation, it was found that 16 collected masses were related to five different species including V.odorata L., V.alba, V.suavis, V.sieheana and V.ignobilis. Ethnopharmacological results showed that in most regions this plant is called by the name of violet or something similar and it is used in the treatment of viral diseases such as colds. The analysis of the results showed that the characteristics of the habitat had a significant effect on all the components of the measured performance and phytochemical traits, so that the amount of chlorophyll and the dry weight of the aerial parts of the samples increased with the increase in the organic matter and nutrients of the soil. Also, with the increase in altitude, the amount of antioxidant compounds by ABTS and DPPH methods, phenolic and flavonoidic compounds increased in aerial parts, and the results of principal component analysis (PCA) also confirmed the obtained results. In the clustering that was done based on the two factors of yield and phytochemical components, it was found that the cluster decomposition for phytochemical characteristics was divided into 14 categories and for yield components into 9 categories at 0.2 Euclidean distance. The results of cluster analysis also showed that the climatic and edaphic characteristics of the habitat have a great effect on the yield components and phytochemical characteristics of the violet plant, which can determine the appropriate cultivation pattern.
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Ahmad, H., Khan, S. M., Ghafoor, S., and Ali, N. 2009. Ethnobotanical study of upper siran. Journal of herbs, spices and medicinal plants. 15(1): 86-97.
Al-Amier, H., and Craker, L.E. 2006. In vitro selection for stress tolerant spearmint. Botanicals and Medicinals. 306-310.
Alibakhshi, M., Mahdavi, Kh., Mahmoudi, J., and Ghelichnia, H. 2014. Phytochemical survey of essential oil of Stachys inflate indifferent regions of Mazandaran. Eco – phytochemical Journal of Medicinal Plants. 6(2):56-68.
Alizadeh Ahmadabadi, A., and khorasaninejad, S. 2016. The effect of humic acid pretreatment on germination of purple cornflower (Echinacea purpurea) plant under drought and salinity conditions. Arid Biome Scientific and Research Journal. 6 (2): 97-107. (In Persian)
Alizade ahmad abadi, A., Khorasaninejad, S., and Hemmati, Kh. 2017. The effect of limited irrigation stress and humic acid on the some morphological and root phytochemical characteristics of Purple coneflower. Journal of Crops Improvement (Journal of Agriculture). 19(1): 1-14. (In Persian)
Alizadeh Behbahani, B., Tabatabaei Yazdi, F., Vasiee, A., and Mortazavi, S.A. 2018. Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection. Microbial Pathogenesis. 114: 449-452.
Al-Jaber, H.I., Al-Qudah, M.A., Barhoumi, L.M., Abaza, I.F., and Afifi, F.U. 2012. Essential oil composition of the aerial parts of fresh and air-dried Salvia palaestina Benth. (Lamiaceae) growing wild in Jordan. Natural Product Research. 26 (13): 1179-1187.
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Beheshti, F., and Khorasaninejad, S. 2023. Effect of silicon on some growth, physiological and phytochemical properties of Cannabis sativa L. in soil and soilless culture. Eco-phytochemical Journal of Medicinal Plants. 10(4): 44-61.
Beheshti, F., Khorasaninejad, S., and Hemmati, K. 2023. Effect of salinity stress on morphological, physiological, and biochemical traits of male and female plants of cannabis (Cannabis sativa L.). Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research. 30(2): 242-262.
Chang, C., Yang, M., Wen, H. and Chern, J. 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Food and Drug Analysis. 10: 178-182.
Chorli, S., Khorasaninejad, S., Hemmati, Kh., and Kashefi, B. 2016. The study of morphological characteristics, antioxidant and essential oil contents of the medicinal plant Stachys lavandulifolia Vahl. in habitats of Semnan, Razavi, and North Khorasan provinces. Journal of Plant Ecophysiological Research. 11(41): 41-52. (In Persian)
Chorli, S., Khorasaninejad, S., Hemmati, Kh., and Kashefi, B. 2020. Ethnopharmacology, quantity, and quality of flower and leaf of Stachys lavandulifolia Vahl. in four habitats in Semnan, Khorasan Razavi, and North Khorasan provinces. Journal of Iranian Plant Ecophysiological Research. 11(59): 72-88. (In Persian)
Ghorbani, M., Khorasaninejad, S., Hemmati, K., and Ghorbani, K. 2022. Feasibility study on some native Iranian Viola spp. domestication. Iranian Journal of Medicinal & Aromatic Plants. 38(4): 632-650. (In Persian)
Gorgini Shabankareh, H., Khorasaninejad, S., Sadeghi, M., and Tabasi, A.R. 2018. The effects of irrigation periods and humic acid on morpho-physiological and biochemical traits of Thyme (Thymus vulgaris). Journal of Plant Ecophysiological Research. 13(51): 67-82. (In Persian)
Gorgini Shabankareh, H., Khorasaninejad, S., Soltanloo, H., and Shariati, V. 2021. Physiological response and secondary metabolites of three lavender genotypes under water deficit. Scientific Reports. 11:19164. https://doi.org/10.1038/s41598-021-98750-x.
Gulcin, I., Topal, F., Sarkaya, S.B.O., Buesal, E., Bilsel, G., and Goren, A.C. 2011. Polyphenol contents and antioxidant properties of medlar (Mespilus germanica L.). Records of Natural Products. 5(3): 158-175.
Hassanpouraghdam, M.B., Ghorbani, H., Esmaeilpour, M., Alford, M.H., Strzemski, M., and Dresler, S. 2022. Diversity and distribution patterns of endemic medicinal and aromatic plants of Iran: implications for conservation and habitat management. International Journal of Environmental Research and Public Health. 19(3):1552.
Hosseini, M., Forouzeh, M.R., and Barani, H. 2019. Identification and Investigation of Ethnobotany of Some Medicinal Plants in Razavi Khorasan Province. Journal of Medicinal Plants. 18(2): 15-29.
Jaffarpour, P., Farokhzad, A., Alirezalou, A., and Najad Habibvash, F. 2018. Phytochemical diversity and antioxidant activity in different salvia species in West Azerbaijan province. Eco-phytochemical Journal of Medicinal Plants. 22(6): 23-38.
Jalali zenur, M.J. 2012. New Principles for Sheep Breeding. 3rd Ed. Publishers Partov Vaghee, Tehran.
Jewell, P., Kauferle, L.D., Gusewell, S., Berry, N.R., Kreuzer, M., and Edwards, P.J. 2007. Redistribution of phosphorus by cattle on a traditional mountain pastures in the Alps. Agriculture, Ecosystems and Environment. 122: 377-386.
Kaghazloo, Z., Hemati, Kh., and Khorasaninejad, S. 2017. Effect of height on some secondary metabolites of different organs of Sambucus ebulus L. in three cities in Golestan province. Journal of Iranian Plant Physiological Research. 12(47): 1-13. (In Persian)
Kargar, M., Jaafarianm Z., Tamartash, R., and Alavi S.J. 2015. The effects of some soil properties and topography on some functional traits of Stachys lavandulifolia Vahl. In Angemar rangeland. Lasem watershed. Journal of Rangeland. 8(4): 342-350.
Khorasaninejad, S., Soltanloo, H., Ramezanpour, S.S., Hadian, J., and Atashi, S. 2015. Effect of drought stress on some morphological characteristics, quantity and quality of essential oil in lavender. Journal of Crops Improvement. 17 (4): 988-979.
Lim, T. 2014. Viola odorata. In Edible Medicinal and Non Medicinal Plants Springer, 795-807.
Majewska, M., Skrzycki, M., Podsiad, M., and Czeczot, H. 2011. Evolution of antioxidant potential of flavonoids and in vitro study. ACTA Ploniae Pharmaceutica- Drug Research. 68(4): 611-615.
Marcussen, T., Ballard, H. E., Danihelka, J., Flores, A. R., Nicola, M. V., and Watson, J. M. 2022. A revised phylogenetic classification for Viola (Violaceae). Plants. 11(17): 2224.
Martens, S., and Mithofer, A. 2005. Flavones and flavone synthases. Phytochemistry. 66: 2399-2407
Matsui, R., Takei, Sh., and Ohga, K. 2013. Morphological and Anatomical Variations in Rheophytic Ecotype of Violet, Viola mandshurica var. ikedaeana (Violaceae). American Journal of Plant Sciences. 4: 859-865.
Mazaraie, A., Mousavi-Nik, S.M., and Leila, F. 2018. Assessments of phenolic, flavonoid and antioxidant activity of aqueous, alcoholic, methanol and acetone extracts of thirteen medicinal plants. Nova Biologica Reperta. 4(4): 299-309.
Moradi, H., Hadadinejad, M., Yavari, A., Mohammadiazni, M., Mosavi, M., and Hoseini, M.A. 2021. Comparison of morphological and phytochemical traits in some endogenous genotypes of sweet violet (Viola odorata L.) in Mazandaran and Golestan provinces. journal of Plant environmental physiology. 16(63): 110-115.
Mozaffari, S., Khorasaninejad, S., and Gorgini Shabankareh, H. 2016. Effects of Irrigation content based on field capacity percent and Hiumic acid on morphophysiological traits on medicinal plant (Portulaca oleracea L.). Journal of Crop Production. 9(3): 153-175. (In Persian)
Mozaffari, S., Khorasaninejad, S., and Gorgini shabankareh, H. 2017. The effects of irrigation regimes and humic acid on some of physiological and biochemical traits of Common Purslane in greenhouse. Journal of Crops Improvement (Journal of Agriculture). 19 (2):401-416. (In Persian)
Najjar Firoozjaee, M., Hemmati, K., Khorasaninejad, S., Daraei Garmekhani, A., and bagherifard, A.A. 2014. The Effect of Height on Morphological Characteristics and Some Secondary Metabolites of Nettle Plant in Mazandaran and Golestan Provinces. Journal of Plant Ecophysiological Research. 9(3): 1-10. (In Persian)
Nikkhah, A., Khayami, M., and Heidari, R. 2010. Evaluation of nitric oxide scavenging activity of anthocyanins from black berry (Morus nigra L.), strawberry (Fragaria vesca L.) and berry (Morus alba L. Var. Nigra) extracts. Scientific and Research Journal of Iranian Medicinal and Aromatic Plants. 25(1): 120-128. 28.
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Omidbaigi, R. 2014. Approaches to production and processing of medicinal plants. Astan Quds Razavi, to be published, 400p.
Robinson, S. I., O’Gorman, E. J., Frey, B., Hagner, M., and Mikola, J. 2022. Soil organic matter, rather than temperature, determines the structure and functioning of subarctic decomposer communities. Global Change Biology. 28(12): 3929-3943.
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Saeidi, K., Sefidkon, F., and Babaei, A. 2014. Determination of carotenoids and lycopene content of Dog-Rose (Rose canina L.) fruit in different regions of Iran. Iranian Journal of Medicinal and Edicinal and Aromatic Plants. 5(67): 833-842.
Safi, Z., Saeidi, K., Lorigooini, Z., and Shirmardi, H.A. 2016. Evaluation of total phenols and antioxidant activity of Mullein (Verbascum songaricum) ecotypes. Journal of Shahrekord University of Medical Sciences. 17: 68-75.
Slazak, B., Kaltenböck, K., Steffen, K., Rogala, M., Rodríguez-Rodríguez, P., Nilsson, A., and Göransson, U. 2021. Cyclotide host-defense tailored for species and environments in violets from the Canary Islands. Scientific reports: 11(1): 1-13.
Sałata, A., Nurzyńska-Wierdak, R., Kalisz, A., Kunicki, E., Ibáñez-Asensio, S., and Moreno-Ramón, H. 2022. Effects of organic cropping on phenolic compounds and antioxidant capacity of globe artichoke herbs. Agronomy. 12(1): 192.
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Ahmad, H., Khan, S. M., Ghafoor, S., and Ali, N. 2009. Ethnobotanical study of upper siran. Journal of herbs, spices and medicinal plants. 15(1): 86-97.
Al-Amier, H., and Craker, L.E. 2006. In vitro selection for stress tolerant spearmint. Botanicals and Medicinals. 306-310.
Alibakhshi, M., Mahdavi, Kh., Mahmoudi, J., and Ghelichnia, H. 2014. Phytochemical survey of essential oil of Stachys inflate indifferent regions of Mazandaran. Eco – phytochemical Journal of Medicinal Plants. 6(2):56-68.
Alizadeh Ahmadabadi, A., and khorasaninejad, S. 2016. The effect of humic acid pretreatment on germination of purple cornflower (Echinacea purpurea) plant under drought and salinity conditions. Arid Biome Scientific and Research Journal. 6 (2): 97-107. (In Persian)
Alizade ahmad abadi, A., Khorasaninejad, S., and Hemmati, Kh. 2017. The effect of limited irrigation stress and humic acid on the some morphological and root phytochemical characteristics of Purple coneflower. Journal of Crops Improvement (Journal of Agriculture). 19(1): 1-14. (In Persian)
Alizadeh Behbahani, B., Tabatabaei Yazdi, F., Vasiee, A., and Mortazavi, S.A. 2018. Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection. Microbial Pathogenesis. 114: 449-452.
Al-Jaber, H.I., Al-Qudah, M.A., Barhoumi, L.M., Abaza, I.F., and Afifi, F.U. 2012. Essential oil composition of the aerial parts of fresh and air-dried Salvia palaestina Benth. (Lamiaceae) growing wild in Jordan. Natural Product Research. 26 (13): 1179-1187.
Barros, L., Carvalho, A.M., Morais, J.S., and Ferreira, I.C.F.R. 2010. Strawberry-tree, blackthorn fruits: detailed characterization in nutrients and phytochemicals with antioxidant properties. Journal of Food Chemistry. 120: 247-254.
Barens, J.D., Balaguer, L., Manrique, E., Elvira, S., and Davison, A.W. 1992. A reappraisal of the use of DMSO for the extraction and determination of chlorophyll a and b in lichens and higher plants. Environmental Exprimental Botany. 32 (2): 85–90.
Beheshti, F., and Khorasaninejad, S. 2023. Effect of silicon on some growth, physiological and phytochemical properties of Cannabis sativa L. in soil and soilless culture. Eco-phytochemical Journal of Medicinal Plants. 10(4): 44-61.
Beheshti, F., Khorasaninejad, S., and Hemmati, K. 2023. Effect of salinity stress on morphological, physiological, and biochemical traits of male and female plants of cannabis (Cannabis sativa L.). Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research. 30(2): 242-262.
Chang, C., Yang, M., Wen, H. and Chern, J. 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Food and Drug Analysis. 10: 178-182.
Chorli, S., Khorasaninejad, S., Hemmati, Kh., and Kashefi, B. 2016. The study of morphological characteristics, antioxidant and essential oil contents of the medicinal plant Stachys lavandulifolia Vahl. in habitats of Semnan, Razavi, and North Khorasan provinces. Journal of Plant Ecophysiological Research. 11(41): 41-52. (In Persian)
Chorli, S., Khorasaninejad, S., Hemmati, Kh., and Kashefi, B. 2020. Ethnopharmacology, quantity, and quality of flower and leaf of Stachys lavandulifolia Vahl. in four habitats in Semnan, Khorasan Razavi, and North Khorasan provinces. Journal of Iranian Plant Ecophysiological Research. 11(59): 72-88. (In Persian)
Ghorbani, M., Khorasaninejad, S., Hemmati, K., and Ghorbani, K. 2022. Feasibility study on some native Iranian Viola spp. domestication. Iranian Journal of Medicinal & Aromatic Plants. 38(4): 632-650. (In Persian)
Gorgini Shabankareh, H., Khorasaninejad, S., Sadeghi, M., and Tabasi, A.R. 2018. The effects of irrigation periods and humic acid on morpho-physiological and biochemical traits of Thyme (Thymus vulgaris). Journal of Plant Ecophysiological Research. 13(51): 67-82. (In Persian)
Gorgini Shabankareh, H., Khorasaninejad, S., Soltanloo, H., and Shariati, V. 2021. Physiological response and secondary metabolites of three lavender genotypes under water deficit. Scientific Reports. 11:19164. https://doi.org/10.1038/s41598-021-98750-x.
Gulcin, I., Topal, F., Sarkaya, S.B.O., Buesal, E., Bilsel, G., and Goren, A.C. 2011. Polyphenol contents and antioxidant properties of medlar (Mespilus germanica L.). Records of Natural Products. 5(3): 158-175.
Hassanpouraghdam, M.B., Ghorbani, H., Esmaeilpour, M., Alford, M.H., Strzemski, M., and Dresler, S. 2022. Diversity and distribution patterns of endemic medicinal and aromatic plants of Iran: implications for conservation and habitat management. International Journal of Environmental Research and Public Health. 19(3):1552.
Hosseini, M., Forouzeh, M.R., and Barani, H. 2019. Identification and Investigation of Ethnobotany of Some Medicinal Plants in Razavi Khorasan Province. Journal of Medicinal Plants. 18(2): 15-29.
Jaffarpour, P., Farokhzad, A., Alirezalou, A., and Najad Habibvash, F. 2018. Phytochemical diversity and antioxidant activity in different salvia species in West Azerbaijan province. Eco-phytochemical Journal of Medicinal Plants. 22(6): 23-38.
Jalali zenur, M.J. 2012. New Principles for Sheep Breeding. 3rd Ed. Publishers Partov Vaghee, Tehran.
Jewell, P., Kauferle, L.D., Gusewell, S., Berry, N.R., Kreuzer, M., and Edwards, P.J. 2007. Redistribution of phosphorus by cattle on a traditional mountain pastures in the Alps. Agriculture, Ecosystems and Environment. 122: 377-386.
Kaghazloo, Z., Hemati, Kh., and Khorasaninejad, S. 2017. Effect of height on some secondary metabolites of different organs of Sambucus ebulus L. in three cities in Golestan province. Journal of Iranian Plant Physiological Research. 12(47): 1-13. (In Persian)
Kargar, M., Jaafarianm Z., Tamartash, R., and Alavi S.J. 2015. The effects of some soil properties and topography on some functional traits of Stachys lavandulifolia Vahl. In Angemar rangeland. Lasem watershed. Journal of Rangeland. 8(4): 342-350.
Khorasaninejad, S., Soltanloo, H., Ramezanpour, S.S., Hadian, J., and Atashi, S. 2015. Effect of drought stress on some morphological characteristics, quantity and quality of essential oil in lavender. Journal of Crops Improvement. 17 (4): 988-979.
Lim, T. 2014. Viola odorata. In Edible Medicinal and Non Medicinal Plants Springer, 795-807.
Majewska, M., Skrzycki, M., Podsiad, M., and Czeczot, H. 2011. Evolution of antioxidant potential of flavonoids and in vitro study. ACTA Ploniae Pharmaceutica- Drug Research. 68(4): 611-615.
Marcussen, T., Ballard, H. E., Danihelka, J., Flores, A. R., Nicola, M. V., and Watson, J. M. 2022. A revised phylogenetic classification for Viola (Violaceae). Plants. 11(17): 2224.
Martens, S., and Mithofer, A. 2005. Flavones and flavone synthases. Phytochemistry. 66: 2399-2407
Matsui, R., Takei, Sh., and Ohga, K. 2013. Morphological and Anatomical Variations in Rheophytic Ecotype of Violet, Viola mandshurica var. ikedaeana (Violaceae). American Journal of Plant Sciences. 4: 859-865.
Mazaraie, A., Mousavi-Nik, S.M., and Leila, F. 2018. Assessments of phenolic, flavonoid and antioxidant activity of aqueous, alcoholic, methanol and acetone extracts of thirteen medicinal plants. Nova Biologica Reperta. 4(4): 299-309.
Moradi, H., Hadadinejad, M., Yavari, A., Mohammadiazni, M., Mosavi, M., and Hoseini, M.A. 2021. Comparison of morphological and phytochemical traits in some endogenous genotypes of sweet violet (Viola odorata L.) in Mazandaran and Golestan provinces. journal of Plant environmental physiology. 16(63): 110-115.
Mozaffari, S., Khorasaninejad, S., and Gorgini Shabankareh, H. 2016. Effects of Irrigation content based on field capacity percent and Hiumic acid on morphophysiological traits on medicinal plant (Portulaca oleracea L.). Journal of Crop Production. 9(3): 153-175. (In Persian)
Mozaffari, S., Khorasaninejad, S., and Gorgini shabankareh, H. 2017. The effects of irrigation regimes and humic acid on some of physiological and biochemical traits of Common Purslane in greenhouse. Journal of Crops Improvement (Journal of Agriculture). 19 (2):401-416. (In Persian)
Najjar Firoozjaee, M., Hemmati, K., Khorasaninejad, S., Daraei Garmekhani, A., and bagherifard, A.A. 2014. The Effect of Height on Morphological Characteristics and Some Secondary Metabolites of Nettle Plant in Mazandaran and Golestan Provinces. Journal of Plant Ecophysiological Research. 9(3): 1-10. (In Persian)
Nikkhah, A., Khayami, M., and Heidari, R. 2010. Evaluation of nitric oxide scavenging activity of anthocyanins from black berry (Morus nigra L.), strawberry (Fragaria vesca L.) and berry (Morus alba L. Var. Nigra) extracts. Scientific and Research Journal of Iranian Medicinal and Aromatic Plants. 25(1): 120-128. 28.
Oki, T., Masuda, M., Kobayashi, M., Nishiba, Y., Furuta, S., and Suda, I. 2002. Polymeric procyanidins as radical-scavenging components in red-hulled rice. Journal of Agricultural and Food Chemistry. 30: 5382-5387.
Omidbaigi, R. 2014. Approaches to production and processing of medicinal plants. Astan Quds Razavi, to be published, 400p.
Robinson, S. I., O’Gorman, E. J., Frey, B., Hagner, M., and Mikola, J. 2022. Soil organic matter, rather than temperature, determines the structure and functioning of subarctic decomposer communities. Global Change Biology. 28(12): 3929-3943.
Rohmanna, N. A., and Mulyawan, R. 2022. The effect of growing media on the vitamin C and chlorophyll content of gelinggang microgreen. In IOP Conference Series: Earth and Environmental Science. 1005 (1): 012005. IOP Publishing.
Saeidi, K., Sefidkon, F., and Babaei, A. 2014. Determination of carotenoids and lycopene content of Dog-Rose (Rose canina L.) fruit in different regions of Iran. Iranian Journal of Medicinal and Edicinal and Aromatic Plants. 5(67): 833-842.
Safi, Z., Saeidi, K., Lorigooini, Z., and Shirmardi, H.A. 2016. Evaluation of total phenols and antioxidant activity of Mullein (Verbascum songaricum) ecotypes. Journal of Shahrekord University of Medical Sciences. 17: 68-75.
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