Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Research Paper

Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to treat cancer and related disorders Farzaneh Naghibi a, Amir Khalaj b, Mahmoud Mosaddegh a,b, Maryam Malek Mohamadi a, Maryam Hamzeloo-Moghadam b,n a

Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, No. 8 Shams Alley, Vali-e-Asr Street, 1516745811, Tehran, Iran

b

art ic l e i nf o

a b s t r a c t

Article history: Received 3 February 2014 Received in revised form 12 April 2014 Accepted 18 May 2014

Ethnopharmacological relevance: By studying the Iranian Traditional Medicine (ITM) Pharmacopoeia, we have collected information about medicinal plants which had been used to manage cancer-like disorders over eight hundred years, from medieval to the early modern era. Exploring the ITM herbal knowledge, the selected species have been subjected to MTT assay for examining their in vitro cyototoxic activity. Materials and methods: Cancer was reviewed through the ITM and some terminologies were acquired. Five ITM Pharmacopoeia in Arabic and Persian languages from 10th to 18th century AD were explored. These Pharmacopoeia contained the ITM plants which were used to cure cancer and cancer-like diseases. The ITM names were matched with the scientific names. Then the medicinal plants were collected, authenticated and were evaluated for cytotoxic activity using MTT assay, against MCF-7, HepG-2, A-549 and HT-29 cell lines. Finally, the apoptosis induction ability of the most cytotoxic medicinal plant was investigated by activated caspase 3 inspection in MCF-7 cell line. Results: Six species of the evaluated medicinal plants revealed cytotoxic activity. The most cytotoxicity was observed in Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech with IC50 values of 28.3 μg mL  1, 53.9 μg mL  1 and 43.3 μg mL  1 against MCF-7, A-549 and HT-29 cell lines, respectively. Caspase 3 activation was also observed in MCF-7 cells by Tanacetum polycephalum subsp. argyrophyllum. This is the first time that the cytotoxic activity of this species has been reported. Conclusion: In the present study, some reliable references of ITM have been introduced and though many technical difficulties, linguistic problems and some other hindrances were encountered during the study, using traditional medicine texts for medicinal plant selection could be considered as a helpful starting point in the field of cancer drug discovery. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cancer Ethnopharmacology Iranian Traditional Medicine MTT assay Apoptosis Caspase 3 Traditional medicine texts

1. Introduction

1.2. Background

1.1. Aim of the study

Despite enormous technological development in recent years, a large number of people are diagnosed with cancer and consequently many deaths are reported. The statistics released by the American Cancer Society and the International Union against Cancer have shown probable 27 million diagnoses and 17 million deaths by 2030 worldwide (Aggarwal et al., 2009). Cancer treatments include surgery, radiotherapy and chemotherapy (Brundage, 2008). Chemotherapy in cancer care, as a term is: “a kind of therapy that uses chemical compounds to fight neoplastic diseases” (Schwab, 2001). In spite of low selectivity of cytotoxic agents to kill normal cells over cancerous ones, which is the cause of limitations, challenges and side effects, using such molecules to eliminate cancer cells and inhibit tumor growth are the original approach to the therapy (Xu and Huang, 2012).

The study has focused on the historical Pharmacopoeia of Iranian Traditional Medicine (ITM) to excerpt data on medicinal plants which had been used to manage cancer and related diseases over eight centuries, from medieval to the early modern era. The obtained data subsequently helped us to choose some medicinal plants and evaluate their cytotoxic activity. We have focused on plants which were used specifically and in a long period of time to manage an especial disorder called cancer. n

Corresponding author. Tel./fax: þ 9821 8877 6027. E-mail address: [email protected] (M. Hamzeloo-Moghadam).

http://dx.doi.org/10.1016/j.jep.2014.05.025 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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A large number of natural and synthetic products are approved as anticancer agents and currently are in use (Kumar et al., 2013). Thus, setting up the beginning time for the treatment of an illness like cancer is too difficult because the herbal and other preparations have been used since antiquity (Baguley, 2002); thus, traditional herbal texts could provide a remarkable resource as the starting point for drug discovery (Buenz et al., 2004). ITM as a main subset of the medieval Islamic Medicine had a strong effect on the people of Iranian plateau for a long period of time and ITM scholars had created some important inscribed information such as the Canon on Medicine by Avicenna (dead 1037 AD) (Naghibi et al., 2014). Moreover, traditional medicine therapies are still attractive and practical and plant based health care systems are taking their parts in different cultures nowadays (Cragg and Newman, 2013). In the present study, the cytotoxic activity of 19 medicinal plants has been evaluated through a micro-culture tetrazolium/formazan (MTT) assay. The medicinal plants were selected among some, which were traditionally used to cure cancer and related illnesses in ITM during 10th to 18th centuries AD. Furthermore, The apoptosis induction ability of the most cytotoxic plant has been evaluated by caspase 3 activation. This study has introduced medicinal plants for further cancer studies based on ITM manuscripts.

names were also studied (Amin, 2005; Dini, 2005, Ghahraman and Okhovvat, 2004, 2009; Mozaffarian, 2007). The goal was to achieve all ITM mentioned medicinal plants as far as possible. The considerations toward selection of every plant will be discussed later in this text. It should be noted that in some cases we had no choice but to substitute some plants because they had failed to grow or could not be found in their habitat due to climate changes, field utilization changes or over grazing.

2. Materials and methods

Dried powdered plants (10 g) were macerated with methanol at room temperature for 24 h for thrice. The extracts were concentrated using a rotary evaporator (40 1C) and the dried extracts were kept in 2–8 1C for future assays. The extracts were dissolved in DMSO: 10 mg mL  1 to make the stock solutions of each sample. Serial dilutions were prepared accordingly from the above stock solution to reach final concentrations (DMSO 1%).

2.1. Materials Dulbecco's modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), (Gibco, Auckland, New Zealand), RPMI1640 medium, Penicillin-Streptomycin, MTT ([3-(4, 5-dimethylthiazol-2-yl) -2, 4-diphenyl tetrazolium bromide]) (Sigma, St. Louis, MO, USA), DMSO (dimethyl sulphoxide) (Merck, Hohenbrunn, Germany) were used in the cytotoxic activity evaluation and methanol (Merck, Hohenbrunn, Germany) was used for plants extraction. NucView™ 488 Caspase 3 Assay Kit for Live Cells (Biotium, Hayward, CA, USA) and 5-FU (Sigma, St. Louis, MO, USA) were used in apoptosis assay. 2.2. Data extraction from ITM Pharmacopoeia According to the defined cancer terminology in previous studies (Table 1), the ITM Pharmacopoeia were explored for medicinal plants that were claimed to cure or relief cancerous states. The selected ITM medicinal plants names were matched to scientific names. Some former studies for identifying the scientific

2.3. Plant material Nineteen plant species were collected from Zanjan, Azerbaijan, Ardabil, Gilan, Mazandaran and Alborz provinces, Iran (2012) and were authenticated by botanists at the Traditional Medicine and Materia Medica Research Center (TMRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran. All voucher specimens were deposited at the TMRC herbarium for future reference. The plants were dried in shadow and ground.

2.4. Extraction and preparation for the assays

2.5. Cell lines MCF-7 (human breast adenocarcinoma), HepG-2 (hepatocellular carcinoma), A-549 (non-small-cell lung carcinoma) and HT-29 (colorectal adenocarcinoma) cells were obtained from the Pasteur Institute, Tehran, Iran. MCF-7 and HT-29 cell lines were cultured in DMEM with 5% FBS for MCF-7 cells and 10% FBS for HT-29 cells while the other two cell lines were cultured in RPMI1640 medium with 10% FBS to maintain the desired growth. All cell lines were treated with 1% penicillin-streptomycin and were kept in a humidified incubator at 37 1C in an atmosphere of 5% CO2.

Table 1 ITM cancer keywords used to search ITM Pharmacopoeia. ITM terms (English translation)a ITM term definitionb Cankers

Horrid ulcers Chronic ulcers The Pigs, Scrofula

Fistula Black bile tumors Crab, Cancer Cold tumor Hard, dense or solid tumor a b

Invasive hard to cure ulcers with disgusting appearance and thick red or green borders inclined to the edge of the lesion. The lesion caused decay. A kind of ulcer that was formed because of waste spoiled humors of the body. These humors could cause ulcers that would not or hardly be cured and might lead to death. Might cause chronic ulcers which would occasionally be cured or turned into the chronic black ugly ulcers and disperse. A kind of tumor, stucked to the neighboring tissues. It might grow and new ones might be formed from the first one, just like warts. It mostly occurred around the neck and armpit. It was called Pigs because of the resemblance of the patient's neck to a pigs’ neck. It had amplitude from benign to malignant tumors. An old sore with thick borders. The progression into the tissue was like a pipe and had permanent discharge. Constructed from black bile, they showed no other signs of cancer except for hardness and dark appearance. A tumor, constructed from black bile. It was painful and fast growing. The blood vessels around the lesion made the appearance like a crab and it had the sense of beating. A tumor constructed from black bile or phlegm. Normally, it was non-invasive and benign. It had a slow growing rate. It was non-sensitive and had cool touch. Made from phlegm, black bile or the combination of both and were classified from benign to malignant tumors.

The endeavor was to find the closest alternative in English language. The definitions were summarized from selected historical manuscripts of ITM (Naghibi et al., 2014).

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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cascade. The excitation/emission wavelength were 450/490 nm, respectively.

2.6. MTT assay Cell viability was assessed through MTT assay, as given earlier (Mosaddegh et al., 2010; Hamzeloo Moghadam et al., 2012) in the absence and presence of different concentrations of the extracts. The cells were seeded in 96-well plates at 6  103 for MCF7, 15  103 for HepG2, and 8.5  103 for A549 cell lines. The cells were incubated under the same condition, afterwards. After 24 h, the medium was replaced with fresh medium containing different concentrations of the extracts to be tested: 100, 50, 25, 12.5, 6.25 and 3.125 (μg mL  1). After 72 h exposure of the cells at 37 1C for each sample, the medium was replaced with fresh medium containing MTT, with a final concentration of 0.5 mg mL  1. The cells were incubated for another 4 h, then the medium containing MTT was removed, and the remaining formazan crystals were dissolved in DMSO. The absorbance was recorded at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader (TECAN), using 5-Flourouracil (5-FU) as the positive control. The relative cell viability (%) related to control wells was calculated by [A] samples/[A] control  100 where [A] samples was the absorbance of the test sample and [A] control was the absorbance of wells containing cells, cell culture medium, and DMSO 1%. The viability vs. Log concentration curves were graphed by the Microsoft Excel program and the IC50 values were assessed.

2.7. Caspase 3 assay in live cells Caspases are normally presented in the inactive form (zymogens) in the cytosol and different pathways would turn them into active forms. The significant member of this family in the execution phase of apoptosis which can activate other caspases is caspase 3 (Logue and Martin, 2009). The apoptosis ability of Tanacetum polycephalum subsp. argyrophyllum has been evaluated by observing active caspase 3 in the test medium. MCF-7 cells were cultured with the same above mentioned method. After 24 h of incubation, they were treated with Tanacetum polycephalum subsp. argyrophyllum methanol extract. Following 20 h of incubation, the medium was replaced with PBS and 10 μL/mL from NucView™ 488 Caspase 3 Assay Kit for Live Cells was added. After 20 min of incubation in the darkness, the results were evaluated with an inverted florescent microscope (HUND) and the images were recorded by a digital camera (Canon 600D). The green florescence at the nuclei documented the presence of active caspase 3 at the cytosol and subsequently the active apoptotic

3. Results 3.1. ITM Pharmacopoeia ITM Pharmacopoeia used in the present study, are listed in Table 2. The route of literature selection was as mentioned in our former study (Naghibi et al., 2014). 3.2. ITM plant names 99 medicinal plant names were acquired from ITM manuscripts for cancer management. Thought not all of them were evaluated in the cytotoxicity assay, these ITM names formed the basic information for continuing the rest of study (Table 3). Table 4 presents selected ITM names besides proposing scientific names that could be related to every medicinal plant and the probable English common names. Most of the time more than one scientific name could be matched with a single medicinal plant. The selected scientific names and a short bibliography about the selected medicinal plants are presented in Table 5. 3.3. Cytotoxic activity evaluation results Nineteen plant species -23 samples due to more than one organ extraction for some plants- were evaluated for cytotoxic activity (10 out of the 19 plants have been evaluated for the first time). The extracts showing the IC50 value more than 100 μg mL  1 were considered as inactive (Table 6). Despite strong statements about nearly all of the tested medicinal plant in the ITM literature, only the total extract of five ITM plants which were related to 6 species demonstrated IC50 values. There have been some previous reports about some of the evaluated species as in Achillea santolinoides subsp. wilhelmsii (K.Koch) Greuter (Asteraceae) which was found to be cytotoxic in some studies. For example, Ali et al. reported its cytotoxic effect in 2011 (against brine shrimps-IC50 2.3 μg mL.  1), as in our study (against MCF-7 with the IC50 value of 60.7 μg mL.  1). This plant is currently used by ethnic people of Kohgiluyeh and Boyerahmad Province-Iran to handle conditions such as bleeding, diabetes, hypertension, kidney stone and constipation (Mosaddegh et al., 2012). The two other cytotoxic plants were also from the Asteraceae family, Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech (against MCF-7, A-549 and HT-29- with the

Table 2 Selected medical literatures of ITM. Author

Original title

Title in English

Date Text used in produced A.D. this study

Language of the literature

The writer ethnicity

(Rhazes), AbūBakr Muhammad ibn Zakarīyā’ al-Razi (da. cab. 925 A.D.)c (Avicenna), Abū ‘Alī al-Husayn ibn ‘AbdAllāh Ibn Sīnā (d 1037 A.D.) Abū Mansūr Muwaffaq bin Ali al-Hirawī (d ca after second half of 11th) Muhammad Mu’min ibn MīrMuhammad Zamān Daylamī Tunakābunī (d. 2nd half of 17th)

Kitāb al-Hāwīfī alTibb Kitāb al-Qānūn fī al-Tibb Kitāb al-Abnia an Haqāyiq al-Adwiya Tuhfat al-mu’minīn

The Comprehensive Book on Medicine or The Continens The Canon on Medicine

Before 925

Arabic

Persian

Before 1037

Arabic

Persian

The Book of buildings of Truths about drugs The Present for the Faithful

Before second half of 11th 1679

Persian

Persian

Persian

Persian

Muhammad Husayn ibn Muhammad Hādī ‘Alavī Shīrāzī (d. 1747 or 1749)

Makhzan al-adviyah

The Storehouse of Medicaments Before 1749

Persian

Persian

(Rhazes, 1962) (Avicenna, 1593a and b) (Hirawī, 2009) (Hakim Mu’min, 2006) (Alavi, 2007)

a

Dead. Circa. c The authors’ names, title of the books and the titles in English have been cited from National Library of Medicine, 2013 except for Abū Mansūr Muwaffaq bin Ali al-Hirawī. b

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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Table 3 ITM medicinal plants used to cure or relief cancer and related diseases. a

No.

ITM medicinal plants name

1 2 3

Abhal Abnous Abou Khalsa

4 5 6

Abroon Aftimoon Agh’howan

/æbuːχælsɑː/ /æbɾuːn/ /æƒʈiːmuːn/ /æɢhævɒːn/

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Aghalifi Aghthee Aklil ol jabal Akte Anjore Annab Anthemes Aqhowan Arghitoon Aslaq Baboonaj Badenjan Baghala mesri Banjakosht Belenjasb Berenjasf

/ æɠɑːliƒe/ /ɒːɢtiː/ /ækliːl ɔːl ʤæbæl/ /ɒːktiː/ /ænʤɔɾe/ /ænnɒːb/ /ɑːnʈemiːs/ /æɠhɔʋɑn/ /ærɢiːtuːn/ /æslæɢ/ /bɒːbuːnæʤ/ /bɒːdænʤɒːn/ /bɒːɢlɒː mesriː/ /bænʤæŋgoʃt/ /belenʤɒːsæb/ /berenʤɒːsæf/

23 24 25

Cababe Cabar Chayi e khataee

/kæɓɑːbe/ /kæɓæɾ/

26 27 28 29 30 31 32

Choub e chini Dar filfil Daroghinoon Defli Drubetares Enab al-salab Esphanakh

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

Fanjangosht Farthanion Fashara Filgoosh Filrahrakh Filzahraj Ghabares Ghantorion Ghatel al-rohban Ghennab Ghersane Ghysoom Habb-e-alban Halboob Harshaf Heltit Hoechoobe Hommaz Horf Hozaz Hugharighoun Huperikon Irs

/sƒɑːnɑːχ/ /fænʤæŋgoʃt/ /ƒæɾɵɑːniːuːn/ /fɒːʃærɒː/ /fiːlguːʃ/ /fiːlræhræx/ /fiːlzæhræʤ/ /ɢɒːbɑːɾes/ /ɢæntuːrjuːn/ /ɢɒːtel ælrohbɒːn/ /ɢennɑb/ /ɢærsæʔne/ /ɢæjsuːm/ /hæbeælbɒːn/ /hælbuːb/ /hærʃæf/ /heltiːt/ /hæveʧuːbe/ /hommɒːz/ /horf/ /hozæz/ /huɢɒːriːɢuːn/ /huperiːkon/ /iːres/

56 57 58 59 60 61 62 63 64 65 66

Irsa Karnab Karnab Khaizaran Khalidonion Khasi ol Kalb Khas-ol-Hemar Khatmi Khomman Khomman-e-kabir Khomman-e-saghir

/iːresɒː/ /kærnæb/ /kærnæb/ /xæjzærɒːn/ /xɒːliːduːnjuːn/ /xesiː æl kælb/ /xæsɔlhemɒːr/ /xætmiː/ /xmmɒːn/ /xomɒːnekæbiːr/ /xomɒːnesæɢiːr/

67 68 69

Khoolanjan Kozbare Luff

/xuːlænʤɒːn/ /kozboræ/ /lævf/

Phonography of medicinal plants names by IPA b

Therapeutic properties of medicinal plants (claimed to relieve cancer and related diseases c)

/æbhel/ /ɑːbnuːs/

Would clear the repulsive and chronic ulcers putrefaction Decoction in wine would remove the Pigs d. It would relieve repulsive ulcers Poultice would dissolve the Pigs and hard tumors

/ʧɑːɔɪ e χæʈɑːiː/ /ʧuːb e ʧɪnɪ/ /dɑːɾƒeɭƒeɭ/ /dæɾuːɠɪnuːn/ /deƒliː/ /dɾuːbeʈɑːɾes/ /enæb ɔɭ sæɭæb/

Poultice would benefit repulsive ulcers and old sores Would clear cancer Intra-vaginal administration would open hard tumors and all phlegmatic situations in the uterus Would clear repulsive ulcers and cancers Would relieve repulsive ulcers Poultice would be useful in chronic tumors Would relive fistula Would relieve cankers and ulcerous cancers Would relieve repulsive ulcers and cankers Would smooth hard tumors Would be beneficial on hard tumors with wax Would benefit repulsive ulcers Hip bath would dissolve uterus and anal tumors Would smooth hard tumors Would be useful on hard tumors Poultice would dissolve repulsive ulcers, the Pigs and hard tumors Hip bath would dissolve uterus and anal tumors Would smooth uterus tumors Would smooth or dissolve the tumors that were formed in testis, uterus, anus, viscera and liver Poultice would dissolve tumors Poultice would benefit repulsive ulcers and would dissolve the Pigs and fistula Poultice would benefit hard tumors Useful to cure repulsive ulcers, fistula, hard tumors and cancer Would dissolve cold tumors Would dissolve tumors Would dissolve hard tumors Would relieve the Pigs Would benefit cankers Cooked poultice would smooth hard tumors Hip bath would dissolve uterus and anal tumors Would relief uterus tumors Would dissolve hard tumors Poultice was a remedy for cancer and nasal fistula Would clear repulsive ulcers Would cure the repulsive ulcers Would dissolve the Pigs and hard tumors Fresh poultice would benefit repulsive ulcers Would dissolve hard tumors The oil would benefit hard tumors Would dissolve hard tumors Fresh poultice would benefit tumors Poultice would benefit hard tumors Would dissolve cold tumors The oil would dissolve hard tumors Was strong tumor dissolver Poultice would dissolve the Pigs and hard tumors Poultice with honey would be useful for cancerous sores Would dissolve spleen tumor Would cure the repulsive ulcers Would relive horrid ulcers and cankers Would relive horrid ulcers and cankers Cooked poultice would relief uterus pain and tumors and other hard and chronic tumors and the Pigs Would relief chronic ulcers and wounds Poultice would benefit cancer Would relief hard tumors Would dissolve the tumors Would dissolve cancers, cankers and the Pigs Phlegmatic tumor dissolver Poultice would dissolve the Pigs and hard tumors Would dissolve tumors and the Pigs Would relive fistula Tumor dissolver Suppository would be useful to relieve uterus pain and fistula. The leaf's poultice would be [useful] for fistula Would be useful to relief the Pigs and cancer Poultice with the rose flowers oil would cure ulcerous or non ulcerous cancers Poultice would be the remedy for cancer and nasal fistula

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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Table 3 (continued ) No.

ITM medicinal plants name

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

Lughion Ousaj Panjankosht Qar Ratm Sakbinaj Salgh Sanidritis Shahesfaram Shankar Sharbin Shaylam Shoniz Soranjan Spharze Susan Termes Thafesia Todari Yatoua Zabib Zafaran Zanb-ol-Khail Zanjabil al-kalab Zaravand Zard choobe Zarir Zatar Zofre Zoghal

a

Phonography of medicinal plants names by IPA b

Therapeutic properties of medicinal plants (claimed to relieve cancer and related diseases c)

/luːɢiːuːn / /ævsæʤ/ /pænʤæŋgoʃt/ /ɢɒːre/ /rætæm/ /sækbiːnæʤ/ /sælɢ/ /sæniːdriːtæjs/ /ʃɒːhesfæræm/ /ʃæŋgæɾ/ /ʃærbiːn/ /ʃæjlæm/ /ʃuːniːz/ /suːrenʤɒːn/ /esfærzæ/ /suːsæn/ /tærmos/ /ɵɒːfesæjɒː/ /tuːdæriː/ /jætuːʔ/ /zæbiːb/ /zæʔfærɒːn/ /zænæboɒːlxæjl/ /zænʤæbiːl æl kelɒːb/ /zærɒːuːnd/ /zærdʧuːbæ/ /zæriːr/ /zæʔtær/ /zofræ/ /zæɢɒːl/

Would relieve repulsive ulcers Would prohibit repulsive ulcers propagation Hip bath would dissolve uterus and anal tumors Would dissolve cold tumors Tumor dissolver Would dissolve hard tumors and the Pigs Would benefit repulsive ulcers Beneficial on Tumors and the Pigs Would dissolve all tumors Poultice would dissolve the Pigs and hard tumors Would dissolve uterus tumors Would dissolve tumors Possess hard tumor dissolving property Tumor dissolving property experienced Poultice with the rose flowers and vinegar would relieve the Pigs and hard tumors Would dissolve phlegmatic tumors. Would refine the uterus tumors and relieve pain Poultice would be a remedy for hard tumors, repulsive ulcers and the Pigs Poultice with sulfur would be useful to open hard tumor Would benefit the inner [organ] cancer Would dissolve anal tumors, horrid ulcers and cankers Would be useful to relieve the cancer Suppository would benefit repulsive ulcers of uterus Poultice would benefit horrid ulcers Leaves would relieve hard tumors Poultice would benefit repulsive ulcers Would dissolve tumors Would dissolve tumors Would dissolve hard tumors Would dissolve fistulas Useful for the chronic tumors and ulcers

a

These names were ITM common names that have been transferred and listed in English by alphabetic order. International Phonetic Alphabet. At least there was one record of ITM therapeutic properties about cancer and related disorders. These statements were summarized, as claimed in selected Pharmacopoeia, presented in Table 2. The phrases have been translated from Arabic or Persian to English, word by word. d The Pigs is a translation of the name of this illness from Arabic to English (see Table 1). b c

IC50 values: 28.3, 53.9 and 43.3 μg mL.  1, respectively) and Achillea vermicularis Trin. (against MCF-7, A-549 and HT-29- with the IC50 values of 38.4, 65.8 and 55.6 μg mL.  1, respectively). Three other cytotoxic species were Alkanna bracteosa Boiss., Hypericum scabrum L. and Vitex agnus-castus var. pseudonegundo Hausskn from Boraginaceae, Guttifereae and Verbenaceae, respectively. 3.4. Caspase 3 assay in live cells To perform this part of study and according to the MTT assay results we decided to choose Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech as the most proper medicinal plant to investigate apoptosis induction ability. The following figure would exhibit the possible apoptosis induction ability of the Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech methanol extract by observing activated caspase 3 in live cells (Fig. 1). The positive control test was also performed by 5-FU (Fig. 2).

4. Discussion One of the most important challenges to perform this study was identifying and matching the scientific names. A plant name mentioned in traditional medicine (TM) could be helpful but, at the same time, it could also be totally confusing because:

 A single plant name in TM could be ascribed to many different species and vice versa. This would happen even when the plants were completely from different botanical families. For



example “FilGoosh” which means elephant's ear was attributed to some Arum spp and Luffa spp mostly, because of the shape of their leaves that looked like an elephant's ear, even though, they were from different families, Solanaceae and Araceae, respectively. A single plant in TM could be called by different names. This was more confusing for distinct geographical and weather conditions, cultural, linguistic, ethnic and even religious considerations. Narrated morphologies (e.g. different parts of the plants’ color, shape, height as well as flowering or growing seasons and resemblance to famous plants), organoleptic (e.g. taste, smell, fragility), linguistic (e.g. expression of the name of the plant in different languages, accents and etymology), ethnic (e.g. people's thought and superstitions) and the plants’ habitat (e.g. growing on mountains, valleys, gardens and farms, in the shadow or sunny places) data in the ITM literatures made us sure about the consistency between these ethnopharmacologic data through the different regions and organized the clues toward tagging scientific names. But sometimes the enigmatic plant's morphological description or even illustrations were confusing.

To decrease such problems, we tried to choose most of our ITM references from restricted geography, the Iranian plateau. The long timeline could make the above mentioned problems worse but it was inevitable for estimating the longevity of the therapeutic properties of medicinal plants. Such limitations forced us to tag more than one scientific name to a single ITM plant name, in some cases. Finally, we tried to choose the most appropriate scientific names to perform the rest of the study.

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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Table 4 Suggested scientific names that were related to every selected medicinal plant. Selected ITM medicinal plants names from Table 3a

English common names Suggested scientific names in the former studies which had tried to match plants’ scientific names to (De Vos, 2010) ITM names

Abou Khalsa, Shankar, Khas olhemar, Hoe choobe Aghalefi, Al-Anjore, Anjore,

Nettle

Akte, Khomman

Dwarf elder,

Antemes, Al-Baboonaj, Baboonaj

Wild Chamomile

Aslaq, Panjankosht, Banjakosht, Fanjangosht Berenjasf

Chaste berry, Monk's pepper Yarrow

Drubetares Farthanion, Al- Agh’hovan, Agh’hovan Ghabares, Al-Kabar, Kabar

Fern Stinking Chamomile Capers

Hugharighoun, Huperikon

St. John’s wort

Irs, Al-Susan, Susan, Irsa

Fleur-de-lys, Iris, Gladwyn

Khalidonion

-

Lughion, Al-Hozaz, Filrahrakh, Filzahraj, Hozaz, Yatoua Zanb ol-Khail

Wolfberry, Buckthorn, Lycion Spurge,, Horsetail

a

Arnebia euchroma (Royle) I.M.Johnst. (Amin, 2005), Alkanna tinctoria Tausch (Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Alkanna sp (Ghahraman and Okhovvat, 2004, 2009) Urtica dioica L., Urtica pillulifera L. (Mozaffarian, 2007, Amin, 2005, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Urtica urens L. (Mozaffarian, 2007, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009) Sambucus niger L., Sambucus ebulus L. (Mozaffarian, 2007, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009) Matricaria recutita L. (Dini, 2005), Tripleurospermum disciform (C.A. May) Shultz-Bip (Amin, 2005), Anthemis sp, Matricaria sp (Mozaffarian, 2007, Ghahraman and Okhovvat, 2004, 2009) Vitex agnus-castus L. (Amin, 2005, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Vitex negundo L., Vitex psudo-negundo (Hausskn.) Hand.Mtz., Vitex trifolia L. (Mozaffarian, 2007) Cyclamen europaeum L., Artemisia vulgaris L. (Dini, 2005), Tanacetum parthenium (L.) Sch.Bip, Artemisia campestris Pursh, Artemisia vulgaris L., Artemisia sp. (Ghahraman and Okhovvat, 2004, 2009), Achillea eriophora DC (Amin, 2005), Artemisia vulgaris L. (Mozaffarian, 2007) Asplenium adiantum-nigrum L. (Dini, 2005, Ghahraman and Okhovvat, 2004, 2009) Tanacetum parthenium (L.) Sch.Bip. (Amin, 2005, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Cotula foetida Poepp. ex DC, Eclipta prostrata (L.) L. (Dini, 2005, Ghahraman and Okhovvat, 2004, 2009) Capparis spinosa L. (Mozaffarian, 2007 and Dini, 2005), Capparis sp (Ghahraman and Okhovvat, 2004, 2009), Capparis cartilaginea Decne., Capparis decidua (Forssk.) Edgew., Capparis mucronifolia Boiss., Capparis parviflora Boiss. (Mozaffarian, 2007) Hypericum perforatum L. (Amin, 2005, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Hypericum barbatum L., Hypericum ciliatum L., Hypericum coris L., Hypericum sp (Ghahraman and Okhovvat, 2004, 2009) Iris florentina L. (Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Iris germanica L. (Mozaffarian, 2007, Amin, 2005, Dini, 2005, Ghahraman and Okhovvat, 2004, 2009), Iris spuria Pall.(Amin, 2005), Iris pseudacorus L. (Amin, 2005), Iris songarica Schrenk (Mozaffarian, 2007) Chenopodium ambrosioides L. (Dini, 2005), Scrophularia auriculata L., Chelidonium majus L. (Ghahraman and Okhovvat, 2004, 2009) Lycium lanceolatum Veill, Rhamnus infectoria Tchich., Lycium afrum L. (Dini, 2005), Lycium sp, Berberis lycium Royle, Rhamnus saxatilis Jacq. (Ghahraman and Okhovvat, 2004, 2009). Euphorbia sp (Dini, 2005, Ghahraman and Okhovvat, 2004, 2009) Equisetum arvense L. (Mozaffarian, 2007, Amin, 2005, Ghahraman and Okhovvat, 2004, 2009), Equisetum hyemale L., Equisetum debile Roxb. ex Vaucher (Ghahraman and Okhovvat, 2004, 2009), Equisetum sp (Mozaffarian, 2007)

As we noted in this study (Section 4) most of the time more than a single ITM plant name had been used to call a single plant name.

Table 5 Plants selected for evaluating cytotoxicitya. ITM name

b

Abou Khalsa, Shankar, Khas-ol-Hemar, Hoechoobe Aghalifi, Anjore Akte, Khomman Anthemes, Baboonaj Aslaq, Panjankosht, Banjakosht, Fanjangosht Belenjasb, Berenjasf Drubetares Farthanion, Agh’howan Ghabares, Cabar Hugharighoun, Huperikon Irs, Al-Susan, Susan, Irsa Khalidonion Lughion, Filzahraj, Hozaz Yatoua Zanb-ol-Khail a b

Scientific name

Family

Alkanna bracteosa Boiss. Urtica dioica L. Sabbucus ebulus L. Tripleurospermum disciforme (C.A.Mey.) Sch.Bip. Vitex psudo-negundo (Hausskn.) Hand.Mtz. which is a synonym for Vitex agnus-castus var. pseudonegundo Hausskn Achillea santolinoides subsp. wilhelmsii (K.Koch) Greuter Achillea vermicularis Trin. Asplenium trichomanes L. Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech Capparis spinosa L. Capparis cartilaginea Decne Hypericum scabrum L. Iris songarica Schrenk Scrophularia variegata M.Bieb. Lycium shawii Roem. & Schult. Lycium ruthenicum Murray Euphorbia marschalliana Boiss., Euphorbia seguieriana Neck. Equisetum telmateia Ehrh.

Boraginaceae Urticaceae Asteraceae

Asteraceae Aspleniaceae Asteraceae Capparidaceae Clusiaceae Iridaceae Scrophulariaceae Solanaceae Euphorbiaceae Equistaceae

The considerations which led us to select the species for cytotoxicity assay, will be discussed later in Section 4. These names are from ITM Pharmacopoeia (Table 3) that have been transferred and listed in English by alphabetic order.

According to ITM morphology of Abou Khalsa, Shankar, Khasol-Hemar or Hoechoobe we decided to accept that Alkanna sp could be matched with this medicinal plant. So, we tried to find and collect the Alkanna species which had not been subjected to cytotoxic assays before. Examples of some Alkanna species which had been examined in cytotoxicity evaluations studies include Alkanna orientale (Mothana et al., 2009), Alkanna cappadocia (Sevimli-Gur et al., 2010) and Alkanna tinctoria (Bogurcu et al., 2011) and . Although Alkanna bracteosa had been investigated for

some biologic effect e.g. anti-inflammatory and analgesic (Mahmoudi et al., 2012) but it had never been investigated for cytotoxic activity. There were strong statements in ITM about Aghalifi or Anjore and the matching led to Urtica species. The most famous Urtica sp is Urtica dioica L. To judge about the cytotoxic effect of Urtica dioica using bibliography was controversial. The direct cytotoxic effect of the essential oil of the aerial part of Urtica dioica was shown to be concentration and time of incubation-dependent (Chrubasik et al.,

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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Table 6 The results of cytotoxicity assay in MCF-7, A-549, HT-29 and HepG-2. Scientific name

Extracted plant part

Alkanna bracteosa Boissb Vitex agnus-castus var. pseudonegundo Hausskn

b

Euphorbia marschalliana Boissb Euphorbia seguieriana Neckb Lycium shawii Roem. & Schult. Lycium ruthenicum Murrayb Asplenium trichomanes Lb Capparis spinosa L. Capparis cartilaginea Decne. Sambocus ebulus L. Scrophularia variegata M.Biebb Equisetum telmateia Ehrh. Hypericum scabrum Lb Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlechb Urtica dioica L. Iris songarica Schrenk Tripleurospermum disciform (C.A. May)b Achillea santolinoides subsp. wilhelmsii (K.Koch) Greuter Achillea vermicularis Trinb a b

Aerial parts Roots Aerial parts Flowers Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Roots Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Roots Rhizomes Aerial parts Aerial parts Aerial parts

ITM suggested plant part

Roots Roots Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Root, Aerial parts and Fruits Roots Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Seeds and aerial parts Roots Aerial parts Aerial parts Aerial parts

IC50 (lg/mL) MCF-7

A-549

HT-29

HepG-2

-a 54 23.8 – – – – – – – – – – – – 54.6 28.3 – – 9 – 60.7 34.8

– – – – – – – – – – – – – – – – 53.9 – – – – – 65.8

– – 53.5 – – – – – – – – – – – – 46.4 43.3 – – – 9 – 55.6

– – – – – – – – – – – – – – – – – – – – – – –

The IC50 values greater than 100 μg mL  1 were considered as inactive. The (b) indicated the species which had been evaluated for cytotoxic activity for the first time in the present study.

Fig. 1. Results of the Caspase 3 assay on the live cells of MCF-7 treated with 20 mg/mL of methanol extract of Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech. The left side figure remarks the light microscope view and the right side remarks fluorescent view of the same field.

Fig. 2. Results of the Caspase 3 assay on the live cells of MCF-7 treated with 0.5 mg/mL of 5-FU.

2007a). Different reports from in vitro cells treated with Nettle's root extracts showed different results, from significant antiproliferative effect to no cytotoxicity (Chrubasik et al., 2007b). In another study, the aqueous extract of Urtica dioica was surveyed for anti-viral properties, in vitro. The anti-viral result of this extract was not clear because cytotoxic effect of the extract

interacted with anti-viral properties (Uncini Manganelli et al., 2005). Akte or Khomman could be matched to both Sambucus ebulus L. and Sambucus niger L. but we could not found Sambucus niger at the designated habitat. Sambucus ebulus L. had been investigated for different biologic effects e.g. antinociceptive (Ahmadiani et al., ,1998) and anti-inflammatory effects (Ahmadiani et al., ,1998 and Schwaiger et al., 2011). Cytotoxic activity of Sambucus ebulus fruits had been evaluated on the normal cell lines CHO and rat fibroblast and cancerous cell lines HepG-2 and CT-26 which had showed cytotoxic effect (Shokrzadeh et al., 2009). We evaluated methanol extract of the aerial parts in the present study against 3 other cancer cell lines, A-549, HT-29 and MCF-7. Some investigations had been carried out about biological activity e.g. the essential oil composition and antimicrobial activity of the plant Tripleurospermum disciforme (C.A.Mey.) Sch.Bip. (Chehregani et al., 2010) and the analgesic properties had been evaluated (Parvini et al., 2007) but there was no report about the cytotoxic property of this species. This plant is a prevalent medicinal plant in the present ITM market which is in use as Baboonaj (Amin, 2005) and that was our motive to choose this species.

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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According to traditional morphology of Aslaq, Panjankosht, Banjakosht or Fanjangosht, we decided to accept that Vitex agnus-castus L could be the most proper scientific name of this medicinal plant. Since the cytotoxic effect of Vitex agnus-castus L. besides the apoptotic effect of this plant had been reported before (Ohyama et al., 2005), we tried to find and collect Vitex sp which had not been subjected to cytotoxic assay before. The ethanolic extract of Vitex negundo had exhibited antiproliferative effect against HepG-2 cell line (Kadir et al., 2013). The attractive biological effect of Vitex agnus-castus var. pseudonegundo Hausskn was the insecticidal effect (Sahaf et al., 2008) and there has been no report about its cytotoxic properties, so we decided to examine this medicinal plant. Complex ITM morphology of medicinal plants which was refered to as Berenjasf made us focus on ethnobotany (Mosaddegh et al., 2012) which led us to Achillea sp. Different biological activity had been attributed to the genus Achillea. For example the essential oil anti-microbial activity of Araneidae eriophora (Ghasemi et al., 2008). Also there were some reports about the cytotoxic activity of different plants of this genus e.g. Achillea talaganica (Saeidnia et al., 2009) and the crude sapponin extract of Achillea wilhelmsii which had been evaluated against KB cell line (Ali et al., 2011). There was been no report about cytotoxic activity evaluation of Achillea vermicularis. According to ITM morphology, Drubetares could be matched with Asplenium adiantum-nigrum and according to our study it could be matched also with some other plants from genus Asplenium, nevertheless, we could find only Asplenium trichomanes L. Some plants of this genus e.g. Asplenium adiantum-nigrum have been used as contraceptive in Indian Traditional Medicine (Kumar et al. 2013) while Asplenium trichomanes has been applied to relive renal and respiratory conditions in Albania and Spain (Gonz´alezTejero et al., 2008). It has also been traditionally used in Italy during pregnancy (Idolo et al., 2010). Asplenium scolopendrium has been used as anticancer (Duke et al., 2002) and Asplenium trichomanes has been investigated for estrogenic properties in vitro (Dall’Acqua et al., 2009). There has been no report about cytotoxic properties of Asplenium trichomanes. The ITM morphology of Farthanion, Al-Agh'hovan or Agh'hovan was confusing and led us to Tanacetum sp. Some plants of this genus have been investigated for biological activities e.g. sesquiterpene lactones isolated from Tanacetum chiliophyllum, Tanacetum corymbosum, Tanacetum densum, Tanacetum vulgare, Tanacetum parthenium, Tanacetum argyrophyllum, Tanacetum argenteum have displayed cytotoxic activity and apoptosis induction against Human myeloid cell lines (HL-60 and U937) (Triana et al., 2013). Antibacterial and antioxidant activity of the essential oil of Tanacetum polycephalum had been previously reported (Amiri, 2007). In the present study, the cytotoxic activity of Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech has been evaluated. There were some strong statements about anti-tumor properties of Ghabares, Al-Kabar or Kabar in ITM and according to the ITM morphology, it was not difficult to match this medicinal plant to Capparis sp. The most famous member of this genus is Capparis spinosa and it has been used as an anticancer, anti-wart and wound healer (Duke et al., 2002). Another member of this genus is Capparis tomentosa which has been used to control diarrhea and jaundice (Duke et al., 2002). The dried fruits of Capparis decidua have taken part in the anti-diabetic formulations of India and Pakistan and have been tested in an animal models in the management of diabetes (Sharma et al., 2010). The essential oil and aqueous extract of Capparis spinosa have been found to show significant antiproliferative (Kulisic-Bilusic et al., 2012). Cytotoxic activity of Capparis cartilaginea, from Yemen, had been investigated and had not demonstrated IC50 below 50 μg mL  1 against

67 MCF-7, A-427 and 5637 cell lines (Mothana et al., 2009). Regarding 68 the strong ITM background, it was decided to evaluate the 69 cytotoxic activity of Capparis spinosa L. and Capparis cartilaginea 70 Decne. that grow in Iran. 71 The ITM morphology of Hugharighoun or Huperikon led us to 72 Hypericum sp. More than 400 plants belong to the genus Hyper73 icum. These plants grow in every continent except Antarctica 74 (Crockett and Robson, 2011). It has been widely used in folk 75 medicine for some therapeutic properties such as antidepressant 76 and wound healing (Ghasemi et al., 2007). There have been some 77 reports about the cytotoxic activity of different Hypericum L. 78 species e.g. Hypericum perforatum, (Hostanska et al., 2003), Hyper79 icum ellipticum (Manning et al., 2011) and Hypericum ascyron 80 (Hashida et al., 2008), but there has been no report about cytotoxic 81 activity evaluation of Hypericum scabrum L. and this was an 82 opportunity for us to evaluate its cytotoxic activity for the 83 first time. 84 The ITM description of Irs, Al-Susan, Susan or Irsa especially the 85 flowers guided us to Iris sp. C-glycosylflavones isolated from the 86 leaves’ extract of Iris tectorum have been evaluated against A-549 87 human lung cancer cell line by MTT assay. The cytotoxic activity 88 has been reported as moderate (Ma et al., 2012). The cytotoxicity 89 of isolated compounds from Egyptian Iris germanica has been also 90 evaluated against Mouse lymphoma (L5178Y), rat brain (PC-12) 91 and human cervix (Hela) cancer cell lines (Mohamed et al. 2013). 92 Also the cytotoxic activity of some isoflavonoids from Iris songarica 93 has been evaluated against HL-60 cell line (Moein et al., 2008) and 94 the cytotoxic activity of this medicinal plants was finally evaluated 95 in our present study. 96 Some plants from the genus Scrophularia, have been evaluated 97 for cytotoxic activity e.g. Scrophularia striata has been evaluated 98 against human astrocytoma cell line (Ardeshiry-lajimi et al., 2010). 99 Scrophularia cryptophila has also showed cytotoxicity against brine 100 shrimps (Valiyari et al., 2012) and Scrophularia oxysepala against 101 MCF-7 cells (Ozbilgin et al., 2014). No data has been reported 102 about cytotoxic activity evaluation of Scrophularia variegata M. 103 Bieb. besides the ethnobotany data of this species in veterinary 104 (Unpublished data) interested us to test this medicinal plant. 105 Lughion, Al-Hozaz, Filrahrakh, Filzahraj or Hozaz was matched 106 with Lycium sp. As a phytotherapeutic agent, fruits of Lycium 107 barbarum have been used as Immunomodulatory and hypotensive 108 medications (Duke et al., 2002). The root bark of Lycium barbarum 109 L. has been officially recorded in Chinese Pharmacopeia (Zhang et 110 al., 2013). This plant has been also known as a medicinal plant in 111 India (Khare, 2007) and it was proposed that it could be con112 sidered as adjunct in combination with doxorubicin in cancer chemotherapy (Amagase and Farnsworth, 2011). The leaves of Q7113 114 Lycium carolinianum and Lycium halmifolium were toxic (Nelson, 115 Shih and Balick, 2007) and water extract of Lycium barbarum had 116 shown anti-inflammatory effects (Zheng et al., 2011). Cytotoxic 117 activity of Lyciumshawii from Yemen has been investigated against 118 two cancer cell lines (5637 and MCF-7) (Mothana et al., 2011). No 119 cytotoxic activity about Lycium ruthenicum has been recorded. 120 Lycium shawii Roem. & Schult. cytotoxic activity comparison from 121 Iran and Yemen localities and no cytotoxic report of Lycium 122 ruthenicum Murray were the encouragements for this plants 123 selection. 124 The matching process for the ITM name Yatoua guided to 125 Euphorbia sp. Some plants from the genus Euphorbia had been 126 investigated for cytotoxic activity before, e.g. Euphorbia wallichii 127 (Ul-Haq et al., 2012), Euphorbia hylonoma (Zhang et al., 2014) and 128 Euphorbia socotrana (Ozbilgin et al., 2014). The chemical constitu129 ents of Euphorbia seguieriana (Jeske et al., 1995) and Euphorbia 130 marschalliana (Jassbi et al., 2004) had been studied but there were 131 no report about cytotoxic activity evaluation of Euphorbia 132 marschalliana and Euphorbia seguieriana. The strong ITM

Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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statement and the lack of data about cytotoxic activity of this two species was the motive for their selection. The ITM morphology of Zanb ol-Khail led to Equisetum sp. Some species of Equisetum have been investigated for cytotoxic activity e.g. Equisetum hyemale against L-1210 cells (Li et al., 2012) and Equisetum arvense against NIH3T3 mouse fibroblast cell line (Uslu et al., 2013). Collected Equisetum telmateia from Serbia have shown significant anti-oxidant activity (Milovanović et al., 2007). The cytotoxicity evaluation of the Equisetum debile (Synonym: Equisetum telmateia) by a Brine Shrimp Lethality Bioassay Method had been carried out (Sarkar et al., 2012) but there was no cytotoxicity report about Equisetum telmateia Ehrh. And it was selected in our study. The apoptotic potential of the methanol extract of Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech was also examined in MCF-7 cells. Evaluating the activity of the activated caspase 3 is a good indicator of apoptosis induction. The activated cytosolic caspase 3 cleaves the substrate and releases the specific nucleus dye which could stain the DNA. Along with the fluorescent shining nuclei of apoptotic cells, the morphologic changes of nucleus during apoptosis such as chromatin condensation and nucleus separation could be observed by fluorescent microscopy. These changes were demonstrated in the reported figures. Fig. 1 shows the brilliant fluorescence caused by activated caspase 3 in the condensed nuclei of MCF-7 cells and the light microscopic field view certified the apoptotic process by obvious shrunk cytoplasm. The positive control on Fig. 2 would confirm the apoptosis induction ability of the methanol extract of Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech. Coming to a conclusion, regarding the reputation of the species, Achillea santolinoides subsp. wilhelmsii (K.Koch) Greuter, Achillea vermicularis Trin., Tanacetum polycephalum subsp. argyrophyllum (K.Koch) Podlech, Alkanna bracteosa Boiss., Hypericum scabrum L. and Vitex agnus-castus var. pseudonegundo Hausskn in ITM for treatment of cancer and related disorders, along with their considerable cytotoxic activity revealed in the present study, these plants could be good candidates for future cancer studies. It seems such method toward medicinal plant selection with an approach to traditional medicine could be helpful for investigating cytotoxic plants as a source for new drugs in the field of cancer drug discovery.

Uncited references (Alavi (Ibn Muhammad Hādī ‘Alavī Shīrāzi) (2007); Avicenna, (IbnSina) (1593); Hirawī (Muwaffaq bin Ali al-Hirawī) (2009); Kumar et al., (2011); Mosaddegh et al., (2006); National Library of Medicine (2013); Rhazes (Razi) (1962)).

Acknowledgments The results were based on a PhD thesis of Traditional Pharmacy (Amir Khalaj, no. 129) granted by the School of Traditional Medicine, Shahid Beheshti University of Medical Sciences. The authors wish to thank for the financial support (Grant no. 10908). The authors also wish to thank Mrs. Bahara Eslami-Tehrani for her assistance in cell culture experiments.

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Please cite this article as: Naghibi, F., et al., Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.025i

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Cytotoxic activity evaluation of some medicinal plants, selected from Iranian traditional medicine Pharmacopoeia to treat cancer and related disorders.

By studying the Iranian Traditional Medicine (ITM) Pharmacopoeia, we have collected information about medicinal plants which had been used to manage c...
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