Evaluation of cytotoxicity and anticonvulsant activity of some Iranian medicinal Ferula species

Abstract

Several Ferula (Umbelliferae) species have been used in Iranian traditional medicine as antiflatulent, antispasmodic, anticonvulsant, expectorant, etc. In the present study, cytotoxicity and anticonvulsant activity of the methanol extracts from several Ferula species were evaluated. Air-dried samples of different parts of these plants (Ferula diversivittata Regel & Schmalh. (roots), Ferula persica Willd. (aerial parts), Ferula ovina (Boiss.) Boiss. (roots), Ferula badrakema Kos.-Pol. (roots), Ferula diversivittata (flowers), Ferula latisecta Rech. F. & Aell. (roots), and Ferula badrakema (fruits)) were macerated with methanol for 3 days. The mixtures were then filtered, concentrated and dried. For determination of the cytotoxicity of the extracts and also the oleo-gum-resin of F. assafoetida L., the brine shrimp (Artemia salina) was employed as a model assay system since it provides a convenient in-house pre-screening method for evaluating general cytotoxicity. The methanol extracts of different Ferula species and the oleo-gum-resin of F. assafoetida exhibited cytotoxic effect with LC₅₀ values in the range of 6-321 μg/mL. For the anticonvulsant testing, seizure was induced by injection of pentylenetetrazole (PTZ), 90 mg/kg intraperitoneally (i.p.). This dose was given to 10 groups, each consisting of 6 mice, which were pretreated i.p. with the extracts (300 mg/kg), Diazepam (10 mL/kg) or saline (10 mL/kg). The results showed that none of the tested Ferula species can prevent PTZ-induced seizure at the used dose. In conclusion, all of the extracts and the oleo-gum resin of F. assafoetida showed dose-dependent cytotoxicity which was highest in F. badrakema fruits and lowest in F. badrakema roots. Our findings also revealed that the methanol extracts and F. assafoetida oleo-gum resin do not possess anticonvulsant activity.

Keywords::

• Ferula
• cytotoxicity
• anticonvulsant activity
• methanol extract

Introduction

The genus of Ferula belongs to the Peucedaneae tribe, a subfamily of Apioideae, family of Umbelliferae with 133 species distributed throughout the Mediterranean area and central Asia, especially in the former USSR and neighboring countries such as Iran (Mozaffarian, 1983; Heywood, 1985; Evans, 1989). The Iranian flora comprises 30 species of Ferula, of which some are endemic (Mozaffarian, 1983, 1996). The popular Persian name for most of these species is “koma” (Mozaffarian, 1996). Several species of this genus have been used in folk medicine for their anticonvulsant, antispasmodic, carminative, digestive, expectorant, sedative, antihysteric, laxative, aphrodisiac, antiseptic, and analgesic activities (Zargari, 1996). Traditional applications of individual Ferula species are shown in Table 1.

Table 1.  Therapeutic applications and biological activities of Iranian medicinal Ferula species.

Ferula species	Part used	Traditional usages	References
F. assafoetida	Oleo-gum-resin	Anticonvulsant, carminative, antispasmodic, diuretic, aphrodisiac, antihelmintic, tonic, laxative, alterative, antiperiodic, antispasmodic, deobstruent, deodorant, expectorant, sedative, circulatory stimulant, treatment of stomach ache and indigestion, bronchitis, bronchial asthma, whooping cough	Eigner & Scholtz, 1990 Zargari, 1996 Plants For A Future, 2008
F. latisecta	Aerial parts	Treatment of infant stomach ache	**
F. badrakema	Resin	Anticonvulsant, tonic, anti-hysteric, decongestant, treatment of neurological disorders, stomach ache	Zargari, 1996
F. diversivittata	Root	Anticonvulsant, tonic, anti-hysteric, decongestant, treatment of neurological disorders, stomach ache	Zargari, 1996
F. ovina*	-	-	-
F. persica	Root	Laxative, carminative, anti-hysteric, treatment of lumbago, diabetes, rheumatism, back ache	Zargari, 1996 Afifi & Abu-Irmaileh, 2000 Plants For A Future, 2008)
F. flabelliloba*	-	-	-

*There is no published evidence for the traditional uses of these two Ferula species.

**Data provided by indigenous people.

The chemistry of this genus has been studied by many investigators (Murray et al., 1982) and is well documented as a good source of biologically active compounds such as sesquiterpene derivatives (Tamemoto et al., 2001; Iranshahi et al., 2004, 2007, 2008; Kogure et al., 2004; Motai et al., 2004) and sulfur containing compounds (Rajanikanth et al., 1984; Al-said et al., 1996; Iranshahi et al., 2003, 2006; Zhi-da et al., 1987).

To our knowledge, there is no comprehensive study on the cytotoxicity and anticonvulsant activity of these Ferula species. In the present study, we investigated the cytotoxicity of the methanol extract of the F. diversivittata (roots), F. persica (aerial parts), F. ovina (roots), F. badrakema (roots), F. diversivittata (flowers), F. latisecta (roots) and F. badrakema (fruits), and the oleo-gum-resin of F. assafoetida by the brine shrimp lethality assay (BSA) method as well as their anticonvulsant activity against the pentylenetetrazole (PTZ) induced seizures.

Materials and methods

Plant material

Ferula persica Willd. was collected from Alborz mountains, north of Tehran, Iran, in May 2004. F. ovina (Boiss.) Boiss. and F. flabelliloba Rech. F. & Aell. were collected from Zoshk valley, Mashhad, Iran, in April 2006. F. diversivittata Regel & Schmalh and F. latisecta Rech. F. & Aell. were collected from Hezarmasjed mountains, Mashhad, Iran, in May 2006. F. badrakema Kos.-Pol. was collected from Tandureh Park, Iran, in August 2007.

The plants were identified by Gholamreza Amin of the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences (TEH), and Mohammadreza Joharchi, Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen of F. persica (Accession number 6523) was deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences. Other voucher specimens were deposited at the Herbarium of the School of Pharmacy, Mashhad University of Medical Sciences (Accession numbers 1002, 1003, 1004, 1005, 1006, and 1007). The oleo-gum-resin of F. assafoetida L. was purchased from the market, Tabas, Iran.

Preparation of the extracts

Different air-dried parts (100 g) of Ferula plants, except for F. assafoetida, were macerated with methanol (2 L) for 3 days. The mixture was filtered and then concentrated with a rotary evaporator apparatus and the residue was dried at room temperature. The extract was maintained at 4°C throughout the experiments.

Chemicals

Methanol was purchased from Mojallali (Tehran, Iran) and distilled before use. PTZ and diazepam were purchased from Sigma (Poole, Dorset). PTZ was dissolved in physiologic saline solution. The extracts were dissolved in DMSO, and then in distilled water containing 5% v/v Tween 80. All drugs and the extracts were administered i.p. in volume of 0.1 mL/10 g of mouse body weight.

Animals

Male NMRI mice weighing 18-28 g were used. The animals were housed in standard cages with free access to food (standard laboratory rodent chow) and water. The animal house temperature was maintained at 23° ± 3°C with a 12 h light/dark cycle (light on from 6:00 a.m. to 6:00 p.m.). The ethical guidelines for the investigation of experimental seizures in conscious animals were conducted in accordance with the internationally accepted principles for laboratory animal use and care. All efforts were made to minimize animal suffering and to reduce the number of animals used.

PTZ-induced seizures

The minimal i.p. dose of PTZ at which 99.9% of the animals showed hind limb tonic extention (HLTE) was determined by a dose-percent effect curve (Litchfield & Wilcoxon, 1949). This dose (90 mg/kg) was then given to 10 groups, each consisting of 6 mice, which were pretreated i.p. with the Ferula extracts (300 mg/kg), diazepam (5 mg/kg as positive control) or saline (10 mL/kg as negative control). The time for the extract to reach its maximum effect was determined as 30 min after i.p. injection. If no HLTE occurred during a 30-min observation, the animals were considered protected.

Brine shrimp lethality assay (BSA)

This method was first described by Mongelli et al. (1996) to study the cytotoxic activity of compounds. Water life brine shrimp (Artemia salina) eggs were purchased from Iran Fisheries Organization (Tehran, Iran). The eggs were hatched in a flask containing 300 mL artificial seawater made by dissolving distilled water. The flask was well aerated with the aid of an air pump, and kept in a water bath at 29°–30°C. A bright light was left on. The nauplii hatched within 48 h. The extracts and pure compounds were dissolved in normal saline. Different concentrations were obtained by serial dilution. Solution of each concentration (500 μL) was transferred into clean 24-well plates via a pipette, and aerated sea water having 10–20 nauplii (500 μL) was added. A check count was performed, and the number of live larvae was noted after 24 h. The mortality end point of the bioassay was determined as the absence of controlled forward motion during 30 sec of observation. The controls consisted of sea water and berberine hydrochloride (LC₅₀ = 26 μg/mL). Lethality percentage was determined and LC₅₀ calculated based on probit analysis with 95% confidence interval.

Results and discussion

In the present study, all of the tested Ferula species (F. diversivittata (roots), F. persica (aerial parts), F. ovina (roots), F. badrakema (roots), F. assafoetida (resin), F. diversivittata (flowers), F. latisecta (roots) and F. badrakema (fruits)) showed dose-dependent cytotoxic activity and some of them were toxic (LC₅₀ < 50 μg/mL, as compared with the LC₅₀ of the positive control berberine, 26 μg/mL) in the brine shrimp bioassay (Table 2). Among these species, F. badrakema fruits exerted significantly higher levels of mortality in brine shrimp larvae than other treatments, whereas F. badrakema roots showed the least activity (Table 2).

Table 2.  Cytotoxic effects of Ferula species on Artemia salina larva.

Plant name	Part used	Concentration (μg/mL)				LC50	CI %95^b
		10	100	500	1000		Lower	Upper
F. diversivittata	Roots	26^a	49	92	100	53	39	72
F. persica	Aerial parts	15	20	92	100	128	88	185
F. ovina	Roots	18	83	100	100	25	18	34
F. badrakema	Roots	2	8	53	100	321	259	398
F. assafoetida	Oleo-gum Resin	25	75	98	100	28	19	42
F. diversivittata	Flowers	46	58	100	100	19	4	98
F. latisecta	Roots	43	92	100	100	13	8	18
F. badrakema	Fruits	62	70	86	100	6	2	18

^aPercentage of dead larvae. ^b Confidence interval.

Our findings also revealed that no tested methanol extracts of the Ferula species, nor oleo-gum-resin of F. assafoetida (300 mg/kg, i.p.) could prevent PTZ-induced seizures in mice and the latency of HLTE was obtained within 2–3 min and the percentage of mice mortality was 100% in PTZ test.

In Iranian traditional medicine, the gum obtained from the aerial parts of F. gumosa, F. assafoetida and F. badrakema, and the roots of F. diversivittata have been mentioned to be used as an antiepileptic remedy (Aqili Khorasani, 1991; Zargari, 1996). To our knowledge, no study has yet been performed about anticonvulsant activity of Ferula species except those by Sayyah et al. (2001, 2002) which reported anti-seizure activity from seed and root acetone extracts and also essential oil of Ferula gumosa (Sayyah & Mandgary, 2003). In the present study, seizure was induced by PTZ. The PTZ test represents a valid model for human generalized myoclonic seizures and also generalized seizures of the petit mal (absence) type (Loscher & Schmidt, 1988).

Our results indicated that although Ferula species were advocated for their anticonvulsant effects in Iranian traditional herbal medicine, their methanol extracts do not possess anticonvulsant effects compared to the untreated animals. This finding is consistent with another study in which the anticonvulsant effect of the seed methanol, ethyl acetate and acetone extracts of F. gumosa was investigated against PTZ-induced seizures where only acetone extract showed a significant and dose-dependent activity (Sayyah et al., 2002). In the mentioned study, it was proposed that the anti-seizure profile of F. gumosa seed acetone extract may be related in part to monoterpenes and terpenoid compounds present in the extract.

In our study, cytotoxicity of the Ferula species was also studied by the brine shrimp (Artemia salina) assay. Interestingly, cytotoxicity of F. badrakema fruits was different from the roots (Table 2). This may be due to the high amount of essential oil in the fruit (4% v/w), because essential oils are usually toxic for biological systems. It has been reported that dried root latex powder of F. assafoetida is an important source of botanical molluscicides which possess time- and concentration-dependent toxicity, and this toxicity is mainly due to the water-soluble components, particularly ferulic acid and umbelliferone (Saleem et al., 2001; Hirotaka et al., 2003). In another study it has been shown that F. communis has toxic effects (Shlosberg & Egyed, 1983), and this toxicity is probably related to the presence of the sesquiterpene prenylated coumarin derivative ferulenol (Fraigui et al., 2002), which has been shown to have biological activities such as antibacterial properties and a dose-dependent cytotoxicity against various human tumor cell lines (Bocca et al., 2002).

In summary, results of the present study revealed that all of the tested methanol Ferula extracts, especially those of F. badrakema fruits and F. latisecta roots, possess cytotoxic activity which may pose their application for therapeutic and/or antiseptic purposes. However, the exact mechanism of this effect remains to be clarified and further investigations including chronic toxicity studies and activity-guided fractionation must be performed in order to assess the real toxicological profile and the active compounds of the extract.

Declaration of interest

This research was supported by a grant from Mashhad University of Medical Sciences Research Council.