Cytotoxic, antioxidant and antimicrobial effects of nine species of woundwort (Stachys) plants

Abstract

Context: Woundwort (Stachys) plants from the Lamiaceae family have been used in folk medicine for various purposes.

Objective: This study was designed to analyze cytotoxic, antioxidant and antimicrobial activities of Stachys plants, because these fields have extensively benefited of drug discovery from natural sources.

Materials and methods: Nine Stachys plants were collected from different regions of Iran. Cytotoxic activities of methanol, 80% methanol and dichloromethane (DCM) extracts of these plants were assessed on three human cancer cell lines (HL-60, K562 and MCF-7 cells) with the MTT assay, while antioxidant and antimicrobial activities were determined on methanol extracts by DPPH and nutrient broth micro-dilution assays, respectively.

Results: DCM extract of St. pilifera Benth. had the lowest IC₅₀ in three cancer cell lines ranging from 33.1 to 48.2 µg/ml, followed by the 80% methanol extract of St. persica S.G.Gmel. ex C.A.Mey. (IC₅₀ range: 62.1–104.1 µg/ml) and DCM extract of St. byzantina C. Koch (IC₅₀ range: 62.7–131.0 µg/ml). St. byzantina. St. lavandulifolia Vahl., St. acerosa Boiss., St. obtusicrena Boiss. and St. persica showed lowest IC₅₀ values in the DPPH scavenging assay (135.1, 162.6, 164.7, 169.4 and 172.4 µg/ml, respectively), while their total phenolic contents were 23.9, 18.2, 18.6, 20.4, 27.8 mg equivalent of gallic acid in 1 g dry plant, respectively. The methanol extracts of St. byzantina and St. persica inhibited all six tested Gram-negative and Gram-positive bacterial strains.

Conclusion: Various Stachys species (especially St. byzantina and St. persica) are valuable sources of natural compounds with important biological properties.

• Cancer
• DPPH assay
• medicinal plants
• MTT assay
• traditional medicine

Introduction

Plants have often provided opportunities for discovery of pharmacologically active compounds (Bakkali et al., 2008; Itokawa et al., 2008). The chemical diversity present in plants, which is the product of many millennia of evolution of defensive mechanisms against their natural enemies, gives us the possibility of finding novel bioactive compounds (Cassady et al., 1990; Pan et al., 2012).

The field of anticancer drugs has especially benefitted from drug discovery from plants; Since the 1940s, 74.8% of all developed anticancer drugs have been from “non-synthetic” origin and 48.6% have been either natural products or a direct derivative of them (Newman & Cragg, 2012). After anticancer drugs, the discovery of anti-infective drugs comes in the second place of dependence on natural products (Newman & Cragg, 2012). Furthermore, plants have also been an important source of natural antioxidants (Firuzi et al., 2011).

Stachys L. with about 300 species is one of the largest genera of the Lamiaceae family (Harley et al., 2004). This genus is widely distributed in the temperate regions covering mainly the Mediterranean and Irano-Turanian area (Bhattacharjee, 1980). In Iran, Stachys with 34 species is the third largest genus of the Lamiaceae family, of which 13 are endemic (Naghibi et al., 2009). In this report, we screened nine Stachys species among which, St. obtusicrena Boiss., St. benthamiana Boiss., St. acerosa Boiss. and St. pilifera Benth. are endemic (Rechinger, 1982).

Different Stachys species, which are known as betony, woundwort or mountain tea in folk medicine, are consumed as herbal teas in Iran and Turkey (Goren et al., 2011). St. byzantina C. Koch and St. lavandulifolia Vahl. have been used in the Iranian folk medicine for treatment of different diseases (Naghibi et al., 2009).

Several investigators have reported various biological activities for the members of this genus, including cytotoxic (Amirghofran et al., 2006; Conforti et al., 2009; Haznagy-Radnai et al., 2008), antioxidant (Bilusic Vundac et al., 2007; Conforti et al., 2009; Firuzi et al., 2010b; Khanavi et al., 2009; Morteza-Semnani et al., 2010; Salehi et al., 2007), antimicrobial (Ebrahimabadi et al., 2010; Goren et al., 2011; Javidnia et al., 2009; Manafi et al., 2010; Salehi et al., 2007), immunomodulatory (Amirghofran et al., 2007) and anti-inflammatory (Khanavi et al., 2005) properties.

In this study, we collected nine different Stachys species from various parts of Iran, including St. acerosa. St. benthamiana. St. byzantina. St. lavandulifolia. St. obtusicrena. St. persica S.G.Gmel. ex C.A.Mey., St. pilifera. St. pubescens Ten. and St. Spectabilis Choisy ex DC. and carried out cytotoxic and antioxidant assays on different extracts of these plants.

Materials and methods

Reagents

Fetal bovine serum (FBS), phosphate buffered saline (PBS), RPMI 1640 and trypsin were purchased from Biosera (Ringmer, UK). Dichloromethane, dimethyl sulfoxide, Folin-Ciocalteu reagent, gallic acid, methanol, nutrient broth and sodium carbonate were purchased from Merck (Darmstadt, Germany). Catechin hydrate, chloramphenicol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrochloric acid 32% and p-iodonitrotetrazolium violet (INT) were obtained from Sigma-Aldrich (St. Louis, MO). Cisplatin, quercetin and penicillin/streptomycin were purchased from EBEWE Pharma (Unterach, Netherlands), Acros Organics (Geel, Belgium) and Invitrogen (San Diego, CA), respectively.

Plant material

Plants studied in this report were collected in June and July 2008 at the full flowering stage from different areas of Iran (Table 1) and identified by Mojtaba Asadollai (botanist). A voucher specimen was deposited in the herbarium of the Medicinal and Natural Products Chemistry Research Center (MNCRC), Shiraz, Iran, for each sample (Table 1). Aerial parts of plants were air-dried at room temperature in the shade and were used for solvent extraction.

Table 1. Location of collection and herbarium number of Stachys species investigated in this study.

Plant name	Location	Herbarium number	Date
St. acerosa Boiss.	Fars province: Dashte Arjan.	PC-88-14	2008
St. benthamiana Boiss.	Fars province: Sepidan, Chelegah.	PC-88-12	2008
St. byzantina K. Koch	Gilan province: Asalem to Khalkhal.	PC-88-16	2008
St. lavandulifolia Vahl.	Hamadan province: Avaj to Razan.	PC-88-13	2008
St. obtusicrena Boiss.	Kohgiluyeh province: Dena Mt., Gardaneh Bijan.	PC-88-15	2008
St. persica S.G.Gmel. ex C.A.Mey.	Gilan province: Asalem to Khalkhal.	PC-88-9	2008
St. pilifera Benth.	Fars province: Sepidan to Margoon waterfall.	PC-88-17	2008
St. pubescens Ten.	East Azarbayjan province: Kaleybar.	PC-88-11	2008
St. spectabilis Choisy ex DC.	Fars province: Sepidan, Chelegah.	PC-88-10	2008

Solvent extraction of plants

Each plant was separately extracted in different solvents as follows: 3 g of dry plant was macerated in 60 ml of solvent for 24 h. The extraction was repeated twice and the two extracts were mixed together, filtered and then concentrated in a rotary evaporator under reduced pressure. The resulting powders were kept at −20 °C until use. Shortly before each experiment, the extract was dissolved in the appropriate solvent and used to test cytotoxic and antimicrobial activities. For antioxidant and total phenolic content determinations, 25 mg of dried ground plant was extracted in 1.5 ml of 80% methanol for 48 h, and the centrifuged or filtered extract was directly used for the assays.

Cell lines and culture

The following human cancer cell lines were purchased from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran: HL-60 (human acute promyelocytic leukemia), K562 (human chronic myelogenous leukemia) and MCF-7 (human breast adenocarcinoma) cells.

The cells were cultured in sterile T25 flasks in RPMI 1640 medium supplemented with fetal bovine serum (20% v/v for HL-60 and 10% v/v for K562 and MCF-7 cells), penicillin (100 units/ml) and streptomycin (100 µg/ml). HL-60 and K562 cell lines were grown in suspension, while MCF-7 cells were grown in monolayer cultures in humidified air containing 5% CO₂ at 37 °C.

Cytotoxicity assay

The inhibitory effect of plant extracts on tumoral cell growth was assessed by the MTT reduction assay (Firuzi et al., 2010a). This colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to the purple formazan by the action of mitochondrial enzyme succinate dehydrogenase in viable cells. Extractions with dichloromethane (DCM), methanol and 80% methanol were separately performed on plants. The powdered extracts were dissolved in dimethylsulfoxide (DMSO), and then diluted in growth medium at least 200 times. Cells were seeded in 96-well plates at the density of 50 000 cells/ml in 100 µl medium and incubated for 24 h. Then, 50 µl of medium was replaced with fresh medium containing three different concentrations of the extracts. After 72 h of incubation, the medium of each well was replaced by RPMI medium without phenol red containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and incubated at 37 °C for 4 h. Dimethylsulfoxide was used to solubilize the formed formazan crystals. The potency of cell growth inhibition for each extract was expressed as IC₅₀ value, defined as the concentration that caused 50% of maximum inhibition of cell viability. The absorbance of different wells was measured at 570 nm, with background correction at 655 nm using a microplate reader.

Free radical scavenging assay

The free radical scavenging activity of the plant extracts was measured by the method of Blois (1958) with some modifications (Jassbi et al., 2004). Twenty-five to 100 μl of the 80% methanol extract were adjusted to 200 μl by methanol and added to 3800 μl of DPPH solution 10⁻⁴M in methanol. After 30 min of shaking in the dark, the absorbance of the solutions was measured at 517 nm by a spectrophotometer. The percentage of reduced DPPH was calculated by the following equation: where A_c and A_s are absorbance of control and sample, respectively. IC₅₀ values were calculated from linear regression of the DPPH inhibition percentage caused by different concentrations of the extracts, using Curve Expert software and expressed as μg extracted plant material with the solvent/1 ml 10⁻⁴M DPPH (µg PM/ml DPPH).

Total phenolic content determination

Total phenolic contents of the plant material were determined by the Folin-Ciocalteu method as described previously with some modifications (Singleton & Rossi, 1965). Briefly, to a 40 µl solution of the plant extract, 3.16 ml water and 200 µl Folin-Ciocalteu reagent were added, and the mixture was shaken well. Six hundred microliters of sodium carbonate 0.25% were added to this mixture after 8.5 min incubation at room temperature. This solution was further incubated at RT for 2 h and its absorbance was measured at 765 nm. The concentration of total phenolics were measured against a series of gallic acid standard solutions and expressed as mg equivalent of gallic acid in 1 g dry plant material (mg EG/g PM).

Antimicrobial minimum inhibitory concentration (MIC) determination

To examine the antibacterial activity of the plant extracts, three Gram-negative bacteria (Escherichia coli: PTCC1330, Klebsiella pneumonia: PTCC1053 and Salmonella typhi: PTCC1609) and three Gram-positive bacteria (Staphylococcus aureus: PTCC1112, Staphylococcus epidermidis: PTCC1114, Bacillus subtilis: PTCC1023) were chosen, and the minimum inhibitory concentrations (MIC) of the extracts were determined using nutrient broth micro-dilution (NBMD). NBMD assay was performed using serial twofold dilution of the plant extracts added to bacterial suspension in nutrient broth as previously described (Eloff, 1998). The plant extracts or positive control were dissolved in DMSO at different concentrations and added (5 µl) to 95 µl of fresh media and 100 µl of bacterial suspension (OD = 0.1 at 600 nm) in a 96-well microplate. The microplates were incubated at 37 °C for 24 h in a shaking incubator and then 10 µl of 0.5% INT solution in water was added to each well and incubated for further 30 min in the same conditions. The MIC was considered as the lowest concentration of the extract or antibacterial standard, which discolored the purple color of the INT solution.

Statistical analysis

Comparisons between values obtained in biological assays for different plants (comparisons among several groups) were performed by one-way analysis of variance (ANOVA). Statistical analyses were performed with SPSS software for Windows version 11.5.0 (SPSS Inc., Chicago, IL).

Results and discussion

Cytotoxic activity

Cytotoxicity of plant extracts was measured on three human cancer cell lines (Table 2). The dichloromethane (DCM) extract of Stachys pilifera was very active and had IC₅₀ values of lower than 50 µg/ml in all three cell lines. However, methanol and 80% aqueous methanol extracts of this plant, except for the methanol extract on K562 cells, were not effective on the cell lines. Methanol 80% extract of St. persica as well as DCM extract of St. byzantina were also active on all three cell lines. It was interesting to note that three different extracts of St. acerosa were selectively effective on K562 cells, which is a chronic myelogenous leukemia cell line, but not on the HL-60 (acute promyelocytic leukemia) and MCF-7 (breast adenocarcinoma) cells.

Table 2. Cytotoxicity of different extracts of Stachys species in human cancer cell lines.

		IC50 (µg/ml)
Plant	Extraction solvent	HL-60 cells	K562 cells	MCF-7 cells
St. acerosa	Dichloromethane	NA	65.3 ± 3.2^bc	160.3 ± 20.4^e
	Methanol	102.8 ± 22.7^abc*	56.1 ± 3.1^b	NA
	Methanol 80%	NA	67.8 ± 11.9^bc	NA
St. benthamiana	Dichloromethane	NA	NA	71.1 ± 19.1^ab
	Methanol	NA	NA	NA
	Methanol 80%	114.2 ± 4.2^bc	107.8 ± 16.9^d	162.4 ± 15.6^e
St. byzantina	Dichloromethane	62.7 ± 14.6^ab	66.9 ± 4.9^bc	131.0 ± 46.0^bcde
	Methanol	NA	78.0 ± 6.5^bc	NA
	Methanol 80%	NA	NA	NA
St. lavandulifolia	Dichloromethane	142.1 ± 57.4^c	NA	81.2 ± 9.5^abc
	Methanol	NA	NA	NA
	Methanol 80%	113.6 ± 12.0^bc	NA	151.7 ± 35.9^de
St. obtusicerna	Dichloromethane	NA	NA	NA
	Methanol	NA	NA	NA
	Methanol 80%	NA	169.6 ± 21.9^e	NA
St. persica	Dichloromethane	NA	NA	NA
	Methanol	NA	NA	NA
	Methanol 80%	62.1 ± 8.3^ab	64.7 ± 11.9^bc	104.1 ± 22.5^bcde
St. pilifera	Dichloromethane	48.2 ± 2.9^a	33.1 ± 4.8^a	40.9 ± 1.3^a
	Methanol	NA	90.1 ± 7.9^cd	NA
	Methanol 80%	NA	NA	NA
St. pubescens	Dichloromethane	NA	NA	103.3 ± 16.5^bcd
	Methanol	107.9 ± 3.5^bc	111.4 ± 1.5^d	146.5 ± 20.0^cde
	Methanol 80%	113.1 ± 10.4^bc	108.0 ± 3.5^d	NA
St. spectabilis	Dichloromethane	NA	99.6 ± 15.3^d	65.2 ± 20.8^ab
	Methanol	NA	NA	NA
	Methanol 80%	NA	NA	NA
Cisplatin		0.24 ± 0.03	0.63 ± 0.07	2.07 ± 0.60

Values are presented as mean ± S.E.M. of 3–5 experiments. Cisplatin was tested as a reference cytotoxic compound. Methanol 80% consisted of 80% methanol in water. NA: not active at tested concentrations (IC₅₀ more than 200 µg/ml).

*Values in a column with no alphabetical letters in common have a statistically significant difference.

On the other hand, St. obtusicrena appeared to be the least active plant, because none of its extracts had any effect on the cell lines, except for the 80% aqueous methanol extract, which had a modest effect on K562 cell line.

It has been reported that essential oils (Conforti et al., 2009) and extracts (Serbetci et al., 2010) of various Stachys species have antitumoral effects. There are also few reports on the cytotoxic effect of some of the Stachys species assessed in our study. Aqueous methanol extract of stems of St. byzantina has shown cytotoxic effect on MCF-7 cells (Haznagy-Radnai et al., 2008), and St. obtusicrena methanol extract was effective on several cell lines (Amirghofran et al., 2006).

Previous studies have demonstrated the presence of aucubin and harpagide (iridoid glycosides) in Stachys plants and have stated that these compounds are probably responsible for the cytotoxic effects observed for these plants (Haznagy-Radnai et al., 2008). Other investigators have suggested that sesquiterpene compounds and carvacrol may be responsible for the cytotoxic activity of various Stachys species (Conforti et al., 2009). The presence of cytotoxic compounds with various polarities in different Stachys species observed by other investigators is in line with our observation that in some plants, such as St. pilifera, DCM extract (that may contain more nonpolar compounds such as terpenoides) had stronger effects, while in some plants like St. persica, 80% methanol extract (that contains more polar agents such as phenolic compounds) exhibited higher cytotoxic potential.

Antioxidant activity and total phenolic content

Antioxidant activity and total phenolic content of the 80% aqueous methanol extracts of the plants were determined by the DPPH radical scavenging and Folin-Ciocalteu assays, respectively (Table 3). Antioxidant activity of studied plant extracts expressed as IC₅₀ values in DPPH assay varied from 454.4 ± 26.8 µg extracted plant/ml in St. spectabilis to 135.1 ± 4.5 in St. byzantina, while total phenolic contents of plant extracts varied from 10.6 ± 0.8 mg equivalent gallic acid/1 g dry plant (mg EG/g) for St. spectabilis to 27.8 ± 1.0 mg EG/g for St. persica.

Table 3. Antioxidant activity and total phenolic content of methanolic extracts of Stachys species.

Plant name	DPPH IC50 (µg/ml)*	Total phenolic content (mg EG/g)**
St. acerosa	164.7 ± 4.7^ab***	18.6 ± 0.7^cd
St. benthamiana	290.6 ± 3.7^d	17.6 ± 0.5^c
St. byzantina	135.1 ± 4.5^a	23.9 ± 0.8^e
St. lavandulifolia	162.6 ± 0.9^ab	18.2 ± 0.7^cd
St. obtusicrena	169.4 ± 3.3^b	20.4 ± 0.5^d
St. persica	172.4 ± 5.7^b	27.8 ± 1.0^f
St. pilifera	214.1 ± 1.1^c	25.0 ± 1.0^e
St. pubescens	374.3 ± 10.7^e	13.7 ± 0.7^b
St. spectabilis	454.4 ± 26.8^f	10.6 ± 0.8^a
Quercetin	1.79 ± 0.046	–

Values are presented as mean ± S.E.M. of 4 experiments.

*µg extracted plant/ml 10⁻⁴M DPPH.

**mg equivalent gallic acid/1 g dry plant.

***Values in a column with no alphabetical letters in common have a statistically significant difference.

Table 4. Antimicrobial activity of methanolic extracts of Stachys species measured by nutrient-broth micro-dilution bioassay.

	Minimum inhibitory concentration (mg/ml)
Plant name	Escherichia coli	Klebsiella pneumoniae	Salmonella typhi	Bacillus subtilis	Staphylococcus epidermidis	Staphylococcus aureus
St. acerosa	NA	NA	10	NA	10	10
St. benthamiana	NA	2.5	1.25	NA	2.5	NA
St. byzantina	2.5	5	2.5	5	10	5
St. lavandulifolia	10	NA	NA	5	NA	NA
St. obtusicrena	2.5	NA	NA	5	NA	NA
St. persica	5	2.5	1.25	5	5	2.5
St. pilifera	NA	5	NA	NA	2.5	2.5
St. pubescens	5	NA	1.25	2.5	5	NA
St. spectabilis	5	5	2.5	2.5	NA	5
Chloramphenicol	0.05	0.05	0.05	0.0125	0.025	0.0125

Minimum inhibitory concentration of the plant extracts was measured by nutrient broth micro-dilution (NBMD) assay and expressed as mg/ml. NA: not active.

A direct relationship between antioxidant activity and total phenolic content could not be observed. This lack of correlation for the members of this genus may be due to the difference in the number and location of the active hydroxyl groups of their phenolic compounds (Seyoum et al., 2006). Free hydroxyl groups in ortho and para positions, such as 3′,4′-hydroxyls in flavonoids, produce stable quinones upon oxidation, a property that render these phenolic compounds efficient antioxidants. Glycosylation, methylation and acetylation of each of these functional groups reduce their antioxidant properties. For example, butrin; 3′,4′,7-trihydroxyflavanone 3′,7-di-O-β-d-glucopyranoside, a flavonoid glycoside with no radical scavenging potential, on acidic hydrolysis produce its aglycone butin, with several-fold enhancement of its activity (Jassbi et al., 2004). Other previous studies on the polar fractions of Stachys species resulted in isolation of several flavonoids some of which partially or totally methylated or glycosylated on their hydroxyl groups (Marin et al., 2004).

In a study performed on the methanol extracts of the woundwort plants, St. persica showed the highest antioxidant activity measured by FRAP assay and also the highest total phenolic content (Khanavi et al., 2009). However, different from our results, St. byzantina had a low activity in this study (Khanavi et al., 2009). In another report about the antioxidant activity of different Stachys species, methanol extracts of St. byzantina and St. lavandulifolia showed moderate activities in inhibition of peroxide formation in sunflower oil (Morteza-Semnani et al., 2010). Water extract of St. byzantina has also shown high antioxidant activity in DPPH and flow injection analysis-luminol chemiluminescence (FIA-CL) assays (Erdemoglu et al., 2006).

The woundwort plants synthesize polyphenolic compounds such as flavonoids (Marin et al., 2004) and phenolic acids in higher quantities (Vundac et al., 2005, 2007) compared to terpenoids such as mono- and sesquiterpenoids, which are found as a blend in their essential oils (Goren et al., 2011). Several Iranian woundworts have been studied for their essential oils rich in mono- and sesquiterpenoids, but the relatively low yield of these types of compounds (usually less than 0.5%) as well as the lack of phenolics in their oils may be a cause of the lower radical scavenging potentials of their volatile oils (Ebrahimabadi et al., 2010). Analysis of the essential oil of Stachys species endemic to Balkan Peninsula has also revealed that oxygenated sesquiterpenes constitute the major part of the oil, which are followed by diterpene hydrocarbons (Cavar et al., 2010).

Our results showed that the polar extracts of the studied plants generally contain both high levels of phenolics (1.8–2.7% of their dried plant material) and low IC₅₀ values in the DPPH radical scavenging assay.

The second common group of phenolics in this species which increase the radical scavenging activity of the plant extracts are caffeic acid derivatives such as different caffeoyl quinic acids, for example, chlorogenic acid and their glycosylated derivatives (Vundac et al., 2005, 2007).

Antimicrobial activity

The lowest MIC value (1.25 mg/ml) was shown by St. benthamiana and St. pubescens against Salmonella typhi. The rest of the plants had higher MIC values against tested microbial species and showed weaker effects (Table 4). Some previous studies have also shown that St. byzantina. St. lavandulifolia and St. persica have antimicrobial activities (Morteza-Semnani & Saeedi, 2009; Saeedi et al., 2008).

Conclusion

Cytotoxic, antioxidant and antimicrobial activities of different extracts of nine Stachys species were studied. St. pilifera dichloromethane (DCM) extract had the highest cytotoxic activity, while St. byzantina and St. persica resulted positive in all biological assays. The results obtained in this study show that various Stachys species, which are used in folk medicine as remedies for different diseases, are valuable sources of natural compounds with important biological properties.

Declaration of interest

The authors report no declarations of interest.

Acknowledgements

This study was supported by Shiraz University of Medical Sciences, Vice-chancellor for Research, Iran (Grants 4678, 89-5496) and Iranian National Science Foundation, Iran (Grant 87041859).