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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
In vitro antioxidant and antiproliferative activities of nine Salvia species a
b
b
Monica Rosa Loizzo , Morteza Abouali , Peyman Salehi , Ali c
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Sonboli , Mohammad Kanani , Francesco Menichini & Rosa Tundis
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Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036Rende, CS, Italy b
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran c
Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran Published online: 21 Jul 2014.
To cite this article: Monica Rosa Loizzo, Morteza Abouali, Peyman Salehi, Ali Sonboli, Mohammad Kanani, Francesco Menichini & Rosa Tundis (2014) In vitro antioxidant and antiproliferative activities of nine Salvia species, Natural Product Research: Formerly Natural Product Letters, 28:24, 2278-2285, DOI: 10.1080/14786419.2014.939086 To link to this article: http://dx.doi.org/10.1080/14786419.2014.939086
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Natural Product Research, 2014 Vol. 28, No. 24, 2278–2285, http://dx.doi.org/10.1080/14786419.2014.939086
In vitro antioxidant and antiproliferative activities of nine Salvia species
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Monica Rosa Loizzoa*, Morteza Aboualib, Peyman Salehib, Ali Sonbolic, Mohammad Kananic, Francesco Menichinia and Rosa Tundisa a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, CS, Italy; bDepartment of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran; cDepartment of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran
(Received 20 May 2014; final version received 24 June 2014) Supported by a growing increase of scientific research attesting the health properties of salvia species, we have decided to investigate nine Salvia namely Salvia sclarea, Salvia atropatana, Salvia sahendica, Salvia hydrangea, Salvia xanthocheila, Salvia macrosiphon, Salvia glutinosa, Salvia chloroleuca and Salvia ceratophylla species for their antioxidant and antiproliferative activities. In order to correlate the bioactivity with their phytochemical content, the total phenol and total flavonoid contents were also determined. S. ceratophylla exhibited the strongest activity against C32 cells with an IC50 value of 20.8 mg mL21, while S. glutinosa exhibited an IC50 value of 29.5 mg mL21 against ACHN cell line. Interestingly, S. glutinosa displayed also the highest DPPH radical-scavenging activity with an IC50 of 3.2 mg mL21. These species are characterised by the highest total phenol and flavonoid contents. The obtained results suggest that Salvia species are healthy plant foods. Keywords: Salvia; phenol content; flavonoids content; antiproliferative activity; antioxidant activity
1. Introduction The Salvia genus belongs to the Lamiaceae family and consists of about 900 species widespread throughout the world. In Iran, 58 species are growing naturally, among which Salvia hypoleuca, Salvia urmiensis, Salvia sahendica and Salvia persepolitana are endemic (Hedge 1986; Walker et al. 2004). In Iranian folk medicine, decoction and powder of the leaves of Salvia mirzayanii (local name: Moor Talkh) were used for treating stomach pain, and infusion of the flowers of Salvia hydrangea (local name: Gol-e Arooneh) as anti-inflammatory, anti-spasmolytic, carminative as well for treating colds (Ghannadi 2002). S. sahendica, an endemic perennial herb, which grows wild in Iran is traditionally used as antibacterial and antifungal agent, as culinary plant for seasoning and flavouring foods (Lotfipour et al. 2007; Salehi et al. 2007; Esmaeili et al. 2009). Based on our ethnobotanical data and on local beliefs, Salvia macrosiphon is locally used for the treatment of cough and cold in southern parts of Iran. Salvia chloroleuca is used for gastrointestinal disorders. Salvia virgata is used for the treatment of skin diseases and cancer (Baytop 1999). Salvia hispanica has a long history of uses such as medicinal, culinary and religious (Cahill 2003). Salvia divinorum is used as antibacterial, antioxidant and antidiabetic agent (Ulubelen et al. 1997). Different Salvia species have been investigated for their antiproliferative activity (Tundis et al. 2011; Zare Shahneh et al. 2013; Zihlif et al. 2013). The GLOBOCAN data (GLOBOCAN 2008) evidenced that in 2012 worldwide, 14.1 million new
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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cancer cases were diagnosed, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis). Natural products provided the only source of pharmaceuticals for thousands of years, and have made enormous contributions to human health through valuable and unique compounds. Several drugs currently used in chemotherapy were isolated from plant species or derived from a natural prototype (Newman & Cragg 2007). Several chronic diseases including cancer involves oxidative damage to cellular components, such as lipids, proteins and DNA, and initiation of subsequent cell death via necrosis or apoptosis (Liangli 2008). In healthy individuals, production of ROS is balanced by natural anti-oxidative defence system. Thus, increased intake of natural antioxidants in the daily diet is recommended. In an effort to address this, we hypothesised that Salvia species exert antiproliferative effects against tumour cell lines. To test our hypothesis, we selected nine Salvia species commonly used as spices and consumed as tea. Thus, in this study, we have evaluated the Salvia ssp. methanol extracts for their in vitro antioxidant and antiproliferative activity. The total phenol and flavonoid contents were also determined in order to correlate the observed bioactivity with the phytochemical content (Table 1). 2. Results and discussion 2.1. Antioxidant properties Several studies demonstrated that plant phenolic compounds exhibit considerable free radicalscavenging activities through their reactivity as hydrogen- or electron-donating agents, and metal ion-chelating properties. Therefore, it would be valuable to determine the total phenol content of the food plant extracts. As shown in Table 2, total phenol content ranged from 5.1 to 42.5 gallic acid g21 of dried extract for Salvia xanthocheila and Salvia glutinosa, respectively. High values were found also in Salvia ceratophylla and Salvia sclarea with 32.7 and 30.4 mg gallic acid g21 dried extract. The same trend was observed also in the results of total flavonoid content where S. glutinosa, S. ceratophylla and S. sclarea displayed the highest content with 36.2, 27.0 and 12.2 mg (þ )-catechin equivalents per g of dried extract. Recently, the total phenolic content S. hydrangea, Salvia lachnocalyx, Salvia macilenta, S. multicaulis, S. sclarea and S. xanthocheila were investigated. S. macilenta displayed the highest total phenol content with 326 mg gallic acid/g dried extract while S. lachnocalyx exhibited the highest total flavonoid content with 253 mg (þ )-catechin equivalents per g of dried extract (Asadi et al. 2009). Tosun et al. (2009) investigated the total phenol content of eight Salvia species from Turkey and determined the values to be in the range 50.3 –167.1 mg GAE g21 of dried weight for Salvia verticillata and Salvia limbata, respectively. Table 1. Localities information and voucher number of studied Salvia species. Species
Locality
S. sclarea L. S. atropatana Bunge S. ahendica (Boiss.) Buhse S. hydrangea DC. S. xanthocheilla Boiss. ex Benth. S. macrosiphon Boiss. S. glutinosa L. S. chloroleuca Rech.f. & Aell. S. ceratophylla L.
Esfahan, Semirom Zanjan, Rudbar East Azarbaijan, Tabriz Fars, Shira West Azarbaijan, Khoy Lorestan, Khorramabad Ardebil, Khalkhal Khorasan, Mashhad Qazvin, Razmian
Altitude Herbarium Month of Extraction (m) no. collection yield (%) 1900 2400 2230 1500 2800 970 1800 1750 1520
MPH-658 MPH-1426 MPH-1321 MPH-1545 MPH-1308 MPH-1543 MPH-1563 MPH-1625 MPH-1520
May June June May June May July June May
5.0 6.0 5.1 5.5 6.4 7.0 5.8 5.8 7.2
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Table 2. Total phenolic and flavonoid contents and antioxidant activities of selected Salvia species.
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Species S. sclarea S. atropatana S. ahendica S. hydrangea S. xanthocheila S. macrosiphon S. glutinosa S. chloroleuca S. ceratophylla Ascorbic acid BHT
Total phenol contenta
Total flavonoid contentb
30.4 ^ 0.3 11.1 ^ 0.8 7.0 ^ 0.4 21.9 ^ 0.1 5.1 ^ 0.7 16.4 ^ 0.1 42.5 ^ 1.0 11.1 ^ 1.5 32.7 ^ 1.7
12.2 ^ 0.2 7.0 ^ 0.4 3.2 ^ 0.3 11.5 ^ 0.1 2.2 ^ 0.2 12.4 ^ 0.9 36.2 ^ 0.5 6.6 ^ 1.0 27.0 ^ 1.0
DPPHc
ABTSd
FRAPe
4.8 ^ 0.1 17.5 ^ 0.3 16.2 ^ 0.3 5.3 ^ 0.1 14.6 ^ 0.3 14.7 ^ 0.1 3.2 ^ 0.3 16.7 ^ 1.0 5.5 ^ 0.1 5.0 ^ 0.8 –
56.1 ^ 0.8 32.4 ^ 0.7 25.3 ^ 1.1 40.1 ^ 1.7 37.2 ^ 1.1 23.9 ^ 1.2 59.1 ^ 0.4 28.6 ^ 4.2 42.0 ^ 0.7 1.0 ^ 0.03 –
160.1 ^ 1.5 20.6 ^ 7.3 8.9 ^ 4.1 195.9 ^ 5.4 14.4 ^ 4.6 10.2 ^ 3.4 422.0 ^ 9.8 12.9 ^ 0.8 290.7 ^ 6.0 – 63.2 ^ 4.5
Notes: Data are expressed as means ^ SD (n ¼ 3). a Milligram of gallic acid equivalents/g dried extract ^ SD. b Milligram of (þ)-catechin equivalents/g of dried extract ^ SD. c IC50 mg/mL ^ SD. d mmol TE/g of dried extract ^ SD. e mmol Feþ þ equivalents/g of dried extract ^ SD. Positive controls: ascorbic acid and BHT. DPPH test: one-way ANOVA analysis: P , 0.0001 (F ¼ 24.76, r 2 ¼ 0.92). Dunnett multiple comparison test: P , 0.05 (S. sclarea, S. hydrangea, S. glutinosa and S. ceratophylla vs. ascorbic acid); P , 0.01 (S. atropatana, S. ahendica, S. xanthocheila, S. macrosiphon, S. chloroleuca vs. ascorbic acid). ABTS test: one-way ANOVA analysis: P , 0.0001 (F ¼ 187.20, r 2 ¼ 0.99). Dunnett multiple comparison test: P , 0.01 (all samples vs. ascorbic acid). FRAP test: one-way ANOVA analysis: P , 0.0001 (F ¼ 111.30, r 2 ¼ 0.99). Dunnett multiple comparison test: P , 0.01 (all samples vs. BHT).
The antioxidant properties of Salvia species were studied by three different methods – DPPH, ABTS and FRAP assays. Results are summarised in Table 2. S. glutinosa extract exhibited the highest DPPH radical-scavenging activity with an IC50 value of 3.2 mg mL21, followed by S. sclarea (IC50 value of 4.8 mg mL21), S. hydrangea (IC50 value of 5.3 mg mL21) and S. ceratophylla (IC50 value of 5.5 mg mL21). A similar trend was also observed in the ABTS assay with the highest activity reported in S. glutinosa (59.1 mmol TE g21 dried extract) followed by S. sclarea (56.1 mmol TE g21 dried extract). This radical-scavenging ability could be related to the nature of phenolic compounds, thus contributing to their electron transfer/hydrogen donating ability. The FRAP method was also used to evaluate the reducing potential of Salvia extracts. Using this assay, S. glutinosa (422.0 mmol FeSO4 eq g21 of dried extract) was found to be the most active followed by S. ceratophylla (290.7 mmol FeSO4 eq g21 of dried extract), S. hydrangea (195.9 mmol FeSO4 eq g21 of dried extract) and S. sclarea (160.1 mmol FeSO4 eq g21 of dried extract). Generally, S. glutinosa exhibited the highest antioxidant activity in all assays. S. sclarea, S. hydrangea and S. ceratophylla extracts were among the best antioxidant sources with a slight deviation. Many Salvia species and their isolated constituents possess significant antioxidant activities. In a comparative study, the antioxidant properties of several Salvia species including S. hydrangea, S. sclarea and S. xanthocheila were investigated (Asadi et al. 2009). In both DPPH and FRAP assays, S. hydrangea displayed the highest antioxidant activity followed by S. sclarea, and S. xanthocheila. Previously, the radical-scavenging activity of Salvia aegyptiaca, Salvia verbenaca and Salvia argentea were determined (Salah et al. 2006). In the DPPH assay, the order of decreasing potency is S. aegyptiaca, S. verbenaca, and then S. argentea. In the ABTS test, the same trend was observed. These results are lower than those found in our Salvia species. 2.2. Antiproliferative effects The antiproliferative effects of Salvia species were investigated in vitro using sulforhodamine B (SRB) assay against cancer cell lines namely C32, ACHN, COR-L23, A375, Caco-2, Huh-7D12,
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A549, MCF-7 and LNCaP. The results are summarised in Table 3. All samples exhibited a concentration – response effect against cancer cell lines. S. ceratophylla extract exhibited the strongest inhibitory activity against C32 and ACHN cell lines with IC50 values of 20.8 and 27.2 mg mL21, respectively. A similar result was observed with S. glutinosa and S. xanthocheila against ACHN cells with IC50 values of 29.5 and 34.0 mg mL21, respectively. Moreover, S. glutinosa exhibited IC50 values of 30.4 and 29.1 mg mL21, against lung carcinoma large cell and human breast cancer, respectively. The IC50 values of 31.2 and 34.3 mg mL21 were found when S. chloroleuca was applied to ACHN and C32 cell lines. S. macrosiphon inhibited COR-L23, MCF-7 and ACHN cell growth with IC50 values of 32.6, 33.7 and 36.1 mg mL21, respectively. Salvia atropatana exhibited the highest inhibitory activity against human Caucasian lung carcinoma A549 (IC50 of 48.1 mg mL21) compared with other tested Salvia species. Interesting results on human breast cancer and hormone-dependent prostate carcinoma were found when S. hydrangea was applied to cell cultures (IC50 values of 37.3 and 31.6 mg mL21, respectively). No antiproliferative activity at the maximum concentration tested (150 mg mL21) was observed with all samples against human skin fibroblast 142BR. Recently, Salvia leriifolia extracts and isolated constituents were investigated for their antiproliferative activity against a panel of human cancer cell lines (Tundis et al. 2011). The n-hexane extract exhibited the strongest antiproliferative activity against the C32 cell line with an IC50 of 11.2 mg mL21. Previously, Kamatou et al. (2008) reported the anticancer activity of indigenous Salvia species. The IC50 values ranged from 9.69 to 43.65 mg mL21 and from 8.72 to 59.12 mg mL21 against MCF-7 and SF-268 cell lines, respectively, with Salvia radula and Salvia africana-caerulea being the most active. Salvia lanceolata displayed the highest antiproliferative activity against HT-29 cell line. Other Salvia species were studied by Fiore et al. (2006). Among them, Salvia menthaefolia exerted a good antiproliferative against WiDr and HT-29 cell lines (IC50 values of 89.6 and 95.3 mg mL21, respectively). The antiproliferative effects towards human cancer cells (MCF7, HT-29 and SF268) and a human kidney epithelial cell line of 17 Salvia species used in the traditional Jordanian medicine were also investigated (Alkofahi et al. 1996). Salvia africana-lutea and Salvia runcinata were the most active against breast cancer cell line. Salvia dominica and Salvia fruticosa exhibited IC50 values of 5.83 and 25.55 mg mL21 against the oestrogen receptorpositive cell line MCF-7 with minimal toxicity against normal human periodontal fibroblasts (Abu-Dahab et al. 2012). Previously, the cytotoxic activities of the fractions prepared from the methanol extract of 23 Salvia species were investigated against HeLa, A431 and MCF7 (Janicsa´k et al. 2011). The n-hexane fractions of S. hispanica, Salvia nemorosa, Salvia albiflora, Salvia pratensis, Salvia recognita and Salvia ringens and the chloroform fraction of Salvia officinalis and S. albiflora caused more than 50% growth inhibition of the skin carcinoma cell line. None of the tested extracts exhibited substantial antiproliferative effects against HeLa and MCF7 cells. S. ringens was the most potent with a 61.8% cell growth inhibitory activity on A431 cells. 3. Experimental 3.1. Chemical Reagents were obtained from Sigma-Aldrich S.p.A. (Milan, Italy). Solvents were purchased from Merck chemical company (Darmstadt, Germany). 3.2. Plant material and extraction procedure The aerial parts of S. sclarea, S. atropatana, S. sahendica, S. hydrangea, S. xanthocheila, S. macrosiphon, S. glutinosa, S. chloroleuca and S. ceratophylla were collected from different
101.8 ^ 6.4 124.4 ^ 4.4 . 150 121.2 ^ 5.3 . 150 . 150 142.8 ^ 5.9 123.4 ^ 2.8 . 150
69.0 ^ 0.6 –
61.2 ^ 2.8 52.3 ^ 2.4 40.3 ^ 1.4 47.1 ^ 2.2 45.5 ^ 3.1 32.6 ^ 1.6 30.4 ^ 1.5 39.9 ^ 2.0 38.9 ^ 1.9
45.5 ^ 0.7 –
S. sclarea S. atropatana S. ahendica S. hydrangea S. xanthocheila S. macrosiphon S. glutinosa S. chloroleuca S. ceratophylla Positive control Vinblastine Taxol 7.2 ^ 0.7 –
107.4 ^ 5.4 68.1 ^ 2.9 60.7 ^ 3.4 67.5 ^ 2.8 97.8 ^ 2.9 138.5 ^ 5.4 123.2 ^ 5.2 120.7 ^ 4.8 120.9 ^ 3.7
A375
3 ^ 0.3 –
51.5 ^ 2.8 55.3 ^ 2.6 58.3 ^ 3.4 43.6 ^ 2.4 40.5 ^ 2.9 64.2 ^ 3.3 55.5 ^ 2.1 34.3 ^ 2.8 20.8 ^ 1.4
C32
67.3 ^ 2.0 –
67.1 ^ 2.4 48.1 ^ 2.7 64.2 ^ 2.9 62.6 ^ 3.1 130.3 ^ 3.4 . 150 . 150 71.6 ^ 2.8 . 150
A549
22.7 ^ 0.14 –
54.8 ^ 2.2 62.0 ^ 2.4 55.9 ^ 2.8 42.3 ^ 1.8 34.0 ^ 1.4 36.1 ^ 1.6 29.5 ^ 2.1 31.2 ^ 2.0 27.2 ^ 2.1
ACHN
45.6 ^ 0.8 –
129.3 ^ 4.4 129.8 ^ 4.7 . 150 111.5 ^ 3.9 . 150 . 150 . 150 . 150 121.3 ^ 4.6
Huh-7D12
– 0.08 ^ 0.004
47.1 ^ 2.1 112.3 ^ 3.5 115.9 ^ 3.7 37.3 ^ 2.8 60.6 ^ 3.9 33.7 ^ 2.2 29.1 ^ 2.1 134.2 ^ 3.6 57.5 ^ 3.1
MCF-7
29.3 ^ 0.86 –
74.8 ^ 2.3 65.6 ^ 3.1 65.7 ^ 3.0 31.6 ^ 2.8 75.5 ^ 2.7 59.3 ^ 2.6 124.4 ^ 3.6 104.8 ^ 3.4 111.1 ^ 4.1
LNCaP
150 150 150 150 150 150 150 150 150 37.00 ^ 0.44 –
. . . . . . . . .
142BR
Notes: Cell lines: COR-L23, human Caucasian lung large cell carcinoma; Caco-2, human Caucasian colon adenocarcinoma; A375, malignant melanoma; C32, amelanotic melanoma; A549, human Caucasian lung carcinoma; ACHN, human renal cell adenocarcinoma; Huh-7D12, human hepatocellular carcinoma; MCF-7, breast adenocarcinoma; LNCaP, prostate carcinoma; 142BR, human skin fibroblast. Data are expressed as means ^ SD (n ¼ 3). Vinblastine and taxol were used as positive control. COR-L23 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 31.64, r 2 ¼ 0.93); Dunnett multiple comparison test: P . 0.05 (S. ahendica, S. hydrangea, S. xanthocheila, S. chloroleuca and S. ceratophylla vs. vinblastine), P , 0.05 (S. atropatana vs. vinblastine), P , 0.01 (S. sclarea, S. macrosiphon and S. glutinosa vs. vinblastine). Caco-2 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 272.0, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). A375 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 650.3, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). C32 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 146.2, r 2 ¼ 0.98); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). A549 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 692.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. sclarea, S. ahendica, S. hydrangea and S. chloroleuca vs. vinblastine), P , 0.01 (S. xanthocheila, S. macrosiphon, S. atropatana, S. glutinosa and S. ceratophylla vs. vinblastine). ACHN cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 692.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. sclarea, S. ahendica, S. chloroleuca, and S. hydrangea vs. vinblastine), P , 0.01 (S. xanthocheila, S. macrosiphon, S. atropatana, S. glutinosa and S. ceratophylla vs. vinblastine). Huh-7D12 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 397.2, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). MCF-7 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 783.6, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). LNCaP cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 367.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. hydrangea vs. taxol), P , 0.01 (S. sclarea, S. atropatana, S. ahendica, S. xanthocheila, S. macrosiphon, S. glutinosa, S. chloroleuca and S. ceratophylla vs. taxol).
Caco-2
COR-L23
Salvia
Cell line
Table 3. In vitro antiproliferative activity [IC50 (mg/mL)] of selected Salvia species on nine cancer cell lines.
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regions in Iran. Voucher herbarium specimens (Table 1) have been deposited in the Medicinal Plants and Drugs Research Institute herbarium, Shahid Beheshti University, Tehran, Iran. The dried aerial parts of Salvia species (10 g) were ground into powder and extracted with methanol (200 mL) for three times for 24 h at room temperature, then filtered and concentrated under reduced pressure. Information about periods of sample collection and extraction yield (%) are reported in Table 1.
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3.3. Total phenol content The total phenol content was measured using the Folin – Ciocalteau method (Singleton et al. 1999). The absorbance was evaluated at 760 nm after incubation at room temperature for 2 h. Gallic acid was chosen as a standard. The total phenolic contents were expressed as mg of gallic acid equivalents g21 dried extract ^ standard deviation (SD). 3.4. Total flavonoid content The total flavonoid content was determined by the procedure previously described by Zhishen et al. (1999). The absorbance was measured against a blank at 510 nm. Results were expressed as mg of (þ )-catechin equivalents per g of dried extract. 3.5. Radical-scavenging activity by DPPH assay The DPPH radical-scavenging capacity was carried out according to the technique previously reported (Loizzo et al. 2009). Ascorbic acid was used as positive control. At the end of the experiment, the IC50 was calculated according to the following equation: Scavenging activity ¼ [(A0 2 A1)/A0 £ 100], where A0 is the absorbance of the blank and A1 is the absorbance in the presence of the extract or standard sample. The blank was prepared with all reagents except extract. 3.6. ABTS radical cation-scavenging activity ABTS antioxidant assay was performed according to the procedure previously described (Loizzo et al. 2009). The absorbance was recorded at 734 nm using Molecular Devices SpectraMax Plus Plate Reader (Molecular Devices, CELBIO, Milan, Italy). The inhibition percent of extracts was evaluated using the following equation: % Inhibition ¼ [(Ab 2 As)/ Ab £ 100], where Ab is the absorbance of blank and As is the absorbance of the sample. Ascorbic acid was used as positive control. 3.7. FRAP assay The FRAP assay was performed using Benzie and Strain method (Benzie & Strain 1996). The standard curve was then prepared by plotting the FRAP value for each standard versus its concentration. BHT was used as positive control. Results were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of mmol Feþ þ equivalent/g of dried extract. 3.8. Antiproliferative activity 3.8.1. Cell culture All media, buffers, trypsin and dyes were filter-sterilised prior to use and warmed to 37 8C. Human amelanotic melanoma (C32, CRL1585), renal cell adenocarcinoma (ACHN, CRL1611),
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lung carcinoma large cell (COR-L23, 92031919), malignant melanoma (A375, 88113005), colorectal adenocarcinoma (Caco-2, HTB37), hepatocellular carcinoma (Huh-7D12, 1042712), human Caucasian lung carcinoma (A549, 86012804), human breast cancer (MCF-7, HTB-D22), hormone-dependent prostate carcinoma (LNCaP, CRL-1740) cancer cell lines and one normal cell line of human skin fibroblast (142BR) were used in our experiments. COR-L23, C32, ACHN and LNCaP cells were cultured in RPMI 1640 medium while 142BR, MCF-7, A549, Huh-7D12, Caco-2 and A375 cells were cultured in DMEM. Both media were supplemented with 10% foetal bovine serum, 1% L -glutamine and 1% penicillin/streptomycin. 3.8.2. SRB assay Salvia extracts were tested to evaluate their potential cytotoxic activity through the SRB assay (Loizzo et al. 2007). Briefly, cells were placed in 96-well plates in a range from 5 £ 104 to 15 £ 104 cells. After 24 h, the cells were treated with 100 mL/well of serial dilutions of the sample. After 48 h of exposure, cells were fixed with ice-cold 40% trichloroacetic acid. The fixed cells were stained for 30 min with 50 mL of 0.4 % (w/v) SRB in 1% acetic acid. The plates were washed with 1% acetic acid and air-dried overnight. For reading plate, the bound dye was solubilised with 100 mL of 10 mM tris(hydroxymethyl)aminomethane (Tris base). The absorbance of each well was read on a Molecular Devices SpectraMax Plus Plate Reader (Molecular Devices, CELBIO, Milan, Italy) at 490 nm. Cell survival was measured as the percentage absorbance compared with the untreated control. Vinblastine sulfate and taxol were used as positive control. 3.9. Statistical analyses All experiments were carried out in triplicate. Data are expressed as means ^ SD. The concentration causing 50% inhibition (IC50) was calculated by non-linear regression with the use of Prism GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). In the antioxidant assay, the concentration – response curve was obtained by plotting the percentage inhibition versus concentration while in the anti-proliferative activity the concentration – response curve was obtained by plotting the percentage of cell viability versus the concentrations. Differences concerning parameters were analysed by the one-way ANOVA test to compare group means (a ¼ 0.05). The r 2 values thus obtained were used to predict relationships. To complete the statistical analysis multi-comparison Dunnett’s test was performed. Differences were considered significant at P , 0.05. 4. Conclusion Several Salvia species are widely used for medicinal purpose and for this reason the genus is largely investigated. Thus, we have screened nine Iranian Salvia species for their potential antioxidant effect and antiproliferative properties against a panel of human cancer cell lines. S. ceratophylla exerted an interesting activity against C32 and ACHN cell lines while S. glutinosa and S. hydrangea exhibited the highest activity against MCF-7 and LNCaP cell lines, respectively. These species also displayed the highest antioxidant activity with high total phenol content and are the best candidates for further in vivo studies and natural sources to find new anticancer compounds. Acknowledgements Financial support by Iran National Science Foundation (INSF; Grant no. 86023.07) is gratefully acknowledged.
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The antimalarial and antiproliferative effects of solvent extracts of South African Salvia species and isolated compounds from S. radula. South Af J Bot. 74:238–243. Liangli Y. 2008. Wheat antioxidants. Hoboken, NJ: Wiley. Loizzo MR, Said A, Tundis R, Hawas UW, Rashed K, Menichini F, Frega NG, Menichini F. 2009. Antioxidant and antiproliferative activity of Diospyros lotus L. extract and isolated compounds. Plant Foods Hum Nutr. 64:264–270. Loizzo MR, Tundis R, Statti GA, Menichini F. 2007. Jacaranone: a cytotoxic constituent from Senecio ambiguus subsp. ambiguus (biv.) DC. against renal adenocarcinoma ACHN and prostate carcinoma LNCaP cells. Arch Pharm Res. 30:701–707. Lotfipour F, Samiee M, Nazemiyeh H. 2007. Evaluation of the antibacterial activity of Salvia sahendica and Phlomiscaucasica: pharmaceutical sciences. J Fac Pharm Tabriz Univ Med Sci. 1:29–34. Newman DJ, Cragg GM. 2007. Natural products as sources of new drugs over the last 25 Years. J Nat Prod. 70:461–477. Salah KB, Mahjoub MA, Ammar S, Michel L, Millet-Clerc J, Chaumont JP, Mighri Z, Aouni M. 2006. Antimicrobial and antioxidant activities of the methanolic extracts of three Salvia species from Tunisia. Nat Prod Res. 20:1110–1120. Salehi P, Sonboli A, Nejad Ebrahimi S, Yousefzadi M. 2007. Antibacterial and antioxidant activities of the essential oils and various extracts of Salvia sahendica in different phenological stages. Chem Nat Compd. 43:328–330. Singleton VL, Orthofer R, Lamuela-Raventos RM. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteau reagent. Meth Enzymol. 299:152–178. Tosun M, Ercisli S, Sengul M, Ozer H, Polat T, Ozturk E. 2009. Antioxidant properties and total phenolic content of eight Salvia species from Turkey. Biol Res. 42:175–181. Tundis R, Loizzo MR, Menichini F, Bonesi M, Colica C, Menichini F. 2011. In vitro antiproliferative activity of Salvia leriifolia Benth. extract and isolated constituents against a panel of human cancer cell lines. Chem Biodiv. 8:1152–1162. Ulubelen A, Topc u G, Bozok-Johansson B. 1997. Norditerpenoids and diterpenoids from Salvia multicaulis with antituberculous activity. J Nat Prod. 60:1275–1280. Walker JB, Sytsma KJ, Treutlein J, Wink M. 2004. Salvia (Lamiaceae) is not monophyletic: Implications for the systematics, radiation, and ecological specializations of Salvia and tribe Mentheae. Am J Bot. 91:1115–1125. Zare Shahneh F, Valiyari S, Baradaran B, Abdolalizadeh J, Bandehagh A, Azadmehr A, Hajiaghaee R. 2013. Inhibitory and cytotoxic activities of Salvia officinalis L. extract on human lymphoma and leukemia cells by induction of apoptosis. Adv Pharm Bull. 3:51–55. Zhishen J, Mengcheng T, Jianming W. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64:555–559. 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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
In vitro antioxidant and antiproliferative activities of nine Salvia species a
b
b
Monica Rosa Loizzo , Morteza Abouali , Peyman Salehi , Ali c
c
a
Sonboli , Mohammad Kanani , Francesco Menichini & Rosa Tundis
a
a
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036Rende, CS, Italy b
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran c
Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran Published online: 21 Jul 2014.
To cite this article: Monica Rosa Loizzo, Morteza Abouali, Peyman Salehi, Ali Sonboli, Mohammad Kanani, Francesco Menichini & Rosa Tundis (2014) In vitro antioxidant and antiproliferative activities of nine Salvia species, Natural Product Research: Formerly Natural Product Letters, 28:24, 2278-2285, DOI: 10.1080/14786419.2014.939086 To link to this article: http://dx.doi.org/10.1080/14786419.2014.939086
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Natural Product Research, 2014 Vol. 28, No. 24, 2278–2285, http://dx.doi.org/10.1080/14786419.2014.939086
In vitro antioxidant and antiproliferative activities of nine Salvia species
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Monica Rosa Loizzoa*, Morteza Aboualib, Peyman Salehib, Ali Sonbolic, Mohammad Kananic, Francesco Menichinia and Rosa Tundisa a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, CS, Italy; bDepartment of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran; cDepartment of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran
(Received 20 May 2014; final version received 24 June 2014) Supported by a growing increase of scientific research attesting the health properties of salvia species, we have decided to investigate nine Salvia namely Salvia sclarea, Salvia atropatana, Salvia sahendica, Salvia hydrangea, Salvia xanthocheila, Salvia macrosiphon, Salvia glutinosa, Salvia chloroleuca and Salvia ceratophylla species for their antioxidant and antiproliferative activities. In order to correlate the bioactivity with their phytochemical content, the total phenol and total flavonoid contents were also determined. S. ceratophylla exhibited the strongest activity against C32 cells with an IC50 value of 20.8 mg mL21, while S. glutinosa exhibited an IC50 value of 29.5 mg mL21 against ACHN cell line. Interestingly, S. glutinosa displayed also the highest DPPH radical-scavenging activity with an IC50 of 3.2 mg mL21. These species are characterised by the highest total phenol and flavonoid contents. The obtained results suggest that Salvia species are healthy plant foods. Keywords: Salvia; phenol content; flavonoids content; antiproliferative activity; antioxidant activity
1. Introduction The Salvia genus belongs to the Lamiaceae family and consists of about 900 species widespread throughout the world. In Iran, 58 species are growing naturally, among which Salvia hypoleuca, Salvia urmiensis, Salvia sahendica and Salvia persepolitana are endemic (Hedge 1986; Walker et al. 2004). In Iranian folk medicine, decoction and powder of the leaves of Salvia mirzayanii (local name: Moor Talkh) were used for treating stomach pain, and infusion of the flowers of Salvia hydrangea (local name: Gol-e Arooneh) as anti-inflammatory, anti-spasmolytic, carminative as well for treating colds (Ghannadi 2002). S. sahendica, an endemic perennial herb, which grows wild in Iran is traditionally used as antibacterial and antifungal agent, as culinary plant for seasoning and flavouring foods (Lotfipour et al. 2007; Salehi et al. 2007; Esmaeili et al. 2009). Based on our ethnobotanical data and on local beliefs, Salvia macrosiphon is locally used for the treatment of cough and cold in southern parts of Iran. Salvia chloroleuca is used for gastrointestinal disorders. Salvia virgata is used for the treatment of skin diseases and cancer (Baytop 1999). Salvia hispanica has a long history of uses such as medicinal, culinary and religious (Cahill 2003). Salvia divinorum is used as antibacterial, antioxidant and antidiabetic agent (Ulubelen et al. 1997). Different Salvia species have been investigated for their antiproliferative activity (Tundis et al. 2011; Zare Shahneh et al. 2013; Zihlif et al. 2013). The GLOBOCAN data (GLOBOCAN 2008) evidenced that in 2012 worldwide, 14.1 million new
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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cancer cases were diagnosed, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis). Natural products provided the only source of pharmaceuticals for thousands of years, and have made enormous contributions to human health through valuable and unique compounds. Several drugs currently used in chemotherapy were isolated from plant species or derived from a natural prototype (Newman & Cragg 2007). Several chronic diseases including cancer involves oxidative damage to cellular components, such as lipids, proteins and DNA, and initiation of subsequent cell death via necrosis or apoptosis (Liangli 2008). In healthy individuals, production of ROS is balanced by natural anti-oxidative defence system. Thus, increased intake of natural antioxidants in the daily diet is recommended. In an effort to address this, we hypothesised that Salvia species exert antiproliferative effects against tumour cell lines. To test our hypothesis, we selected nine Salvia species commonly used as spices and consumed as tea. Thus, in this study, we have evaluated the Salvia ssp. methanol extracts for their in vitro antioxidant and antiproliferative activity. The total phenol and flavonoid contents were also determined in order to correlate the observed bioactivity with the phytochemical content (Table 1). 2. Results and discussion 2.1. Antioxidant properties Several studies demonstrated that plant phenolic compounds exhibit considerable free radicalscavenging activities through their reactivity as hydrogen- or electron-donating agents, and metal ion-chelating properties. Therefore, it would be valuable to determine the total phenol content of the food plant extracts. As shown in Table 2, total phenol content ranged from 5.1 to 42.5 gallic acid g21 of dried extract for Salvia xanthocheila and Salvia glutinosa, respectively. High values were found also in Salvia ceratophylla and Salvia sclarea with 32.7 and 30.4 mg gallic acid g21 dried extract. The same trend was observed also in the results of total flavonoid content where S. glutinosa, S. ceratophylla and S. sclarea displayed the highest content with 36.2, 27.0 and 12.2 mg (þ )-catechin equivalents per g of dried extract. Recently, the total phenolic content S. hydrangea, Salvia lachnocalyx, Salvia macilenta, S. multicaulis, S. sclarea and S. xanthocheila were investigated. S. macilenta displayed the highest total phenol content with 326 mg gallic acid/g dried extract while S. lachnocalyx exhibited the highest total flavonoid content with 253 mg (þ )-catechin equivalents per g of dried extract (Asadi et al. 2009). Tosun et al. (2009) investigated the total phenol content of eight Salvia species from Turkey and determined the values to be in the range 50.3 –167.1 mg GAE g21 of dried weight for Salvia verticillata and Salvia limbata, respectively. Table 1. Localities information and voucher number of studied Salvia species. Species
Locality
S. sclarea L. S. atropatana Bunge S. ahendica (Boiss.) Buhse S. hydrangea DC. S. xanthocheilla Boiss. ex Benth. S. macrosiphon Boiss. S. glutinosa L. S. chloroleuca Rech.f. & Aell. S. ceratophylla L.
Esfahan, Semirom Zanjan, Rudbar East Azarbaijan, Tabriz Fars, Shira West Azarbaijan, Khoy Lorestan, Khorramabad Ardebil, Khalkhal Khorasan, Mashhad Qazvin, Razmian
Altitude Herbarium Month of Extraction (m) no. collection yield (%) 1900 2400 2230 1500 2800 970 1800 1750 1520
MPH-658 MPH-1426 MPH-1321 MPH-1545 MPH-1308 MPH-1543 MPH-1563 MPH-1625 MPH-1520
May June June May June May July June May
5.0 6.0 5.1 5.5 6.4 7.0 5.8 5.8 7.2
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Table 2. Total phenolic and flavonoid contents and antioxidant activities of selected Salvia species.
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Species S. sclarea S. atropatana S. ahendica S. hydrangea S. xanthocheila S. macrosiphon S. glutinosa S. chloroleuca S. ceratophylla Ascorbic acid BHT
Total phenol contenta
Total flavonoid contentb
30.4 ^ 0.3 11.1 ^ 0.8 7.0 ^ 0.4 21.9 ^ 0.1 5.1 ^ 0.7 16.4 ^ 0.1 42.5 ^ 1.0 11.1 ^ 1.5 32.7 ^ 1.7
12.2 ^ 0.2 7.0 ^ 0.4 3.2 ^ 0.3 11.5 ^ 0.1 2.2 ^ 0.2 12.4 ^ 0.9 36.2 ^ 0.5 6.6 ^ 1.0 27.0 ^ 1.0
DPPHc
ABTSd
FRAPe
4.8 ^ 0.1 17.5 ^ 0.3 16.2 ^ 0.3 5.3 ^ 0.1 14.6 ^ 0.3 14.7 ^ 0.1 3.2 ^ 0.3 16.7 ^ 1.0 5.5 ^ 0.1 5.0 ^ 0.8 –
56.1 ^ 0.8 32.4 ^ 0.7 25.3 ^ 1.1 40.1 ^ 1.7 37.2 ^ 1.1 23.9 ^ 1.2 59.1 ^ 0.4 28.6 ^ 4.2 42.0 ^ 0.7 1.0 ^ 0.03 –
160.1 ^ 1.5 20.6 ^ 7.3 8.9 ^ 4.1 195.9 ^ 5.4 14.4 ^ 4.6 10.2 ^ 3.4 422.0 ^ 9.8 12.9 ^ 0.8 290.7 ^ 6.0 – 63.2 ^ 4.5
Notes: Data are expressed as means ^ SD (n ¼ 3). a Milligram of gallic acid equivalents/g dried extract ^ SD. b Milligram of (þ)-catechin equivalents/g of dried extract ^ SD. c IC50 mg/mL ^ SD. d mmol TE/g of dried extract ^ SD. e mmol Feþ þ equivalents/g of dried extract ^ SD. Positive controls: ascorbic acid and BHT. DPPH test: one-way ANOVA analysis: P , 0.0001 (F ¼ 24.76, r 2 ¼ 0.92). Dunnett multiple comparison test: P , 0.05 (S. sclarea, S. hydrangea, S. glutinosa and S. ceratophylla vs. ascorbic acid); P , 0.01 (S. atropatana, S. ahendica, S. xanthocheila, S. macrosiphon, S. chloroleuca vs. ascorbic acid). ABTS test: one-way ANOVA analysis: P , 0.0001 (F ¼ 187.20, r 2 ¼ 0.99). Dunnett multiple comparison test: P , 0.01 (all samples vs. ascorbic acid). FRAP test: one-way ANOVA analysis: P , 0.0001 (F ¼ 111.30, r 2 ¼ 0.99). Dunnett multiple comparison test: P , 0.01 (all samples vs. BHT).
The antioxidant properties of Salvia species were studied by three different methods – DPPH, ABTS and FRAP assays. Results are summarised in Table 2. S. glutinosa extract exhibited the highest DPPH radical-scavenging activity with an IC50 value of 3.2 mg mL21, followed by S. sclarea (IC50 value of 4.8 mg mL21), S. hydrangea (IC50 value of 5.3 mg mL21) and S. ceratophylla (IC50 value of 5.5 mg mL21). A similar trend was also observed in the ABTS assay with the highest activity reported in S. glutinosa (59.1 mmol TE g21 dried extract) followed by S. sclarea (56.1 mmol TE g21 dried extract). This radical-scavenging ability could be related to the nature of phenolic compounds, thus contributing to their electron transfer/hydrogen donating ability. The FRAP method was also used to evaluate the reducing potential of Salvia extracts. Using this assay, S. glutinosa (422.0 mmol FeSO4 eq g21 of dried extract) was found to be the most active followed by S. ceratophylla (290.7 mmol FeSO4 eq g21 of dried extract), S. hydrangea (195.9 mmol FeSO4 eq g21 of dried extract) and S. sclarea (160.1 mmol FeSO4 eq g21 of dried extract). Generally, S. glutinosa exhibited the highest antioxidant activity in all assays. S. sclarea, S. hydrangea and S. ceratophylla extracts were among the best antioxidant sources with a slight deviation. Many Salvia species and their isolated constituents possess significant antioxidant activities. In a comparative study, the antioxidant properties of several Salvia species including S. hydrangea, S. sclarea and S. xanthocheila were investigated (Asadi et al. 2009). In both DPPH and FRAP assays, S. hydrangea displayed the highest antioxidant activity followed by S. sclarea, and S. xanthocheila. Previously, the radical-scavenging activity of Salvia aegyptiaca, Salvia verbenaca and Salvia argentea were determined (Salah et al. 2006). In the DPPH assay, the order of decreasing potency is S. aegyptiaca, S. verbenaca, and then S. argentea. In the ABTS test, the same trend was observed. These results are lower than those found in our Salvia species. 2.2. Antiproliferative effects The antiproliferative effects of Salvia species were investigated in vitro using sulforhodamine B (SRB) assay against cancer cell lines namely C32, ACHN, COR-L23, A375, Caco-2, Huh-7D12,
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A549, MCF-7 and LNCaP. The results are summarised in Table 3. All samples exhibited a concentration – response effect against cancer cell lines. S. ceratophylla extract exhibited the strongest inhibitory activity against C32 and ACHN cell lines with IC50 values of 20.8 and 27.2 mg mL21, respectively. A similar result was observed with S. glutinosa and S. xanthocheila against ACHN cells with IC50 values of 29.5 and 34.0 mg mL21, respectively. Moreover, S. glutinosa exhibited IC50 values of 30.4 and 29.1 mg mL21, against lung carcinoma large cell and human breast cancer, respectively. The IC50 values of 31.2 and 34.3 mg mL21 were found when S. chloroleuca was applied to ACHN and C32 cell lines. S. macrosiphon inhibited COR-L23, MCF-7 and ACHN cell growth with IC50 values of 32.6, 33.7 and 36.1 mg mL21, respectively. Salvia atropatana exhibited the highest inhibitory activity against human Caucasian lung carcinoma A549 (IC50 of 48.1 mg mL21) compared with other tested Salvia species. Interesting results on human breast cancer and hormone-dependent prostate carcinoma were found when S. hydrangea was applied to cell cultures (IC50 values of 37.3 and 31.6 mg mL21, respectively). No antiproliferative activity at the maximum concentration tested (150 mg mL21) was observed with all samples against human skin fibroblast 142BR. Recently, Salvia leriifolia extracts and isolated constituents were investigated for their antiproliferative activity against a panel of human cancer cell lines (Tundis et al. 2011). The n-hexane extract exhibited the strongest antiproliferative activity against the C32 cell line with an IC50 of 11.2 mg mL21. Previously, Kamatou et al. (2008) reported the anticancer activity of indigenous Salvia species. The IC50 values ranged from 9.69 to 43.65 mg mL21 and from 8.72 to 59.12 mg mL21 against MCF-7 and SF-268 cell lines, respectively, with Salvia radula and Salvia africana-caerulea being the most active. Salvia lanceolata displayed the highest antiproliferative activity against HT-29 cell line. Other Salvia species were studied by Fiore et al. (2006). Among them, Salvia menthaefolia exerted a good antiproliferative against WiDr and HT-29 cell lines (IC50 values of 89.6 and 95.3 mg mL21, respectively). The antiproliferative effects towards human cancer cells (MCF7, HT-29 and SF268) and a human kidney epithelial cell line of 17 Salvia species used in the traditional Jordanian medicine were also investigated (Alkofahi et al. 1996). Salvia africana-lutea and Salvia runcinata were the most active against breast cancer cell line. Salvia dominica and Salvia fruticosa exhibited IC50 values of 5.83 and 25.55 mg mL21 against the oestrogen receptorpositive cell line MCF-7 with minimal toxicity against normal human periodontal fibroblasts (Abu-Dahab et al. 2012). Previously, the cytotoxic activities of the fractions prepared from the methanol extract of 23 Salvia species were investigated against HeLa, A431 and MCF7 (Janicsa´k et al. 2011). The n-hexane fractions of S. hispanica, Salvia nemorosa, Salvia albiflora, Salvia pratensis, Salvia recognita and Salvia ringens and the chloroform fraction of Salvia officinalis and S. albiflora caused more than 50% growth inhibition of the skin carcinoma cell line. None of the tested extracts exhibited substantial antiproliferative effects against HeLa and MCF7 cells. S. ringens was the most potent with a 61.8% cell growth inhibitory activity on A431 cells. 3. Experimental 3.1. Chemical Reagents were obtained from Sigma-Aldrich S.p.A. (Milan, Italy). Solvents were purchased from Merck chemical company (Darmstadt, Germany). 3.2. Plant material and extraction procedure The aerial parts of S. sclarea, S. atropatana, S. sahendica, S. hydrangea, S. xanthocheila, S. macrosiphon, S. glutinosa, S. chloroleuca and S. ceratophylla were collected from different
101.8 ^ 6.4 124.4 ^ 4.4 . 150 121.2 ^ 5.3 . 150 . 150 142.8 ^ 5.9 123.4 ^ 2.8 . 150
69.0 ^ 0.6 –
61.2 ^ 2.8 52.3 ^ 2.4 40.3 ^ 1.4 47.1 ^ 2.2 45.5 ^ 3.1 32.6 ^ 1.6 30.4 ^ 1.5 39.9 ^ 2.0 38.9 ^ 1.9
45.5 ^ 0.7 –
S. sclarea S. atropatana S. ahendica S. hydrangea S. xanthocheila S. macrosiphon S. glutinosa S. chloroleuca S. ceratophylla Positive control Vinblastine Taxol 7.2 ^ 0.7 –
107.4 ^ 5.4 68.1 ^ 2.9 60.7 ^ 3.4 67.5 ^ 2.8 97.8 ^ 2.9 138.5 ^ 5.4 123.2 ^ 5.2 120.7 ^ 4.8 120.9 ^ 3.7
A375
3 ^ 0.3 –
51.5 ^ 2.8 55.3 ^ 2.6 58.3 ^ 3.4 43.6 ^ 2.4 40.5 ^ 2.9 64.2 ^ 3.3 55.5 ^ 2.1 34.3 ^ 2.8 20.8 ^ 1.4
C32
67.3 ^ 2.0 –
67.1 ^ 2.4 48.1 ^ 2.7 64.2 ^ 2.9 62.6 ^ 3.1 130.3 ^ 3.4 . 150 . 150 71.6 ^ 2.8 . 150
A549
22.7 ^ 0.14 –
54.8 ^ 2.2 62.0 ^ 2.4 55.9 ^ 2.8 42.3 ^ 1.8 34.0 ^ 1.4 36.1 ^ 1.6 29.5 ^ 2.1 31.2 ^ 2.0 27.2 ^ 2.1
ACHN
45.6 ^ 0.8 –
129.3 ^ 4.4 129.8 ^ 4.7 . 150 111.5 ^ 3.9 . 150 . 150 . 150 . 150 121.3 ^ 4.6
Huh-7D12
– 0.08 ^ 0.004
47.1 ^ 2.1 112.3 ^ 3.5 115.9 ^ 3.7 37.3 ^ 2.8 60.6 ^ 3.9 33.7 ^ 2.2 29.1 ^ 2.1 134.2 ^ 3.6 57.5 ^ 3.1
MCF-7
29.3 ^ 0.86 –
74.8 ^ 2.3 65.6 ^ 3.1 65.7 ^ 3.0 31.6 ^ 2.8 75.5 ^ 2.7 59.3 ^ 2.6 124.4 ^ 3.6 104.8 ^ 3.4 111.1 ^ 4.1
LNCaP
150 150 150 150 150 150 150 150 150 37.00 ^ 0.44 –
. . . . . . . . .
142BR
Notes: Cell lines: COR-L23, human Caucasian lung large cell carcinoma; Caco-2, human Caucasian colon adenocarcinoma; A375, malignant melanoma; C32, amelanotic melanoma; A549, human Caucasian lung carcinoma; ACHN, human renal cell adenocarcinoma; Huh-7D12, human hepatocellular carcinoma; MCF-7, breast adenocarcinoma; LNCaP, prostate carcinoma; 142BR, human skin fibroblast. Data are expressed as means ^ SD (n ¼ 3). Vinblastine and taxol were used as positive control. COR-L23 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 31.64, r 2 ¼ 0.93); Dunnett multiple comparison test: P . 0.05 (S. ahendica, S. hydrangea, S. xanthocheila, S. chloroleuca and S. ceratophylla vs. vinblastine), P , 0.05 (S. atropatana vs. vinblastine), P , 0.01 (S. sclarea, S. macrosiphon and S. glutinosa vs. vinblastine). Caco-2 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 272.0, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). A375 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 650.3, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). C32 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 146.2, r 2 ¼ 0.98); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). A549 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 692.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. sclarea, S. ahendica, S. hydrangea and S. chloroleuca vs. vinblastine), P , 0.01 (S. xanthocheila, S. macrosiphon, S. atropatana, S. glutinosa and S. ceratophylla vs. vinblastine). ACHN cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 692.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. sclarea, S. ahendica, S. chloroleuca, and S. hydrangea vs. vinblastine), P , 0.01 (S. xanthocheila, S. macrosiphon, S. atropatana, S. glutinosa and S. ceratophylla vs. vinblastine). Huh-7D12 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 397.2, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). MCF-7 cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 783.6, r 2 ¼ 0.99); Dunnett multiple comparison test: P , 0.01 (all samples vs. vinblastine). LNCaP cell line: one-way ANOVA analysis: P , 0.0001 (F ¼ 367.7, r 2 ¼ 0.99); Dunnett multiple comparison test: P . 0.05 (S. hydrangea vs. taxol), P , 0.01 (S. sclarea, S. atropatana, S. ahendica, S. xanthocheila, S. macrosiphon, S. glutinosa, S. chloroleuca and S. ceratophylla vs. taxol).
Caco-2
COR-L23
Salvia
Cell line
Table 3. In vitro antiproliferative activity [IC50 (mg/mL)] of selected Salvia species on nine cancer cell lines.
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regions in Iran. Voucher herbarium specimens (Table 1) have been deposited in the Medicinal Plants and Drugs Research Institute herbarium, Shahid Beheshti University, Tehran, Iran. The dried aerial parts of Salvia species (10 g) were ground into powder and extracted with methanol (200 mL) for three times for 24 h at room temperature, then filtered and concentrated under reduced pressure. Information about periods of sample collection and extraction yield (%) are reported in Table 1.
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3.3. Total phenol content The total phenol content was measured using the Folin – Ciocalteau method (Singleton et al. 1999). The absorbance was evaluated at 760 nm after incubation at room temperature for 2 h. Gallic acid was chosen as a standard. The total phenolic contents were expressed as mg of gallic acid equivalents g21 dried extract ^ standard deviation (SD). 3.4. Total flavonoid content The total flavonoid content was determined by the procedure previously described by Zhishen et al. (1999). The absorbance was measured against a blank at 510 nm. Results were expressed as mg of (þ )-catechin equivalents per g of dried extract. 3.5. Radical-scavenging activity by DPPH assay The DPPH radical-scavenging capacity was carried out according to the technique previously reported (Loizzo et al. 2009). Ascorbic acid was used as positive control. At the end of the experiment, the IC50 was calculated according to the following equation: Scavenging activity ¼ [(A0 2 A1)/A0 £ 100], where A0 is the absorbance of the blank and A1 is the absorbance in the presence of the extract or standard sample. The blank was prepared with all reagents except extract. 3.6. ABTS radical cation-scavenging activity ABTS antioxidant assay was performed according to the procedure previously described (Loizzo et al. 2009). The absorbance was recorded at 734 nm using Molecular Devices SpectraMax Plus Plate Reader (Molecular Devices, CELBIO, Milan, Italy). The inhibition percent of extracts was evaluated using the following equation: % Inhibition ¼ [(Ab 2 As)/ Ab £ 100], where Ab is the absorbance of blank and As is the absorbance of the sample. Ascorbic acid was used as positive control. 3.7. FRAP assay The FRAP assay was performed using Benzie and Strain method (Benzie & Strain 1996). The standard curve was then prepared by plotting the FRAP value for each standard versus its concentration. BHT was used as positive control. Results were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of mmol Feþ þ equivalent/g of dried extract. 3.8. Antiproliferative activity 3.8.1. Cell culture All media, buffers, trypsin and dyes were filter-sterilised prior to use and warmed to 37 8C. Human amelanotic melanoma (C32, CRL1585), renal cell adenocarcinoma (ACHN, CRL1611),
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lung carcinoma large cell (COR-L23, 92031919), malignant melanoma (A375, 88113005), colorectal adenocarcinoma (Caco-2, HTB37), hepatocellular carcinoma (Huh-7D12, 1042712), human Caucasian lung carcinoma (A549, 86012804), human breast cancer (MCF-7, HTB-D22), hormone-dependent prostate carcinoma (LNCaP, CRL-1740) cancer cell lines and one normal cell line of human skin fibroblast (142BR) were used in our experiments. COR-L23, C32, ACHN and LNCaP cells were cultured in RPMI 1640 medium while 142BR, MCF-7, A549, Huh-7D12, Caco-2 and A375 cells were cultured in DMEM. Both media were supplemented with 10% foetal bovine serum, 1% L -glutamine and 1% penicillin/streptomycin. 3.8.2. SRB assay Salvia extracts were tested to evaluate their potential cytotoxic activity through the SRB assay (Loizzo et al. 2007). Briefly, cells were placed in 96-well plates in a range from 5 £ 104 to 15 £ 104 cells. After 24 h, the cells were treated with 100 mL/well of serial dilutions of the sample. After 48 h of exposure, cells were fixed with ice-cold 40% trichloroacetic acid. The fixed cells were stained for 30 min with 50 mL of 0.4 % (w/v) SRB in 1% acetic acid. The plates were washed with 1% acetic acid and air-dried overnight. For reading plate, the bound dye was solubilised with 100 mL of 10 mM tris(hydroxymethyl)aminomethane (Tris base). The absorbance of each well was read on a Molecular Devices SpectraMax Plus Plate Reader (Molecular Devices, CELBIO, Milan, Italy) at 490 nm. Cell survival was measured as the percentage absorbance compared with the untreated control. Vinblastine sulfate and taxol were used as positive control. 3.9. Statistical analyses All experiments were carried out in triplicate. Data are expressed as means ^ SD. The concentration causing 50% inhibition (IC50) was calculated by non-linear regression with the use of Prism GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). In the antioxidant assay, the concentration – response curve was obtained by plotting the percentage inhibition versus concentration while in the anti-proliferative activity the concentration – response curve was obtained by plotting the percentage of cell viability versus the concentrations. Differences concerning parameters were analysed by the one-way ANOVA test to compare group means (a ¼ 0.05). The r 2 values thus obtained were used to predict relationships. To complete the statistical analysis multi-comparison Dunnett’s test was performed. Differences were considered significant at P , 0.05. 4. Conclusion Several Salvia species are widely used for medicinal purpose and for this reason the genus is largely investigated. Thus, we have screened nine Iranian Salvia species for their potential antioxidant effect and antiproliferative properties against a panel of human cancer cell lines. S. ceratophylla exerted an interesting activity against C32 and ACHN cell lines while S. glutinosa and S. hydrangea exhibited the highest activity against MCF-7 and LNCaP cell lines, respectively. These species also displayed the highest antioxidant activity with high total phenol content and are the best candidates for further in vivo studies and natural sources to find new anticancer compounds. Acknowledgements Financial support by Iran National Science Foundation (INSF; Grant no. 86023.07) is gratefully acknowledged.
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