Introduction
Numerous studies have been focused on natural anticarcinogenic agents. Many antioxidants have been identified as anticarcinogens. Antimutagens have also been proposed as cancer chemopreventive agents. Therefore, the regular consumption of antimutagens can reduce genotoxic effects of mutagenic and carcinogenic factors (1, 2).
The use of natural products as anticancer has a long history that began with traditional medicine. Several drugs in chemotherapy were isolated from plant species or derived from a natural prototype. They include vinblastine, vincristine, taxanes, etoposide and teniposide, the semisynthetic derivatives of epipodophyllotoxin, camptothecin, irinotecan, and topotecan and several others. Over 50% of the drugs in clinical trials for anticancer activity were isolated from natural sources or were related to them (3).
The brine shrimp lethality assay is a general bioassay that seems to be capable of detecting a wide spectrum of bioactivity present in crude extracts. The commercial availability of inexpensive brine shrimp eggs, the low cost, the safety and ease of performing the assay, as well as no special technology requirement make this a very helpful bench-top tool for the phytochemistry laboratory. The lethality to brine shrimp is recommended as an effective prescreen to existing cytotoxicity and antitumor assays. A number of studies have established the use of the brine shrimp assay to screen plants commonly used as pesticides, anticancer, and with molluscicidal, larvicidal, fungicidal, and cytotoxic activity. Lastly, this assay has been used successfully to biomonitor the isolation of cytotoxic, antineoplastic, antimalarial, insecticidal, and antifeedant compounds from plants (4).
Antioxidants are usually added to foods to prevent the radical chain reactions of oxidation. They act through inhibiting the initiation and generation step leading to the termination of reaction and delay the oxidation process. However, synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxy toluene are restricted because of their potential for toxic and carcinogenic effects. Therefore, there has been a significant interest to find natural antioxidants to replace the synthetic compounds in food applications and a growing tendency in consumer preferences for natural antioxidants, all of which has given more impetus to explore the natural sources of antioxidants (1).
The present study aims to provide data on the cytotoxic potential of 23 plant species of Leguminosae family from different regions of Iran on developing brine shrimp nauplii. The antioxidant activity of the species with potent cytotoxicity effects have been evaluated using the spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging method.
Experimental
Plant material
Twenty-three plant species of Leguminosae family were collected in May-June 2009 from different regions of Iran. The plant species were identified and voucher specimens have been deposited at Institute of Medicinal Plants Herbarium (IMPH) (Table 1). The plant parts were air-dried under the shade and ground using a laboratory mill and a kitchen blender.
Table 1
List of plant species collected from different regions of Iran and yields of the methanol extracts
| No | Plant species voucher no. | Time of collection | Area of collection | Part used | Yield (%w/w) |
|---|---|---|---|---|---|
| 1 | Astragalus squarrosus Bunge (IMPHa 611) | May 2009 | The road of Quhin to Loshan, Qazvin province | Aerial parts | 24 |
| 2 | Caesalpinia gilliesii (Hook.) D.Dietr. (IMPH 610) | May 2009 | Chabahar, Sistan and Baloochestan province | Aerial parts | 12 |
| 3 | Crotalaria burhia Buch.-Ham. ex Benth. (IMPH 612) | May 2009 | The road of Chabahar to Baris, Sistan and Baloochestan province | Stems | 10 |
| 4 | Gleditschia caspica Desf. (IMPH 613) | May 2009 | Chaparpardzaman, The road of Zibakenar to Golshan, Gilan province | Leaves, Fruits | 32 |
| 5 | Glycyrrhiza glabra L. var. glabra L. (IMPH 614) | June 2009 | The road of Moshkan to Kashan, Esfahan province | Aerial parts | 34 |
| 6 | Glycyrrhiza glabra L. var. glandulifera (Waldst. and Kit.) Boiss. (IMPH 615) | June 2009 | The road of Esafahan to Naiin, Esfahan province | Aerial parts | 24.5 |
| 7 | Indigofera articulata Gouan. (IMPH 616) | May 2009 | Tis, The road of Chabahar to Baris, Sistan and Baloochestan province | Aerial parts | 20 |
| 8 | Indigofera intricata Boiss. (IMPH 617) | June 2009 | Bandar Abbas, Hormozgan province | Aerial parts | 13.5 |
| 9 | Lathyrus annus L. (IMPH 618) | May 2009 | Galangoodeh, Bandar Anzali, Gilan province | Aerial parts | 18.5 |
| 10 | Lathyrus sativus var. stenophyllus Boiss. (IMPH 619) | May 2009 | Galangoodeh, Bandar Anzali, Gilan province | Aerial parts | 30 |
| 11 | Lotus corniculatus L. subsp. corniculatus (IMPH 620) | May 2009 | Bandar Anzali, Gilan province | Aerial parts | 16.9 |
| 12 | Medicago rigidula (L.) All. (IMPH 621) | May 2009 | Hashtgerd, Tehran province | Leaves, Fruits | 13.5 |
| 13 | Melilotus indicus (L.) All. (IMPH 622) | May 2009 | Talesh mahalleh, Gilan province | Aerial parts, Roots | 19 |
| 14 | Onobrychis altissima Grossh. (IMPH 623) | May 2009 | Mehrshahr, Tehran province | Aerial parts | 17.8 |
| 15 | Sophora alopecuroides L. (IMPH 624) | June 2009 | The road of Natanz to Moorcheh khort, Esfahan province | Aerial parts | 29 |
| 16 | Sophora pachycarpa C.A. Mey. (IMPH 625) | June 2009 | Natanz, Esfahan province | Aerial parts | 22.5 |
| 17 | Taverniera cuneifolia Arn. (IMPH 626) | June 2009 | Chooj, The road of Bandar Abbas to Minab, Hormozgan province | Leaves, Flowers | 20 |
| 18 | Taverniera spartea DC. (IMPH 627) | June 2009 | The road of Bandar Abbas to Minab, Hormozgan province | Stem | 26 |
| 19 | Tephrosia persica Boiss. (IMPH 628) | June 2009 | The road of Bandar Abbas to Minab, Hormozgan province | Aerial parts | 19 |
| 20 | Trifolium campestre Schreb. (IMPH 629) | May 2009 | Golshan, Bandar Anzali, Gilan province | Leaves, Flowers | 13.1 |
| 21 | Trifolium repens L. (IMPH 630) | May 2009 | Galangoodeh, Bandar Anzali, Gilan province | Aerial parts | 21 |
| 22 | Trigonella spruneriana Boiss. (IMPH 631) | May 2009 | The road of Quhin to Loshan, Qazvin province | Aerial parts | 10 |
| 23 | Vicia peregrina L. var. peregrine (IMPH 632) | May 2009 | The road of Quhin to Loshan, Qazvin province | Aerial parts | 23.5 |
Preparation of the crude extracts and fractions
The shade-dried powdered plant samples (20 g) were extracted with methanol in soxhlet apparatus for 12 h. The methanol extracts were concentrated using rotary vacuum distillation below 50°C under reduced pressure to get the crude extracts (Table 1) (5).
Among the screened extracts, anyone showing potent activity against the brine shrimp was resuspended in water and partitioned with chloroform (CHCl3) to separate less polar, water-insoluble compounds (5).
Toxicity testing against the brine shrimp
Hatching shrimp
Brine shrimp was obtained from Salt Creek, Inc. (Salt Lake City, UT 84104, USA). Brine shrimp eggs, Artemia salina Leach, were hatched in artificial seawater prepared through dissolving 38 g of sea salt in 1 L of distilled water. After 48 h of incubation at room temperature (27-29°C), the larvae (nauplii) were attracted to one side of the vessel with a light source and collected with pipette. Nauplii were separated from eggs through being aliquoted three times in small beakers containing seawater (6).
Brine shrimp assay
The bioactivity of the extracts was monitored with the brine shrimp lethality assay (7); 50 mg of methanol extracts were exactly measured and dissolved in 10 mL of DMSO to get a concentration of 5 mg/mL. From the stock solutions, different volumes were placed in 22 different vials making the volume up to 5 mL by the NaCl solution. The final concentration of the samples in the vials became 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 5, to 140 μg/mL (ppm), respectively. Serial dilutions were made in triplicate (8).
Ten brine shrimp nauplii were then placed in each vial. Both positive (thymol) (9, 10) and negative (seawater containing DMSO) control assays were carried out in order to verify the susceptibility of Artemia under the assay conditions employed. For the negative control test of each vial, one vial containing the same volume of DMSO plus seawater up to 5 mL was used. After 24 h of incubation, the vials were observed using a magnifying glass and the numbers of survivors in each vial were counted and noted (8). In cases where control deaths occurred, the data was corrected using Abbott’s formula (%deaths = [(test – control)/ control] × 100) described by Rasoanaivo and Ratsimamanga-Urverg (11). The LC50 values were determined from the 24 h counts. In cases where data were insufficient for this technique, the dose-response data were transformed into a straight line by means of a logit transformation; the LC50 values were derived from the best-fit line obtained by linear regression analysis (7). The extract or isolated compounds were considered bioactive when LC50 value was lower than 30 μg/mL (6).
Radical scavenging activity using DPPH method
The DPPH radical-scavenging activity of the nine plants with LC50 value lower than 40 μg/mL was determined using the method proposed by Afolayan et al. A solution of 0.135 mM DPPH in methanol was prepared and 1.5 mL of this solution was mixed with 1.5 mL of the extract in methanol containing 2-1000 μg/mL concentration of the extract. The reaction mixture was vortexed completely and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured through spectrophotometry at the 517 nm. These tests were carried out in triplicate and ascorbic acid was used as the reference (12).
Lower absorbance of the reaction mixture indicates higher DPPH° scavenging activity. DPPH° scavenging activity was calculated using the following formula: DPPH° scavenging activity (%) = [1 – (S – SB)/(C – CB)] × 100%, where S, SB, C and CB were the absorbances of the sample, the blank sample (1.5 mL of methanol plus 1.5 mL of sample at different concentrations), the control (1.5 mL of DPPH° solution plus 1.5 mL of methanol), and the blank control (methanol), respectively (13).
A percent inhibition versus the concentration curve was plotted and the concentration of the sample required for 50% inhibition was determined and expressed as IC50 value (14).
Statistical analysis
All the experimental results were as mean ± SD of three parallel measurements. The data was entered into a Microsoft Excel© database and analyzed using SPSS (version 15.0). The LC50 and IC50 values were obtained by the linear regression analysis. Extracts giving LC50 values lower than 30 μg/mL were considered to be cytotoxic. The extracts with IC50 values lower than 50 μg/mL showed antioxidant activity.
Results and Discussion
Yield of plant extracts
The percent yield of crude extracts following the removal of solvent using a rotary evaporator, were 10% for Crotalaria burhia and Trigonella spruneriana to 34% for Glycyrrhiza glabra var. glabra (Table 1).
Cytotoxicity of plant extracts
Results of the toxicity of the extracts against brine shrimp (LC50 values) are shown in Table 2. A total of 23 methanol extracts were tested for their toxicity against the brine shrimp using the brine shrimp lethality assay. The extracts of Taverniera spartea and Tephrosia persica showed significant cytotoxicity against brine shrimp (LC50 < 30 μg/mL) with LC50 values of 0.34 and 2.43 μg/mL, respectively, whereas the positive control, thymol, showed a LC50 value of 1.37 μg/mL. Chloroform fraction of these two species (Taverniera spartea and Tephrosia persica) represented different cytotoxicity against the brine shrimp with LC50 values of 113.79 and 1.23 μg/mL, respectively. These results suggested that the total extract of Taverniera spartea was more cytotoxic than its less polar fraction. But in the case of Tephrosia persica, chloroform fraction had cytotoxic effect as well as total methanol extract (Table 2).
Table 2
Brine shrimp lethality assay results of extracts or fractions of 23 plant species of the Leguminosae family
| No. | Plant species | Solvent | LC50value (μg/mL)a |
|---|---|---|---|
| 1 | Astragalus squarrosus | Methanol | 54.91 ± 0.38 |
| 2 | Caesalpinia gilliesii | Methanol | 36.67 ± 0.59 |
| 3 | Crotalaria burhia | Methanol | 44.45 ± 0.39 |
| 4 | Gleditschia caspica | Methanol | 37.10 ± 0.16 |
| 5 | Glycyrrhiza glabra var. glabra | Methanol | 50.41 ± 0.73 |
| 6 | Glycyrrhiza glabra var. glandulifera | Methanol | 44.51 ± 0.48 |
| 7 | Indigofera articulata | Methanol | 42.43 ± 0.23 |
| 8 | Indigofera intricata | Methanol | 44.08 ± 0.45 |
| 9 | Lathyrus annus | Methanol | > 90 |
| 10 | Lathyrus sativus var. stenophyllus | Methanol | 45.42 ± 0.31 |
| 11 | Lotus corniculatus subsp. corniculatus | Methanol | 32.00 ± 0.14 |
| 12 | Medicago rigidula | Methanol | 35.48 ± 0.17 |
| 13 | Melilotus indicus | Methanol | 72.52 ± 0.80 |
| 14 | Onobrychis altissima | Methanol | 51.38 ± 0.89 |
| 15 | Sophora alopecuroides | Methanol | 73.11 ± 1.20 |
| 16 | Sophora pachycarpa | Methanol | 56.73 ± 0.55 |
| 17 | Taverniera cuneifolia | Methanol | 39.39 ± 0.55 |
| 18 | Taverniera spartea | Methanol | 0.34 ± 0.01 |
| 19 | Taverniera spartea | Chloroform | 113.79 ± 1.43 |
| 20 | Tephrosia persica | Methanol | 2.43 ± 0.03 |
| 21 | Tephrosia persica | Chloroform | 1.23 ± 0.03 |
| 22 | Trifolium campestre | Methanol | 32.97 ± 0.17 |
| 23 | Trifolium repens | Methanol | 36.35 ± 0.59 |
| 24 | Trigonella spruneriana | Methanol | 52.41 ± 1.24 |
| 25 | Vicia peregrina var. peregrina | Methanol | 92.58 ± 1.07 |
| 26 | Thymolb | - | 1.37 ± 0.005 |
The extracts of twelve species including Caesalpinia gilliesii, Crotalaria burhia, Gleditschia caspica, Glycyrrhiza glabra var. glandulifera, Indigofera articulata, Indigofera intricata, Lathyrus sativus var. stenophyllus, Lotus corniculatus subsp. Corniculatus, Medicago rigidula, Taverniera cuneifolia, Trifolium campestre and Trifolium repens presented moderate cytotoxicity (LC50 between 30 and 50 μg/mL) against the brine shrimp. The extracts of Lathyrus annus and Vicia peregrina var. peregrina did not show any significant cytotoxicity (LC50 > 90 μg/mL) (Table 2). Since in most cases the toxicity is associated with pharmacological properties, it was deduced that the extracts of Taverniera spartea and Tephrosia persica had the best bioactivity.
Antioxidant activity of the plant extracts
IC50 values of DPPH percent scavenging activity of extracts and LC50 value of brine shrimp assay are given in Table 3, as calculated from the percent inhibition versus the concentration of extract curves. Gleditschia caspica and Taverniera spartea showed significant antioxidant activity (IC50 < 50 μg/mL) with IC50 values of 14.54 and 20.32 μg/mL, respectively, whereas the positive control, ascorbic acid, showed an IC50 value of 8.22 μg/mL. Five species including Taverniera cuneifolia, Lotus corniculatus subsp. corniculatus, Trifolium campestre, Tephrosia persica and Trifolium repens presented moderate antioxidant activity (IC50 between 50 and 200 μg/mL). Two species including Caesalpinia gilliesii and Medicago rigidula represented the highest IC50 value (205.41 and 423.13 μg/mL, respectively).
Table 3
IC50 values related to the DPPH assays of extracts with brine shrimp lethality assay LC50 value less than 40 μg/mL
| No. | Plant species | Brine shrimp assay LC50(μg/mL)a | DPPH assay IC50(μg/mL)a |
|---|---|---|---|
| 1 | Medicago rigidula | 35.48 ± 0.17 | 423.13 ± 0.05 |
| 2 | Caesalpinia gilliesii | 36.67 ± 0.59 | 205.41 ± 0.04 |
| 3 | Trifolium repens | 36.35 ± 0.59 | 180.78 ± 0.13 |
| 4 | Tephrosia persica | 2.43 ± 0.03 | 117.46 ± 0.09 |
| 5 | Trifolium campestre | 32.97 ± 0.17 | 71.88 ± 0.04 |
| 6 | Lotus corniculatus subsp. corniculatus | 32.00 ± 0.14 | 70.95 ± 0.01 |
| 7 | Taverniera cuneifolia | 39.39 ± 0.55 | 56.29 ± 0.02 |
| 8 | Taverniera spartea | 0.34 ± 0.01 | 20.32 ± 0.01 |
| 9 | Gleditschia caspica | 37.10 ± 0.16 | 14.54 ± 0.01 |
| 10 | Thymolb | 1.37 ± 0.005 | - |
| 11 | Ascorbic acidb | - | 8.22 ± 0.001 |
Cancer is a big challenge in the world as the suitable remedy is very expensive and even impossible in some cases. Many scientists are now engaged to find a potent remedy for cancer through the discovery of new and effective chemotherapeutic agents from plants and other sources (8).
A number of studies have reported the antioxidant or cytotoxic activity of some medicinal plants. For instance, 16 selected plants, which were collected from different localities of Yemen, have been evaluated for antimicrobial, antioxidant and cytotoxic activity and phytochemical screening (15). In another study, the cytotoxic potential of the different solvent extracts of the Sapium baccatum leaves, six column fractions of petroleum ether extract and three pure compounds have been determined by using brine shrimp lethality assay. The LC50 of all the tested samples were showed to be lethal to brine shrimp nauplii (16). Radical scavenging activity of the essential oils of Zataria multiflora from different parts of Iran has been determined. In the DPPH antioxidant assay, all samples exhibited a remarkable activity (IC50 = 19.7 μg/mL) almost similar to BHT (IC50 = 18.1 μg/mL) (17). Bioassay screening of the essential oil and various extracts of Heracleum persicum fruits and rhizomes of Zingiber officinale have been studied using brine shrimp cytotoxicity assay (18). The extracts of Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas have been screened for their antioxidant and antibacterial activity, as well as their general toxicity towards brine shrimps (19).
Brine shrimp lethality assay is a primary assay to detect cytotoxic property of plant extract and, further studies are required to establish the cytotoxicity of the plant extracts against human cancer cell lines. However, our results in this study may predict that which species of Leguminosae family will give better results on cancer cell lines. Although the crude extract or chloroform fraction have been examined in the present study, further investigations using single components from these extracts may explore potent cytotoxic properties.
This is the first report on cytotoxicity screening of these twenty-three plant species of Leguminosae family from different regions of Iran. However, according to the criteria of the American National Cancer Institute, the LC50 limit to consider a crude extract promising for further purification is lower than 30 μg/mL (20). Thus, only two species among 23 tested species of plants presented significant cytotoxicity against brine shrimp. The extracts of Taverniera spartea and Tephrosia persica could be considered as the potential sources of anticancer compounds.
Another mechanism of cancer prevention might be the radical scavenging of free radical oxygen and other species associated with cancer cell development. However, the results of radical scavenging activity with DPPH showed that two samples out the 9 tested samples were more active (Gleditschia caspica and Taverniera spartea; IC50 < 50 μg/mL).
Among the 23 tested plant species, Taverniera spartea had the most cytotoxic and antioxidant activity and was the best candidate for these effects. Further investigations are necessary for the chemical characterization of the active compounds and more comprehensive biological assays.
