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Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats
© Ismail and Mirza. 2015
Received: 4 December 2014
Accepted: 23 June 2015
Published: 27 June 2015
Lactuca sativa (lettuce) has been traditionally used for relieving pain, inflammation, stomach problems including indigestion and lack of appetite. Moreover, the therapeutic significance of L. sativa includes its anticonvulsant, sedative-hypnotic and antioxidant properties.
In the present study, the MC (methanol and chloroform; 1:1) and aqueous extracts of seed and leaf along with cell suspension exudate were prepared. These extracts were explored for their analgesic, anti-inflammatory, antidepressant and anticoagulant effects by hot plate analgesic assay; carrageenan induced hind paw edema test, forced swimming test and capillary method for blood clotting respectively in a rat model. The results were analyzed using one-way Analysis of Variance (ANOVA) followed by Turkey multiple comparison test.
Interestingly, the extracts and the cell suspension exudate showed dual inhibition by reducing pain and inflammation. The results indicated that the aqueous extracts of leaf exhibited highest analgesic and anti-inflammatory activities followed by leaf MC, cell suspension exudate, seed aqueous and seed MC extracts. The current findings show that aqueous and MC extracts of seed have the least immobility time in the forced swimming test, which could act as an anti-depressant on the central nervous system. The leaf extracts and cell suspension exudate also expressed moderate anti-depressant activities. In anticoagulant assay, the coagulation time of aspirin (positive control) and MC extract of leaf was comparable, suggesting strong anti-coagulant effect. Additionally, no abnormal behavior or lethality was observed in any animal tested.
Taken together, L. sativa can potentially act as a strong herbal drug due to its multiple pharmaceutical effects and is therefore of interest in drug discovery and development of formulations.
Lactuca sativa L. (lettuce) is a leafy vegetable and belongs to the Asteraceae family and genus Lactuca. It is a valuable dietary source of vitamin K, E and C as well as carotenoids . Traditionally, it is well-known for its use as folk remedy for inflammation, pain, stomach problems including indigestion and lack of appetite . Previously, considerable pharmacological studies have been conducted to evaluate therapeutic significance of the crude extracts of Lactuca sativa which showed its anticonvulsant, sedative-hypnotic and antioxidant properties . In addition, anti-inflammatory and anti-nociceptive activities of the seed extracts have also been evaluated . Cell suspension cultures are utilized as appropriate tool for the evaluation of an extensive variety of phenomena, bypassing the structural intricacy of the plant. An efficient method for the regeneration of shoots directly from cell suspensions of lettuce has also been previously described . Moreover, cell suspensions offer appropriate platform for the production of high esteem secondary metabolites and different substances of commercial and economic interest.
So far, no study has been carried out to evaluate the combined effects of L. sativa on inflammation, analgesia, blood clotting and depression. Therefore, we have examined the analgesic, anti-inflammatory, antidepressant and anticoagulant effects of leaf extracts, seed extracts and cell suspension exudate of L. sativa (CV. Grand Rapids) by hot plate analgesic assay, carrageenan induced hind paw edema test, forced swimming test and capillary method for blood clotting respectively in a rat model.
The hot plate assay is a simple method of the pain reaction in animals by which effectiveness of analgesics can be tested by measuring the heat induced pain response. The application of such stimuli evokes a behavioral response which results in the withdrawal of foot which varies inversely with the intensity of the stimulus. If an increase in latency is observed in the experimental treatment, then the treatment is said to be anti-nociceptive or analgesic. Conversely, if a condition such as an inflammation of the paw, serves to decrease the response latency, it is said to induce hyperalgesia . Inflammation is a condition involving confined increase in the amount of leukocytes and various complex mediator molecules . The most common screening method of acute inflammation has been the prevention of edema in rats by induction of carrageenan. It is believed that the COX (cyclooxygenase) enzyme involved in inflammation can be inhibited by NSAIDs (Nonsteroidal anti-inflammatory drugs) to reduce the edema. But NSAIDs have some side effects like renal and gastric toxicity . Medicinal plants are believed to be cost-effective and harmless source of novel biochemical constituents with strong therapeutic properties.
The forced swimming test (FST), described initially by Porsolt , is the frequently used model for evaluating antidepressant property. The basic principle of the assay is that rats ultimately develop immobility when they are released in water containing cylinder after they stop active escape behaviors, like swimming or climbing. During the FST, reduction in the immobility time, delay of its onset and increase of escape activities can be observed after the treatment of antidepressant agents .
Anticoagulants play an essential role as mediators for the treatment and prevention of thromboembolic disorders . Due to their pharmacological possessions, plants can serve as the sources for the investigation of new compounds with anticoagulant properties. There is convincing scientific indications representing that the use of phytochemicals with anticoagulant effects and dietary anticoagulants can eventually eliminate or reduce the risks of thromboembolic diseases .
The aim of present study extracts and cell suspension culture of Lactuca sativa (CV. Grand Rapids) were investigated for their analgesic, anti-inflammatory, antidepressant and anticoagulant effects in rat model. The plant extracts had shown significant activities which represent its significance as potential herbal drug.
Preparation of the extract
The seeds and leaves of Lactuca sativa (CV. Grand Rapids) were purchased from local market in Pakistan. The plant was identified by Dr. Muhammad Zafar (taxonomist) in Plant Sciences Department Quaid-i-Azam University (QAU). A voucher specimen (number 128085) was deposited in the “Herbarium of medicinal Plants of Pakistan” in QAU Islamabad, Pakistan. The plant leaves were air dried under shade for five weeks at room temperature. The seeds and leaves were then ground to powder and were macerated in portions of 100 g, with the 250 ml solvent consisting of MC (methanol: chloroform (1:1)) and water (250 ml) for 7 days. The mixture was then shaken thoroughly and filtered with Whatman filter paper 1. Then rotaevaporator was used to concentrate the filtrate. The concentrate was dried at room temperature and these crude extracts were stored at 4 °C for pharmacological studies.
Preparation of cell suspension culture
Seeds of L. sativa were germinated on half strength MS  medium after surface sterilization with 15 % sodium hypochlorite for 45 s followed by three times washing with sterile distilled water. Callus was produced by placing the cotyledon explants on MS medium supplemented with 4.4 μM BA and 2.7 μM NAA . After callus generation cell suspension culture was prepared by previously reported method .
Preparation of standard and test drugs
For the experiment, extracts were prepared as 100 mg/ml (10 % DMSO) and rest of the drugs were solubilized in saline (1 mg/ml) and were administered orally in volumes of 1 ml/100 g of rat’s body weight. In this study, one optimal dose (1 g/Kg) was selected which showed maximum effect without any lethal outcome to animals as described previously .
Animals and treatment groups
Adult albino rats of weights between 150-200 g of either sex were used for the study. They were housed in standard aluminum cages and bred with standard diet with water ad libitum in the Primate facility of Faculty of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan. The study was legitimated by the Institutional Animal Ethics Committee and all precautions were followed to diminish animal suffering. In each experiment, rats were divided into seven groups having five rats in each treatment and test samples were administered orally in each group.
Group-I: used for negative control in which 10 % DMSO (1 ml/Kg) in distilled water.
Group-II: used for positive control in which activity specific standard drug (10 mg/Kg) was used.
Group-III: cell suspension exudate of L. sativa was administered (1 ml/Kg).
Group-IV: Leaf MC extract of L. sativa was administered (1 g/Kg).
Group-V: Seed MC extract of L. sativa was administered (1 g/Kg).
Group-VI: Aqueous leaf extract of L. sativa was administered (1 g/Kg).
Group-VII: Aqueous seed extract of L. sativa was administered (1 g/Kg).
Hot plate analgesic assay
Carrageenan induced hind paw edema test
Where “X” is edema of control rats and “Y” is edema of treatment rats.
Forced swimming test
The anti-depressant activity of L. sativa was determined by the forced swimming test described previously . The rats were enforced to swim inside a vertical cylinder filled with water. Deprived of getting away, the ensuing anxiety produces vigorous swimming action to escape by climbing or diving the walls of the cylinder. When the rats stop all activities except those essential for survival (keeping the head above the water). This was assigned as provoked depression. On the first day rats were placed in the cylinder individually and were forced to swim inside a vertical cylinder (18 cm diameter, 40 cm height and 15 cm holding of water maintained at 25 °C). After 5-6 min the animal becomes stable and stays stationary for almost 80 % of the time. After 15 min in the water the rats were evacuated and permitted to dry before being come back to their home enclosures. This process is called pre-swimming. On the next day, oral dose was given to rats, according to their respective groups as mentioned earlier. DMSO (10 %) was used as negative control and Fluoxetine HCl (1 mg/Kg) was used as positive control. After 30 min of dosage, rats were again placed in the water filled cylinder maintained at 25 °C and the camera was placed to the side of the cylinder. Video recording was started before placing the rats into the cylinder and then the rats were placed in the cylinder and the timer was switched on. After 6 min, recording was stopped; the rats were removed from the water, dried with towels and then placed back to their cages. The water was changed after every 2 rats so that the image produced should be clear and sharp to distinguish the rats behaviors. Using a cumulative stopwatch total immobility time was recorded during the last 4 min of a total 6 min video.
Anticoagulant activity is the time required for blood to clot without the presence of any substance. Anticoagulant activity was determined by the capillary tube method as described previously . The tail of the rat was cleaned with spirit and then was pricked by the lancet. The tail was squeezed to obtain a larger drop of blood to be filled into a capillary tube. The time of appearance of the drop of the blood on the cut tail was noted. The capillary tubes were sealed and immersed in water bath at 37 °C. After one minute the tube was taken out and small pieces of the capillary tube were broken at every 10 s until a fibrin thread is seen between the two broken ends. The time interval between the appearance of the blood drop and the thread formation was the clotting time.
The data was analyzed using one-way Analysis of Variance (ANOVA) followed by Turkey multiple comparison test. Results are represented as mean ± S.D. and p < 0.05 is considered to be significant.
Results and discussion
Here we report the L. sativa a herbal drug for its analgesic, anti-inflammatory, anti-depressant and anti-coagulant activities of the seeds and leaf extracts along with the cell suspension exudate. We obtained very interesting and promising results which elucidate the importance of lettuce as a traditional medicine.
Hot plate analgesic assay
Latency period in hot plate analgesic assay
Treatment Groups (n = 5)
Latency period (sec) ± S.D.
Negative control (saline)
5.3 ± 0.51
5.6 ± 0.49
5.4 ± 0.55
5.1 ± 0.53
9.4 ± 0.61
14.9 ± 0.81**
17.8 ± 0.89**
15.4 ± 0.68**
Cell suspension exudate
8.9 ± 0.48
12.7 ± 0.61*
15.3 ± 0.58*
13.1 ± 0.49*
9.7 ± 0.56
13.9 ± 0.78*
17.5 ± 0.85*
15.8 ± 0.64*
6.8 ± 0.35
9.2 ± 0.28**
11.4 ± 0.62*
10.1 ± 0.71**
9.5 ± 0.55
14.6 ± 0.59**
17.6 ± 0.43*
15.7 ± 0.75*
7.2 ± 0.51
9.9 ± 0.29*
12.2 ± 0.48*
10.9 ± 0.62*
Carrageenan induced hind paw edema test
Edema volume in anti-inflammatory assay
Treatment Groups (n = 5)
Edema volume (ml) a ± S.D.
Negative control (saline)
0.49 ± 0.02
0.6 ± 0.03
0.68 ± 0.02
1.15 ± 0.02
0.38 ± 0.01**
0.25 ± 0.02**
0.17 ± 0.01**
0.06 ± 0.01**
Cell suspension exudate
0.49 ± 0.04*
0.47 ± 0.05*
0.35 ± 0.05*
0.34 ± 0.05*
0.48 ± 0.05*
0.49 ± 0.05*
0.38 ± 0.06**
0.33 ± 0.04**
0.48 ± 0.08*
0.47 ± 0.08**
0.42 ± 0.06*
0.40 ± 0.07*
0.48 ± 0.02*
0.42 ± 0.03*
0.28 ± 0.05*
0.26 ± 0.03*
0.47 ± 0.08*
0.44 ± 0.05*
0.42 ± 0.07*
0.39 ± 0.08*
Forced swimming test
The present experimental findings of different extracts suggest that L. sativa is a broad spectrum pharmaceutical crop conforming significant analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties that has potential to replace synthetic drugs. More interestingly, cell suspension exudate showed prominent results in all the assays which is the main point of interest because valuable secondary metabolites and economically important substances can be produced in bulk from plant cell suspensions in simple, cost-effective and reproducible way. However, advance study is needed to explore the precise mechanism of action the active components.
The authors are highly grateful (with permission) to Dr. Muhammad Tahir Waheed for critically analyzing the manuscript for scientific writing. HI acknowledges the Higher Education Commission (HEC), Islamabad, Pakistan for provision of the scholarship during the study.
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