|Iranian Journal of Pharmaceutical Research
(2009), 8 (3): 223-226
Received: July 2008
Accepted: November 2008
Copyright ? 2009 by School of Pharmacy
Anti-nociceptive Activity of Aqueous-methanolic
Extract of Phytolacca americana Growing in Iran
Mohammad Karamia*, Soodabeh Saeidniab, Naghi Shahabi majdc,
Mohammad Ali Ebrahimzadeha, Neda Omrania and Asieh Salariand
aPharmaceutical Sciences Research Center and School of Pharmacy, Medical Sciences University of Mazandaran, Sari, Iran. bMedical Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran. cPhysiology and Pharmacology Department, School of Medicine, Medical Sciences University of Mazandaran, Sari, Iran. dResalat Health Center, Medical Sciences University of Mazandaran, Sari, Iran.
Abstract Anti-nociceptive activity of aerial parts of Phytolacca americana were investigated, using the hot plate method in mice. Results of the present study showed that the aqueous methanolic extract of aerial parts of P. americana produced a statistically significant increase in pain threshold after 30 min, in comparison with the control, at adose of 190 mg kg-1 (P<0.001). The activity was comparable to that of morphine (30 mg kg-1 i.p., p> 0.05). The anti-nociceptive activity of P. americana increased until the 60th min (P<0.05 compared to morphine). The results of this study support the extensive use of P. americana in Western Asia and America. The LD50 of extract following a 14 days acute toxicity study was calculated to be 208 mg kg-1 i.p.
Pain is still one of the main health problems of the world?s populations (1). Many bioactive substances are involved in the modulation of pain sensation (2). Eclectic physicians relied upon herbal medicines and natural remedies to treat disease (3). The pain relief composition is prepared from roots of the Burdock plants family and in particular from at least one of the species, such as Arctium lappa, or as Arctium minus in combination with roots of the Phytolaccaceae family and in particular the species Phytolacca americana (4). Pokeweed, P. americana, is a perennial plant native to North America. The boiled leaves are used in a popular salad (called grandmother?s salad) in the American diet (5-7). P. americana grows widely in northwestern parts of Iran, mainly in the coastal areas and forest lands (8, 9). It is well known for several medicinal properties, despite its toxicity, especially hepatotoxicity (10, 11).
P. americana has been most commonly used as laxative. It has been shown to possess pain relieving, anti-inflammatory, anti-rheumatism and anti-arthritic activities. Also, it is suitable for the treatment of various skin diseases (12, 13). Nowadays, pokeweed is still used cautiously by some herbalists to treat the above-mentioned conditions. There are a few reports on its anti-viral, anti-cancer, anti-fungal and immunostimulant activities (14, 15). One hundred and fifty species of Phytolacca dried flowers are available in Eastern North America, northwestern areas of Iran and other parts of the world, they have been used to relive pain and for the reduction of fever (16-21). They are used alone or as a mixture.
The purpose of this study was the evaluation of anti-nociceptive activity of an aqueous methanolic extract of the aerial parts (flowered browse) of P. americana growing in Iran, using the hot plate method, as well as the determination of its median lethal dose (LD50).
Morphine was purchased from the American Peptide Company (Sunnyvale, CA, USA).The experimental protocol used in this study was carried out in accordance with the ethics committee of the Mazandaran University of Medical Sciences.
Male Swiss albino mice weighing between 25-30 g were used for these studies. They were housed in groups of five, under standard light (7.00 to 19.00) and temperature (22 ? 1?C), with food and water ad libitum. The animals were transferred to the laboratory at least 1h before the start of the experiment. Experiments were performed during the day (08:00-16:00 h). Each animal was used only once.
Preparation of the plant extract
Aerial parts (flowered browse) of Phytolacca americana were collected from Mazandaran (a northern state in Iran) in April 2006, identified and confirmed by Dr. Saeidnia at the Department of Pharmacognosy, School of pharmacy, mazandaran university of medical sciences. Aerial parts were dried at room temperature and coarsely ground before extraction. One hundred grams of the powdered sample was extracted at room temperature by percolation with methanol/ water (80:20, 400 mL?3 times). The resulting extract was concentrated over a rotary vacuum evaporator, until a solid extract sample was obtained. The resulting crude extract was freeze-dried. The extract was prepared in phosphate buffer (pH 7.4) and tween 80 (4:1) for pharmacological studies.
Hot plate method
Morphine was injected intraperitoneally (i.p.) to mice, as a single dose of 30 mg kg-1 (as a positive control). Solvent was injected to the negative control group (10 mL kg-1, i.p.). An aqueous methanolic extract of the aerial parts of P. americana was given at a dose of 190 mg kg-1 i.p. to the animals, as a single dose. Anti-nociceptive activity was assessed by measuring the hot plate latency to heat, as described by Leimbach and Eddy (22). A minimum of three trials was recorded for each animal and toxicity studies carried out in mice, according to the method stated by Reddy and Byahatti (23). Mice were placed in a thermostatically controlled hot plate apparatus (Harvard, UK), maintained at 52 ? 0.5?C and the reaction time (time elapsed between placing the mouse on the hot plate and appearance of signs of acute discomfort) for licking or kicking of the fore-or hind paws was recorded using a stop watch. The controlled reaction time in was recorded before the start of experiment. Mice, which did not show any reaction after 15 sec, were discarded. Reaction time (in sec) before and at 0, 30 and 60th min after administration of the drugs was recorded. A cut-off time of 45 sec was imposed to avoid tissue damage.
The median lethal dose (LD50)
Extract was dissolved in phosphate buffer (pH 7.4) and Tween 80 (4:1) (2) and was given as a single dose to mice intraperitoneally. Acute toxicity assays were conducted based on our recently published method (24). Briefly, doses in the tested dose-interval were progressively increased such that each dose was 50% higher than the previous one (0, 12.5, 25, 50, 100, 200, 400, 800 mg kg-1), until the dose lethal to half of the test population had been attained. The animals were observed during a 14 days study period and deaths were recorded.
Analyses of the data
Statistical analysis was performed using the SPSS software for Windows (Ver.10, SPSS Inc., Chicago, USA). Data were analyzed by one-way analysis of variance (ANOVA) and presented as mean?sem. Student-Newman-Keuls test was used for statistical analysis and p<0.05 was considered to be significant.
Results and Discussion
Results of the present study showed that the aqueous methanolic extract of the aerial parts (flowered browse at 190 mg kg-1) of P. americana produced a statistically significant increase in the pain threshold, after 30 min, in comparison with the control (Figure 1). The effect or activity was rather low, however enough for treatment and blocking the pain. This activity was comparable to that of morphine (30 mg kg-1 i.p., p> 0.05). The anti-nociceptive activity of extract increased until the 60th min. The P-value was greater than 0.05, compared to morphine (Figure 2). Poke root is an herbal medicine used to treat inflammation (swelling) of the mouth, throat, nose, and breast. It is also used to treat skin infections and stop pain (25). The pain relief composition is prepared from roots of the Phytolacca family and in particular the species Phytolacca americana (4). On other hand, the anti-cancer effects appear to work primarily based upon anti-tumor and anti-inflammatory properties, along with immuno-stimulant functions (14, 15). Furthermore, it contains aromatase inhibitors and has antioxidant properties (25). A number of anti-inflammatory components have also been reported in P. americana (15, 16, 21). Among the constituents, oleanolic acid appears to be the most significant, with it?s anti-inflammatory and prostaglandin synthesis inhibitory properties (25). The results of this study support the extensive use of this plant in western Asia and America (18-20). It is possible that the same components could lead to anti-nociceptive activity in our extract. This needs to be justified in future studies. Based on our results, P. americana could be candidated as an analgesic agent. Although, the mechanism of plant action to increase anti-nociceptive activity in mice is unclear. On the other hand, phytolaccatoxin and the related triterpene saponins, believed to be the primary toxic constituents, are present in the berry juice and other plant parts (26-28). Other toxic constituents have also been identified, including the alkaloids phytolaccine and phytolaccotoxin, as well as a glycoprotein and histamines. When pokeweed is used as food, the water in which it is boiled, must be discarded (26-28). The lethal dose 50% (LD50) is most frequently used to characterize the response of animals, such as rats and mice, as a general indicator of an agent acute toxicity test (29). Based on our data, the LD50 values, after the 14 days acute toxicity study was calculated to be 208 mg kg-1 i.p.
This work was supported by a grant from the research council of the Medical Sciences University of Mazandaran, Iran.
(1) Basbaum AI and Field HL. Endogenous pain control
systems: brainstem spinal pathways and endorphin circuitry. Ann. Rev. Neurosci.
(1984) 7: 309-338.
(2) Ebrahimzadeh MA, Mahmoudi M and Salimi E. Antiinflammatory activity of Sambucus ebulus hexane extracts. Fitoterapia (2006) 77: 146- 48
(3) Winston D. The use of botanicals in eclectic pediatrics. J. Am. Herbalists Guide (2004) 3: 59-64.
(4) Wren, R.C. Potter?s Encyclopedia of Botanical Drugs & Preparations. Potter & Clarke, Ltd., London (1900) 381-391.
(5) Santillo H. Natural Healing with Herbs. Hohm Press, Arizona (1993) 100.
(6) Duke JA. Handbook of Medicinal Herbs. CRC Press, Florida (1991) 581.
(7) Murray MT and Pizzorno JE. Procyanidolic oligomers. In: Pizzorno JE and Murray MT. (eds.) The Textbook of Natural Medicine. Vol. 1, 2nd ed. Churchill Livingston, London (1999) 899-902.
(8) Zargari A. Medicinal Plants. 5th ed. Tehran University Press, Tehran (1981) 208.
(9) Mirhaydar H. Plant Information: Plant Usage in Disease Treatment. Vol.2 2nd ed. Farhang Islami Press, Tehran (1994) 244.
(10) Barker BE, Farnes P and Fanger H. Mitogenic activity in Phytolacca americana (pokeweed). Lancet (1965) 1: 170.
(11) Stein ZL. Pokeweed-induced gastroenteritis: B.D. Toxicity of pokeberries (fruit of Phytolacca americana Large) for Turkey poults. Am. J. Hosp. Barnett. (1975) 54: 1215-17.
(12) Goldestein SW, Jenkins GL and Thompson MR. A chemical and pharmacological study of Phytolacca americana. J. Am. Pharm. Assoc. (1973) 26: 306-12
(13) Macht DI. A pharmacological study of Phytolacca. J. Am. Pharm. Assoc. Sci. (1937) 26: 594-599.
(14) Heinrich M, Branes J, Gibbons S. Williamson EM. Fundamentals of Pharmacognosy and Phytotherapy. Churchill Livingstone, Edinburgh (2004) 166.
(15) Johnson A and Shimizu Y. Phytolacinic acid: a new triterpene from Phytolacca americana. Tetrahedron (1974) 30: 2033-2036.
(16) Kang SS and Woo WS. Triterpenes from the berries of Phytolacca americana. J. Nat. Prod. (1980) 43: 510-3.
(17) Kang SK and Woo WS. Two new saponins from Phytolacca americana. Planta Med. (1987) 53: 338-4.
(18) Huseini HF, Alavian SM, Heshmat R, Heydari MR, Abolmaali K. The efficacy of liv-52 on liver cirrhotic patients: A randomized, double-blind, placed-controlled first approach. Phytomedicine (2005) 12: 619-24.
(19) Goldestein SW, Jenkins GL and Thompson MR. A chemical and pharmacological study Phytolacca americana. J. Am. Pharm. Assoc. (1973) 26: 306-12.
(20) Woo WS and Kang SS. Phytolaccoside B: triterpene glycoside from Phytolacca americana. Photochem. (1976) 15: 1315-17.
(21) Woo WS and Shin KH. Antiinflammatory action of Phytolacca saponin. J. Pharm. Soc. Korea (1976) 20: 149-55.
(22) Eddy NB and Leimback D. Diethyl buteryl and diethienyl butyl amines. J. Pharmacol. Exper. Ther. (1953) 107: 385-93.
(23) Reddy BM, Byahatti AVN and Ramesh M. Anti-inflammatory activity of Stapelia nobilis and Caralluma stalagmifera. Fitoterapia (1996) 6: 545-47.
(24) Ebrahimzadeh MA, Mahmoudi M and Karami M. Separation of active and toxic portions in Sambucus ebulus. Pakistan J. Biol. Sci. (2007) 10: 4171-73.
(25) Newall C, Anderson LA and Phillipson JD. Herbal Medicines: A Guide for Health-Care Professionals. Pharmaceutical Press, London (1996) 176.
(26) Jeong SI, Kim KJ, Choo YK Keum KS, Choi BK, Jung KY. Phytolacca americana inhibits the high glucose-induced mesangial proliferation via suppressing extracellular matrix accumulation and TGF-beta production. Phytomedicine (2004) 11: 175-81.
(27) Larson K. God?s Free Harvest. Rhema Publishing, Suwanee (1995) 231.
(28) Armstrong W. Pokeweed: An interesting American vegetable. http://waynesword.palomar.edu/ ecoph24.htm. Accessed July 28, (2009) 1.
(29) Klaassen CD. (ed.) Casarett and Doull?s Toxicology, the Basic Science of Poisons. 6th ed. McGraw-Hill, New York (2001) 772.