Journal of Ethnopharmacology 129 (2010) 280–282 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Ethnopharmacological communication Erythropoietic activity of Asteracantha longifolia (Nees.) in rats Rajesh Singh Pawar a , Alok Pal Jain b , Santram Lodhi b , Abhay K. Singhai b,∗ a b VNS Institute of Pharmacy, Vidhya Vihar, Neelbud, Bhopal 462044, MP, India Department of Pharmaceutical Sciences, Dr. Harisingh Gour Central University, Sagar 470003, MP, India a r t i c l e i n f o Article history: Received 8 September 2006 Received in revised form 9 February 2010 Accepted 13 March 2010 Available online 27 March 2010 Keywords: Asteracantha longifolia Erythropoietic activity Iron deficiency anemia a b s t r a c t Ethnopharmacological relevance: Asteracantha longifolia Nees. (Family-Acanthaceae) is a wild herb commonly used in traditional ayurvedic medicine as Kokilaaksha and the Unani drug as Talimakhana in India and Srilanka for various medicinal uses as aphrodisiac, tonic, sedative and blood diseases etc. Aim of the study: The aim of the current study was to validate and explore the folk use of Asteracantha longifolia Nees. (AL) (Leaf part) on pharmacological grounds using haloperidol induced iron deficiency anemia for the assessment of erythropoietic activity. Materials and methods: Determination of iron in plant extracts was carried out using spectrophotometric method. Plant extract was obtained from crude drugs using extraction with ethanol. In vivo study, haloperidol induced iron deficiency anemia model was used in experimental studies. Results: An administration of ethanolic extract of AL at the doses of 100 mg/kg and 200 mg/kg body weight, i.p., demonstrated a significant (P < 0.05) increase in erythrocyte count, haemoglobin count, serum iron and serum protein etc. This effect may be due to the presence of iron (622 ␮g/50 mg) in extract estimated by spectrophotometric method. Conclusion: An ethanolic extract of AL effectively restored the hematological parameters, serum iron and serum protein and normalized the microcytic (smaller in size), anisocytosis (disturbed shape) and hypochromic RBCs. These observations could justify the inclusion of this plant in the management of iron deficiency anemia due the presence of iron and other constituents as flavonoids, terpenoids, steroids, lupeol and betulin. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Asteracantha longifolia (AL) Nees. (syn. Hygrophila spinosa T. Anders.; Hygropihila auriculata [K. Schum.] Heine) (known as Kokilaksa in Sanskrit and Talmakhana in Hindi, Family-Acanthaceae) is a common weed growing in marshy and water logged areas. The plant is an important medicinal herb, widely distributed in India and used by local population for different medicinal purposes. It is also used commercially as ingredient of some over the counter (OTC) formulations used in liver disorder and those prescribed as general tonic. The herb has been reported to contain chemical constituents such as ␤-sitosterol, lupeol (Saleem et al., 2005; Mazumdar and Sengupta, 1978; Tiwari et al., 1967), both have been reported to have antipyretic (Ali, 1967; Sunita and Abhishek, 2008) and hepatoprotective activity (Shailajan et al., 2005; Singh and Handa, 1995). The plant contains flavonoids, terpenoids, steroids and betulin. It is reported to possess hepatoprotective property and is used as a liver tonic (Shivashangari et al., 2004). Roots are used as a diuretic (Joshi, 2000; Warrier, 1995), seeds as aphrodisiac tonic in Unani medicine (Rastogi and Mehrotra, 1991) and leaves are popularly used for hepatoprotection against anaemia and in female reproductive dysfunction. Many medicinal plants classified as “Rasayana” in ayurveda are believed to be useful in strengthening the erythropoietic and immune system of an individual (Kirtikar and Basu, 1987). We have investigated the haematopoietic activity of the chloroform and petroleum ether extract of Asteracantha longifolia Nees. (Pawar et al., 2006a). As this plant has been reported to have haematopoietic activity, it was hypothesized that it should also be able to treat anaemia. The present investigation was designed to explore the haematological, serum iron and serum protein profile on the experimental rats exposed to the ethanolic extract of AL for erythropoietic activity. 2. Materials and methods ∗ Corresponding author at: Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003 (MP), India. Tel.: +91 7582264125. E-mail address: abhay singhai@rediffmail.com (A.K. Singhai). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.03.015 2.1. Extraction and preparation of dosage form The collection of the plant of Asteracantha longifolia (AL) was done in the month of December (2003) in villages of Sagar dis- R.S. Pawar et al. / Journal of Ethnopharmacology 129 (2010) 280–282 trict, Madhya Pradesh. A voucher specimen no. Bot/Herbarium/781 has been deposited in the Department of Botany, University of Sagar (MP). The powdered aerial parts (350 gm) were successively extracted in a soxhlet apparatus for 24 h with ethanol (95%) which was then concentrated and dried under reduced pressure. The yield was found to be 4.15% (w/w) as semisolid mass. Two grams of semisolid ethanolic extract was dispersed in 30 mL of propylene glycol (Hewawasam et al., 2003). 2.2. Identification and determination of iron content A qualitative test was performed for the identification of iron in plant extract. TLC profile was developed for detection of iron in plant extract and was compared with ferrous ammonium sulphate as standard (Kastelan-Macan et al., 1987). The iron content in the plant extract was determined by visible spectrophotometer (Omolo et al., 1997; Adams, 1995). Briefly, 50 mg of extract was dissolved in 5 mL of 6 M HCl, which was boiled on a hot plate until a clear solution was obtained. The solution was transferred to a 50 mL volumetric flask and pH was adjusted to 3.5 followed by addition of 1.4 mL of hydroquinone and 0.93 mL of o-phenanthroline solution; finally the volume was made up to 50 mL with distilled water and analyzed in a calibrated visible spectrophotometer (GBC CINTRA –10 UV). 2.3. Preliminary phytochemical screening Ethanolic extract of AL was subjected to various qualitative tests for the identification of various plant constituents present in this plant (Harborne, 1998). 2.4. Animals Animal study was performed at Pharmacology Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar with due permission from Institutional Animal Ethics Committee (Registration No. 397/01/ab/CPCSEA). Swiss albino rats 90–110 g and 35–45 g of male/female sex, maintained in standard conditions of temperature, relative humidity, light/day cycles and fed with normal diet and water ad libitum, were used. Haloperidol (Serenace, RPG, Life Sciences Ltd.) and all other chemicals were used of analytical grade. 2.5. Toxicological studies In toxicity study, no adverse effect or mortality was detected in albino rats up to a dose of 750 mg/kg, i.p. of the plant extract when administered separately during the 24 h observation period (Ghosh, 1984). 2.6. Haloperidol induced iron deficiency anaemia The chronic administration of typical antipsychotic drug (butyrophenone) may cause blood dyscrasias and iron deficiency anaemia. The iron deficiency anaemia was produced by haloperidol (0.2 mg/kg body weight) given intraperitoneally in rats within 4 days and it was accompanied by a lowering of the haemoglobin concentration, erythrocyte count as well as significant lowering of serum iron and serum protein. The rats were treated with haloperidol showed microcytic and hypochromic type blood cells. The test animals were divided into five groups of six rats in each group. Group I was kept as control (administered vehicle only), Group II was haloperidol control (0.2 mg/kg body weight i.p.), Group III was treated with ethanolic extract of AL alone (200 mg/kg body weight i.p.), Group IV was given haloperidol along with ethanolic extract of AL (0.2 mg/kg and 100 mg/kg body weight i.p.), and 281 Group V was given haloperidol along with ethanolic extract of AL (0.2 mg/kg and 200 mg/kg body weight i.p.). Haematological parameters were evaluated in the anaemic animal models. Iron deficiency anaemia was induced by haloperidol (0.2 mg/kg body weight) given intraperitoneally for 4 days. On day 4, blood samples were collected from the retro-orbital plexus vein of rat eye in vials containing EDTA as anticoagulant and evaluated for haematological parameters (erythrocyte count, leukocyte count, haemoglobin count and haematocrit value) using haematology cell counter (manufactured by ERMA, Japan) repeatedly (five times) to check the reproducibility of results (Pawar et al., 2006b) and serum iron, serum protein were estimated by Ferrozine Method and Direct Biuret Test Method (Siedel et al., 1984). In recovery period study, after 4 days, haloperidol was withdrawn from all the extract treated groups and groups were treated with their respective test extracts once a day, at the doses of 100 mg/kg and 200 mg/kg body weight i.p., continuously up to 15 days. Blood samples were collected on 19th day and evaluated for haematological parameters, serum iron and serum protein (Gomes et al., 2001). 2.7. Statistical analysis Data were expressed as mean ± standard error mean (S.E.M.). The statistical significance between groups was analyzed by one way ANOVA followed by Bonferroni post hoc for intergroup comparisons. The level for statistical difference was set at P < 0.05. 3. Results and discussion Present study was carried out mainly to determine erythropoietic activity of AL, an important plant in Indian traditional medicine, so as to evaluate it as an adjuvant during cancer chemotherapy. Hence in this study, haloperidol induced model was used to assess the effect of ethanolic extract on various haematological parameters, serum iron and serum protein etc. The percentage of extract yield was found to be 4.18% (w/w) and the iron content in ethanolic extract was found to be 622 ␮g/50 mg of plant extract. TLC profile was established for iron identification in plant extract and optimal solvent system was found to be 9:2:1 (v/v/v), ethanol:nitric acid:hydrochloric acid and the locating reagent was Na-diethyldithiocarbamate. The chronic administration of typical antipsychotic drug (butyrophenone) causes blood dyscrasias. Haloperidol act as antipsychotic drug and induce iron deficiency anaemia at a particular dose (Wasti et al., 2004). The iron deficiency anaemia was induced by haloperidol (0.2 mg/kg body wt.) given i.p. in rats for 4 days, assigned as haloperidol control (HP). Studies on HP control (Group II) showed significant (P < 0.001) decrease in haematological parameters as compared to that of control animals (Group I, administered vehicle only). It was accompanied by a lowering of the erythrocyte count, haemoglobin concentration as well as significant lowering of serum iron and serum protein (P < 0.001) as shown in Table 1. Haloperidol induced iron deficiency anaemia was reduced in a dose dependent fashion by the treatment of ethanolic extract as shown in Table 1. Comparison of Group III, IV and V with Group II exhibited significant (P < 0.05) increase in haematological parameters and serum protein except serum iron after 4 days as shown in Table 1. Recovery period observations (on 19th day) of HP control group indicated that HP induced anaemia was not restored to normal counts of haematological values, serum iron and serum protein level even after discontinuation of haloperidol treatment after 4 days when compared to control group I (P < 0.001) recorded in Table 1. 282 R.S. Pawar et al. / Journal of Ethnopharmacology 129 (2010) 280–282 Table 1 Effect of ethanolic extract (EE) of Asteracantha longifolia (AL) extract in haloperidol treated albino rats. Treatment/parameters Mean RBCs (106 /␮L) Mean Hb (g/dL) Mean WBCs (103 /␮L) Mean HCT (%) Serum iron (␮g/dL) Serum protein (g/dL) Day-4 I—Control (vehicle) II—HP control (0.2 mg/kg) III—EE (200 mg/kg) IV—HP + EE (100 mg/kg) V—HP + EE (200 mg/kg) 8.85 4.27 6.7 5.1 5.9 ± ± ± ± ± 0.750 0.445* 0.190* 0.156* 0.392* 14.5 8.1 13.5 9.4 10.4 ± ± ± ± ± 0.230 0.558*** 0.838* 0.947* 1.42* 9.4 3.7 7.6 4.9 5.5 ± ± ± ± ± 0.254 0.249** 0.675* 1.64* 0.985* 46.5 33.48 44.85 35.50 37.78 ± ± ± ± ± 1.28 2.25*** 2.38* 2.96* 3.76* 60 14 49 35 43 ± ± ± ± ± 6.521 1.958*** 8.352*** 8.698*** 9.124*** 6.5 2.8 5.1 3.9 4.3 ± ± ± ± ± 0.158 0.201 0.196* 0.258* 0.136* Day-19 I—Control (Vehicle) II—HP control (0.2 mg/kg) III—EE (200 mg/kg) IV—HP + EE (100 mg/kg) V—HP + EE (200 mg/kg) 8.90 5.12 7.5 6.2 7.0 ± ± ± ± ± 0.620 0.185 0.185* 0.226* 0.295* 14.9 9.2 14.2 10.4 13.9 ± ± ± ± ± 0.282 0.127 0.948** 0.842*** 1.32*** 9.9 4.9 8.0 5.9 7.5 ± ± ± ± ± 0.214 0.152 0.575*** 1.34*** 0.935*** 47.9 35.85 46.85 39.94 45.84 ± ± ± ± ± 1.11 1.87*** 2.88 2.29 4.16 61 22 50 43 52 ± ± ± ± ± 5.410 2.458*** 5.178*** 4.857*** 6.258*** 6.8 3.2 5.5 4.4 5.3 ± ± ± ± ± 0.200 0.285 0.326*** 0.247*** 0.259*** n = 6 albino rats per group; values are represents mean ± SEM. * P < 0.05; (comparison of I with II and III, IV, V with II) on days 4 and 19. ** P < 0.01; (comparison of I with II and III, IV, V with II) on days 4 and 19. *** P < 0.001; (comparison of I with II and III, IV, V with II) on days 4 and 19. In continuation to this study, an administration of ethanolic extract of AL at the dose of 100 mg/kg and 200 mg/kg body weight was given intraperitoneally for next 15 days after discontinuation of haloperidol treatment in all the groups. Comparison of Group II with Group III, IV and V demonstrated significant (P < 0.001) increase in haematological parameters as shown in Table 1. The synthetic drugs used in the treatment of anaemia have various side effects such as gastrointestinal disturbances, liver damages, poor absorption etc. Use of plants as the source of immunomodulatory material mainly to protect erythropoietic system is still in its infancy (Mazumdar et al., 1996). The presence of iron is usually indicated in extracts with polar solvents viz. ethanol and water whereas it is generally absent in extracts with non-polar solvents such as petroleum ether, benzene, chloroform etc. The plant extracts (AL) obtained by extraction with polar solvents as well as their ash showed the presence of iron in crude drugs. The results showed that ethanolic extract possesses good erythropoietic activity. This was demonstrated by a significant increase in erythrocytes count, haemoglobin content, leukocytes count and haematocrit values etc. as well as increase (P < 0.05) in serum iron and serum protein level was also observed. This study highlights the importance of iron content and other constituents (Mishra et al., 2001) of Asteracantha longifolia that could be a potential new source for erythropoietic activity as well as a scientific proof of its ethnopharmacological use in blood disorders. 4. 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