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| Year : 2008 | Volume
: 2
| Issue : 1 | Page : 43-45 |
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| Anti-oxidant activity of ethyl acetate extract of Aquilaria agallocha on nitrite-induced methemoglobin formation |
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PB Miniyar1, TS Chitre2, SS Karve2, HJ Deuskar2, KS Jain2
1 Sinhgad Institute of Pharmacy, Narhe, Pune - 411 041, Maharashtra, India
2 Sinhgad College of Pharmacy, Vadgaon BK, Pune - 411 041, Maharashtra, India
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| Date of Submission | 15-Aug-2007 |
| Date of Acceptance | 20-Sep-2007 |
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 Abstract | | |
Aquilaria agallocha Roxb, family Thymelaeaceae, is an evergreen plant of India, China and Tibet, commonly described as aloe wood or agar wood. Traditionally, bark, root and heartwood are used for their medicinal properties as a folk medicine to treat inflammation, arthritis, vomiting, cardiac disorders, cough, asthma, leprosy, anorexia, headache and gout. The present study was carried out to investigate the antioxidant activity of ethyl acetate extract of Aquilaria agallocha (EAA). EAA was tested in vitro at different concentrations for inhibitory effect on nitrite-induced oxidation of haemoglobin in human blood hemolysate. Results indicate a strong antioxidant effect of EAA in a concentration range of 500-3500 µg/ml. However, pro-oxidant activity was observed at higher concentrations of these compounds. Keywords: Aquilaria agallocha, antioxidant, nitrite-radical, methemoglobin
How to cite this article:
Miniyar P B, Chitre T S, Karve S S, Deuskar H J, Jain K S. Anti-oxidant activity of ethyl acetate extract of Aquilaria agallocha on nitrite-induced methemoglobin formation. Int J Green Pharm 2008;2:43-5 |
How to cite this URL:
Miniyar P B, Chitre T S, Karve S S, Deuskar H J, Jain K S. Anti-oxidant activity of ethyl acetate extract of Aquilaria agallocha on nitrite-induced methemoglobin formation. Int J Green Pharm [serial online] 2008 [cited 2013 May 25];2:43-5. Available from: http://www.greenpharmacy.info/text.asp?2008/2/1/43/39164 |
| Introduction | |  |
Aquilaria agallocha (Thymelaceceae) Roxb is a native evergreen plant of India, China and Tibet. It is commonly described as aloe wood or agar wood. Traditionally, bark, root and heartwood are used for their medicinal properties. It is reported to contain sesquiterpenes (Bhandari, 1982; Ishihara, 1991). Agar wood is highly charged with resinous matter and contains 48% of alcohol-soluble matter. After saponification of the alcoholic extract of Aquilaria agallocha , benzyl acetone, an unidentified ketone of molecular formula C 14 H 20 O 2 , a sesquiterpene alcohol and some acids (including hydrocinnamic acid) are obtained. The sesquiterpene alcohol possesses the characteristic odour of the wood (Manjunath, 1988). It is reported for the treatment of anaphylactic reactions and also possesses anti-histaminic properties (Kim, 1997). The plant is traditionally used to treat inflammation, arthritis, vomiting, cardiac disorders, cough, asthma, leprosy and anorexia (Iyer, 1994). Since the plant has been used in folk medicines in the treatment of headache, inflammation, gout and arthritis (Kirtikar, 1999; Nadkarni, 1976), an investigation was undertaken to explore the antioxidant activity of ethyl acetate extract of the heartwood of Aquilaria agallocha (EAA) .
Haemoglobin undergoes oxidation, when haemoglobin binds to molecular oxygen. There is an accompanied risk of superoxide formation along with the oxidation of haemoglobin to methemoglobin (Miles, 1994). There are several inherent antioxidant defence mechanisms, which prevent methemoglobin formation. Superoxide dismutase, catalase, ascorbic acid and glutathione peroxidase constitute a few of these endogenous antioxidants (Doyle, 1982). In spite of this, oxidation of haemoglobin to methemoglobin occurs in response to a variety of chemical stimuli, which include drugs like primaquine and dapsone and environmental pollutants like nitrogen dioxide. The endogenous antioxidant defence system present in our body maintains the level of methemoglobin within one percent. Many antioxidants such as ascorbic acid, uric acid, 3-ribosyluric acid and glutathione have been found to protect haemoglobin in two stages, viz. a slow stage followed by a rapid autocatalytic stage involving superoxide anion, hydrogen peroxide and nitrogen dioxide. Curcumin, an established free-radical scavenger, protects haemoglobin against nitrite-induced oxidation (Unnikrishnan, 1995).
The chief objective of this work was to identify new uses for Aquilaria agallocha , a drug currently in use. An investigation was undertaken to study the effect of EAA at different concentrations, on nitrite-induced oxidation of haemoglobin in blood hemolysate to methemoglobin.
| Materials and Methods | | |
Plant Material
Fresh heartwood of Aquilaria agallocha , obtained from a commercial source in Pune, was identified and authenticated by Dr. H.B. Singh, Head, Raw Materials, Herbarium and Museum Division, New Delhi, India. The specimen voucher is preserved in the Department of Pharmacognosy, Sinhgad College of Pharmacy, Pune.
Preparation of Extract
The heartwood of Aquilaria agallocha was dried under shade and powdered by a mechanical grinder. The powdered heartwood (100 g) was extracted with ethyl acetate (60-80°C) using Soxhlet apparatus for 72 h. The extract was concentrated under vacuum and dried at room temperature. The extract (5.8 g) thus obtained was resinous in nature and of brownish black colour. Various qualitative tests were performed for EAA to confirm the chemical constituents, viz. triterpenoids, tannins, phenolic compounds and glycosides.
Preparation of Hemolysate
Blood samples were centrifuged (2500 rpm for 20 min) to remove the plasma and buff coat of white cells. Erythrocytes thus obtained were washed three times with phosphate-buffered saline and were hemolysed by suspending them in 20 volumes of 0.5% w/v sodium chloride solution for 30 min. The hemolysate thus obtained was then centrifuged at 2500 rpm for 1 h to remove membrane and cell debris. The resulting solution was diluted with 0.9% w/v sodium chloride solution to yield a final concentration of oxyhaemoglobin (0.5 at 577 nm), suitable for spectrophotometric analysis.
Procedure of Experiment
Hemolysate prepared, as per the procedure mentioned above, was incubated with different concentrations of test compounds (viz. 500, 1000, 1500, 2000, 2500, 3000 and 3500 µg/ml) for different time intervals (0-10 min). Curcumin was used at a concentration of 20 µm. Compounds were added to the reaction mixture by dissolving them in ethanol. Controls were prepared with an equivalent amount of ethanol. Haemoglobin exhibits maximum absorption at 577 and 560 nm, while methemoglobin absorbs at 631 nm. Formation of methemoglobin (induced by sodium nitrite at a final concentration of 300 mm) was estimated by monitoring the absorbance at 631 nm using Jasco UV 430 double-beam spectrophotometer.
Preliminary investigations indicate that EAA inhibits nitrite-induced oxidation of hemoglobin. [Figure - 1] shows a dose dependant decrease in absorbance at 631 nm in the hemolysate treated with EAA. The EAA gave a significant inhibition at concentration 500 µg/ml. The results are consistent at different time intervals tested, viz. 0-10 min. However, curcumin showed a more significant activity even at lower concentration levels than EAA. (The results are not included because the concentration used was not comparative. Curcumin concentration was kept low in the range of 20 µm because of solubility problems.)
| Results | | |
EAA in different concentrations was tested against the standard drug curcumin. Results indicate the inhibitory role of EAA in a concentration range from 500 to 3000 µg/ml. The pro-oxidant activity of EAA was higher at higher concentration.
| Discussion | | |
Aquilaria agallocha is used to treat inflammation in folk medicine. The purpose of the study was to establish scientific evidences for the usage of this plant as an antioxidant. The EAA was studied for its inhibitory effects on methemoglobin, the oxidation product of haemoglobin, produced by the treatment with sodium nitrite. The antioxidant effect of EAA was tested at different concentrations. It was observed that EAA showed antioxidant activity at a lower concentration range. However, the action was reversed at higher concentrations.[11]
| References | | |
| 1. | Bhandari P, Pant P, Rastogi RP, Aquillochin: A coumarinolignan from Aquilaria agallocha, Phytochemistry, 21, 1982, 2147-2149.  |
| 2. | Doyle MP, Pickering RA, Dykstra RL, Nelson CL, Boyer RF, Biochem. Biophysics. Res. Commun, 105, 1982, 127. |
| 3. | Ishihara M, Tsuneya T, Shiga M, Uneyama K, Sequiterpenes from agar wood, Phytochemistry, 30, 1991, 563-566. |
| 4. | Iyer SR, Indian Medicinal Plants - a compendium of 500 species. Orient Longman, Hyderabad, India, 1994. |
| 5. | Kim YC, Lee EH, Lee YM, Kim HK, Song BK, Lee EJ, Kim HM, Effect of aqueous extract of Aquilaria agallocha stems on the immediate hypersensitive reactions, J. Ethanpharmcol, 58, 1997, 31-38. |
| 6. | Kirtikar KR, Basu BD, Plate volume IV. In: Indian Medicinal Plants. International Book Distributors, Dehradun, India, 1999, 836. |
| 7. | Manjunath BL, The Wealth of India - A dictionary of Indian raw materials and Industrial Products, CSIR, New Delhi, India, 1988. |
| 8. | Miles AM, Grisham MB, Methods in Enzymology, 234, 1994, 555. |
| 9. | Nadkarni KM, Nadkarni KA, Indian Materia Medica, Popular Prakashan Private Limited, Mumbai, India, 1976. |
| 10. | Unnikrishnan MK, Rao MNA, Inhibition of nitrite induced oxidation of hemoglobin by curcuminoids, Pharmazie, 50, 1995, 490. |
| 11. | Unnikrishnan MK, Rao MNA, Curcumin inhibits nitrogen dioxide induced oxidation of hemoglobin, Mol. Cell. Biochem, 146, 1995, 35. |
Correspondence Address:
P B Miniyar
Sinhgad Institute of Pharmacy, Narhe, Pune - 411 041, Maharashtra
India
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DOI: 10.4103/0973-8258.39164
[Figure - 1] |
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