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Evaluation of anti inflammatory activity of Indigofera aspalathoides Vahl in Swiss albino mice
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Evaluation of anti inflammatory activity of Indigofera aspalathoides Vahl in Swiss albino mice N. Boopala Bhagavan a, S. Arunachalam b, P. Dhasarathan b, N.D. Kannan a,* a b
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India Department of Biotechnology, Sri Kaliswari College, Sivakasi 626130, India
article info
abstract
Article history:
Background: The objective of this study was to evaluate the anti inflammatory activity of
Received 19 December 2012
Indigofera aspalathoides in Swiss albino mice.
Accepted 16 February 2013
Methods: The anti inflammatory activity was evaluated by carrageenan induced paw edema
Available online 30 March 2013
model. Biochemical parameters such as Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT) and ESR counting were studied.
Keywords:
High Performance Liquid Chromatography (HPLC) analysis was performed to identify the
Anti inflammatory
bioactive compounds present in I .aspalathoides.
Indigofera aspalathoides
Results: The acute toxicity (LD50 value) of the Extract I. aspalathoides (EIA) was standardized
Carrageenan
as 250 mg/kg in orally treated mice. EIA found to have significant anti inflammatory activity
HPLC
in carrageenan induced paw edema compared to standard Indomethacin. It was evidenced
Indomethacin
by remarkable reduction in serum lysosomal enzymes and ESR as compared with control. HPLC chromatogram revealed the presence of five different bio active compounds in EIA. Conclusion: The result strongly suggested that I. aspalathoides has potent anti inflammatory property. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Inflammation is a severe response by living tissue to any kind of injury. There can be four primary indicators of inflammation: pain, redness, heat or warmness and swelling.1 Recent studies indicate that the mediators and cellular effectors of inflammation are important constituents of the local environment of tumors.2 Medicinal plants in particular, are believed to be an important source of new chemical substances with potential therapeutic efficacy.3 Inflammation plays an important role in various diseases with high prevalence within populations such as rheumatoid arthritis,
atherosclerosis and asthma. In recent years, plant materials continue to play a major role as therapeutic remedies in many developing countries.4 Plants represent still a large source of structurally novel compounds that might serve as lead for the development of novel drugs.5 Indigofera aspalathoides Vahl (Family: Leguminaceae) is a low under shrub commonly distributed in South India. It is commonly known as Sivanar Vembu in Southern Western Ghats of Tamil Nadu. In Indian system of herbal medicine, I. aspalathoides is specifically used for treating for Psoriasis, secondary syphilis, and viral hepatitis hepato-protective activity, kidney disorders.6 It was reported that stem extracts of
* Corresponding author. Tel.: þ91 452 2458273; fax: þ91 452 2459103. E-mail address: [email protected] (N.D. Kannan). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.02.018
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
I. aspalathoides has significant anti tumor, anti inflammatory, anti viral and antimicrobial activity.7 Global demand for herbal medicine is increasing at a rapid rate owing to their low cost and no side effects. However, proper phytochemical and pharmacological investigation should be carried out prior to the commercialization of plant based drug as many health hazards were reported with inadvertent use of plant drugs.8 Amala et al9 conducted a preliminary study to confirm the anti inflammatory activity of I. aspalathoides tender shoots. However, no systematic approach has been done so far to analyze phytochemical constituent that contribute anti inflammatory activity of I. aspalathoides. In the present study, the systemic study combining phytochemical and pharmacological aspects was carried out to evaluate the anti inflammatory of I. aspalathoides using Swiss albino mice.
2.
Materials and methods
2.1.
Plant collection and extraction
Plant sample was collected from Gopalasamy Hills in Viruthunagar district, Tamil Nadu, India. This plant was authenticated and voucher specimens were deposited in the Department of Biotechnology, Sri Kaliswari College, Sivakasi. The stems were shade-dried and pulverized. The powder was treated with petroleum ether to remove wax and chlorophyll and extracted with methanol. The extracts were concentrated by distilling the solvent in a rotary flash evaporator. Methanol was evaporated and dried extracts were dissolved in water.
3.
Evaluation of anti inflammatory activity
3.1.
Experimental animals
Swiss albino mice, (20e32 g) aged 8e12 weeks were used for anti inflammatory studies. They were kept hygienically in polypropylene cages in a controlled environment (Temperature 25 2 C, relative humidity 65 10%, and 12 h dark/light cycle) with standard laboratory diet and water ad libitum. This study was conducted after obtaining institutional animal ethical committee clearance (Number: 1086/AC/07/CPESEA).
3.2.
Acute toxicity studies
The acute toxicity (LD50) of the EIA was determined in mice by Lorke method.10 The study was carried out as per the guidelines of OECD (Number: 1086/AC/07/CPESEA).
3.3. Determination of inhibition of carrageenan induced mice paw edema The anti inflammatory activity was assessed using Winter et al method.11 The selected Swiss albino mice were divided into five groups of six animals (n ¼ 6) each and housed under laboratory condition. Group 1: controlecarrageenan (0.1 mL of 1%) only Group 2: carrageenan þ MEIA (250 mg/kg p.o)
351
Group 3: carrageenan þ aqueous EIA (250 mg/kg p.o) Group 4: carrageenan þ Indomethacin (10 mg/kg p.o) The percentage of inhibition of paw edema was calculated using following formula, Percentage of inhibition of paw edema volume ð%Þ ¼ 1 ðVt =Vc Þ 100 Vt ¼ Paw edema volume of drug treated group Vc ¼ Paw edema volume of control group
3.4.
Bio chemical assay
Biochemical changes in carrageenan induced paw edema were estimated in an interval of 6 h. The blood was collected from anaesthetized mice by cardiac puncture. The serum was separated from blood sample. The separated serum was analyzed for lysosomal enzymes such as SGOT and SGOT described by Woessner method.12 The activity of SGOT and SGPT were expressed in U/mL.
3.5.
Estimation of erythrocyte sedimentation rate (ESR)
The 0.8 mL of blood was collected from each group of mice by using sterile syringe via cardiac puncture and put into the tube which contained EDTA and mixed with the 0.2 mL of 3.8% of sodium citrate solution in a test tube. The Westergren tube is filled up to ‘0’ mark with citrated blood and plasma vertically in the stand. The sedimentation of RBC in mm in 1 h is observed and compared with other groups.
3.6.
High Performance Liquid Chromatography analysis
300 mL of water were added to the stem powder of I. aspalathoides (15 g) and heating was carried out a micro oven. After boiling, gentle heating was continued until the volume reduced to less than 30 mL. The mixture was filtered and frozen at 30 C for further use. The final concentration of the EIA was equivalent to 0.5 g/mL. The plant sample was analyzed by HPLC apparatus, equipped with a pump LC-10AT (Shimadzu, Corporation, Kyota, Japan), a Photodiode Array (PDA) detector SPD- M10 AVP (Shimadzu, Japan). The stationary phase of the column was a Diamonsil C18 (4.6 250 mm, 5-mm particle size). The plant sample (25 mL) was injected in column in an isocratic mobile phase comprising of Acetonitrile: 0.1% acetic acid (80: 20) at flow rate of 1 mL/min. The elution time was 15 min and detection was carried out at 240 nm. Column (C 18) was maintained at 25 C. The data acquisition was performed by ChemStation version A 08.03.
3.7.
Statistical analysis
The experimental results were expressed as the mean standard deviation. The level of significance was tested using one way analysis of variance (ANOVA) and Dunnett’s test at p < 0.05.
352
4.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
Result and discussion
Sub lethal concentration of the EIA was found to be 250 mg/kg b.w. and no mortality was detected upto this concentration during 24 h observation period. It was reported that during inflammation, over expression of the inducible forms of cyclooxygenase (COX) and the lipoxygenase (LO) enzymes cause the generation of the lipid mediators and damaging free radicals.13 Variations of paw edema volume in response to various treatments imposed to carrageenan induced paw volume were shown in Table 1. As expected, purified standard drug (Indomethacin) showed maximum reduction of paw edema volume. Nonetheless, both methanolic and aqueous extract of I. aspalathoides reduced paw edema volume significantly ( p < 0.05). Methanolic and aqueous extract of I. aspalathoides recorded 37.5% and 31.6% of inhibition of paw edema respectively. Then it was revealed that both extracts of I. aspalathoides has effective anti inflammatory activity. Moderately higher rate of inhibition by methanolic extract may be attributed to the high solubility of phytochemicals in methanol rather than water.14 Carrageenan induced paw edema test is a significant tool for the assessment of anti inflammatory profile of natural products.15 Carrageenan induced paw edema was observed to be progressively increased after the initial phase (0e1.5 h). In the second phase (1.5e5 h), various factors that are responsible for inflammation such as vasoactive amines (histamine, serotonin), arachidonic acids (prostaglandins, leukotrienes) and cytokines (tumor necrosis factor and interleukin-1) were produced due to the action of carrageenan.16,17 Since aqueous and methanolic extract of I. aspalathoides has significantly reduced the paw edema volume, it could be believed that EIA suppress the production of above mentioned factors. Lysosomal enzymes were reported to play crucial role during the development of inflammation.18 During the treatment with carrageenan, the level of SGOT and SGPT were elevated significantly. Interestingly the increased levels of these enzymes were reduced to the normal range after 6 h, since the administration of EIA (Fig. 1). Pharmacological action of most of the anti inflammatory activity is either based on inhibition of lysosomal membrane.19 Hence it can be assume that EIA may possibly be acting either by inhibiting the lysosomal enzyme or by stabilizing the membrane. The ESR count has been used for staging the inflammatory disease.20 In order to find out the response of both extracts of
Fig. 1 e Effect of EIA on serum enzymes in carrageenan induced hind paw edema in mice. Treatment and dose: methanol [ 250 mg/kg b.w; water [ 250 mg/kg b.w; indomethacin [ 10 g/kg b.w.
Table 2 e Effect of I. aspalathoides on ESR counting on the carrageenan induced mice. S. no
Treatment and dose (mg/kg body weight)
Erythrocyte sedimentation rate (mm/hr)
1 2 3
Normal mice (Control) Inflammed mice Methanolic extract treated mice (250) Aqueous extract treated mice (250) Indomethacin (10) (a standard drug)
8 0.03 15 0.04** 10 0.05*
4 5
12 0.09* 7.5 0.02
Results expressed as mean SEM, n ¼ 6 in each group. The statistical analyzes were performed by one way ANOVA followed by Dunnett’s test. *Significant at (P < 0.05); **Significant at (P < 0.001) compared with control group.
I. aspalathoides against inflammation, ESR counting was done. The results were given in Table 2. The result showed that both EIA have the ability to reduce ( p < 0.05) the elevated levels of ESR to normal levels at the stage of inflammation. Identification of bioactive principles from medicinal plants is crucial for the standardization of herbal drugs. High Performance Liquid Chromatography is widely employed for screening the phytoconstituents for the quality management of herbal medicines. HPLC analysis was carried out for EIA and
Table 1 e Effects of I. aspalathoides on mice carrageenan induced hind paw edema. S. no
Treatment
Measurement of paw edema volume by vernier caliper (mm) Initial
1 2 3 4
Control Methanol Water Indomethacin (a standard drug)
0.16 0.08 0.10 0.10
0.05 0.02** 0.04* 0.01*
1h 0.30 0.12 0.23 0.13
0.03 0.04** 0.01** 0.03**
2h 0.43 0.13 0.25 0.14
0.02 0.03** 0.05** 0.04**
Inhibition of paw edema (%)
3h 0.44 0.19 0.26 0.16
0.01 0.03** 0.02** 0.01**
e 37.5 31.6 53.4
Results were expressed as mean SEM, n ¼ 6 in each group. The statistical analyzes were performed by one way ANOVA followed by Dunnett’s test. *Significant at (P < 0.05); **Significant at (P < 0.001) compared with control group.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
353
A.P. Selvarajan, Secretary, Sri Kaliswari College, Sivakasi to providing all facilities for my research.
references
Fig. 2 e HPLC chromatogram of extract of I. aspalathoides obtained by PDA detector at 240 nm.
Table 3 e Peak table for each compound in HPLC chromatogram. S. no 1 2 3 4 5
Retention time
Area percentage (%)
Height percentage (%)
2.828 3.12 3.393 37.292 49.707
10.535 3.635 6.764 69.906 9.16
35.644 17.502 29.104 15.62 5.131
found five different bioactive principles with retention time of 2.828, 3.120, 3.393, 37.292, 49.707 respectively (Fig. 2 and Table 3). The identified compounds were expected to belong to the family of pterocarpan which are the major active compounds in I. aspalathoides. It was supported by the previous finding that indigocarpan and mucronulatol, isolated from I. aspalathoides has high anti inflammatory activity.21 The further research will be performed to identify the specific compounds by preparative HPLC.
5.
Conclusion
The present study strongly justified that the stem of I. aspalathoides possess significant anti inflammatory activity. However, further studies focusing on the purification of bioactive compounds and their pharmacological action are required for developing effective anti inflammatory drug from I. aspalathoides.
Conflicts of interest All authors have none to declare.
Acknowledgments The authors are grateful to NRCBS-MKU for providing HPLC analysis facility & DST-PURSE for financial support and Mr.
1. Burke A, Smyth E, FitzGerald GA. Analgesic, antipyretic agents; pharmacotherapy of gout. In: Brunton LB, Lazo JS, Parker KL, eds. Goodman & Gilman’s the Pharmacological Basis of Therapeutics. New York: McGraw-Hill; 2005:671e715. 2. Mantovani A, Allavena P, Sica A. Cancer-related inflammation. Nature. 2008;454:436e444. 3. Ameh SJ, Obodozie OO, Afolabi EK. Some basic requirements for preparing an anti sickling herbal medicine e NIPRISAN. Afr J Pharm Pharmacol. 2009;3(5):259e264. 4. Khan AL, Gilani SA, Fuji Y, Watanabe KN. Monograph on Inula Britannica L. vol. 978. Tokyo-Japan: Mimatsu Corporation; 2008. 24e28. 5. Marston A, Hostettann. Natural product analysis over the last decades. Plant effect thermal stimulus was not dose. Planta Med. 2009;75(7):672e682. 6. Gupta M, Mazumder UK, Haldar PK, Kandar CK, Manikandan L, Senthil GP. Hepato protective activity of Indigofera aspalathoides against carbon tetrachloride induced liver damage in rats. Orient Pharm Exp Med. 2004:100e103. 7. Rajkapoor B, Murugesh N, Kaviman S, Krishna DR, Ravichandran V. Anti microbial, Anti viral and cytotoxic studies of Indigofera aspalathoides Vahl. Pharmacogn Mag. 2007;3(11):163e166. 8. Mosihuzzaman MM, Choudhary IM. Protocols on safety, efficacy, standardization and documentation of herbal medicine. Pure Appl Chem. 2008;80(10):2195e2230. 9. Amala Bhaskar E, Ganga N, Arivudainambi R, Santhanam G. Anti inflammatory activity of Indigofera aspalathoides Vahl. Indian J Med Res. 1982;76:115e118. 10. Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol. 1983;54:275e287. 11. Winter CA, Risley GA, Nuss GW. Carrageenan induced edema in hind paw of the rat as assay for anti inflammatory drugs. Proc Soc Exp Bio Med. 1962;111:544e547. 12. Woessner JR. The determination of hydroxy proline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys. 1961;93:440e443. 13. Guimaraes R, Barros L, Carvalho AM. Ferreira ICFR infusions and decoctions of mixed herbs used in folk medicine: synergism in antioxidant potential. Phytother Res. 2011;25:1209e1214. 14. Negi Bhawna Sunil, Bharti Deve P. Invitro anti microbial activity of Acacia catechu and its phytochemical analysis. Indian J Microbiol. 2010;50(4):369e374. 15. Alqasoumi SI, Soliman G, Awaad AS, Donia AERM. Antiinflammatory activity, safety and protective effects of Leptadenia pyrotechnica, Haloxylon salicornicum and Ochradenus baccatus in ulcerative colitis. Phytopharmacol. 2012;2:58e71. 16. Khan I, Nisar M, Ebad F, Nadeem S, Saeed M, Khan H. Antiinflammatory activities of Sieboldogenin from Smilax china Linn. Experimental and computational studies. J Ethnopharmacol. 2009;121(1):175e177. 17. Boura ALA, Svolmanis AP. Converting enzyme inhibition in the rat by captopril is accompanied by potentiation of carrageenan induced inflammation. Br J Pharmacol. 1984;82:3e8. 18. Anderson AJ, Bocklehurst WE, Wills AL. Evidence for the role of lysosomes in the formation of prostaglandins during carrageenan induced inflammation in rat. Pharmacol Res Comm. 1971;3:13e17. 19. Nair RB, Ravishankar B, Vijayan NP, Sasikala CK, Saraswathy VN. Anti-inflammatory effect of Strbilanthus
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heyneanus leaves e a biochemical study. J Res Ay Sid. 1988;9:46e50. 20. Weinstein A, Del Guidice J. The erythrocyte sedimentation rate: time honored and tradition bound. J Rheumatol. 1994;21:1177e1178.
21. Selvam C, Jackak Sanjay M, Oli Gnana R, et al. A new cyclooxygenase (COX) inhibitory pterocarpan from I. aspalathoides structure elucidation and determination of binding orientation in the active sites of the enzyme by molecular docking. Tetrahedron Lett. 2004;45(22):4311e4314.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Evaluation of anti inflammatory activity of Indigofera aspalathoides Vahl in Swiss albino mice N. Boopala Bhagavan a, S. Arunachalam b, P. Dhasarathan b, N.D. Kannan a,* a b
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India Department of Biotechnology, Sri Kaliswari College, Sivakasi 626130, India
article info
abstract
Article history:
Background: The objective of this study was to evaluate the anti inflammatory activity of
Received 19 December 2012
Indigofera aspalathoides in Swiss albino mice.
Accepted 16 February 2013
Methods: The anti inflammatory activity was evaluated by carrageenan induced paw edema
Available online 30 March 2013
model. Biochemical parameters such as Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT) and ESR counting were studied.
Keywords:
High Performance Liquid Chromatography (HPLC) analysis was performed to identify the
Anti inflammatory
bioactive compounds present in I .aspalathoides.
Indigofera aspalathoides
Results: The acute toxicity (LD50 value) of the Extract I. aspalathoides (EIA) was standardized
Carrageenan
as 250 mg/kg in orally treated mice. EIA found to have significant anti inflammatory activity
HPLC
in carrageenan induced paw edema compared to standard Indomethacin. It was evidenced
Indomethacin
by remarkable reduction in serum lysosomal enzymes and ESR as compared with control. HPLC chromatogram revealed the presence of five different bio active compounds in EIA. Conclusion: The result strongly suggested that I. aspalathoides has potent anti inflammatory property. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Inflammation is a severe response by living tissue to any kind of injury. There can be four primary indicators of inflammation: pain, redness, heat or warmness and swelling.1 Recent studies indicate that the mediators and cellular effectors of inflammation are important constituents of the local environment of tumors.2 Medicinal plants in particular, are believed to be an important source of new chemical substances with potential therapeutic efficacy.3 Inflammation plays an important role in various diseases with high prevalence within populations such as rheumatoid arthritis,
atherosclerosis and asthma. In recent years, plant materials continue to play a major role as therapeutic remedies in many developing countries.4 Plants represent still a large source of structurally novel compounds that might serve as lead for the development of novel drugs.5 Indigofera aspalathoides Vahl (Family: Leguminaceae) is a low under shrub commonly distributed in South India. It is commonly known as Sivanar Vembu in Southern Western Ghats of Tamil Nadu. In Indian system of herbal medicine, I. aspalathoides is specifically used for treating for Psoriasis, secondary syphilis, and viral hepatitis hepato-protective activity, kidney disorders.6 It was reported that stem extracts of
* Corresponding author. Tel.: þ91 452 2458273; fax: þ91 452 2459103. E-mail address: [email protected] (N.D. Kannan). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.02.018
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
I. aspalathoides has significant anti tumor, anti inflammatory, anti viral and antimicrobial activity.7 Global demand for herbal medicine is increasing at a rapid rate owing to their low cost and no side effects. However, proper phytochemical and pharmacological investigation should be carried out prior to the commercialization of plant based drug as many health hazards were reported with inadvertent use of plant drugs.8 Amala et al9 conducted a preliminary study to confirm the anti inflammatory activity of I. aspalathoides tender shoots. However, no systematic approach has been done so far to analyze phytochemical constituent that contribute anti inflammatory activity of I. aspalathoides. In the present study, the systemic study combining phytochemical and pharmacological aspects was carried out to evaluate the anti inflammatory of I. aspalathoides using Swiss albino mice.
2.
Materials and methods
2.1.
Plant collection and extraction
Plant sample was collected from Gopalasamy Hills in Viruthunagar district, Tamil Nadu, India. This plant was authenticated and voucher specimens were deposited in the Department of Biotechnology, Sri Kaliswari College, Sivakasi. The stems were shade-dried and pulverized. The powder was treated with petroleum ether to remove wax and chlorophyll and extracted with methanol. The extracts were concentrated by distilling the solvent in a rotary flash evaporator. Methanol was evaporated and dried extracts were dissolved in water.
3.
Evaluation of anti inflammatory activity
3.1.
Experimental animals
Swiss albino mice, (20e32 g) aged 8e12 weeks were used for anti inflammatory studies. They were kept hygienically in polypropylene cages in a controlled environment (Temperature 25 2 C, relative humidity 65 10%, and 12 h dark/light cycle) with standard laboratory diet and water ad libitum. This study was conducted after obtaining institutional animal ethical committee clearance (Number: 1086/AC/07/CPESEA).
3.2.
Acute toxicity studies
The acute toxicity (LD50) of the EIA was determined in mice by Lorke method.10 The study was carried out as per the guidelines of OECD (Number: 1086/AC/07/CPESEA).
3.3. Determination of inhibition of carrageenan induced mice paw edema The anti inflammatory activity was assessed using Winter et al method.11 The selected Swiss albino mice were divided into five groups of six animals (n ¼ 6) each and housed under laboratory condition. Group 1: controlecarrageenan (0.1 mL of 1%) only Group 2: carrageenan þ MEIA (250 mg/kg p.o)
351
Group 3: carrageenan þ aqueous EIA (250 mg/kg p.o) Group 4: carrageenan þ Indomethacin (10 mg/kg p.o) The percentage of inhibition of paw edema was calculated using following formula, Percentage of inhibition of paw edema volume ð%Þ ¼ 1 ðVt =Vc Þ 100 Vt ¼ Paw edema volume of drug treated group Vc ¼ Paw edema volume of control group
3.4.
Bio chemical assay
Biochemical changes in carrageenan induced paw edema were estimated in an interval of 6 h. The blood was collected from anaesthetized mice by cardiac puncture. The serum was separated from blood sample. The separated serum was analyzed for lysosomal enzymes such as SGOT and SGOT described by Woessner method.12 The activity of SGOT and SGPT were expressed in U/mL.
3.5.
Estimation of erythrocyte sedimentation rate (ESR)
The 0.8 mL of blood was collected from each group of mice by using sterile syringe via cardiac puncture and put into the tube which contained EDTA and mixed with the 0.2 mL of 3.8% of sodium citrate solution in a test tube. The Westergren tube is filled up to ‘0’ mark with citrated blood and plasma vertically in the stand. The sedimentation of RBC in mm in 1 h is observed and compared with other groups.
3.6.
High Performance Liquid Chromatography analysis
300 mL of water were added to the stem powder of I. aspalathoides (15 g) and heating was carried out a micro oven. After boiling, gentle heating was continued until the volume reduced to less than 30 mL. The mixture was filtered and frozen at 30 C for further use. The final concentration of the EIA was equivalent to 0.5 g/mL. The plant sample was analyzed by HPLC apparatus, equipped with a pump LC-10AT (Shimadzu, Corporation, Kyota, Japan), a Photodiode Array (PDA) detector SPD- M10 AVP (Shimadzu, Japan). The stationary phase of the column was a Diamonsil C18 (4.6 250 mm, 5-mm particle size). The plant sample (25 mL) was injected in column in an isocratic mobile phase comprising of Acetonitrile: 0.1% acetic acid (80: 20) at flow rate of 1 mL/min. The elution time was 15 min and detection was carried out at 240 nm. Column (C 18) was maintained at 25 C. The data acquisition was performed by ChemStation version A 08.03.
3.7.
Statistical analysis
The experimental results were expressed as the mean standard deviation. The level of significance was tested using one way analysis of variance (ANOVA) and Dunnett’s test at p < 0.05.
352
4.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
Result and discussion
Sub lethal concentration of the EIA was found to be 250 mg/kg b.w. and no mortality was detected upto this concentration during 24 h observation period. It was reported that during inflammation, over expression of the inducible forms of cyclooxygenase (COX) and the lipoxygenase (LO) enzymes cause the generation of the lipid mediators and damaging free radicals.13 Variations of paw edema volume in response to various treatments imposed to carrageenan induced paw volume were shown in Table 1. As expected, purified standard drug (Indomethacin) showed maximum reduction of paw edema volume. Nonetheless, both methanolic and aqueous extract of I. aspalathoides reduced paw edema volume significantly ( p < 0.05). Methanolic and aqueous extract of I. aspalathoides recorded 37.5% and 31.6% of inhibition of paw edema respectively. Then it was revealed that both extracts of I. aspalathoides has effective anti inflammatory activity. Moderately higher rate of inhibition by methanolic extract may be attributed to the high solubility of phytochemicals in methanol rather than water.14 Carrageenan induced paw edema test is a significant tool for the assessment of anti inflammatory profile of natural products.15 Carrageenan induced paw edema was observed to be progressively increased after the initial phase (0e1.5 h). In the second phase (1.5e5 h), various factors that are responsible for inflammation such as vasoactive amines (histamine, serotonin), arachidonic acids (prostaglandins, leukotrienes) and cytokines (tumor necrosis factor and interleukin-1) were produced due to the action of carrageenan.16,17 Since aqueous and methanolic extract of I. aspalathoides has significantly reduced the paw edema volume, it could be believed that EIA suppress the production of above mentioned factors. Lysosomal enzymes were reported to play crucial role during the development of inflammation.18 During the treatment with carrageenan, the level of SGOT and SGPT were elevated significantly. Interestingly the increased levels of these enzymes were reduced to the normal range after 6 h, since the administration of EIA (Fig. 1). Pharmacological action of most of the anti inflammatory activity is either based on inhibition of lysosomal membrane.19 Hence it can be assume that EIA may possibly be acting either by inhibiting the lysosomal enzyme or by stabilizing the membrane. The ESR count has been used for staging the inflammatory disease.20 In order to find out the response of both extracts of
Fig. 1 e Effect of EIA on serum enzymes in carrageenan induced hind paw edema in mice. Treatment and dose: methanol [ 250 mg/kg b.w; water [ 250 mg/kg b.w; indomethacin [ 10 g/kg b.w.
Table 2 e Effect of I. aspalathoides on ESR counting on the carrageenan induced mice. S. no
Treatment and dose (mg/kg body weight)
Erythrocyte sedimentation rate (mm/hr)
1 2 3
Normal mice (Control) Inflammed mice Methanolic extract treated mice (250) Aqueous extract treated mice (250) Indomethacin (10) (a standard drug)
8 0.03 15 0.04** 10 0.05*
4 5
12 0.09* 7.5 0.02
Results expressed as mean SEM, n ¼ 6 in each group. The statistical analyzes were performed by one way ANOVA followed by Dunnett’s test. *Significant at (P < 0.05); **Significant at (P < 0.001) compared with control group.
I. aspalathoides against inflammation, ESR counting was done. The results were given in Table 2. The result showed that both EIA have the ability to reduce ( p < 0.05) the elevated levels of ESR to normal levels at the stage of inflammation. Identification of bioactive principles from medicinal plants is crucial for the standardization of herbal drugs. High Performance Liquid Chromatography is widely employed for screening the phytoconstituents for the quality management of herbal medicines. HPLC analysis was carried out for EIA and
Table 1 e Effects of I. aspalathoides on mice carrageenan induced hind paw edema. S. no
Treatment
Measurement of paw edema volume by vernier caliper (mm) Initial
1 2 3 4
Control Methanol Water Indomethacin (a standard drug)
0.16 0.08 0.10 0.10
0.05 0.02** 0.04* 0.01*
1h 0.30 0.12 0.23 0.13
0.03 0.04** 0.01** 0.03**
2h 0.43 0.13 0.25 0.14
0.02 0.03** 0.05** 0.04**
Inhibition of paw edema (%)
3h 0.44 0.19 0.26 0.16
0.01 0.03** 0.02** 0.01**
e 37.5 31.6 53.4
Results were expressed as mean SEM, n ¼ 6 in each group. The statistical analyzes were performed by one way ANOVA followed by Dunnett’s test. *Significant at (P < 0.05); **Significant at (P < 0.001) compared with control group.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 5 0 e3 5 4
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A.P. Selvarajan, Secretary, Sri Kaliswari College, Sivakasi to providing all facilities for my research.
references
Fig. 2 e HPLC chromatogram of extract of I. aspalathoides obtained by PDA detector at 240 nm.
Table 3 e Peak table for each compound in HPLC chromatogram. S. no 1 2 3 4 5
Retention time
Area percentage (%)
Height percentage (%)
2.828 3.12 3.393 37.292 49.707
10.535 3.635 6.764 69.906 9.16
35.644 17.502 29.104 15.62 5.131
found five different bioactive principles with retention time of 2.828, 3.120, 3.393, 37.292, 49.707 respectively (Fig. 2 and Table 3). The identified compounds were expected to belong to the family of pterocarpan which are the major active compounds in I. aspalathoides. It was supported by the previous finding that indigocarpan and mucronulatol, isolated from I. aspalathoides has high anti inflammatory activity.21 The further research will be performed to identify the specific compounds by preparative HPLC.
5.
Conclusion
The present study strongly justified that the stem of I. aspalathoides possess significant anti inflammatory activity. However, further studies focusing on the purification of bioactive compounds and their pharmacological action are required for developing effective anti inflammatory drug from I. aspalathoides.
Conflicts of interest All authors have none to declare.
Acknowledgments The authors are grateful to NRCBS-MKU for providing HPLC analysis facility & DST-PURSE for financial support and Mr.
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