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In vitro antioxidant activity and in vivo alpha glucosidase activity of endophytic actinomycetes isolated from Catharanthus roseus (l.) G. Don
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 ) 6 7 4 e6 7 8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Review Article
In vitro antioxidant activity and in vivo alpha glucosidase activity of endophytic actinomycetes isolated from Catharanthus roseus (l.) G. Don D. Jacintha Jasmine*, P. Agastian Professor, Department of Plant Biology and Biotechnology, School of Life Sciences, Loyola College, Chennai 600 034, Tamil Nadu, India
article info
abstract
Article history:
Aim: Antioxidant compounds in food play an important role as a health protecting factor.
Received 28 November 2012
Scientific evidence suggests that antioxidants reduce the risk for chronic diseases
Accepted 4 June 2013
including cancer and heart disease. The present study was to evaluate the free radical
Available online 21 June 2013
scavenging activity and alpha glucosidase activity of Ethyl acetate extracts of endophytic actinomycetes (EEA) isolated from the roots of Catharanthus roseus (l.) G. Don.
Keywords:
Method: The EEA was screened for its capability in lowering blood sugar level and radical
Antioxidant activity
scavenging activity against Hydroxyl (HO-), Hydrogen peroxide (H2O2), Nitric oxide (NO) and
EEA
scavenging of stable radical 2,2-diphenyl-1-picrylhydrazyl or DPPH and using standard
DPPH
procedures.
Radical scavenging activity
Result: Significant free radical scavenging activity and marked alpha glucosidase activity
Alpha glucosidase activity
were exhibited by EEA isolated from the roots of Catharanthus roseus (l.) G. Don. Conclusion: The extract used in the study possesses significant antioxidant activity as well as an alpha glucosidase inhibition and have the potential for application in pharmaceuticals. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Catharanthus roseus (Madagascar periwinkle) is a native and endemic to Madagascar. This herb is now common in many tropical and subtropical regions worldwide, including the southern United States.1 It is used to treat gastrointestinal disorders, rheumatisms, cold, skin illnesses and inflammations. Endophytes are present in almost all plant species and have been recognized as a potential source of novel medicinal compounds.2 As reviewed,3 51% of the biologically active substances
isolated from endophytes were previously unknown. Although a number of bio-pharmacological compounds with antimicrobial, antitumor, anti-inflammatory, and antiviral activities have been previously isolated from entophytes,4 information related to their antioxidant activities is very scanty.5 Endophytic populations, like rhizospheric populations, are conditioned by biotic and abiotic factors.6 Actinomycetes have been looked upon as a separate group of microorganisms occupying a position between the true fungi and the true bacteria. The actinomycetes are
* Corresponding author. Tel.: þ91 7829090872. E-mail address: [email protected] (D.J. Jasmine). 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.2013.06.007
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 ) 6 7 4 e6 7 8
noteworthy as antibiotic producers, making 75% of all known products and the Streptomyces are especially profilic.7 They are the major microbes in the soil micro-ecosystem8 and an amount of actinomycetes have already been isolated and identified.9 Many efforts have been made to select and isolate actinomycetes from other biotopes, such as lake water,10 marine sediments,11 plant surface and plant tissues.12
2.
Materials & methods
2.1.
Plant materials
Roots of healthy Catharanthus roseus plants were collected from Loyola College campus located in the Garden, they were taken to the laboratory and processed immediately after collection. The species was identified and authenticated by Dr. Agastian, Head, Department of Plant Biology and Biotechnology, Loyola College, India.
2.2.
Isolation of actinomycetes
The procedure for surface sterilization was done according to the standard reference method proposed by Fisher and Petrini.13 Roots of Catharanthus roseus (0.5e1.0 cm in diameter) were washed in running tap water to remove soil particles. After washing, it was followed by surface sterilization with 3e5% sodium hypochlorite for 3 min, followed by rinsing with sterile distilled water and then treated with ethanol 70% for 30 s. Then each root was split into pieces of 1.0 cm to expose cortex and vascular bundles. They were then aseptically transferred to petri dishes containing starch casein agar medium for actinomycetes and 2.5% water agar medium for fungal isolation. Nalidixic acid and Actidione (50 mg/ml) were added to starch casein agar medium to suppress fungal growth. Streptomycin (250 mg/L) was added to water agar medium to suppress bacterial growth. Plates were incubated at 28 C for a maximum of three weeks. Actinomycetes and fungi growing on the medium were isolated, subcultured and identified.
2.3. Mass production of crude extracts for antioxidant property analysis and alpha glucosidase activity The isolated actinomycetes and fungi were mass produced by inoculating them in Modified Nutrient Glucose broth (MNGB) and Potato dextrose broth (Himedia, Mumbai) respectively. The inoculated actinomycetes flasks were kept in rotatory shaker at 120 rpm about 10 days for its growth and as still culture for inoculated fungus. Full growth occurred after 10 days and then the broth was centrifuged at 8000 rpm for 10 min at 4 C. The supernatant was collected and dissolved in equal volume of ethyl acetate and the organic layer was separated using the separating funnel. The solvent was subjected to Rota vacuum evaporator for getting concentrated crude extracts and stored at 4 C until further use.
2.4.
Antioxidant activities
DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity of EEA was determined using the method proposed by
675
Mahesh Ramalingam.14 The ability of EEA to scavenge the hydroxyl radical generated by the Fenton reaction was measured according to the modified method described by Manish et al.15 The ability of the endophytic extract to scavenge hydrogen peroxide was determined according to the standard method described by Arulmozhi et al.16 Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which was measured by the Griess reaction proposed by Seyyed et al.17 Butylated hydroxytoluene and Ascorbic acid were used as a positive control. The absorbance was recorded using a UV-VIS spectrophotometer (Jasco V-530, Japan Servo Co. Limited., Japan). Radical scavengingð%Þ ¼
2.5.
OD control OD test sample 100 OD control
a-Glucosidase inhibitory assay
In order to investigate the inhibitory effect of EEA, an in vitro aglucosidase inhibition test was performed. a-Glucosidase from yeast is used extensively as a screening material for a-glucosidase inhibitors, but the results do not always agree with those obtained in mammals. Therefore, we used the rat small intestine homogenate as a-glucosidase (Maltose a-glucosidase) solution because we speculated that it would better reflect the in vivo state. The inhibitory effect was measured using the method slightly modified by Dahlqvist.18 The assay mixture consisted of 100 mM maleate buffer (pH 6.0), 2% (w/v) each sugar substrate solution (100 ml), and the extract (50e1000 mg/mL) and acarbose was used as reference drug as a-glucosidase inhibitor. It was preincubated for 5 min at 37 C, and the reaction was initiated by adding the crude a-glucosidase solution (50 ml) to it, followed by incubation for 10 min at 37 C. The glucose released in the reaction mixture was determined with the kit (Accuzyme, GOD-POD); OD was read at 505 nm. The rate of carbohydrate decomposition was calculated as percentage ratio to the amount of glucose obtained when the carbohydrate was completely digested. The rate of prevention was calculated by the following formula: All the OD values must by divided by standard value and then multiplied by 100 which gives rise to glucose in (mg/dl) % Inhibition :
2.6.
Control Test 100 Control
In vivo study
Based on the results obtained from in vitro study, it was checked in vivo at 500 mg/kg. We had followed the standard procedure proposed by Abesundara, Matsui and Matsumoto.19 Briefly, the animals (male albino rats) were fasted for 24 h. Then zero hour blood samples were taken from orbital sinus. Then, the animals were treated with extract or vehicle. Ten minutes after the treatment with the extracts, maltose solution (2 G/Kg) was given to the animals. 30, 60 and 120 min after the administration of maltose, plasma glucose levels were estimated using GOD-POD method. Acarbose (3 mg/kg) was used as positive control. All tests were performed after approval by the animals ethical committee of Entomology Research Institute, Loyola College, Chennai and in accordance
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with the disciplinary principles and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
2.7.
HPLC
High performance liquid chromatography fingerprint of alkaloids in EEA was performed using Waters HPLC system (Waters HPLC, USA) equipped with two pumps (Waters Pump 515) and a UVeVisible detector (Waters 2489), operated by Empower 2 software. A reversed phase C18 column (Symmetry, 250 4.6 mm; particle size ¼ 5 mm). The column temperature was maintained at 30 C and the injection volume was 10 ml. The elution was isocratic in the solvent mixture of acetonitrile: acetic acid: water (18:2:80) at the flow rate of 0.8 ml/min. The run time was less than 20 min High Performance Liquid Chromatography (HPLC) is one mode of chromatography; the most widely used analytical technique. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.
3.
Table 2 e Hydroxyl radical scavenging activity. Concentration (mg/mL)
% inhibition of Hydroxyl free radical EEA
BHT
Ascorbic acid
50 100 200 400 500 1000
34.68 42.72 47.63 50.84 61.5 71.15
86.88 89.08 91.45 92.55 98.73 99.06
87.73 89.42 89.42 93.31 96.27 99.23
Table 3 e Hydrogen peroxide radical scavenging activity. Concentration (mg/mL)
50 100 200 400 500 1000
Antioxidant activity performed using EEA is listed in Table 1. In DPPH free radical scavenging activity, EEA was found to show high percentage of inhibition (54.29%) at 1000 mg/ml and a moderate percentage of inhibition (47.81%) at 500 mg/ml respectively. It is evident from the study, that the investigated extracts have the ability to quench free radicals. The extract showed dose dependent DPPH radical scavenging activity. Hydroxyl radical scavenging activity of EEA is shown in Table 2. EEA showed high activity 71.15% at 1000 mg/ml followed by a second high activity 61.5% at 500 mg/ml. Hydroxyl radical is an extremely reactive species formed in biological systems implicated as highly damaging in free radical pathology, capable of damaging almost every molecule found in the living cells. This radical has the capacity to join nucleotides in DNA and cause strand breakage, contributing to aging,
EEA
BHT
Ascorbic acid
12.09 19.08 25.53 36.55 43.41 67.31
46.63 56.85 59.81 65.72 73.65 84.27
47.84 56.58 61.29 67.47 73.11 85.34
Table 4 e Nitric oxide radical scavenging activity. Concentration (mg/mL)
Results and discussion
% inhibition of hydrogen peroxide free radical
50 100 200 400 500 1000
% inhibition of DPPH free radical EPA
BHT
Ascorbic acid
5.21 22.37 27.17 35.40 49.96 69.24
22.80 30.26 45.77 55.74 63.97 68.83
35.40 42.96 44.92 50.80 63.46 73.43
carcinogenesis, mutagenesis, cytotoxicity and several other diseases.20 In the case of hydrogen peroxide free radical scavenging activity Table 3, EEA showed a high inhibition percentage, 67.31% at 1000 mg/ml, followed by a moderate inhibition percentage and 43.41% at 500 mg/ml respectively. Hydrogen peroxide itself is not reactive, as it can sometimes be toxic to
Table 1 e DPPH radical scavenging activity.
Table 5 e In vitro a-glucosidase inhibition using EEA of C. roseus.
Concentration (mg/mL)
Concentration (mg/ml)
50 100 200 400 500 1000
% inhibition of DPPH free radical EEA
BHT
Ascorbic acid
17.66 25.28 20.09 38.08 47.81 54.29
68.88 73.58 78.11 85.08 90.92 94.81
72.60 78.60 84.11 89.78 93.19 95.46
50 100 200 400 500 1000
% of inhibition of alpha glucosidase EPA
Acarbose reference
5.84 16.95 34.79 45.02 55.55 83.33
74.56 81.28 88.01 91.81 94.15 96.78
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 ) 6 7 4 e6 7 8
Table 6 e In vivo lowering of maltose level in the blood using EEA of C. roseus. Tested material
‘0’ h
30 min
60 min
120 min
Control Treated Acarbose
87.08 94.79 97.22
96.25 93.12 97.55
109.37 93.12 95.02
109.79 108.54 96.33
Table 7 e In vivo lowering of sucrose level in the blood using EEA of C. roseus. Tested material
‘0’ h
30 min
60 min
120 min
Control Treated Acarbose
90.86 94.06 95.58
104.68 96.13 94.88
118.81 105.24 101.79
100.43 106.21 99.585
cell because it may give rise to OH radical in the cells. Addition of hydrogen peroxide to cells in culture can lead to transition metal ion dependent OH radicals mediated DNA damage. Scavenging of hydrogen peroxide by our crude endophytic extract may be attributed to their phenolic nature, which can donate electrons to H2O2, thus neutralizing it to water.21 Nitric oxide scavenging activity of EEA is listed Table 4. In case of nitric oxide scavenging activity, EEA showed high activity 69.24% at 1000 mg/ml followed by a moderate activity 35.40% at 400 mg/ml. BHT and Ascorbic acid were used as the positive control. Nitric oxide is a diffusible free radical, which plays many roles as an effector molecule including neuronal signaling, and regulation of cell mediated toxicity. Nitric oxide (NO) is generated in different cell types by at least three isoforms of NO synthase (NOS). Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed and their enzymatic activity is Ca2þ/calmodulin-dependent.22 Suppression of NO released may be partially attributed to direct NO scavenging, as the extract decreased the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro.
677
Based on the results obtained from the in vitro a-glucosidase inhibition, EEA was found to show high activity. Hence in vivo studies were carried out using EEA on lowering maltose and sucrose levels in the blood. At 30 min after maltose load, the normal control animals had shown an increase in plasma glucose level; whereas the EEA treated as well as the Acarbose treated animals had not shown any significant rise in plasma glucose level. As shown in Table 5 incubation of the EEA at different concentrations with intestinal alpha glucosidase enzyme caused an increased activity with 83.33% inhibition when incubated at 1000 mg/ml concentration. However, the inhibitory effect was equally comparable to that of the acarbose, which is well known alpha glucosidase inhibitor. With the interesting result obtained using EEA, further in vivo study of a-glucosidase inhibition was carried out. The study reveals that there is no significant rise in the plasma glucose level. At 30 min after administration of maltose and sucrose orally, the normal control animals had shown an increase in plasma glucose level 109.79 mg/dl at 120 min; whereas the EEA treated as well as the Acarbose treated animals had not shown any significant rise in plasma glucose level. At 60 min after sucrose load, the control animals had shown an increase in plasma glucose level 118.81 mg/dl whereas the EEA treated as well as the Acarbose treated animals had not shown any rise in plasma glucose level Tables 6 and 7. Hence EEA has a potential ability to inhibit the alpha glucosidase enzyme thereby causing partial digestion and keeping the blood glucose level normal. Here we report the ability of EEA to inhibit alpha glucosidase. HPLC analysis revealed that the major constituents of the extracts are vinblastine an alkaloid compound which showed a sharp peak at 2.850 mV respectively (Fig. 1). EEA was able to inhibit alpha glucosidase inhibitory activities in vitro in dose dependent manner. It has been recently reported that tea polyphenols inhibited glucose transporter of small intestine epithelial cells. Ethyl acetate extracts showed better activity than acarbose with smallest IC50 values was 73.64 mg/mL. The most active extract showed competitive inhibition. Chemical analysis indicated that the a-glucosidase inhibitor was flavonoid.23 In addition, polyphenols controlled the rise in blood glucose level when humans fed with fixed amount of
Fig. 1 e HPLC profile of ethyl acetate extract of endophytic actinomycetes.
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carbohydrates with food, because a negative correlation was indicated by the polyphenolic content and glycemic index.24 The enzyme inhibitors impede digestion through their action of digestive enzymes which play a key role in the digestion of plant starch and portions. Our results showed strong inhibition of alpha glucosidase activity. Higher inhibitory activities of EEA against alpha glucosidase that our results confirmed suggest its potential in prevention and therapy of obesity and diabetes. In most of the cases the mechanism of inhibition occurs through the direct blockage of the active center at several sub sites of the enzyme. EEA has a good free radical scavenging activity against all the four radicals. Maximum percentage inhibition was found against hydroxyl radical (71.15%). Alpha glucosidase activity performed under in vitro conditions showed an interesting result of 83.33% inhibition further in vivo study of a-glucosidase inhibition was carried out in lowering maltose and sucrose levels in blood. EEA treated and Acarbose treated animals did not show any change in the plasma glucose level. Hence EEA has a potential ability to inhibit the alpha glucosidase enzyme thereby causing partial digestion and keeping the blood glucose level normal.
Conflicts of interest All authors have none to declare.
references
1. Chevallier. Encyclopedia of Medicinal Plants Edition. 1996;vol. 1:220e221. 2. Tan RX, Zou WX. Endophytes: a rich source of functional metabolites. Nat Prod Rep. 2001;18:448e459. 3. Schulz B, Boyle C, Draeger S, Ro¨mmert AK. Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res. 2002;106:996e1004. 4. Aly AH, Edrada-Ebel R, Wray V, Muller WE. Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides. Phytochemistry. 2008;69:1716e1725. 5. Strobel G, Ford E, Worapong J, Harper JK. Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities. Phytochemistry. 2002;60:179e183. 6. Seghers D, Wittebolle L, Top EM, Verstraete W, Siciliano SD. Impact of agricultural practices on the Zea mays L. endophytic community. Appl Environ Microbiol. 2004;70:1475e1482. 7. Thakur D, Bora TC, Bordoloi DC, Mazumdar S. Influence of nutrition and culturing conditions for optimum growth and antimicrobial metabolite production by Streptomyces sp. 201. J Med Mycol. 2009;19:161e167.
8. Thangapandian VP, Ponmurugan, Ponmurugan S. Actinomycetes diversity in the rhizosphere soils of different medicinal plants in Kolly Hills-Tamilnadu, India, for secondary metabolite production. Asian J Plant Sci. 2007;6:66e70. 9. Muiru WM, Mutitu, Mukunya DM. Identification of selected actinomycete isolates and characterization of their antibiotic metabolites. J Boil Sci. 2008;8:1021e1026. 10. Singh LS, Baruah I, Bora TC. Actinomycetes of loktak habitat: isolation and screening for antimicrobial activities. Biotechnology. 2006;5:217e221. 11. Kumar KS, Haritha R, Mohan YSYVJ, Ramana T. Screening of marine actinobacteria for antimicrobial compounds. Res J Microbiol. 2011;6:385e393. 12. Zin NM, Sarmin K, Getha, Tan GY. Anti-trypanosomal activity of endophytic Streptomycete. Curr Res Bacteriol. 2011;4:1e8. 13. Fisher PJ, Petrini O. Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytol. 1992;120:137e143. 14. Ramalingam Mahesh, Yong-Ki Park. Free radical scavenging activities of Cnidium officinale Makino and Ligusticum chuanxiong Hort. methanolic extracts. Pharmacognosy Mag. 2010;6:323e330. 15. Manish S, Lavhale, Mishra SH. Evaluation of free radical scavenging activity of Butea Monospora Lam. Indian J Exp Biol. 2007;47:376e384. 16. Arulmozhi S, Mazumder Papiya Mitra, Ashok Purnima. In vitro antioxidant and free radical scavenging activity of Alstonia scholaris Linn R.Br. Iranian J Pharmacol Ther. 2008;6:191e196. 17. Nabavi Seyed Mohammad, Ebrahimzedh Mohammad Ali, Navabi Seyed Fazel, Fazelian Mojtaba, eslami Bahman. Pharmacognosy Mag. 2009;4:122e126. 18. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem. 1964;7:18e25. 19. Abesundara KJ, Matsui T, Matsumoto K. Alfa-glucosidase inhibitory activity of some Sri Lanka plant extracts, one of which, Cassia auriculata, exerts a strong antihyperglycemic effect in rats comparable to therapeutic drug acarbose. J Agric Food Chem. 2004;52:2541e2545. 20. Selim KA, El-Beih AA, AbdEl-Rahman TM, El-Diwany AI. Biology of endophytic fungi. Curr Res Environ Appl Mycol. 2012;2:31e82. 21. Wettasinghe M, Shahidi F. Scavenging of reactive-oxygen species and DPPH free radicals by extracts of Borrage and evening primrose meals. Food Chem. 2000;70:17e26. 22. Taechowisan Thongchai, Chuaychot Nantiya, Chanaphat Srisakul, Wanbanjob Asawin, Shen Yuemao. Anti-oxidative and inhibitory activity on nitric oxide production of flavonoids from Streptomyces sp. Tc052. J Pharm Res. 2009;2:313e316. 23. Ramdanis R, Soemiati A, Munim A. Isolation and aglucosidase inhibitory activity of endophytic fungi from mahogany (Swietenia macrophylla King) seeds. Int J Med Aromatic Plants. 2012;2:447e452. 24. Payan F. Structural basis for the inhibition of mammalian and insect a-amylase by plant protein inhibitors. Biochemica et Biophisica Acta (BBA) e Proteins and Proteomics. 2004;2:171e180.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Review Article
In vitro antioxidant activity and in vivo alpha glucosidase activity of endophytic actinomycetes isolated from Catharanthus roseus (l.) G. Don D. Jacintha Jasmine*, P. Agastian Professor, Department of Plant Biology and Biotechnology, School of Life Sciences, Loyola College, Chennai 600 034, Tamil Nadu, India
article info
abstract
Article history:
Aim: Antioxidant compounds in food play an important role as a health protecting factor.
Received 28 November 2012
Scientific evidence suggests that antioxidants reduce the risk for chronic diseases
Accepted 4 June 2013
including cancer and heart disease. The present study was to evaluate the free radical
Available online 21 June 2013
scavenging activity and alpha glucosidase activity of Ethyl acetate extracts of endophytic actinomycetes (EEA) isolated from the roots of Catharanthus roseus (l.) G. Don.
Keywords:
Method: The EEA was screened for its capability in lowering blood sugar level and radical
Antioxidant activity
scavenging activity against Hydroxyl (HO-), Hydrogen peroxide (H2O2), Nitric oxide (NO) and
EEA
scavenging of stable radical 2,2-diphenyl-1-picrylhydrazyl or DPPH and using standard
DPPH
procedures.
Radical scavenging activity
Result: Significant free radical scavenging activity and marked alpha glucosidase activity
Alpha glucosidase activity
were exhibited by EEA isolated from the roots of Catharanthus roseus (l.) G. Don. Conclusion: The extract used in the study possesses significant antioxidant activity as well as an alpha glucosidase inhibition and have the potential for application in pharmaceuticals. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Catharanthus roseus (Madagascar periwinkle) is a native and endemic to Madagascar. This herb is now common in many tropical and subtropical regions worldwide, including the southern United States.1 It is used to treat gastrointestinal disorders, rheumatisms, cold, skin illnesses and inflammations. Endophytes are present in almost all plant species and have been recognized as a potential source of novel medicinal compounds.2 As reviewed,3 51% of the biologically active substances
isolated from endophytes were previously unknown. Although a number of bio-pharmacological compounds with antimicrobial, antitumor, anti-inflammatory, and antiviral activities have been previously isolated from entophytes,4 information related to their antioxidant activities is very scanty.5 Endophytic populations, like rhizospheric populations, are conditioned by biotic and abiotic factors.6 Actinomycetes have been looked upon as a separate group of microorganisms occupying a position between the true fungi and the true bacteria. The actinomycetes are
* Corresponding author. Tel.: þ91 7829090872. E-mail address: [email protected] (D.J. Jasmine). 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.2013.06.007
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 ) 6 7 4 e6 7 8
noteworthy as antibiotic producers, making 75% of all known products and the Streptomyces are especially profilic.7 They are the major microbes in the soil micro-ecosystem8 and an amount of actinomycetes have already been isolated and identified.9 Many efforts have been made to select and isolate actinomycetes from other biotopes, such as lake water,10 marine sediments,11 plant surface and plant tissues.12
2.
Materials & methods
2.1.
Plant materials
Roots of healthy Catharanthus roseus plants were collected from Loyola College campus located in the Garden, they were taken to the laboratory and processed immediately after collection. The species was identified and authenticated by Dr. Agastian, Head, Department of Plant Biology and Biotechnology, Loyola College, India.
2.2.
Isolation of actinomycetes
The procedure for surface sterilization was done according to the standard reference method proposed by Fisher and Petrini.13 Roots of Catharanthus roseus (0.5e1.0 cm in diameter) were washed in running tap water to remove soil particles. After washing, it was followed by surface sterilization with 3e5% sodium hypochlorite for 3 min, followed by rinsing with sterile distilled water and then treated with ethanol 70% for 30 s. Then each root was split into pieces of 1.0 cm to expose cortex and vascular bundles. They were then aseptically transferred to petri dishes containing starch casein agar medium for actinomycetes and 2.5% water agar medium for fungal isolation. Nalidixic acid and Actidione (50 mg/ml) were added to starch casein agar medium to suppress fungal growth. Streptomycin (250 mg/L) was added to water agar medium to suppress bacterial growth. Plates were incubated at 28 C for a maximum of three weeks. Actinomycetes and fungi growing on the medium were isolated, subcultured and identified.
2.3. Mass production of crude extracts for antioxidant property analysis and alpha glucosidase activity The isolated actinomycetes and fungi were mass produced by inoculating them in Modified Nutrient Glucose broth (MNGB) and Potato dextrose broth (Himedia, Mumbai) respectively. The inoculated actinomycetes flasks were kept in rotatory shaker at 120 rpm about 10 days for its growth and as still culture for inoculated fungus. Full growth occurred after 10 days and then the broth was centrifuged at 8000 rpm for 10 min at 4 C. The supernatant was collected and dissolved in equal volume of ethyl acetate and the organic layer was separated using the separating funnel. The solvent was subjected to Rota vacuum evaporator for getting concentrated crude extracts and stored at 4 C until further use.
2.4.
Antioxidant activities
DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity of EEA was determined using the method proposed by
675
Mahesh Ramalingam.14 The ability of EEA to scavenge the hydroxyl radical generated by the Fenton reaction was measured according to the modified method described by Manish et al.15 The ability of the endophytic extract to scavenge hydrogen peroxide was determined according to the standard method described by Arulmozhi et al.16 Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which was measured by the Griess reaction proposed by Seyyed et al.17 Butylated hydroxytoluene and Ascorbic acid were used as a positive control. The absorbance was recorded using a UV-VIS spectrophotometer (Jasco V-530, Japan Servo Co. Limited., Japan). Radical scavengingð%Þ ¼
2.5.
OD control OD test sample 100 OD control
a-Glucosidase inhibitory assay
In order to investigate the inhibitory effect of EEA, an in vitro aglucosidase inhibition test was performed. a-Glucosidase from yeast is used extensively as a screening material for a-glucosidase inhibitors, but the results do not always agree with those obtained in mammals. Therefore, we used the rat small intestine homogenate as a-glucosidase (Maltose a-glucosidase) solution because we speculated that it would better reflect the in vivo state. The inhibitory effect was measured using the method slightly modified by Dahlqvist.18 The assay mixture consisted of 100 mM maleate buffer (pH 6.0), 2% (w/v) each sugar substrate solution (100 ml), and the extract (50e1000 mg/mL) and acarbose was used as reference drug as a-glucosidase inhibitor. It was preincubated for 5 min at 37 C, and the reaction was initiated by adding the crude a-glucosidase solution (50 ml) to it, followed by incubation for 10 min at 37 C. The glucose released in the reaction mixture was determined with the kit (Accuzyme, GOD-POD); OD was read at 505 nm. The rate of carbohydrate decomposition was calculated as percentage ratio to the amount of glucose obtained when the carbohydrate was completely digested. The rate of prevention was calculated by the following formula: All the OD values must by divided by standard value and then multiplied by 100 which gives rise to glucose in (mg/dl) % Inhibition :
2.6.
Control Test 100 Control
In vivo study
Based on the results obtained from in vitro study, it was checked in vivo at 500 mg/kg. We had followed the standard procedure proposed by Abesundara, Matsui and Matsumoto.19 Briefly, the animals (male albino rats) were fasted for 24 h. Then zero hour blood samples were taken from orbital sinus. Then, the animals were treated with extract or vehicle. Ten minutes after the treatment with the extracts, maltose solution (2 G/Kg) was given to the animals. 30, 60 and 120 min after the administration of maltose, plasma glucose levels were estimated using GOD-POD method. Acarbose (3 mg/kg) was used as positive control. All tests were performed after approval by the animals ethical committee of Entomology Research Institute, Loyola College, Chennai and in accordance
676
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with the disciplinary principles and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
2.7.
HPLC
High performance liquid chromatography fingerprint of alkaloids in EEA was performed using Waters HPLC system (Waters HPLC, USA) equipped with two pumps (Waters Pump 515) and a UVeVisible detector (Waters 2489), operated by Empower 2 software. A reversed phase C18 column (Symmetry, 250 4.6 mm; particle size ¼ 5 mm). The column temperature was maintained at 30 C and the injection volume was 10 ml. The elution was isocratic in the solvent mixture of acetonitrile: acetic acid: water (18:2:80) at the flow rate of 0.8 ml/min. The run time was less than 20 min High Performance Liquid Chromatography (HPLC) is one mode of chromatography; the most widely used analytical technique. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.
3.
Table 2 e Hydroxyl radical scavenging activity. Concentration (mg/mL)
% inhibition of Hydroxyl free radical EEA
BHT
Ascorbic acid
50 100 200 400 500 1000
34.68 42.72 47.63 50.84 61.5 71.15
86.88 89.08 91.45 92.55 98.73 99.06
87.73 89.42 89.42 93.31 96.27 99.23
Table 3 e Hydrogen peroxide radical scavenging activity. Concentration (mg/mL)
50 100 200 400 500 1000
Antioxidant activity performed using EEA is listed in Table 1. In DPPH free radical scavenging activity, EEA was found to show high percentage of inhibition (54.29%) at 1000 mg/ml and a moderate percentage of inhibition (47.81%) at 500 mg/ml respectively. It is evident from the study, that the investigated extracts have the ability to quench free radicals. The extract showed dose dependent DPPH radical scavenging activity. Hydroxyl radical scavenging activity of EEA is shown in Table 2. EEA showed high activity 71.15% at 1000 mg/ml followed by a second high activity 61.5% at 500 mg/ml. Hydroxyl radical is an extremely reactive species formed in biological systems implicated as highly damaging in free radical pathology, capable of damaging almost every molecule found in the living cells. This radical has the capacity to join nucleotides in DNA and cause strand breakage, contributing to aging,
EEA
BHT
Ascorbic acid
12.09 19.08 25.53 36.55 43.41 67.31
46.63 56.85 59.81 65.72 73.65 84.27
47.84 56.58 61.29 67.47 73.11 85.34
Table 4 e Nitric oxide radical scavenging activity. Concentration (mg/mL)
Results and discussion
% inhibition of hydrogen peroxide free radical
50 100 200 400 500 1000
% inhibition of DPPH free radical EPA
BHT
Ascorbic acid
5.21 22.37 27.17 35.40 49.96 69.24
22.80 30.26 45.77 55.74 63.97 68.83
35.40 42.96 44.92 50.80 63.46 73.43
carcinogenesis, mutagenesis, cytotoxicity and several other diseases.20 In the case of hydrogen peroxide free radical scavenging activity Table 3, EEA showed a high inhibition percentage, 67.31% at 1000 mg/ml, followed by a moderate inhibition percentage and 43.41% at 500 mg/ml respectively. Hydrogen peroxide itself is not reactive, as it can sometimes be toxic to
Table 1 e DPPH radical scavenging activity.
Table 5 e In vitro a-glucosidase inhibition using EEA of C. roseus.
Concentration (mg/mL)
Concentration (mg/ml)
50 100 200 400 500 1000
% inhibition of DPPH free radical EEA
BHT
Ascorbic acid
17.66 25.28 20.09 38.08 47.81 54.29
68.88 73.58 78.11 85.08 90.92 94.81
72.60 78.60 84.11 89.78 93.19 95.46
50 100 200 400 500 1000
% of inhibition of alpha glucosidase EPA
Acarbose reference
5.84 16.95 34.79 45.02 55.55 83.33
74.56 81.28 88.01 91.81 94.15 96.78
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 ) 6 7 4 e6 7 8
Table 6 e In vivo lowering of maltose level in the blood using EEA of C. roseus. Tested material
‘0’ h
30 min
60 min
120 min
Control Treated Acarbose
87.08 94.79 97.22
96.25 93.12 97.55
109.37 93.12 95.02
109.79 108.54 96.33
Table 7 e In vivo lowering of sucrose level in the blood using EEA of C. roseus. Tested material
‘0’ h
30 min
60 min
120 min
Control Treated Acarbose
90.86 94.06 95.58
104.68 96.13 94.88
118.81 105.24 101.79
100.43 106.21 99.585
cell because it may give rise to OH radical in the cells. Addition of hydrogen peroxide to cells in culture can lead to transition metal ion dependent OH radicals mediated DNA damage. Scavenging of hydrogen peroxide by our crude endophytic extract may be attributed to their phenolic nature, which can donate electrons to H2O2, thus neutralizing it to water.21 Nitric oxide scavenging activity of EEA is listed Table 4. In case of nitric oxide scavenging activity, EEA showed high activity 69.24% at 1000 mg/ml followed by a moderate activity 35.40% at 400 mg/ml. BHT and Ascorbic acid were used as the positive control. Nitric oxide is a diffusible free radical, which plays many roles as an effector molecule including neuronal signaling, and regulation of cell mediated toxicity. Nitric oxide (NO) is generated in different cell types by at least three isoforms of NO synthase (NOS). Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed and their enzymatic activity is Ca2þ/calmodulin-dependent.22 Suppression of NO released may be partially attributed to direct NO scavenging, as the extract decreased the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro.
677
Based on the results obtained from the in vitro a-glucosidase inhibition, EEA was found to show high activity. Hence in vivo studies were carried out using EEA on lowering maltose and sucrose levels in the blood. At 30 min after maltose load, the normal control animals had shown an increase in plasma glucose level; whereas the EEA treated as well as the Acarbose treated animals had not shown any significant rise in plasma glucose level. As shown in Table 5 incubation of the EEA at different concentrations with intestinal alpha glucosidase enzyme caused an increased activity with 83.33% inhibition when incubated at 1000 mg/ml concentration. However, the inhibitory effect was equally comparable to that of the acarbose, which is well known alpha glucosidase inhibitor. With the interesting result obtained using EEA, further in vivo study of a-glucosidase inhibition was carried out. The study reveals that there is no significant rise in the plasma glucose level. At 30 min after administration of maltose and sucrose orally, the normal control animals had shown an increase in plasma glucose level 109.79 mg/dl at 120 min; whereas the EEA treated as well as the Acarbose treated animals had not shown any significant rise in plasma glucose level. At 60 min after sucrose load, the control animals had shown an increase in plasma glucose level 118.81 mg/dl whereas the EEA treated as well as the Acarbose treated animals had not shown any rise in plasma glucose level Tables 6 and 7. Hence EEA has a potential ability to inhibit the alpha glucosidase enzyme thereby causing partial digestion and keeping the blood glucose level normal. Here we report the ability of EEA to inhibit alpha glucosidase. HPLC analysis revealed that the major constituents of the extracts are vinblastine an alkaloid compound which showed a sharp peak at 2.850 mV respectively (Fig. 1). EEA was able to inhibit alpha glucosidase inhibitory activities in vitro in dose dependent manner. It has been recently reported that tea polyphenols inhibited glucose transporter of small intestine epithelial cells. Ethyl acetate extracts showed better activity than acarbose with smallest IC50 values was 73.64 mg/mL. The most active extract showed competitive inhibition. Chemical analysis indicated that the a-glucosidase inhibitor was flavonoid.23 In addition, polyphenols controlled the rise in blood glucose level when humans fed with fixed amount of
Fig. 1 e HPLC profile of ethyl acetate extract of endophytic actinomycetes.
678
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 ) 6 7 4 e6 7 8
carbohydrates with food, because a negative correlation was indicated by the polyphenolic content and glycemic index.24 The enzyme inhibitors impede digestion through their action of digestive enzymes which play a key role in the digestion of plant starch and portions. Our results showed strong inhibition of alpha glucosidase activity. Higher inhibitory activities of EEA against alpha glucosidase that our results confirmed suggest its potential in prevention and therapy of obesity and diabetes. In most of the cases the mechanism of inhibition occurs through the direct blockage of the active center at several sub sites of the enzyme. EEA has a good free radical scavenging activity against all the four radicals. Maximum percentage inhibition was found against hydroxyl radical (71.15%). Alpha glucosidase activity performed under in vitro conditions showed an interesting result of 83.33% inhibition further in vivo study of a-glucosidase inhibition was carried out in lowering maltose and sucrose levels in blood. EEA treated and Acarbose treated animals did not show any change in the plasma glucose level. Hence EEA has a potential ability to inhibit the alpha glucosidase enzyme thereby causing partial digestion and keeping the blood glucose level normal.
Conflicts of interest All authors have none to declare.
references
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