The antioxidant potential of methanolic extracts of the flower, leaves, fruit, stem and root of Prinsepia utilis (PU) were tested using standard invitro models. The percentage of the phenolic compounds present was also determined. Among the methanolic extracts tested the successive methanol extract of Prinsepia utilis root (MPUR) exhibited strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radical, nitric oxide, hydroxyl radical by p-nitroso dimethyl aniline (p-NDA) method, hydrogen peroxide (H2O2), 2,2'- azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt (ABTS) radical cation with IC50 value of 5.38Â±0.02, 5.04Â±0.11, 900Â±0.06, 37.5Â±0.07, 0.414Â±0.45 Âµg/mL respectively. MPUR was further fractionated based on their polarity to check which fraction is most active in them. Hence all the methanolic extracts and fractions of PU were taken for invitro hepatoprotectivity study by using BRL-3A cell culture. Ethyl acetate fraction of PU (EAFPUR) at 125 and 250 ïg/ml showed good activity in invitro. Based on the above result EAFPUR was selected for in vivo antioxidant and hepatoprotective potential using carbon tetrachloride induced hepatitis rat model. The hepatoprotective and antioxidant activity of the EAFPUR was estimated by measuring the different parameters and histopathological study was also carried out. Rats treated with 100 and 400 mg/kg. b.wt. of EAFPUR along with CCl4 (1 ml / kg. b. wt.) showed significant restoration of all the biochemical parameters when compared with CCl4 treated rats.
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Free radicals are chemical species, which contains one or more unpaired electrons due to which they are highly unstable and cause damage to other molecules by extracting electrons from them in order to attain stability. Currently, there is great interest in finding antioxidants from natural sources to minimize oxidative damage (OD)to cells. OD is caused by free radicals and reactive oxygen species (ROS) (Leong, Tako, Hanashiro, & Tamaki, 2008). Free radical induced oxidative stress which will involve in preventive mechanisms, repair mechanisms, physical defenses and antioxidant defenses (Shahin et al., 2008). Many vegetable extracts show strong antioxidant activity that is linked to the presence of substances arising from the secondary metabolism and whose function in the plant is not always known. In herbal products, phenolic compounds have been shown to be the effective antioxidant constituents. Many polyphenolics exert a more powerful antioxidant effect than vitamin E in invitro and inhibit lipid peroxidation by chain breaking radical scavenging (Pilaipark et al., 2008).
Recently great importance is being given to naturally occurring antioxidants, which may play important roles in inhibiting both free radicals and oxidative chain reactions within tissues and membranes (Robert, Hiroe, & Yotaro, 2008). Antioxidants help organisms to deal with oxidative stress caused by free radical damage. It is commonly accepted that in a situation of oxidative stress, ROS such as superoxide (O.-, OOH.), hydroxyl (OH.) and peroxyl (ROO.) radicals are generated (Shahin et al., 2008) were primary targets are major intracellular and extracellular components, protein, lipids, and nucleic acids (Hiromasa, Takuya, Terumi, Yukari, Naoto, & Toshihiko, 2008). The ROS plays an important role in the pathogenesis of various disease conditions, such as neurodegenerative disorders, cancer, cardiovascular diseases, atherosclerosis, cataracts and inflammation (Filomena et al., 2008; Srinivasan, Chandrasekar, Nanjan, & Suresh, 2007; Leong, Tako, Hanashiro, & Tamaki, 2008). Antioxidants provide protection to living organisms from damage caused by uncontrolled production of ROS and the concomitant lipid peroxidation, protein damage and DNA strand breaking (Srinivasan, Chandrasekar, Nanjan, & Suresh, 2007). The use of traditional medicine is widespread and plants still represent a large source of natural antioxidant that might serve as leads for the development of novel drugs (Filomena et al., 2008; Srinivasan, Chandrasekar, Nanjan, & Suresh, 2007).
2. Materials and methods
2.1. Plant material
The PU was collected from the government horticulture garden, Ootacamund, in September 2006. The plant was authenticated by Botanical Survey of India, Medicinal Plant Survey and Collection Unit, Government Arts College, Ootacamund, Tamilnadu, India. A voucher specimen has been deposited for further reference at JSS College of Pharmacy herbarium, Ootacamund, India.
DPPH and ABTS were obtained from Sigma- Aldrich Co. St. Louis, USA. Rutin and p-NDA were obtained from Acros Organics, NJ, USA. Naphthyl ethylene diamine dihydrochloride (NEDD) was obtained from Roch-Light Ltd. Suffolk, UK. Ascorbic acid, Nitro Blue Tetrazolium (NBT) and Butylated Hydroxyl Anisole (BHA) was obtained from SD Fine Chemicals Ltd. Mumbai, India. Liv-52 from Himalay's drugs Pvt. Ltd. mumbai, India. Sodium Nitro Prusside (SNP) was from Ranbaxy Laboratories Ltd. Mohali, India. Sulphonic acid from E-Merck (India) Ltd. Mumbai, India. All chemicals used were of analytical grade.
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2.3. Extraction procedure
Stem, Leaves, Fruit, Flower and Root were separated, and shade dried. The dried parts were chopped and coarsely powdered, and they were extracted separately using methanol by Soxlation. The extracts were concentrated to dryness under reduced pressure and controlled temperature to yield deep brown-dark brown semi solids.
2.4. Preparation of test and standard solutions
All the extracts of PU and the standard antioxidants (ascorbic acid, rutin, BHA and Î±-tocopherol) were dissolved in Dimethyl sulfoxide (DMSO) separately and used for the in vitro antioxidant assay using eight different methods. For H2O2 method (where DMSO interferes with the method), the extracts and the standards were dissolved in methanol. The stock solutions were serially diluted with the respective solvents to obtain lower dilutions.
2.5. Total phenolic (TP) compound estimation
400 ÂµL of the extracts (1 mg/mL to 0.5 mg/mL) were separately mixed with 2 mL of Folin-Ciocalteu reagent and 1.6 mL of sodium carbonate. After shaking, it was kept for 2 h at room temperature for reaction time. The absorbance was measured at 750 nm in a spectrophotometer (Shimadzu UV-160 A Spectrophotometer, Shimadzu Corporation, Japan). Using gallicacid monohydrate, standard curve was plotted and linearity obtained was in the range of 2.5 to 25 Âµg/mL and the TP content of extracts was calculated (Sadasivam, & Manickam, 1992). The TP content was expressed as gallicacid equivalent in mg/g or %w/w of the extracts (Mills, & Bone, 2000).
2.6. Total flavonol (TF) compound estimation
0.5 mL of the extract was separately mixed with 1.5 mL methanol, 0.1 mL of 10 % aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm using Shimadzu UV-160A Spectrophotometer. Using rutin, standardcurve was plotted and linearity was obtained in the range of 1-10 Âµg/mL. The TF content of the plant extracts was calculated by interpreting the sample value in the standardcurve (Woisky, & Salatino, 1998). The TF content was expressed as rutin equivalent in mg/g or %w/w of the extracts. (Kaufman, Cseke, Sarawarber, Duke, & Brielmamm, 1999; Mills, & Bone, 2000).
2.7. In vitro antioxidant activity
All the extracts were tested for their in vitro antioxidant activity using the standard methods. In all these methods, a particular concentration of the extract or standard solution was used, which gave a final concentration of 1000- 0.45 Î¼g/mL after all the reagents were added. Absorbance was measured against a blank solution containing the extract or standard, but without the reagents. A control test was performed without the extract or standards. The Percentage of inhibition and IC50 values Â± Standard error mean (SEM) (IC50 values are the concentration of the sample required to inhibit 50 % of radical) were calculated.
2.7.1. DPPH radical scavenging method
The assay was carried out in a 96 well micro titre plate. To 200ÂµL of DPPH solution, 10ÂµL of each of the test samples or the standard solution was added separately in wells of the micro titre plate. The plates were incubated at 37Â°C for 20 min and the absorbance of each well was measured at 490 nm, using ELISA reader against the corresponding test and standard blanks and the remaining DPPH was calculated. (Gudda, Jayaprakasha, & Lingamallu, 2004; Pongtip, Roongtawan, & Wandee, 2005; Sanchez, 2002).
2.7.2. Nitric oxide radical inhibition assay
The reaction mixture (6 mL) containing sodium nitroprusside (10 mM, 4 mL), Phosphate buffer saline (1 mL) and 1 mL of extract in DMSO were incubated at 25Â°C for 150 min. After incubation 0.5 mL of the reaction mixture containing nitrate was removed and 1 mL of sulfanilic acid reagent was added, mixed well and allowed to stand for 5 min for completion of diazotization, then 1 mL of NEDD was added, mixed and allowed to stand for 30 minutes in diffused light at room temperature. The absorbance of these solutions was measured at 540 nm using ELISA reader against corresponding blank solution. IC50 value obtained is the concentration of the sample required to inhibit 50 % nitric oxide radical. (Gudda, Jayaprakasha, & Lingamallu, 2004; Sanchez, 2002).
2.7.3. Scavenging of hydroxyl radical by p-NDA method
To a reaction mixture containing ferric chloride (0.1 mM, 0.5 mL), ethylenediamine tetra acetic acid (EDTA) (0.1 mM, 0.5 mL), ascorbic acid (0.1 mM, 0.5 mL), H2O2 (2 mM, 0.5 mL) and p-NDA (0.01 mM, 0.5 mL) in a phosphate buffer pH 7.4 (20 mM) various concentrations of extracts or standards (0.5 mL) were added to give a final volume of 3 mL. Sample blank was prepared by adding 0.5 mL sample and 2.5 mL of phosphate buffer pH 7.4. Absorbance was measured at 440 nm,percentage scavenging was calculated from the control, where instead of extract,DMSO was present (Elizabeth, & Rao, 1990).
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2.7.4. Scavenging of hydrogen peroxide
H2O2 (20 mM) was prepared in Phosphate buffer saline (PBS) (pH 7.4). Various concentrations of 1 mL of the extracts or standards in methanol were added to 2 mL of H2O2 and the absorbance was measured at 230 nm (Gudda, Jayaprakasha, & Lingamallu, 2004; Sanchez, 2002).
2.7.5. Scavenging of ABTS radical cation
To 0.2 mL of various concentrations of the extract or standards, 1 mL of distilled DMSO and 0.16 mL of ABTS solution were added to make a final volume of 1.36 mL and incubated at room temperature and the absorbance was measured spectrophotometrically at 734 nm using an ELISA reader. Blank is maintained without ABTS. IC50 value obtained is the concentration of the sample required to inhibit 50 % ABTS radical mono cation. (Roberta, Nicoletta, Anna, & Ananth, 1999; Sanchez, 2002).
2.7.6. Scavenging of super oxide radical by alkaline DMSO method
To the reaction mixture containing 1 mL of alkaline DMSO, 0.3 mL of the extracts in DMSO at various concentrations were added to 0.1 mL of NBT (0.1 mg) to give a final volume of 1.4 mL. The absorbance was measured at 560 nm. (Elizabeth, & Rao, 1990; Sanchez, 2002).
2.7.7. Lipid peroxidation (LPO) inhibitory assay
The test samples (100 ÂµL) of different concentrations were added to 1 mL of egg lectin mixture; control was without test sample. LPO was induced by adding 10 ÂµL FeCl3 (400 mM) and 10 ÂµL ascorbic acids (200 mM). After incubation for 1 h at 37Â°C, the reaction was stopped by adding 2 mL of 0.25 N HCl containing 15 % Trichloro acetic acid (TCA) and 0.375 % Thiobarbituric acid (TBA) and the reaction mixture was boiled for 15 min, cooled, centrifuged and the absorbance of the supernatant was measured at 532 nm (Dhu, Yen, & Chang, 2001).
2.7.8. Scavenging of hydroxyl radical by the deoxyribose method
Various concentrations of the extracts, and standard in DMSO (0.2 mL) were added to the reaction mixture containing deoxyribose (3 mM, 0.2 mL), ferric chloride (0.1 mM, 0.2 mL), EDTA (0.1 mM, 0.2 mL), ascorbic acid (0.1 mM, 0.2 mL) and H2O2 (2 mM, 0.2 mL) in phosphate buffer (pH, 7.4, 20 mM) to give a total volume of 1.2 mL. The solutions were then incubated for 30 min at 37Â°C. After incubation, ice-cold TCA (0.2 mL, 15% w/v) and TBA (0.2 mL, 1% w/v in 0.25 N HCl) were added. The reaction mixture was kept in a boiling water bath for 30 min, cooled and the absorbance was measured at 532 nm (Halliwell, Gutteridge, & Aruoma, 1987).
2.7.9. Estimation of total antioxidant capacity (TAC) by Phosphomolybdenum method
100 ÂµL of extract is dissolved in 1 mL of TAC reagent. Blank is maintained with distilled water replacing the TAC reagent. Absorbance was seen at 695 nm. (Gudda, Jayaprakasha, & Lingamallu, 2004; Pongtip, Roongtawan, & Wandee, 2005).
2.8. Fractionation of active extracts based on its polarity
Among the five methanolic extracts of PU tested for invitro antioxidant studies, the root extracts showed potent activity. Hence this extract was fractionated based on their polarity to check which fraction is most active in them. The Root extracts (40g) were extracted with different solvent (Petroleum ether, Chloroform, Ethyl acetate, n-Butane and Water) by using separating funnel and separated, filtered, and dried.
2.9. Invitro hepatoprotective evaluation of methanolic extracts and fraction of prinsepia utilis using BRL-3A cell culture.
All the fraction of root extracts of Prinsepia utilis was tested for their invitro hepatoprotective activity using established liver cell culture BRL-3A and different biological parameter were estimated.
The mono layer of the cell culture (BRL-3A) was trypsinized, and the cell count was adjusted to 1.0 Ã-105 cells/ ml using a medium containing 10% calf serum.
To each well of the 96 well micro titer plates, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. The plates were incubated at 37Â° C in 5% carbon dioxide CO2.
After 24 hours, when the partial monolayer was formed, the supernatant was flicked off, washed the monolayer once.
All the wells, except cell control wells were intoxicated with 50 Âµl of 1% carbon tetrachloride (CCl4), followed by addition of 50 Âµl of different concentrations of test drugs.
The cell control wells received 100 Âµl of a maintenance medium. Then the plates were incubated at 37Â° C in 5% CO2 atmosphere for 24 hours.
Toxicant control with CCl4 intoxicated wells without drug treatment, and standard (silymarin) were also maintained.
After incubation, toxicant and drug/ A standard treated cell supernatants was pooled and collected separately in Eppendorf tubes and centrifuged at 4,000 rpm for 15 minutes.
Supernatants were collected and liver enzymes such as Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (ALP), and Total Bilirubin were estimated.
2.10. Invivo antioxidant and hepatoprotective studies on the most potent fraction using CCl4 intoxicated rat model.
In vitro antioxidant andhepatoprotective activity conducted on a different part of methanol extracts and different root fractions of Prinsepia utilis revealed that the ethyl acetate fraction of root extracts had maximum antioxidant content in it. Hence this fraction was selected for invivo antioxidant andhepatoprotective activity by CCl4 intoxicated rat model.
Experimental model: Wistar rats both sex weighing (180-220 g) was used for the experimental study. The animals were divided into 6 groups of six animals each. Group I served as solvent control (normal animals). Group II served as toxicant control and received 1% CMC 0.2 ml for 100 g rat for 7 days followed bythe single administration of CCl4 (1 ml/kg, i.p) on the 8th day. Group III served as positive control and received Liv-52, 2 ml /kg for 7 days followed bythe single administration of the toxicant on the 8th day. Group IV received the low dose of ethyl acetate fraction 25mg/kg b.w, which was dissolved in 1% CMC and 0. 1ml was administered orallyfor 100g rat. Group V received the middle dose of ethyl acetate fraction 100mg/kg b.w, which was dissolved in 1% CMC and 0.1 ml were administered orallyfor 100 g rat. Group VI received the high dose of ethyl acetate fraction 400 mg/kg b.w, which was dissolved in 1% CMC and 0. 1ml was administered orally for 100g rat. On the 7th day after one hour of extract administration, the toxicant CCl4 (1 ml/kg, i.p) was administered to group IV, V and VI. After 24 hours the animals were anaesthetized and blood was collected by Sino-orbital puncture on the 8th day. The blood was centrifuged at 2,000 rpm for 15- 20 minutes. The plasma was separated and was used biochemical estimations like ASAT, ALAT, ALP and bilirubin. Then the liver was removed, homogenized and was used estimations of catalase, SOD and Lipid peroxidase.
2.11. Histopathological Studies
The livers were removed from the animals, and the tissues were fixed in 10% formalin for at least 24 h. Then the paraffin sections were prepared (automatic tissue processor, Autotechnique) and cut into 5 Âµm thick sections in a rotary microtome. The sections were stained with Haematoxylin-Eosin dye and were studied for histopathological changes (Mukherjee, 1988).
2.12. Statistical analysis
Results are expressed as mean Â± SEM. Comparisons among the groups were tested by one-way ANOVA using Graph Pad Prism, Version 4.0 (Graph Pad Software, San Diego, CA. USA).
3.1. TP and TF content
Table 1 reports the results of TP and TF contents of the different parts of PU. The percentage of TP varied widely and ranged from 0.461-10.38 %w/w of extract. Among the plant extracts, MPU of the flower (MPUF) contained the highest amount of phenolics (10.38 %w/w) followed by MPU of leaves (MPUL) (10.25 %w/w), MPU of stem (MPUS) (7.70 %w/w), MPU of fruit (MPUFR) (4.99 %w/w), whereas the lowest level was found in MPU of root extract (MPUR) (0.461 %w/w). Among the extracts, MPUF contained highest amount of flavonoids (560 mg/g), whereas the lowest level was found in MPUR and MPUFR extracts (280 mg/g) (Table 1).
3.2. In vitro antioxidant activity
In this study, the antioxidant potential of the various extracts was estimated by nine methods viz., scavenging of ABTS radical cation, DPPH radical scavenging method, scavenging of H2O2, LPO inhibitory activity, nitric oxide radical inhibition assay, scavenging of hydroxyl radical by deoxyribose method, scavenging of hydroxyl radicalby p-NDA method, scavenging of super oxide radicalby alkaline DMSO method and the estimation of total antioxidant capacity by Phosphomolybdenum method. Among the methanolic extracts tested for in vitro antioxidant activity, MPUR exhibited potent antioxidant activity in DPPH radical scavenging method, nitric oxide radical inhibition assay, scavenging of hydroxyl radical by p-NDA method, scavenging of H2O2, scavenging of ABTS radical cation and the IC50 value were found to be 5.38Â±0.02, 5.04Â±0.11, 900Â±0.06, 37.5Â±0.07, 0.414Â±0.45 Î¼g/mL respectively (Table 2). The values were found to be comparable to the standards used. However, MPUR was found to be poor in LPO inhibitory method with IC50 value of 400Â±0.57 Î¼g/mL and almost in active in alkaline DMSO method (Table.2). The MPUF showed moderate antioxidant activity in DPPH, nitric oxide radical inhibition assay, scavenging of H2O2, ABTS with IC50 value of 48.29Â±0.02, 46Â±0.11, 150Â±0.08, 4.14Â±0.03 Î¼g/mL respectively. It was found to be potent in LPO method. In all in vitro antioxidant methods, MPUS showed moderate activity in all the methods, and it was found to be in active in p-NDA, alkalineDMSO and Deoxyribose method (Table 2). MPUL extracts exhibited moderate activity in DPPH, nitric oxide radicalinhibition assay with IC50 value of 29Â±0.74, 26.2Â±0.12 Î¼g/mL respectively and poorantioxidant activity in H2O2, ABTS and LPO method with IC50 value of 202.5Â±0.11, 15.62Â±0.05, and 400Â±0.57 Î¼g/mL respectively, and it was found to be in active in alkalineDMSO (Table 2). MPUFR showed poorantioxidant activity in DPPH, nitric oxide radicalinhibition assay with IC50 value of 73.03Â±0.01, 68.5Â±0.06 Î¼g/mL respectively and moderate activity in H2O2, ABTS, and LPO with IC50 value of 37.5Â±0.07, 0.414Â±0.45, 237.5Â±1.36 Î¼g/mL respectively. MPUFR was found to be inactive in p-NDA and alkaline DMSO method (Table 2).
There are several methods for the determination of antioxidant activities. The chemical complexity of extracts, often a mixture of dozens of compounds with different functional groups, polarity and chemical behavior could lead to scattered results, depending on the test employed. Therefore, an approach with multiple assays for evaluating the antioxidant potential of extracts would be more informative and even necessary (Mehmet, Fatma, Mehmet, & Gulactu, 2007).
The data presented in this study demonstrate that almost all the reported species possess antioxidant and free radical scavenging activity. Among the methanolic extracts tested for in vitro antioxidant activity, MPUR showed potent activity in most of the methods. Other extracts showed moderate antioxidant activity in all the methods. None of the extracts showed activity in scavenging of superoxide radical assay and only MPUR, showed activity in p-NDA method. Thedifferent behavior of MPUR extracts probably due to thedifferent mechanism involved in the steps of an oxidation process. The observed in vitro activities suggest that the MPUR could exert protective effects also in vivo against oxidative and free radical injuries occurring in different pathological conditions. It is well known that phenolic compounds on the plant kingdom are mainly responsible for the antioxidant potential of plants (Ozsoy, Can, Yanardag, & Akev, 2008). The antioxidant activity of plant extracts is due primarily to phenolic compounds. Several studies evaluated the relationships between antioxidant activity of plant products and their phenolic content. Some authors found a correlation between the phenolic, flavonolic content and antioxidant activity, while others found no relationship (Filomena et al., 2008). In this study, the findings did not show any relationship between antioxidant activity and TP and TF content. Among all the extracts analyzed MPUFL had higher phenolic and flavonol content when compared to other extracts. MPUR extracts was found to be a potent antioxidant activity in most of the antioxidant method but had very less phenol content and flavonol content. The relatively high antioxidant and free radical scavenging activity of extracts containing low phenolic content suggests that the type of phenolics is the determinant for their activities rather than their quantity. These results agree with those of Robert, Hiroe, & Yotaro, 2008; Filomena et al., 2008. WHO reported that differences in antioxidant activities of plant extracts could be due to different quantitative and qualitative composition of their phenolic constituents, from phenolic acids to flavonoids and their derivatives. For instance, the antioxidant activities of phenolic acid and their derivatives, such as esters depend on the number of hydroxyl groups in the molecules (Filomena et al., 2008). The reported plant is a throny shrub; grow up to 3-5 m in height, distributed through the Himalaya, Khasi and jaintia hills of assam. It has run wild in the Nilgiris since its introduction. It is reported to be suitable hydrogenation and soap making. The oil posses rubifacient properties and applied rheumatism and pains resulting from over fatigue (Anon., 1989). In conclusions, this study clearly reveals that MPUR has potent invitro free radicals scavenging effect in different invitro models. MPUR is, therefore, a potential therapeutic, thus making it an excellent candidate for more detailed investigations.