5.1 Introduction 5.2 Materials and methods Study of free radical scavenging activity of the plant extracts by DPPH

Transcription

1 5.1 Introduction 5.2 Materials and methods Study of free radical scavenging activity of the plant extracts by DPPH Evaluation of Reducing Power (RP) of the extracts Photochemilunescence (PCL) Thiobarbituric acid reactive substances (TBARS) Total phenolic content Development of callus from young leaves of S. pulcherrima 5.3. Results and Discussion Callus induction: Antioxidant Activity 5.4. Conclusion References

2 5. Antioxidant activity of medicinal plants 5.1 Introduction Apart from major macro and micronutrients there exist more than 300 nutrients, which are vital for the our body. In recent years, phytonutrients (nutrients in edible plants having antioxidant and anti-inflammatory) have acquired centre stage in the field of nutrition. Phytonutrients in the foods have biological property for disease prevention and health promotion. Truly nutritious diet is one that promotes health and prevents diseases. Free radicals have attracted a great deal of attention in recent years. Free radical production occurs continuously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in causing many diseases. Antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them or by promoting their decomposition (Young and Woodside 2001). Prasad and Kumar (1999) mentioned that supplementation with antioxidants may prevent Whereas, supplementation with multiple antioxidants at appropriate doses is essential because various types of free radicals are produced, antioxidants vary in their ability to quench different free radicals and cellular environments vary with respect to their lipid and aqueous phases. Maritim et al., (2003) mentioned that increased production of high levels of oxygen free radicals contribute to the development of diabetic complications. Nature has endowed us with protective antioxidant mechanisms such as superoxide dismutase (SOD), catalase, glutathione peroxidases and reductase apart from many dietary components (Ahmed 2005). The problem arises when there is imbalance between generation of free radicals and the protection system. Medicinal plants are considered to be an important source of antioxidant compounds and the therapeutic benefit of many medicinal plants is often attributed to their antioxidant properties (Hasani et al., 2007). Herbs and spices are the most important targets to search for 71

3 natural antioxidants. (Yanishlieva et al., 2006). Natural products from dietary components are known to possess antioxidant activity. There are epidemiological evidences correlating higher intake of foods with antioxidant abilities to lower incidence of various human morbidities or mortalities. Current research has unveiled various potential applications of antioxidant/free radical manipulations in prevention or control of disease. (Arnao et al., 2001; Kaur and Kapoor 2002; Mencherini et al., 2007; Miglio et al., 2008 ; Vinson et al.,2005). Devasagayam et al.,(2004) suggested inclusion of newer approaches such as gene therapy to produce more antioxidants in the body, genetically engineered plant products with higher level of antioxidants, synthetic antioxidant enzymes (SOD mimics), novel biomolecules and the use of functional foods enriched with antioxidants. A number of methods have been widely employed in the evaluation of antioxidant activity. Koleva et al.,(2002) compared t -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, static headspace gas chromatography (HS-GC) and - carotene bleaching test (BCBT), and concluded that, DPPH method is rapid, simple and independent of sample polarity, therefore, very convenient for the quick screening of many samples for radical scavenging activity. The free radicals and lipid peroxidation have been reported to be increased in the aged brain of rats. Lipid peroxidation is an important marker of oxidative stress. Shivakumar et al., (1991) recorded that, the levels of thiobarbituric acid reactive products, indicative of lipid peroxidation, were very low at birth and increased to adult levels by the 16th day after birth. Therefore, the present study was undertaken to put forward the possibility of exploring antioxidant phytochemicals in the five selected plant species abundantly grown in Cachar district of Assam, India and available in Sonitpur, Dhemaji and Tinsukia District of Assam. In addition, the antioxidant activity of the methanolic extract from callus developed from leaf explant of one of the cultivated sample of S. pulcherrima was performed. This will indeed 72

4 support in taking advantage of antioxidant phytochemical as an addition of a brick in the wall to defend against problem arising from the free radical in health. 5.2 Materials and methods Plants were selected based on the traditional information and personal field survey. Five plants namely, Clerodendron colebrookianum Walp. (Leaf), (Verbenaceae), Gnetum gnemon L. (Leaf), (Gnetaceae), Sarcochlamys pulcherrima (Roxb.) Gaud. (Leaf), (Urticaceae), Garcinia lancifolia (Don) Roxb (Leaf), (Clusiaceae) and Euryale ferox Salisb. (Seed), (Nymphaeaceae) were selected based on ethnobotanical information (cf. Chapter 3) and a common criteria of edibility to stay away to a greater extent from possible toxic nature of the active compounds. Preliminary screening for antioxidant activity of the plants samples, collected from Cachar District, Assam was done using standard protocol as mentioned below. Based on the findings, S. pulcherrima was selected for further evaluation. Different samples of this plant were collected from different hilly and plain locations of Assam, viz., Cachar (N1), Tinsukia (N2), Dhemaji (N3) and Sonitpur District (C1, C2, C3 and C4), consisting both cultivated (C) and naturally grown (N) plants. In vitro antioxidant activity of the callus, developed from young leaf explants of one cultivated sample from Sonitpur district was carried out Study of free radical scavenging activity of the plant extracts by DPPH Radical scavenging activity of the sample extracts were measured by colorimetric assay using 2,2-diphenyl picryl hydrazyl radical (DPPH, a stable free radical)) as a source of free radical in accordance with the method of Blois (1958). One ml of the test extract solution ( kept the reaction mixture for 35 min at room temperature in dark and absorbance was measured at 517nm using a UV-visible spectrophotometer. L-Ascorbic acid was used as the 73

5 positive control. A mixture of 1ml DPPH solution and 1ml methanol was used as blank. The DPPH radical scavenging activity was calculated using the following formula: % Inhibition = [(A 0 A s /A 0 ) 100] A 0 : absorbance of the control, A s : absorbance of test solution or the standard sample. EC 50 value i. e., the concentration of extract or standard to scavenge 50% of the initial DPPH was obtained. (Lesser the EC 50 value denotes higher the activity of the extract) Evaluation of Reducing Power (RP) of the extracts The antioxidant activity of the test extract is concomitant with the reducing power. Here, the ability of the test extracts to reduce Fe 3+ to Fe 2+ was estimated. Reducing power of the extracts was determined by using potassium ferricyanide-ferric chloride system. One ml 6.6), mixed with 2.5ml of potassium ferricyanide (0.1%) and then the mixture was incubated at 50 C for 20 min. Trichloroacetic acid of 10% concentration (2.5ml) was added to the reaction mixture and centrifuged at 10000g for 10 min. The supernatant (2.5ml) was mixed with equal volume of distilled water, followed by addition of 0.5ml of 0.1% ferric chloride solution. The absorbance of the resultant mixture was measured at 700nm using UV Visible spectrophotometer. Increased absorbance of the reaction mixture indicated the increased reducing power Photochemilunescence (PCL) Total antioxidant activity was determined by photochemilunescence method using integral ACL (antioxidative capacity of lipid soluble substances) kit in the antioxidant analyser. A photosensitizer substance in standardised volume, acts as a source of superoxide anion radical which produces the radicals by optical excitation. Residual radicals, remained 74

6 after partly reacting with the antioxidants, present in the sample cause the detector substance luminol to luminesce. The luminescence is then determined. Results are expressed in nmol equivalent of Trolox Thiobarbituric acid reactive substances (TBARS) Lipid peroxidation is an important marker of oxidative stress. Thiobarbituric acid (TBA) reaction was used to detect lipid oxidation. When lipids are oxidized, malondialdehyde is formed. TBA reacts with malondialdehyde in hot acid to form a red complex of malonaldehyde: TBA (1:2) with an absorbance maximum of 532nm. (The 1:1 intermediate is colorless). BALB/c mice were used in the present study. Methanolic extract and its ethyl acetate and n-butanol fractions were dissolved in water at the concentration of 20mg/ml by sonication (53 khz) for 10min with addition of 1% tween-40. Animals were administered with 0.1ml each test solution. The animals were sacrificed after 14 days of treatment. Isolated different organs and portion of it is kept in saline for estimation of malondiadehyde. About 100mg tissue was homogenized in 500µl of 0.1M phosphate buffer saline (ph 7.4) and added with 1ml of 28% trichloroaceticacid (TCA), vortexed and centrifuged at 2000g (4ºC) for 10 min. One ml of the supernatant was mixed with 900µl of 1% Thiobarbituric acid (TBA), volume was adjusted to 3ml by PBS, heated at 60ºC for half an hour, cooled and absorbance taken at 532nm. TBARS were calculated by following formula: L =Path length= 1cm g = Amount of tissue taken in gram. E 532 = Molar extinction coefficient = 1.56 X 10 5 m -1 cm -1 75

7 Total phenolic content Total Phenolic content were measured by Folinml -1 ) was added with 25ml distilled water and 5ml of Folinphenol reagent followed by 15ml of Na 2 CO 3 (20%, w/v) and the volume was made up to 100ml followed by incubation at room temperature for 2 hrs. The absorbance of all samples was measured at 760nm using a UV vis spectrophotometer. Results were expressed as milligram of gallic acid equivalent per milligram of dry weight (mg GAE/g dw) of extract Development of callus from young leaves of S. pulcherrima Fresh young leaves of S. pulcherrima were collected from Sonitpur district, Assam. The leaves were washed thoroughly for 30 minutes under running tap water, then washed properly with extran (1.0%) and again washed thoroughly under running tap water. In laminar flow air cabinet this leaves were surface sterilized with 70% aqueous ethanol (v/v) for 30 seconds followed by 0.1% HgCl 2 solution for 15 minutes and then rinsed thoroughly thrice with sterile double distilled water. Modified MS (Murashige and Skoog 1962) medium supplemented with 36 different combinations of growth regulators, NAA and BAP (Table...) and sucrose (30g L -1 ) were used. Table 5.1 Different combinations of hormone tested Hormones (Growth regulator) (mg/l) NAA BAP

8 The ph of the medium was adjusted at before autoclaving and the medium was gelled with agar (8g/L). The sterilized leaves were trimmed (1cm long) and inoculated onto the culture medium under aseptic condition. The cultures were incubated at 25±2ºC under 16 hours photoperiod of Lux fluorescence light intensity. Callus initiation was observed and after two months the callus was harvested. The harvested callus was air dried, powdered and extracted with methanol for further antioxidant study Results and Discussion Callus induction: For callus formation, the MS medium with NAA (2.0mg/L), BAP (1.5mg/L) was found to be the best (Figure 5.1). Callus initiation was observed after 10 days of inoculation. In other treatments, Mass culture of Callus Callus formed Harvested callus Figure 5.1: Callus formation and harvesting from S. pulcherrima leaf

9 callus was either not formed or delayed. The extractive value of the methanolic extract from two months old callus was recorded as 1.76%. The extract was further subjected to antioxidant test and phytochemical analysis Antioxidant Activity The results of in vitro antioxidant activity of the methanolic extracts from the test plant samples are depicted in table 5.2. In all the assays, S. pulcherrima was found to be the most potent plant as per antioxidant activity is concerned. Table 5.2: In vitro antioxidant activity of the methanolic plant extracts Plant Method DPPH EC50 $ PCL (RSD %) RP TPC C. colebrookianum ±1.09 a (0.03) a ± ± 1.92 E. ferox 4.20 ± 0.84 n.d ± ± 10.0 G. gnemon ± (2.53) 8.37 ± ± 3.85 G. lancifolia ± (0.39) 7.02 ± ± 4.14 S. pulcherrima 9.70 ±1.51 a (0.84) a ±3.10 a 0.33 ± a a Results are expressed as mean ±SD (n=6), except PCL data. The significant difference was analysed by one-way ANOVA followed by Tukeys post hoc test. p<0.05 was considered significant. Comparison is made between G. lancifolia with other plants. $ EC 50 In nmol equivalent of Trolox/g of dry extract, In mg Gallic Acid equivalent/mg of dried extract, Absorbance given by 1000ppm solution of extract 100 n.d. - not determined Considering the promising antioxidant activities, shown by the methanolic extract from S. pulcherrima, it was further fractionated to get the hexane, ethyl acetate and n-butanol fractions. These fractions were investigated for antioxidant activities and total phenolic 78

10 contents. The results, as shown in table 5.3 revealed that n-butanol fraction was the most potent with highest total phenolic content of ±6.89. The total phenolic content detected in methanolic extract was comparable with that of n-butanol fraction. Table 5.3. In vitro antioxidant activity and total phenolic content of different fractions of S. pulcherrima. Fraction Extractive value (%) DPPH EC 50 (ppm) RP TPC (mg/g GAE) Hexane ± ± ±3.62 Ethyl acetate ± ± ±7.63 n-butanol ± ± ±6.89 The results of the antioxidant and phytochemical study of the different samples of S. pulcherrima and the callus are shown in table 4. The samples from natural habitat exhibited higher activity (EC 50 : ppm) followed by the samples from cultivated one (EC 50 : ppm) and callus (EC 50 : 50.41ppm). Result of reducing power assay supported the activity profile in the DPPH assay. The extract from callus showed very less antioxidant activity compared to the most potent extract from natural S. pulcherrima sample. The probable reason for less antioxidant activity of the callus might be due to lack of optimum nutritional requirements for callus formation under in vitro conditions. Moreover, the inhibition pattern of the callus extract clearly indicated the dissimilarity with the extract from natural sample in terms of their antioxidant activity (Figure 5.2 and 5.3). Extract from the natural sample contains fast reacting substances which reacts immediately after adding DPPH solution while the extract from callus contains slow reacting substances, that unable to decolourise DPPH immediately. This indicated lack of biosynthesis of fast reacting chemical entity in the callus. 79

11 Absorbance Inhibition pattern of S. pulcherrima Callus (More absorbance less inhibition) Time (min) 75ppm 50ppm 25ppm Figure 5.2: Inhibition pattern of S. pulcherrima callus extract Absorbance Inhibition pattern of natural S. pulcherrima (More absorbance less Inhibition) Time (min) 20ppm 10ppm 5ppm Figure 5.3: Inhibition pattern of S. pulcherrima of natural habitat from different places of Assam was , which signified strong correlation between these two parameters (Table 4). The negative sign is for negative correlation i. e. lower value of EC 50 corresponds to higher value of TPC. this is in accordance with the resullt obtained by by Miliauskas et al., (2004). Flavonoids and saponins were detected in the extracts from natural and callus, whereas acidic compound was found only in the extract from natural sample (Table 5.4 and 5.5). 80

12 Table 5.4: Phytochemical analysis, in vitro antioxidant activity and total phenolic content of S. pulcherrima collected from different location. Samples DPPH EC 50 TPC* Flavonoid Saponin (ppm) N1 9.7± ± N2 17.4± ± N ± ± C1 30.3± ± C2 29.6± ± C3 35.7± ± C4 38.4± ± Callus 50.41±5.21 n. d N-Natural habitat, C-Cultivated, + -present, * in mg GAE/mg of dry extract N1- Cachar District, N2-Tinsukia, N3-Dhemaji, C1, C2, C3 and C4-Sonitpur District *Data of callus was not considered during correlation. Table 5.5: Comparison of antioxidant activity of S. pulcherrima sample and Callus S. pulcherrima Callus sample. Concentration (ppm) % Inhibition Concentration (ppm) % Inhibition Growth medium Not significant EC 50 =9.70 EC 50 =50.41 The results of the antioxidant activity of the methanol extract of S. pulcherrima and its ethyl acetate and n-butanol fractions, in lipid peroxidation experiment are displayed in table 5.6. According to the results obtained, the methanolic extract and n-butanol fraction significantly inhibited the formation of TBARS in liver homogenates. These results further indicated higher antioxidant activity of the methanolic extract over n-butanol fraction. Better antioxidant activity of the extract could be inferred due to synergistic action of the various components present in methanolic extract (Table 5.6). 81

13 Table 5.6: TBARS (Molg -1 tissue x 10-5 ) data in different organs of animals treated with methanolic extract from S. pulcherrima leaves and control group Extract (E)/ Organ Fraction (F) Lungs Heart Kidney Liver Methanol (E) ± ± ± ±0.12 Ethyl acetate (F) ± ± ± ±0.21 n-butanol (F) ± ± ± ±0.23 Control ± ± ± ±0.86 E- Extarct, F-Fraction Although the Methanolic extracts showed highest antioxidant activity in TBARS assay, but overall performance of n-butanol fraction in all the experiments were more promising. Hence, the n-butanol fraction was considered for further investigation to isolate and characterize the active compounds. Activity of the isolated compounds The isolated compound, B41showed EC µg ml -1, while the compound 8722 showed EC µg ml -1 in DPPH assay Conclusion The findings of this study substantiated that S. pulcherrima is the most potent antioxidant plant among the plants screened during this study. Methanolic extract from S. pulcherrima, collected from natural habitat showed higher antioxidant activity as compared to the extract from callus, which could be due to particular chemical entity in naturally grown samples. Flavonoids and saponins were detected in the extracts from natural samples and callus, whereas acidic compound was found only in the extract from natural sample. On the whole, the n-butanol fraction showed the highest antioxidant activity. Compounds isolated from the n-butanol fraction - B41and 8722 showed EC µg ml -1, µg ml -1 respectively. 82

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