4.1. ENZYMIC ANTIOXIDANT ACTIVITIES IN M. hortensis LEAVES

Transcription

1 4. RESULTS Free radicals are constantly generated in vivo for physiological purposes. They can be over produced in pathological conditions, causing oxidative stress. A large number of diseases such as autoimmune diseases, inflammation, cardiovascular, neurological diseases and cancer are attributed to oxidative stress. An adequate intake of natural antioxidants is believed to protect the macromolecules against this oxidative damage in cells (Riaz et al., 2011). The body is endowed with both endogenous (catalase, superoxide dismutase, glutathione peroxidase / reductase) and exogenous (vitamins C and E, carotene, uric acid) defense systems against free radicals generated within it. These systems are, however, not sufficient in certain situations in which the production of free radicals significantly increases. The beneficial effects of phytochemicals in this direction are associated with a number of their biological activities including antioxidant and free radical scavenging properties (Oyebanji and Saba, 2011). There is currently a strong interest in plants as pharmaceuticals, especially from edible plant parts, because these compounds play an important role in preventing free radical induced diseases such as cancer. This interest focused not only on the discovery of new biologically active molecules by the pharmaceutical industry, but also on the adoption of the crude extract of the plants, such as infusions for self medication by the public (Haripyaree et al., 2010). The free radical neutralizing property of the extracts from a number of medicinal plants is gaining a lot of importance. They are known to have some biologically active principles and are used in Ayurvedic preparations (Mandade et al., 2011). Many synthetic drugs protect against oxidative damage but they have adverse side effects. An alternative solution to the problem is to consume natural antioxidants from food supplements and traditional medicines. Recently, many natural antioxidants have been isolated from different plant materials (Hazra et al., 2008). In the present study, we have studied the antioxidant activity of M. hortensis leaves. The results obtained are presented below. 1

2 PHASE I The antioxidant contents present in the leaves of M. hortensis were analyzed. Both enzymic and non-enzymic antioxidants were quantified and the values obtained are presented below ENZYMIC ANTIOXIDANT ACTIVITIES IN M. hortensis LEAVES The enzymic antioxidants analysed in the leaves of M. hortensis were superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR), glutathione S-transferase (GST) and polyphenol oxidases (PPO). The activities obtained are presented in Table 4.1. The results revealed that the leaves of M. hortensis possess considerable activities of all the enzymic antioxidants studied. It is evident from the above tabulated values that the leaf of M. hortensis is a good source of enzymic antioxidants NON-ENZYMIC ANTIOXIDANT LEVELS IN M. hortensis LEAVES The levels of non-enzymic antioxidants, namely ascorbic acid, tocopherol, reduced glutathione, total phenols, total flavonoids, total chlorophyll, total carotenoids and lycopene are presented in Table 4.2. The results revealed that the leaves of M. hortensis exhibited appreciable amounts of all the non-enzymic antioxidants analysed. Therefore, it is evident that the leaves of the candidate plant are a rich source of antioxidants, both enzymic and non-enzymic. PHASE II Knowing that the M. hortensis leaves (Plate 4.1) are rich in antioxidants, further analysis was carried out to assess the free radical scavenging activity of the same. In order to identify the active principle and the solvents into which the maximum amount of antioxidants got extracted, the leaves of M. hortensis were serially extracted into solvents of increasing polarity (petroleum ether, benzene, chloroform, ethyl acetate and methanol) using a Soxhlet apparatus. An aqueous extract was also prepared (as mentioned in the methodology chapter). These extracts were then tested for their radical scavenging effects. 2

3 TABLE 4.1 ENZYMIC ANTIOXIDANT ACTIVITIES IN M. hortensis LEAVES ENZYMES ACTIVITIES Superoxide dismutase (U/g leaf) # ± 0.86 Catalase (U/g leaf) $ ± 3.50 Peroxidase (U/g leaf)* ± 0.02 Glutathione reductase (U/g leaf) ± 0.39 Glutathione S-transferase (U/g 0.16 ± 0.04 Catechol oxidase (Units X 10-3 / g leaf) 0.54 ± 0.19 Laccase (Units X 10-3 / g leaf ) 0.50 ± 0.05 The values are mean ± S.D of triplicates. # 1 Unit = Amount of enzyme that causes 50% reduction in NBT oxidation $ 1 Unit = Amount of enzyme required to decrease the absorbance at 240nm by 0.05 units/minute * 1 Unit = Change in absorbance at 430 nm/minute + 1 Unit = mmoles of NADPH 1 Unit = nmoles of CDNB conjugated/minute 1 Unit = Amount of catechol oxidase/laccase enzyme which transforms 1 unit of dihydrophenol to quinine /minute TABLE 4.2 NON-ENZYMIC ANTIOXIDANT LEVELS IN M. hortensis LEAVES * = catechin equivalents ^= catechol equivalents PARAMETERS LEVELS Ascorbic acid (mg/g leaf) 1.70 ± 0.01 Tocopherol (µg/g leaf) 3.59 ± 0.25 Reduced glutathione (nmoles/g leaf) ± 15.1 Total phenols (mg*/g leaf) ± 0.14 Total flavonoids (mg^/g leaf) 5.08 ± 0.51 Total carotenoids (mg/g leaf) ± 0.39 Lycopene (mg/g leaf) 4.26 ± 0.01 Total chlorophyll (mg/g leaf) 3.96 ± 0.22 The values are mean ± S.D. of triplicates 3

4 PLATE 4.1 Majorana hortensis LEAVES Radical Scavenging Effects of M. hortensis Leaf Extracts The extracts were tested for their radical scavenging effects against a battery of oxidant moieties that included the radicals DPPH, ABTS, H 2 O 2 (non-radical), OH, SO and NO. The ability of the different leaf extracts to scavenge DPPH was tested in a rapid dot blot screening and quantified using a spectrophotometric assay. The picture obtained in the dot blot screening is shown in Plate 4.2, where all the extracts showed significant free radical scavenging ability. The maximum activity was observed in the methanolic extract. 1 Petroleum ether 4 Ethyl Acetate 2 Benzene 5 Methanol 3 Chloroform 6 Water PLATE 4.2 DPPH DOT BLOT Assay DPPH and ABTS Radical Scavenging Activity of M. hortensis Leaf Extracts The per cent extent of DPPH and ABTS scavenging by the M. hortensis leaf extracts were carried out spectrophotometrically and the results are presented in Figure 4.1. It was observed that M. hortensis leaf extracts effectively reduced the stable radical DPPH to the yellow-coloured compound diphenylpicryl hydrazine. The maximum extent of both DPPH 4

5 and ABTS radical scavenging was elicited by the methanolic extract, followed by the aqueous extract. DPPH and ABTS scavenging effects of the other solvent extracts were found to be moderate. The minimum radical scavenging activity was exhibited by the petroleum ether extract Hydrogen Peroxide Scavenging Activity of M. hortensis Leaf Extracts The ability of M. hortensis leaf extracts to scavenge H 2 O 2 in an in vitro system was studied and the results are also expressed in Figure 4.1. All the different solvent extracts of M. hortensis leaves exhibited strong H 2 O 2 -scavenging effects. Though the extents of scavenging varied, the methanolic extract showed the maximum scavenging activity, followed by the aqueous extract. The least scavenging activity was observed in the petroleum ether extract Hydroxyl Radical Scavenging Activity of M. hortensis Leaf Extracts The hydroxyl radical has high reactivity and is short-lived. The extent of TBARS produced in the reaction is taken as a measure of hydroxyl radical production. The inhibition of TBARS production is, thus, considered as a measure of hydroxyl radical scavenging efficiency. The exposure to H 2 O 2 caused the maximum damage, which was very effectively reduced by the presence of the leaf extracts. The methanolic extract exhibited the maximum extent of radical scavenging (Figure 4.2). The other solvent extracts also showed a varied percent of free radical scavenging activity though not as much as the methanolic extract of the M. hortensis leaf Effect of M. hortensis Leaf Extracts on the in vitro Generation of Superoxide and Nitric Oxide Radicals The per cent inhibition of SO and NO generation in the presence of the leaf extracts was calculated and the values are depicted in Figure 4.3. All the different solvent extracts of the leaves were found to be very good scavengers of superoxide in vitro, with the maximal inhibitory effect found in the methanolic extract followed closely by the aqueous, chloroform and ethyl acetate extracts. A reduction in NO generation was also observed with all the different extracts of M. hortensis leaves. The methanolic extract showed the maximum inhibition of nitric oxide generation, closely followed by the aqueous extract. 5

6 Percent Radical Scavenged Petroleum ether Benzene Chloroform Ethyl acetate Methanol Water The values are Mean ± S.D. of triplicates FIGURE 4.1 : DPPH, ABTS AND H 2 O 2 SCAVENGING EFFECTS OF M. hortensis LEAVES Percent TBARS Formed No Extract Petroleum ether Without DPPH ABTS H 2 O 2 Benzene Chloroform Ethyl acetate With H 2 O 2 H 2 O 2 Methanol Water The values are Mean ± S.D. of triplicates The value of H 2 O 2 -treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups FIGURE 4.2 : HYDROXYL RADICAL SCAVENGING EFFECT OF M. hortensis LEAVES Percent Inhibition of SO and NO generation Superoxide Nitric Oxide Petroleum ether Benzene Chloroform Ethyl acetate Methanol Water The values are Mean ± S.D. of triplicates The extent of inhibition of nitric oxide generation in vitro was found to be almost similar to that of the extent of inhibition of SO generation FIGURE 4.3 : SUPEROXIDE AND NITRIC OXIDE SCAVENGING EFFECTS OF M. hortensis LEAVES 6

7 The results of all the above revealed that the methanolic extract exhibited the maximum scavenging activity of all the radicals tested, compared to all the other extracts. Therefore, only this extract was taken forward for the further studies. Once the extract with the maximum scavenging activity was identified, the minimum concentration at which this extract would evoke the maximum antioxidant response was analyzed in order to decide on the dose to be used in the further experiments. For this purpose, a set of dose-response experiments were conducted. Different concentrations of the methanolic extract of the leaves, ranging from 0.1 to 0.4 mg were subjected to a battery of radical quenching assays (DPPH, ABTS, H 2 O 2, SO, NO and OH scavenging). The results obtained are depicted in Tables 4.3 and 4.4. Leaf Extract (mg) TABLE 4.3 M. hortensis LEAF EXTRACT DOSE OPTIMIZATION Percent Inhibition of in vitro Percent Radical Scavenging Generation DPPH ABTS H 2 O 2 SO NO ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.28 The values are Mean ± S.D. of triplicates TABLE 4.4 HYDROXYL RADICAL SCAVENGING EFFECTS OF M. hortensis LEAF EXTRACT FOR DOSE OPTIMIZATION Percent TBARS formed Dose of Extract Without H 2 O 2 With H 2 O ± mg ± ± mg ± ± mg ± ± mg ± ± 0.57 The values are Mean ± S.D. of triplicates The value of H 2 O 2 -treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups 7

8 The values obtained showed that the extent of scavenging increased upto the dose of 200 µg, and thereafter exhibited a plateau. This clearly indicated that 200 µg was the optimal dose that could be employed for further study. Therefore, only this dose level was used in subsequent experiments EFFECT OF M. hortensis LEAVES ON OXIDATIVE DAMAGE TO BIOMOLECULES Normal aerobic metabolism is associated with ROS that can damage cellular macromolecules (Breimer and Mikhailidis, 2011). Free radicals are by-products of metabolism, which, in regard to their chemical structure, readily react with biomolecules namely, DNA, lipids, proteins and carbohydrates, and cause changes in their structure and function (Kupczyk et al., 2010). Hence, it is very crucial to study the extent of oxidative damage to biomolecules by standard oxidants in the presence of the component under study for antioxidant activity. For this, the methanolic extract (0.2 mg) of M. hortensis was studied on the extent of oxidative damage to lipids, DNA and proteins Extent of Inhibition of in vitro Lipid Peroxidation The damage to lipids and the extent to which the leaf extract inhibited this process was quantified by measuring the extent of lipid peroxidation (LPO). To ascertain the damage to lipids, three different membrane models were studied. They were RBC ghosts (plasma membrane devoid of intracellular membranes), liver homogenate (a mixture of plasma membrane and internal membranes) and precision-cut liver slices (intact cells). The extent of inhibition of this LPO was studied in the presence of the leaf extract. The per cent inhibition of in vitro lipid peroxidation by the leaf extract in all the three membrane systems is presented in Table 4.4. The maximum inhibition of LPO was observed in the goat liver homogenate, followed by the liver slices and then the RBC ghosts. These results indicated that the lipid components of the liver homogenate, which constitute both the plasma and internal membranes, can be protected from oxidative damage by the leaf extract to a higher magnitude compared to the other lipid preparations in the presence of the leaf extract. 8

9 Percent Inhibition RBC ghosts Liver homogenate Liver slices The values are Mean ± S.D. of triplicates FIGURE 4.4 : INHIBITION OF LIPID PEROXIDATION IN DIFFERENT MEMBRANE PREPARATIONS BY M. hortensis LEAVES Protective Effects of the M. hortensis Leaves on Oxidative Damage to DNA The ultimate biomolecular target of the oxidative assault is DNA. The extent of protection rendered by the leaf extract to DNA exposed to oxidants was studied. Here, different sources of DNA, belonging to various evolutionary hierarchical levels, were used for the analysis. Both the commercially available DNA preparations and DNA from intact cells were used. They were, Lambda DNA (linear, viral phage) puc18 DNA (plasmid, circular, bacterial) Herring sperm DNA (genomic, haploid, fish) Calf thymus DNA (genomic, diploid, mammal ) Human peripheral blood lymphocytes (intact human cells) i) Protective Effects of the Leaf Extract to λ DNA and puc18 DNA The extent of damage induced by H 2 O 2 to DNA from these sources and the protective effects of the extract were studied by viewing the migration pattern of the DNA in agarose gels. The results are presented in Plate 4.3. H 2 O 2 caused a significant extent of damage to 9

10 both λ and puc18 DNA. This was evident by the absence of the specific bands in lane 2, wherein the DNA was treated with oxidant alone. The weakening of the bands in lane 2 suggested that the DNA was severely damaged resulting in very small fragments that cannot be visualized sharply on the gel. M. hortensis leaf extract reversed this damage, which could be seen in lane 4, as indicated by the intact bands. The leaf extract, by itself, did not cause any DNA damage. This observation was reiterated by the Integrated Density Values (IDV) of the bands, recorded using a digital gel documentation software (Alpha Ease FC of Alpha Digidoc 1201), the values of which are presented in Table 4.5. (a) Lambda DNA (b) puc18 DNA Lane 1: Control; Lane 2: H 2 O 2 ; Lane 3: Leaf extract; Lane 4: Leaf extract + H 2 O 2 PLATE 4.3 MIGRATION PATTERNS OF λ DNA AND puc18 DNA TREATED WITH H 2 O 2 WITH AND WITHOUT M. hortensis LEAF EXTRACT Among the two DNA preparations from the lower organisms, the bacterial plasmid DNA was more susceptible to oxidative damage and was also more receptive to the protective effect by the leaf extract. The extent of damage by H 2 O 2 in the DNA from the viral source was lower; however, the extent of protection was also lower in λ DNA. The IDV recorded clearly proved this observation. 10

11 TABLE 4.5 IDV OF THE BANDS IN THE AGAROSE GEL OF DNA DAMAGE IN λ DNA and puc18 DNA Sample IDV of the bands of λ DNA IDV of the bands puc18 DNA Without H 2 O 2 With H 2 O 2 Without H 2 O 2 With H 2 O 2 No Extract Leaf Extract ii) Protective Effect of M. hortensis Leaf Extract on H 2 O 2 Induced Damage to Herring Sperm and Calf Thymus DNA The results of the quantification of oxidative damage to herring sperm DNA is schematically presented in Figure 4.5. It was found that H 2 O 2 caused an increased extent of damage to herring sperm DNA. The extent of damage decreased markedly in the presence of the leaf extract. This indicated the protective effect rendered by the leaf extract against the oxidant. Similar results were observed with calf thymus DNA as well (Figure 4.6). This proved that M. hortensis leaf extract possess good protective effect against oxidative damage to DNA. iii) Effect of M. hortensis Leaf Extract on the Damage Induced by H 2 O 2 to DNA in Intact Cells The DNA damaging effect of H 2 O 2 was studied by following the formation of comets in human peripheral blood cells exposed to the oxidant in vitro. The effect of the leaf extract on this process was followed, and the results are presented in Table 4.6. Plate 4.4. The photographic record of the comets in each of the treatment groups is depicted in 11

12 120 PERCENT TBARS PRODUCED No Extract Leaf Extract Without H 2 O 2 With H 2 O 2 The values are Mean ± S.D. of triplicates. The value of H 2 O 2 -treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups FIGURE 4.5 : INHIBITION OF OXIDANT-INDUCED DAMAGE TO HERRING SPERM DNA BY M. hortensis LEAF EXTRACT 120 PERCENT TBARS PRODUCED No Extract Leaf Extract Without H 2 O 2 With H 2 O 2 The values are Mean ± S.D. of triplicates The value of H 2 O 2 -treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups FIGURE 4.6 : INHIBITION OF OXIDANT-INDUCED DAMAGE TO CALF THYMUS DNA BY M. hortensis LEAF EXTRACT 12

13 TABLE 4.6 EFFECT OF M. hortensis LEAF EXTRACT ON DNA DAMAGE INDUCED BY H 2 O 2 IN HUMAN PERIPHERAL BLOOD CELLS Treatment Groups No. of cells with comets/100 cells Without H 2 O 2 With H 2 O 2 No Extract 5 ± 1 29 ± 1 a Leaf Extract 11 ± 1 a 18 ± 2 a,b,c The values are Means ± SD of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group H 2 O 2 exposure caused a steep increase in the number of cells with comets. In the positive control (cells treated only with H 2 O 2 ), the DNA was severely damaged. The Majorana hortensis leaf extract also showed the presence of a significant number of comets with reference to the untreated controls. The simultaneous treatment with the leaf extract and H 2 O 2 decreased the number of cells expressing the DNA damage significantly. Thus, the results indicated that the leaf extract was able to protect the DNA in peripheral blood cells against oxidative damage (Plate 4.4). These observations suggest that the leaf extract is effective in counteracting the DNA damage Protective Effect of M. hortensis Leaf Extract on Oxidative Damage to Proteins i) Effect of M. hortensis Leaf Extract on Protein Carbonyl Formation Figure 4.7 clearly depicts that there is a significant increase in the formation of protein carbonyl in the presence of the oxidant. When the methanolic extract of M. hortensis leaves was co-administered, there was a decrease in the oxidation of proteins when compared to the oxidant alone treated group. This proves the protective effect of the leaf extract against the oxidation of proteins. 13

14 Control Hydrogen peroxide Methanolic Extract Methanolic Extract + H 2 O O 2 PLATE 4.4 COMET BEARING PERIPHERAL BLOOD LYMPHOCYTES ii) Effect of M. hortensis Leaf Extract on Protein Migration on 1D Gel In an attempt to study the effect of the leaf extract on a mixture of proteins subjected to oxidative stress in vitro, a mixture of bovine serum albumin and ovalbumin was prepared in PBS and incubated in the presence and/or absence of H 2 O 2 and lesaf extract. An aliquot of the reaction mixture was loaded onto polyacrylamide gel and electrophoresed in the presence of SDS. The results of this SDS-PAGE showed five distinct bands. The intensity of the bands in the H 2 O 2 -exposed group (lane 2) showed a clear decrease when compared to that of the control (lane 1). The leaf extract was able to prevent this decrease to a large extent (lane 4) (Plate 4.5). The results thus far obtained, clearly indicated the strong free radical scavenging and biomolecule-protecting effects of the leaf extract. Further to this, an extensive study was formulated to analyze the effects of the leaf extract on live cells and tissues, in the presence and the absence of induced oxidative stress. 14

15 The in vitro models adapted were goat liver slices, Saccharomyces cerevisiae cells, primary chick embryo fibroblasts and cancer cell lines. The goat liver slicess were taken as a model that simulates in vivo conditions. The model was validated in earlier studies in our research group (Sumathi, 2007; Vidya, 2007; Nirmaladevi, 2008; Radha 2010; Sreelatha and Padma, 2010) STUDIES ON THE ANTIOXIDANT STATUS IN LIVER SLICES EXPOSED TO OXIDANT AND LEAF EXTRACT in vitro The liver is a key metabolic organ and it plays an important role in the homeostasis ( Precision-cut liver slices constitute an in vitro model representing the liver under in vivo conditions. Hence, the precision-cuboth in the presence and in the absence of the extracts of M. hortensis leaves. The enzymic goat liver slices were challenged with an oxidant (H 2 O 2 ) (SOD, CAT, Px, GST and GR) and non-enzymic (vitamin C, vitamin E, vitamin A and reduced glutathione) antioxidants were analyzed in the homogenate of the liver slices. Protein carbonyl (nmol/mg) No Extract Leaf Extract Without H 2 O 2 With H 2 O 2 FIGURE 4.7 EFFECT OF M. hortensis LEAF EXTRACT ON PROTEIN CARBONYL FORMATION 15

16 IDV of bands Control H 2 O 2 Leaf Leaf Extract+H 2 O 2 Band Band Band Band Band PLATE 4.5 EFFECT OF M. hortensis LEAF EXTRACT ON THE MIGRATION OF PROTEINS SUBJECTED TO OXIDATIVE STRESS Enzymic Antioxidan Status in Liver Slices Exposed to Oxidant and Leaf Extract in vitro The activities of the major enzymic antioxidants were assayed in the liver slice homogenate prepared after exposure to H 2 O 2 in the presence/absence of the leaf extract. The enzymes assayed were SOD, CAT, Px, GST and GR (Table 4.7). Upon exposure to the oxidant (H 2 O 2 ), all the enzymic antioxidants showed significantly (P<0.05) reduced activities. The treatment with M. hortensis leaf extract significantly (P<0.05) elevated all the enzyme activities, which remained elevated even in the presence of the oxidant. The enzymic antioxidant activities in the group treated with the leaf extract, in the presence of the oxidant, were well over the activities in the untreated group. 16

17 TABLE 4.7 EFFECT OF M. hortensis LEAF EXTRACT ON ENZYMIC ANTIOXIDANTS ACTIVITIES IN GOAT LIVER SLICES EXPOSED in vitro TO H 2 O 2 Enzymic Antioxidants SOD (units#/g tissue) CAT (units $ /g tissue) Px (units * /g tissue) GST / g tissue) GR (units + /g tissue) Untreated control H 2 O 2 treated Groups Leaf extract treated H 2 O 2 + leaf extract treated 9.09 ± ± 0.44 a ± 0.55 a ± 0.79 a,b,c ± ± 2.86 a ± 3.19 a ± 2.14 a,b,c 6.69 ± ± 1.40 a ± 0.84 a ± 0.62 a,b,c 0.27 ± ± 0.01 a 0.56 ± 0.05 a 0.50 ± 0.05 a,b,c 2.48 ± ± 0.10 a 2.60 ± 0.08 a 2.26 ± 0.50 a,b,c Values are mean ± S.D. of triplicates # 1 Unit = 50% inhibition of NBT reduction in one minute $ 1 Unit = Amount of enzyme required to decrease the absorbance at 240nm * 1 Unit = Changes in absorbance at 430 nm/minute + 1 Unit = mmoles of NADPH 1 Unit = nmoles of CDNB conjugated/minute a statistically significant (p<0.05) compared to untreated control b statistically significant (p<0.05) compared to H 2 O 2 control c statistically significant (p<0.05) compared to the respective plant control The non-enzymic antioxidant levels in the group treated with the leaf extract in the presence of the oxidant, were well over the levels in the untreated group Non-Enzymic Antioxidant Levels in Liver Slices Exposed to Oxidant and Leaf Extract in vitro The levels of vitamins C, E, A and reduced glutathione were estimated in the liver slices exposed to H 2 O 2 in the presence or the absence of M. hortensis leaf extract. The levels of these non-enzymic antioxidants in the various treatment groups are presented in Table 4.8. Upon exposure to the oxidant (H 2 O 2 ), all the non-enzymic antioxidants showed significantly (P<0.05) reduced values. The treatment with M.hortensis leaf extract significantly (P<0.05) elevated all the non-enzymic levles, which remained elevated even in the presence of the oxidant. 17

18 TABLE 4.8 EFFECT OF M. hortensis LEAF EXTRACT ON NON-ENZYMIC ANTIOXIDANT LEVELS IN GOAT LIVER SLICES EXPOSED in vitro TO H 2 O 2 Non-enzymic Antioxidant Vitamin C (mg/g tissue) Vitamin E (µg/g tissue) Vitamin A (µg/g tissue) Reduced Glutathione (nmoles/g tissue) Untreated control H 2 O 2 treated Group Leaf extract treated H 2 O 2 + leaf extract treated ± ± 0.55 a ± 1.01 a ± 1.30 a,b,c 7.28 ± ± 0.15 a 9.91 ± 0.12 a 8.81 ± 0.11 a,b,c ± ± 0.01 a ± 4.98 a ± 4.98 a,b,c a a,b The values are mean ± S.D. of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant control Thus, the results showed that the leaf extract of M. hortensis can improve the antioxidant status in the goat liver slices exposed in vitro to oxidative stress. Since the model was carefully planned to simulate in vivo conditions, it is perceivable that the effects will be the same in the intact system too HISTOPATHOLOGY OF THE LIVER SLICES SUBJECTED TO OXIDATIVE STRESS Histopathological analysis (Plate 4.6) was carried out to confirm the spectrophotometric analysis. In the control group, the liver tissue showed normal architecture with intact portal triad. In the presence of the oxidant, the liver tissue showed cellular edema. This is presumably due to the presence of stress, which caused the normal cells to swell and exhibit edema, thereby blocking the sinusoids or minimizing the sinusoidal spaces. The liver tissue in the presence of the plant extract alone showed a lower extent of cellular edema. These results 18

19 Control Hydrogen Peroxide Methanolic Extract Methanolic Extract+H 2 O 2 PLATE 4.6 HISTOPATHOLOGICAL ARCHITECTURE OF THE GOAT LIVER SLICES When the oxidant was co-administered along with the leaf extract, notable damage was observed in certain areas, but some parts of the liver tissue showed recovered areas with normal architecture. The periportal areas showed some edema but the peripheral areas showed preserved architecture. This indicated that the methanolic extract of the M. horntesis leaves is effective in protecting the liver tissue from oxidative damage. PHASE III The results of the first two phases of the study clearly indicated the antioxidant potential of the M. hortensis leaf extract. Therefore, the next phase was initiated to validate the effect of the leaf extract on oxidative cell death. For this, the apoptosis modulating effects of the leaf extract were studied under unstressed and oxidatively stressed conditions. 19

20 This effect was analyzed on both untransformed Saccharomyces cerevisiae and primary chick embryo fibroblasts (non-cancerous) and transformed Hep2 (cancerous) cells. For Saccharomyces cerevisiae cells, H 2 O 2 was used to induce oxidative stress. In the primary cells and in the cancer cell line, etoposide (a standard cancer chemotherapeutic agent that induces cell death via oxidative stress) was used as the oxidant EFFECT OF M. hortensis LEAF EXTRACT ON H 2 O 2 -INDUCED APOPTOSIS IN S. cerevisiae CELLS Yeast cells were used to study the effects of the plant extract on the apoptotic events associated in the presence of the oxidant, H 2 O 2. In order to understand the nature of the cellular death process and the molecular events involved in the process, studies were conducted on morphological and nuclear changes that occur during apoptotic death Morphological Changes of Apoptosis Observed in S. cerevisiae Cells The characteristic morphological changes in apoptotic cells were analysed by Giemsa staining in the presence and the absence of the leaf extract and/or H 2 O 2. The number of apoptotic and non-apoptotic cells was counted under a phase contrast microscope (Table 4.9). TABLE 4.9 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL CHANGES IN S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY GIEMSA STAINING TREATMENT GROUPS Number of Apoptotic Apoptotic Ratio Cells /100 Cells Control H 2 O 2 treated Control H 2 O 2 treated No Extract 8 ± 2 75 ± 1 a Leaf Extract 16 ± 1 a 26 ± 1 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group It was noted that the oxidative stress induced group shows a significant (P<0.05) increase in the number of apoptotic cells (Plate 4.7a) compared to the untreated (control) group. In the presence of the leaf extract, the number of apoptotic cells decreased 20

21 significantly, indicating the anti-apoptotic activity rendered by the leaf extract. The apoptotic ratios in the treated and untreated cells were calculated and the values obtained are represented in Table Nuclear Changes of Apoptosis Observed in S. cerevisiae Cells The apoptotic nuclei stain strongly with the fluorescent dyes, which allow the nonapoptotic cells to be discriminated from the apoptotic ones. The nuclear changes associated with apoptosis were followed after staining the treated cells with the fluorescent dyes, namely EtBr (Plate 4.7b), PI (Plate 4.7c) and DAPI (Plate 4.7d) the number of apoptotic cells counted in the various staining experiments are presented in Tables 4.10 to 4.12 respectively. The apoptotic ratios were also calculated for each group and are listed alongside the number of apoptotic cells in Tables 4.10 to 4.12 respectively. The results of EtBr, PI and DAPI staining indicated that the number of apoptotic cells was highest in the oxidant treated group, which was significantly (P<0.05) reduced when coadministered with the leaf extract. This indicates the protective effect rendered by the M. hortensis leaf extract showing the strong apoptotis inhibiting effect. TABLE 4.10 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY EtBr STAINING Treatment Groups Number of Apoptotic Cells /100 Cells Apoptotic Ratio Control H 2 O 2 treated Control H 2 O 2 treated No Extract 10 ± 2 72 ± 1 a Leaf Extract 15 ± 1 a 23 ± 1 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group 21

22 Control H 2 O 2 Methanol Extract Methanol+H 2 O 2 (a) S. cerevisiae cells stained with Giemsa Control H 2 O 2 Methanol Extract Methanol+H 2 O 2 (b) S. cerevisiae cells stained with EtBr Control H 2 O 2 Methanol Extract Methanol+H 2 O 2 (c) S. cerevisiae cells stained with PI Control (d) H 2 O 2 Methanol Extract Methanol+H 2 O 2 S. cerevisiae cells stained with DAPI PLATE 4.7 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL AND NUCLEAR CHANGES INDUCED BY H 2 O 2 IN S.cerevisiae CELLS 22

23 TABLE 4.11 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY PI STAINING Treatment Groups Number of Apoptotic Cells / 100 Cells Control H 2 O 2 treated Apoptotic Ratio Control H 2 O 2 treated No Extract 11 ± 2 74 ± 1 a Leaf Extract 20 ± 1 a 30 ± 1 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group TABLE 4.12 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY DAPI STAINING Treatment Groups Number of Apoptotic Cells / 100 Cells Apoptotic Ratio Control H 2 O 2 treated Control H 2 O 2 treated No Extract 9 ± 2 78 ± 1 a Leaf Extract 17 ± 1 a 31 ± 1 a,b,c The values are mean ± S.D. of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to H 2 O 2 alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group Effect of M. hortensis Leaves on the Viability of S. cerevisiae Cells The extent of cell survival and the influence of the leaf extract of M. hortensis on the survival was studied by MTT and the per cent cell viability was quantified (Figure 4.8). It is evident from the values that H 2 O 2 exposure markedly decreased the viability of S. cerevisiae cells. The co-administration of the leaf extract showed improved viability of the cells subjected to oxidative stress. This indicates that a significant protection was rendered by the methanolic extract of M. hortensis leaf extract. The plant extract, by itself, also caused cell death to very slight extent in the yeast cells. 23

24 Sulphorhodamine B assay was used as an additional parameter to calculate the cell viability and proliferative potential of S. cerevisiae cells in the presence and the absence of H 2 O 2 and/or the leaf extract of M. hortensis. Figure 4.9 illustrates the results obtained for the cell viability study in yeast using SRB. The viability of the cells decreased in the H 2 O 2 treated group due to the effect of the oxidant. The groups that were simultaneously treated with the leaf extract showed improved cell viability indicating the protective effect of the leaf extract Effect of M. hortensis Leaves on the Viability of S. cerevisiae Cells Subjected to Oxidative Stress (LDH Assay) LDH release has been considered as a very reliable marker of membrane damage due to cell lysis, indicating cytotoxicity. This is indicative of the protection rendered by the leaf extract towards the cytotoxicity. The cytotoxicity in the cells treated with or without H 2 O 2, in the presence or absence of M. hortensis leaf extract was also determined by the LDH release. The results are depicted in Figure H 2 O 2 exposure caused a steep rise in the extent of apoptosis in yeast cells. When administered along with H 2 O 2, the leaf extract resulted in a significant decrease in the LDH release DNA Fragmentation in Yeast DNA fragmentation in apoptotic S. cerevisiae cells were assayed using diphenylamine in a spectrophotometric assay and the per cent extent of fragmentation obtained is shown in Figure The exposure of H 2 O 2 to the S. cerevisiae cells caused significant DNA damage. The co-administration of the methanolic extract of M. hortensis leaves reduced the extent of DNA damage indicating the anti-apoptotic effect of M. hortensis leaf extract. 24

25 Percent Cell Viability No Extract Without Leaf Extract H With H FIGURE 4.8 Effect of M. hortensis leaf extract on the viability of S. cerevisiae cells subjected to oxidative stress as determined by MTT assay The values are mean ± SD of triplicates The values of the untreated (negative) control group were fixed as 100% and the per cent viabilities in the other groups were calculated relative to this FIGURE 4.9 Effect of M. hortensis leaf extract on the viability of S. cerevisiae cells subjected to oxidative stress as determined by SRB assay The values are mean ± SD of triplicates The values of the untreated (negative) control group were fixed as 100% and the per cent viabilities in the other groups were calculated relative to this Percent Cell Viability No Extract Without Leaf Extract With H H Percent Cytotoxicity No Extract Without Leaf Extract H With H FIGURE 4.10 Effect of M. hortensis leaf extract on percent cytotoxicity in S. cerevisiae cells as determined by LDH release The values are means ± S.D. of triplicates 100 FIGURE 4.11 Effect of M. hortensis leaf extract on DNA damage in S. cerevisiae cells subjected to oxidative stress The values are means ± S.D. of triplicates Percent DNA Damage No Extract Leaf Extract Without H With H

26 4.7. EFFECT OF M. hortensis LEAF EXTRACT IN ETOPOSIDE INDUCED STRESS IN PRIMARY CHICK EMBRYO FIBROBLASTS AND Hep2 CELLS It is evident from the results obtained from the S. cerevisiae cells that M. hortensis leaves can render protection to these cells against oxidative stress. As the next step of the study, it was felt necessary to study the effect of the leaf extract on cancer cells. This was done because cancer is recognized to be a result of oxidative genotoxicity. As a control to the cancerous cells, non-cancerous primary cultured chick embryo fibroblasts were used. was employed as the oxidant to induce the oxidative stress. The influence of the etoposide in the presence and the absence of the M. hortensis leaf extract in both chick embryo fibroblasts (Plate 4.8a) and Hep2 cells (Plate 4.8b) were evaluated by various (membrane and nuclear) staining techniques and the cytotoxicity assays Effect of M. hortensis Leaf Extract on the Morphological Changes in Induced Stress in Primary Chick Embryo Fibroblasts and Hep2 Cells The morphological changes observed in primary chick embryo fibroblasts and Hep2 cells stained with Giemsa are depicted in Table 4.13 and Table 4.14 respectively. caused a steep increase in the number of cells (cancerous) showing apoptotic morphology in both chick embryo fibroblasts and Hep2 cells (Plates 4.9a and 4.10a). However, the effect of M. hortensis leaf extract in the two types of cells, showed a markedly differential response. In the chick embryo fibroblasts, the presence of the extract, along with the oxidant showed a recovery in survival, with a decrease in the apoptotic cells (Plate 4.9a). Whereas, in the case of Hep2 cells, the administration of the leaf extract alone increased the number of apoptotic cells compared to control, indicating anticancer activity of the leaf extract. Co-exposure of the Hep2 cells with leaf extract and etoposide caused a further increase in the number of apoptotic cells. This observation indicates that the M.hortensis leaf extract augments the cytotoxicity of the chemotherapeutic agent (etoposide) only in the cancer cells, while protecting the non-cancerous cells from its cytotoxicity. 26

27 a) Primary Chick Embryo Fibroblasts b) Hep2 cells PLATE Effect of M. hortensis Leaf Extract on the Nuclear Changes of Apoptosis Observed Primary Chick Embryo Fibroblasts and Hep2 Cells The nuclear changes in the cancerous and non-cancerous cells by etoposide and its modulation in the presence/absence of leaf extract were studied using the nuclear stains, namely EtBr, PI and DAPI. 27

28 TABLE 4.13 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL CHANGES IN CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY GIEMSA STAINING Treatment Groups Number of Apoptotic Cells / 100 Cells Without With Without Apoptotic Ratio With No Extract 5 ± 1 71 ± 3 a Leaf Extract 9 ± 1 a 25 ± 4 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group TABLE 4.14 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL CHANGES IN Hep2 CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY GIEMSA STAINING Treatment Groups Number of Apoptotic Cells/100 Cells Without With Apoptotic Ratio Without With No Extract 11 ± 2 68 ± 1 a Leaf Extract 20 ± 1 a 76 ± 2 a,b,c The values are mean ± S.D. of triplicates. a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group In both, the primary chick embryo fibroblasts and Hep2 cells, there was an increase in the number of cells showing nuclear apoptotic morphology when subjected to etoposide treatment. The co-administration of the M. hortensis leaf extract with etoposide, showed a striking increase in the number of surviving cells, when compared to the cells treated with etoposide alone, in chick embryo fibroblasts. In Hep2 cell, however, the presence of the leaf extract, along with etoposide, increased the proportion of dying cells, when compared to the group treated with etoposide alone. The observation of this differential effect is very 28

29 significant, as the M. hortensis leaf extract protects noncancerous cells from oxidative death, at the same time rendering the cancerous cells more susceptible to the chemotherapeutic agent-induced oxidative death. The photographic evidence of the noncancerous and cancerous cells showing nuclear changes is presented in Plates 4.9 and 4.10 respectively. The individual cell numbers are the calculated apoptotic ratios of both types of cells are listed in Tables 4.15 to 4.20 respectively. TABLE 4.15 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY EtBr STAINING Treatment Groups Number of Apoptotic Cells / 100 Cells Without With Apoptotic Ratio Without With No Extract 3 ± 1 78 ± 1 a Leaf Extract 10 ± 1 a 23 ± 1 a,b,c The values are mean ± S.D. of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group TABLE 4.16 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN Hep2 CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY EtBr STAINING TREATMENT GROUPS Number of Apoptotic Cells/100 Cells Without With Apoptotic Ratio Without With No Extract 10 ± 2 75 ± 1 a Leaf Extract 22 ± 1 a 79 ± 1 a,b,c The values are mean ± S.D. of triplicates. a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group 29

30 TABLE 4.17 EFFECT OF M. hortensis LEAF EXTRACT ON NUCLEAR CHANGES IN CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY PI STAINING Treatment Groups Number of Apoptotic Cells/100 Cells Without With Apoptotic Ratio Without With No Extract 5 ± 1 78 ± 2 a Leaf Extract 12 ± 2 a 23 ± 4 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group TABLE 4.18 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN Hep2 CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY PI STAINING Treatment Groups Number of Apoptotic Cells /100 Cells Without With Apoptotic Ratio Without With No Extract 10 ± 2 73 ± 1 a Leaf Extract 18 ± 1 a 81 ± 2 a,b,c The values are mean ± S.D. of triplicates. a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group 30

31 Leaf Extract + Control Leaf Extract (a) Primary Chick Embyro Fibroblasts stained with Giemsa Control Leaf Extract + Leaf Extract (b) Primary Chick Embyro Fibroblasts stained with EtBr Control Leaf Extract + Leaf Extract (c) Primary Chick Embyro Fibroblasts stained with PI Control Leaf Extract + Leaf Extract (d) Primary Chick Embyro Fibroblasts stained with DAPI PLATE 4.9 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL AND NUCLEAR CHANGES IN PRIMARY CHICK EMBRYO FIBROBLASTS 31

32 Control Leaf Extract (a) Hep2 Cells Stained With Giemsa Leaf Extract + Control Leaf Extract (b) Hep2 Cells Stained With Etbr Leaf Extract + Control Leaf Extract (c) Hep2 Cells Stained With PI Leaf Extract + Control Leaf Extract (d) Hep2 cells stained with DAPI Leaf Extract + PLATE 4.10 EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL AND NUCLEAR CHANGES IN Hep2 CELLS 32

33 TABLE 4.19 EFFECT OF M. hortensis LEAF EXTRACT ON NUCLEAR CHANGES IN CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY DAPI STAINING Treatment Groups Number of Apoptotic Cells/100 cells Without With Apoptotic Ratio Without With No Extract 4 ± 1 68 ± 4 a Leaf Extract 8 ± 1 a 26 ± 2 a,b,c The values are mean ± S.D of triplicates a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group TABLE 4.20 EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN Hep2 CELLS SUBJECTED TO OXIDATIVE STRESS AS DETERMINED BY DAPI STAINING Treatment Groups Number of Apoptotic Cells/100 Cells Without With Apoptotic Ratio Without With No Extract 12 ± 2 71 ± 1 a Leaf Extract 19 ± 1 a 80 ± 2 a,b,c The values are mean ± S.D. of triplicates. a Statistically significant (P<0.05) compared to untreated control b Statistically significant (P<0.05) compared to etoposide alone treated group c Statistically significant (P<0.05) compared to the respective plant extract treated group 33

34 Effect of M. hortensis Leaf Extract on the Cell Viability of Primary Chick Embryo Fibroblasts and Hep2 Cells Cell viability was deduced by the MTT and SRB assays. In the chick embryo cells, the cytotoxicity of etoposide was reduced by the presence of the leaf extract of M. hortensis. The leaf extract, by itself, also caused a decrease in the viability of Hep2 cells, which was decreased further in the presence of etoposide (Figure 4.12). These observations suggest that the plant extract enhances the action of etoposide on Hep2 cells, while inhibiting the toxicity of etoposide to the primary cells (Figure 4.13). Similar results were obtained for the SRB assay, which was done to confirm the results of MTT. The figures 4.14 and 4.15 show the cell viability results Effect of M. hortensis Leaf Extract on the LDH Release in Primary Chick Embryo Fibroblasts and Hep2 Cells In the present study, the extent of cell death due to oxidative stress and the effect of the leaf extract on this process was confirmed by another assay, namely the release of LDH. This enzyme is a very reliable marker of membrane damage, which, in turn, is indicative of cell death. Hence, the extent of release of LDH from the primary as well as cancer cells, subjected to various treatments, was analyzed. The cytotoxicity in the untransformed cells, i.e., primary chick embryo fibroblasts, when treated with etoposide, showed a higher extent of LDH release due to the cytotoxic effect of the standard chemotherapeutic drug, etoposide. This value was drastically reduced in the presence of the plant extract due to the protection rendered by the M.hortensis leaf extract (Figure 4.16). In the case of the cancerous cells, Hep2, the cytotoxicity was increased in the group of cells that were treated with etoposide. Additionally, in the cells treated with both the drug and the leaf extract, the extent of LDH release increased further. This observation showed the anticancer activity rendered by the leaf extract (Figure 4.17). Thus, the LDH release assay also reiterated the differential effect of the M. hortensis leaf extract on the different types of cells. 34

35 Percent Cell Viability No Extract Leaf Extract Without With Figure 4.12 Effect of M.hortensis Leaf Extract on the Viability of Chick Embryo Fibroblasts Subjected to Oxidative Stress as Determined by MTT Assay Percent Cell Viability No Extract Without Figure 4.14 Effect of M.hortensis Leaf Extract on the Viability of Primary Chick Embryo Fibroblasts Subjected to Oxidative Stress as Determined by SRB Assay Percent Cytotoxicity No Extract Without Leaf Extract With Leaf Extract With Percent Cell Viability No Extract Without Figure 4.13 Effect of M.hortensis Leaf Extractt on the Viability of Hep2 Cells Subjected to Oxidative Stress as Determined by MTT assay Percent Cell Viability No Extract Without Figure 4.15 Effect of M.hortensis Leaf Extractt on the Viability of Hep2 Cells Subjected to Oxidative Stress as Determined by SRB Assay Percent Cytotoxicity No Extract Without Leaf Extract With Leaf Extract With Leaf Extract With Figure 4.16 Figure 4.17 Effect of M. hortensis leaf extract on percent Effect of M. hortensis leaf extract on percent cytotoxicity in primary chick embryo fibroblasts cytotoxicity in Hep2 cells as determined by LDH as determined by LDH release release The values are mean ± SD of triplicates The values of the untreated (negative) control group were fixed as 100% and the per cent viabilities in the other groups were calculated relative to this. 35

36 Effect of M. hortensis Leaf Extract on the DNA Fragmentation in Primary Chick Embryo Fibroblasts and Hep2 Cells against Induced Oxidative Stress The phenomenon of apoptosis is well characterized by DNA fragmentation. In the present study, the extent of DNA fragmentation in the chick embryo fibroblasts and Hep2 cells subjected to the various treatments was analyzed by agarose gel electrophoresis. Exposure of the primary chick embryo fibroblasts and Hep2 cells to the oxidant, etoposide, caused DNA damage as evidenced by a faint band (Lane 2). M. hortensis leaf extract by itself, did not cause a major damage (Lane 3). When the leaf extract was co- caused by administered along with the oxidant, it decreased the extent of DNA damage etoposide (lane 4) in the primary cells, (Plate 4.11a) whereas, in Hep2 cells, the administration of the plant extract augmented the extent of damage caused by etoposide (lane 4) (Plate 4.11b). The IDV quantifying the intensities of the DNA bands are tabulated (Table 4.21). This clearly depicts the differential role played by M. hortensis leaf extract; in the untransformed cells, it exhibited anti-apoptotic activity and in transformed cells, it augmented the activity of the anticancer agent. a) Chick Embryo Fibroblasts b) Hep2 Cells Lane 1 Untreated group Lane 2 treated group Lane 3 Majorana hortensis leaf extract treated group Lane 4 Majorana hortensis leaf extract + PLATE 4.11 EFFECT OF M. hortensiss LEAF EXTRACT ON THE DNA FRAGMENTATION PATTERN OF PRIMARY CHICK EMBRYO FIBROBLASTS AND Hep2 CELLS 36

37 TABLE 4.21 IDV OF THE BANDS IN THE AGAROSE GEL OF DNA FRAGMENTATION Assay OF PRIMARY CHICK EMBRYO FIBROBLASTS AND Hep2 CELLS IDV OF THE BANDS Sample Primary Chick Embryo Fibroblasts Without With Hep2 Cells Without With No Extract Methanolic Extract PHASE IV The first three phases of the study evidenced that M. hortensis leaves are a good source of antioxidants, and show very good anti-apoptotic activity in non-cancerous cells and pro-apoptotic activity in cancer cells. These properties are presumably rendered by the chemical substances or the secondary metabolites present in the leaves that get extracted into methanol. Therefore, it becomes highly essential to identify the active principle(s) rendering the protective effects of M. hortensis leaves. Hence, the fourth phase of this study emphasized on the qualitative identification of the chemical nature of the active component present in the candidate plant. This was followed by spectral studies such as UV absorption spectrum, TLC, HPTLC, HPLC, IR and GC-MS to identify the major components present in the leaves of M. hortensis PRELIMINARY QUALITATIVE PHYTOCHEMICAL ANALYSIS The fresh leaves of M. hortensis were subjected to phytochemical analysis to identify the presence of the major phytochemicals. The qualitative test showed the presence of alkaloids, phenols, flavonoids, saponins, sterols and tannins (Table 4.22). From these results, it can be inferred that the active components in M. hortensis leaves may be alkaloids, phenols, flavonoids, sterols, saponins or tannins. Hence, these phytochemical fractions were isolated and subjected to UV absorption. 37

38 TABLE QUALITATIVE PHYTOCHEMICAL ANALYSIS OF M. hortensis LEAVES S. No. COMPONENTS RESULT 1. ALKALOIDS Mayer s test + Dragondroff s test + Wagner s test + 2. PHENOLS Ferric chlride + Lead acetate + 3. FLAVONOIDS Aqueous NaOH test + Concentrated sulfuric acid test + Schinado s test + 4. STEROIDS Leibermann-Buchard test + Salkowski test + 5. SAPONINS Froth test + Haemolytic test + 6. TANNINS Braemer s test UV ABSORPTION SPECTRUM OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis LEAVES The absorption spectrum of the different fractions namely, alkaloids, phenols, flavonoids, sterols and tannins of the M. hortensis leaves were evaluated in the UV range which gave specific absorption spectrum. 38

39 The alkaloid fraction of M. hortensis leaves (Figure 4.18) showed several major and minor peaks, beginning with a sharp peak at 225 nm, followed by another major peak at 250 nm. A few more well-defined peaks were noted at 300, 320, 330 and 360 nm respectively. Figure 4.19 shows the UV absorption spectrum of the phenolic fraction, which revealed a minor peak at 200 nm, a major peak at 220 nm. Also, 350, 380 and 395 nm exhibited sharp major peaks, indicating the presence of major active principles present in the leaf extract. A few other minor peaks were also noted. The UV absorption spectrum of the flavonoid fraction indicated well defined major peaks at 230, 300 and 325 nm. At 310 nm also a peak was observed, though not well defined. A few minor peaks were also noticed beginning with 205 nm as indicated in Figure The UV absorption spectrum of the saponin fraction was determined and the spectrum is presented in Figure The peaks observed here were similar to the flavonoids and a few peaks coincided. At 230, 310 and 325 nm saponins also exhibited peaks as in flavonoids. Figure 4.22 showed the peaks obtained from the steroid fraction by UV absorption. Several major peaks at 195, 240, 250, 275, 290 and 305 nm were observed and a minor peak at 230 nm. The UV absorption pattern of tannins (Figure 4.23) showed well-defined peaks at 200, 225, 235, nm. Major peaks were noted at nm and 370 nm. Other well defined peaks were observed at 360, 380 and 395 nm TLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis LEAVES The TLC plate, when detected with the alkaloid-specific Dragendroff s spraying reagent, showed six major bands with R f vales 0.83, 0.74, 0.65, 0.53,0.40 and 0.26 (Plate 4.12a). The presence of phenolics was analysed using Folin-Ciocalteau reagent as the spraying reagent. The results are shown in Plate 4.12b, wherein five major spots with R f values 0.68, 0.66, 0.53, 0.50 and 0.48 were visualized. The investigation of flavonoids separated by TLC, sprayed with 10% vanillin in sulphuric acid, showed four major bands 39

40 with R f values 0.81, 0.73, 0.62 and 0.50 as seen in Plate 4.12c. The saponinn fraction showed three major bands as indicated in Plate 4.12d with R f values 0.75, 0.71 and The sterols were also subjected to TLC analysis and the chromatogram was sprayed with 10% sulphuric acid which showed five distinct bands with R f values 0.80, 0.78, 0.63, 0.49, and 0.44 (Plate 4.12e). The number of tannin bands was found to be five, with R f values 0.76, 0.73, 0.67, 0.48 and 0.19 respectively, which were developed by spraying 10% sulphuric acid (Plate 4.12f). FIGURE 4.18 UV ABSORPTION SPECTRUM OF THE ALKALOID FRACTION OF M. hortensis LEAVES FIGURE 4.19 UV ABSORPTION SPECTRUM OF THE PHENOLIC FRACTION OF M. hortensis LEAVES 40

41 FIGURE 4.20 UV ABSORPTION SPECTRUM OF THE FLAVONOID FRACTION OF M. hortensis LEAVES FIGURE 4.21 UV ABSORPTION SPECTRUM OF THE SAPONIN FRACTION OF M. hortensis LEAVES 41

42 FIGURE 4.22 UV ABSORPTION SPECTRUM OF THE STEROID FRACTION OF M. hortensis LEAVES FIGURE 4.23 UV ABSORPTION SPECTRUM OF THE TANNIN FRACTION OF M. hortensis LEAVES 42

43 a) Alkaloids b) Phenolics c) Flavonoids d) Saponins e) Sterols f) Tannins PLATE 4.12 TLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis LEAVES HPTLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis LEAVES The methanolic extract of M. hortensis leaves was subjected to HPTLC analysis for the presence of alkaloids, phenols, flavonoids, saponins, steroids and tannins. The alkaloid profile of the methanolic extract was done with the refernce standard colchicine and the developed plate was sprayed with Dragendroff s reagent. Orange-brown coloured zone at day light mode present in the given standard and sample tracks obser observed in 43

44 the chromatogram after derivatization confirmed the presence of six alkaloids in the leaves (Plate 4.13). The peak table (Table 4.23) and peak densitogram (Figure 4.24) were recorded. The phenolics present in the methanolic extract of M. hortensis leaves were analysed using quercetin as the reference standard. Orange-brown colored zones at visible light mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of phenolics in M. hortensis leaves (Plate 4.14). The peak table (Table 4.24) and peak densitogram (Figure 4.25) showed the presence of four phenols. The flavonoid profile of the methanolic extract of M. hortensis leaves was analysed using rutin as the standard. Yellow and yellow green fluoresecenc zone at UV 366 nm was seen from the chromatogram, which confirmed the presence of flavonoids (Plate 4.15). There were six different flavonoids identified in the methanolic extract of M. hortensis leaves as shown in the peak table (Table 4.25) and peak densitogram (Figure 4.26). Plate 4.16 confirmed the presence of saponins in the methanolic extract of M.hortensis leaves where saponin standard was used. Blue, yellowish brown coloured zones in the visible light mode were present in the track observed from the chromatogram after derivatization, which confirmed the presence of saponins in the given samples. The peak table (Table 4.26) and the peak densitogram (Figure 4.27) represented 9 different saponins. The steroid profile of the methanolic extract of M. hortensis leaves was analyzed using solasodine as the standard. Blue-violet coloured zones in the day light mode present in the given standard and sample tracks observed in the chromatogram after derivatization confirmed the presence of sterols in the M. hortensis leaves (Plate 4.17). The peak table (Table 4.27) and the peak densitogram (Figure 4.28) confirmed the presence of 5 different steroids. Using tannic acid as the standard, the tannin profile of the methanolic extract of M. hortensis leaves was analyzed by spraying with 5% ferric chloride. Bluish brown coloured zones in the day-light mode confirmed the presence of two tannins (Plate 4.18). The peak table (Table 4.38) and peak densitogram (Figure 4.29) showed the second tannin to be tannic acid. 44

45 Before derivatization After derivatization Day light UV 366 nm UV 254 nm Day light PLATE 4.13 HPTLC OF ALKALOIDS FIGURE 4.24 HPTLC PEAK DENSITOGRAM OF ALKALOIDS IN M. hortensis LEAVES TABLE 4.23 HPTLC PEAK TABLE FOR THE ALKALOIDS IN THE M. hortensis LEAVES Track Peak R f Height Area Assigned substance A Unknown A Alkaloid 1 A Alkaloid 2 A Alkaloid 3 A Alkaloid 4 A Unknown A Alkaloid 5 A Unknown A Unknown A Alkaloid 6 A Unknown COL Colchicine standard 45

46 Before derivatization After derivatization Day light UV 366 nm UV 254 nm Day light PLATE 4.14 HPTLC OF PHENOLICS FIGURE 4.25 HPTLC PEAK DENSITOGRAM OF PHENOLICS IN M. hortensiss LEAVES TABLE HPTLC PEAK TABLE FOR THE PHENOLICS IN M. hortensis LEAVES Z Peak R f Height Area Assigned substance QUER Quercetin standardd MH Unknown MH Unknown MH Phenolics 1 MH Phenolics 2 MH Unknown MH Phenolics 3 MH Unknown MH Phenolics 4 MH Unknown MH Unknown 46

47 Before derivatization After derivatization Day light 366 nm 254 nm Day light 366 nm PLATE 4.15 HPTLC OF FLAVONOIDS FIGURE 4.26 HPTLC PEAK DENSITOGRAM OF FLAVONOIDS IN M. hortensis LEAVES TABLE 4.25 HPTLC PEAK TABLE FOR THE FLAVONOIDS IN M. hortensis LEAVES Track Peak R f Height Area Assigned substance RUT Rutin standard MH Flavonoid 1 MH Unknown MH Unknown MH Flavonoid 2 MH Flavonoid 3 MH Flavonoid 4 MH Flavonoid 5 MH Unknown MH Flavonoid 6 MH Unknown 47

48 Before derivatization After derivatization Day light UV 3666 nm UV 254 nm Day light UV 366 nm PLATE 4.16 HPTLC OF SAPONINS FIGURE 4.27 HPTLC PEAK DENSITOGRAM OF SAPONINS IN M. hortensis LEAVES TABLE 4.26 HPTLC PEAK TABLE FOR THE SAPONINS OF THE M. hortensis LEAVES Track Peak R f Height Area Assigned substance SAP Saponin standardd MH Saponin 1 MH Saponin 2 MH Saponin 3 MH Saponin 4 MH Saponin 5 MH Unknown MH Saponin 6 MH Saponin 7 MH Saponin 8 MH Saponin 9 MH Unknown 48

49 Before derivatization Day light UV 366 nm UV 254 nm After derivatization Day light UV 366 nm PLATE 4.17 HPTLC OF STEROIDS FIGURE 4.28 HPTLC PEAK DENSITOGRAM OF STEROIDS IN M. hortensis LEAVES TABLE 4.27 HPTLC PEAK TABLE OF STEROIDS IN M. hortensis LEAVES Track Peak R f Height Area Assigned substance SOL Solasodine standard A Unknown A Unknown A Unknown A Sterol 1 A Sterol 2 A Unknown A Sterol 3 A Sterol 4 A Sterol 5 A Unknown 49