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Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae
Phytomedicine 10: 536–543, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae B. C. Behera, B. Adawadkar, and U. Makhija Plant Science Division, Agharkar Research Institute, Pune, India
Summary Results on the screening of species of the lichen family Graphidaceae for superoxide-scavenging activity (SSA) and xanthine-oxidase inhibitory (IXO) activity have been presented. The potential of the extracts for scavenging of superoxide and inhibition of xanthineoxidase under various physiological conditions has been evaluated. The methanolic extracts of the species of family Graphidaceae showed inhibitory properties of xanthine oxidase (IC50 = 2.0 to 5.26 µg/ml) with an additional superoxide scavenging capacity (IC50 = 3.63 to 13.88 µg/ml). The potential of the methanolic extracts for scavenging of superoxide and inhibition of xanthine oxidase remained stable at 4 °C. Thus the extracts can be maintained for longer periods for their therapeutic uses. Key words: Graphidaceae, inhibition of xanthine oxidase, Superoxide-scavenging activity, lichens, IC50
Introduction A majority of disease conditions like atherosclerosis, hypertension, ischemic diseases, Alzheimer’s disease, parkinsonism, cancer and inflammatory conditions are being considered to be primarily due to the imbalance between pro-oxidant and anti-oxidant homeostasis. Anti-oxidants from natural resources possess multifacetedness and magnitude of the activities provide enormous scope in correcting the imbalance. Therefore, much attention is being directed in harnessing and harvesting antioxidants from natural resources (Tiwari, 2001). Biological and chemical pro-oxidants are considered to be important for the provocation of free radical mediated diseases in an individual. Although free radicals are considered to be important for normal physiology, they cause cellular damage when produced in excess. The radicals initiate a chain reaction of lipid and protein peroxidation by attacking the double bonds of these molecules. About 40 diseases are now being
considered as free radical-mediated. Most of them are metabolic, nervous or other old age diseases (Tripathy and Upadhyaya, 2001). The involvement of free radicals and other oxidants in aging and in several diseases has been investigated in detail recently (Khanom et al. 2000, Niki, 1995, Toda et al. 1991). For example, active oxygen damages the skin directly and forms lipid peroxides which result in the formation of insoluble pigments such as eumelanins and phaeomelanins (Miao et al. 1997). Much physiological damage may be directly imputable to the hydroxyl radical, because it is highly reactive, and so any hydroxyl radical produced in vivo would react at or close to its site of formation. Eliminating superoxide would reduce H2O2 and thereby production of hydroxyl radicals. Xanthine-oxidase catalyses the oxidation of hypoxanthine and xanthine to uric acid, the excess of which triggers the symptomatology of gout in humans (Schmeda Hirschmann 0944-7113/03/10/06–07-536 $ 15.00/0
Inhibitory activity of xanthine oxidase et al. 1987). Furthermore, significant increments in the serum level of xanthine oxidase are clinically observed in patients who suffer from hepatitis, brain tumor, hyperuricemia and gout. Xanthine oxidase has been reported as a key enzyme associated with the incidence of hyperuricemia-related disorders (Kong et al. 1999). Therefore, it is important and urgent to search for superoxide scavengers and xanthine oxidase inhibitors from natural resources to enable the development of new drugs and the prevention of several diseases. Lichens have long been recognized as containing bioactive compounds, but little attempts have been made to screen them. Successful isolation and screening programs have been undertaken in Japan and UK (Hawksworth, 1994). Many natural lichens and cultured lichens have been screened for their biological activities and several novel compounds were also isolated and identified. There is little information available relating to their chemical constituents and biological properties like anti-oxidative activity and inhibition of xanthine oxidase (Yamamoto et al. 1993, 1998). We have recently reported the inhibition of reduction of nitro-blue tetrazolium and xanthine oxidase by the extract of natural thallus of a foliose lichen species Bulbothrix setschwanensis and of its cultures obtained in the laboratory (Behera and Makhija, 2002). There have been no comprehensive studies of a systematic group of lichens reporting the superoxide-scavenging activity (SSA) and the inhibition of xanthine oxidase (IXO). Therefore, the aim of this study was to search for superoxide scavenging compounds and inhibitors of xanthine oxidase in 77 species of different genera Graphina, Graphis, Phaeographina, Phaeographis and Phaeographopsis in the lichen family Graphidaceae which is one of the most abundantly and widely occuring groups of lichens in India to develop new drugs for the prevention of free radical-mediated diseases and hyperuricemia related disorders.
Materials and Methods Dried and identified herbarium specimens of the members (77 species) of the lichen family Graphidaceae collected from various parts of India and deposited in the Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune, Maharashtra, India have been used for the superoxide scavenging activity and inhibition of xanthine oxidase assays (Table 1). The specimens were soaked overnight in distilled water to ensure removal of other particles deposited on the thallus. Since all specimens were crustose and collected on barks of trees, care has also been taken to remove bark portions from the lichen thallus. After re-
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moving bark, 10 mg of wet lichen thallus of each specimen was crushed and suspended in a 10 ml screw cap bottle containing 5 ml of 10% dimethyl sulphoxide in distilled water (DMSO/DW) and kept in the freezer at 4 °C for 48 hours. The extract was then filtered and the filtrate was dried in vacuo for 8 hours at room temperature. The residue was weighed and then tested for its superoxide scavenging activity (SSA) and inhibition of xanthine oxidase activity (IXO). Samples showing SSA and IXO (10% or more) were further extracted with 10 ml of 10% aqueous solution of MeOH containing dimethyl sulphoxide (10%; w/v) and again tested for its IXO and SSA. The xanthine oxidase inhibitory activity was assayed spectrophotometrically at 290 nm (Noro et al. 1983). Test solutions were prepared by adding xanthine (final concentration 50 µM), hydroxylamine (final concentration 0.2 mM); EDTA (final concentration 0.1 mM) and extracts in various concentrations. The reaction was started by the addition of 0.6 ml of xanthine-oxidase (S.D. Fine-Chem Ltd.), 12.5 mU/ml in potassium phosphate buffer (0.1 M, pH 7.8). The mixture (total 3 ml) was incubated for 30 minutes at 37 °C prior to the measurement of uric acid production at 290 nm. The reaction was stopped by adding 0.3 ml of HCl (0.58 M). The uric acid production was calculated from the differential absorbance with a blank solution in which xanthine oxidase was replaced by buffer solution. Each test material initially dissolved in DMSO and then diluted with the same buffer was incorporated in the enzyme assay to assess the inhibitory activity. To measure accurately the superoxide-scavenging activity, assays were carried out using two different methods because certain redox-active compounds in the extracts may disturb the superoxide-generating reactions. The enzymatic method with cytochrome c (cyt. c); (Bayer and Fridovich, 1987) and non enzymatic method with nitro blue tetrazolium (NBT); ( Zhang and Lu, 1990) were therefore used for the evaluations. With the cyt. c method, superoxide anions were generated by a xanthine and xanthine oxidase system. The superoxide-scavenging activity was calculated by measuring the reduction rate of ferricytochrome c. Briefly the assay was performed in 3 ml of reaction mixture in a 1.0 cm cuvette maintained at 25 °C. The reaction mixture contained 0.3 ml of a sample solution, 0.5 ml of ferricytochrome c (1 × 10–5 M), 0.5 ml of xanthine (5 × 10–5 M) and 1.0 ml distilled water and 0.5 ml of a 0.1M potassium phosphate buffer (pH 7.8) containing EDTA (10–4 M). A solution of 0.2 ml of xanthine oxidase (0.5 × 10–8 M) was added to the reaction mixture to produce a rate of reduction of ferricytochrome c at 550 nm using 0.025 absorbance unit per 1.5 minutes, in a total volume of 3.0 ml reaction mixture. Data on the resulting straight line were taken for calculation by an
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established formula. With the NBT method, superoxide was generated by potassium peroxide. The inhibition rate was calculated by measuring the amount of the formazan which was reduced from NBT by superoxide. The same potassium phosphate buffer was used as that used in the cyt. c assay, and the total reaction mixture was again adjusted to 3 ml. The reaction mixture contained 0.5 ml of a potassium phosphate buffer, 0.3 ml
of a sample solution, 0.15 ml of NBT (0.5 × 10–4 M), 1.85 ml of water, and 0.2 ml of potassium peroxide (suspended in dimethyl sulfoxide; 0.33 × 10–3 M). After adding potassium peroxide, the optical density of the reaction mixture was measured at 550 nm, whereas, in the control sample, no potassium peroxide was used. The inhibitory rate was calculated using an established formula.
Table 1. Inhibition of xanthine oxidase (IXO) and superoxide-scavenging activities (SSA) showed by methanolic extracts of various species and the IC50 values of the extracts. Name of the species
Graphina G. acharii (Fée) Müll. Arg. G. adscribens (Nyl.) Müll. Arg. G. glaucorufa (Vainio) Zahlbr. G. multistriata Müll. Arg. G. norlabiata Patw. & Kulk. G. nylenderi Patw. & Kulk. G. perstriatula (Nyl.) Zahlbr. G. salacinilabiata Patw. & Kulk. G. simulans (Leight.) Müll. Arg.
Acc.no
Extract yield (µg)
IXO (%)a
SSA (%)a
IC50, µg/ml a
Cyt.c
NBT
SSA
IXO
75.167 77.1914 77.1715 77.538 76.295 74.2068 85.256 77.51 85.1124
13.2 17.2 21.5 21.6 11.4 8.9 25.6 65.3 17.3
37.2 29.7 44.4 69.4 37.6 41.3 38.5 68.6 34.1
25.7 13.2 26.3 56.9 22.2 18.6 36.3 83.9 33.2
14.4 14.1 8.1 38.2 14.7 26.4 12.7 76.5 41.5
– 11.4 5.7 4.8 6.8 8.1 – 2.9 –
– 5.1 3.4 2.2 4.0 3.6 – 2.2 –
Graphis G. assamensis Nagarkar & Patw. G. exalbata Nyl. G. garoana Nagarkar & Patw. G. glauconigra Vainio G. guimarana Vaino G. hossei Vaino G. inamoena Zahlbr. G. inquinata (Kn. & Mitten) J.D. Hook G. nakanishiana Patw. & Kulk. G. patwardhanii Kulk. G. persulcata Stirton G. persicina May & Flot. G. pyrrhocheiloides Zahlbr. G. sikkimensis Nagarkar & Patw. G. sorediosa Nagarkar & Patw.
77.1334 75.5 78.387 73.2479 74.543 75.175 77.1331 85.107
77.5 13.6 6.7 8.7 14.4 26.1 10.8 14.3
54.3 56.7 33.4 44.3 28.6 48.2 58.3 44.8
61.2 27.4 10.8 38.5 53.3 34.4 23.4 29.5
47.4 11.2 19.3 21.3 17.5 28.5 31.5 17.4
6.8 5.5 13.9 – 4.8 – 6.4 5.1
2.8 2.6 4.5 – 5.2 – 2.6 3.3
74.1646 73.3041 85.415 73.1208 77.1851 77.1806 78.364
16.3 61.6 7.1 13.7 11.3 9.4 21.9
28.5 59.8 13.5 19.4 72.4 49.5 21.2
17.5 44.2 27.6 27.4 14.7 63.3 14.2
21.3 27.3 34.4 31.6 21.4 48.7 13.9
8.6 5.6 – – 10.2 – –
5.3 2.5 – – 2.1 – –
Phaeographina P. caesioradians (Leight.) Redg. P. noralboradians Patw. & Kulk.
85.507 74.2044
27.1 12.4
18.2 66.3
24.2 27.3
16.5 31.2
– 5.5
– 2.3
Phaeographis P. angulosa Müll. Arg. P. submaculata Zahlbr. P. subtigrina (Vainio) Zahlbr.
73.3116 76.472 73.1984
15.9 13.7 8.8
53.7 49.1 51.2
12.4 13.8 22.9
8.2 16.3 9.6
12.1 10.9 6.6
2.8 3.1 2.9
77.127
24.8
33.5
41.2
58.4
3.6
4.5
Phaeographopsis P. indica (Patw. & Nagrkar) Sipman & Aptroot a
Data represent the mean of three independent measurements
Inhibitory activity of xanthine oxidase The superoxide-scavenging activity is expressed as the percentage inhibition (I%) of reduction of cyt. c or NBT in the presence of a sample, relative to that of the control (without the sample) using the following equation: Absorbance sample × 100 I% = 100 – –––––––––––––––––––––––– Absorbance control sample Physiological experiments
The methanolic extracts of seven species namely, Graphina multistriata, G. salacinilabiata, Graphis assamensis, G. guimarana, G. patwardhanii, G. sikkimensis, and Phaeographopsis indica, which showed 50% or more scavenging of superoxide and inhibition of xanthine oxidase, were selected for further physiological studies. In order to know the effect of pH on the activities the reaction mixtures were adjusted to pH 3, 5, 6, and 9 by adding 1 N NaOH or 1 N HCl before adding the extract. After addition of the extract absorbance was measured at the wavelengths 290 nm and 550 nm. To study the effect of temperature on the potential of scavenging of superoxide and IXO, the reaction mixture was incubated at three temperatures of 4°, 40° and 100 °C after adding the sample for one hour and then the absorbance was measured at 290 nm and 550 nm. Further, to find the stability of scavenging of superoxide and IXO activity, extracts were left in a screwed vial wrapped with aluminum foil for one month, and then the absorbance was measured at wavelengths 290 nm and 550 nm. They were also scanned in UV and
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visible regions to compare the absorption spectra of the extracts obtained before and after the incubation. In order to determine the rate of scavenging of superoxide and IXO by the methanolic extracts under various incubation periods, the reaction mixture with the extract was incubated for one hour. The absorbance was measured at 290 and 550 nm at different time intervals of 5, 20 and 60 minutes. The effect of concentration of extracts on the scavenging of superoxide and IXO was tested at concentrations of 2, 5 and 20 µg dried extract /ml of each sample added to the reaction mixture and then the absorbance was measured at 290 nm and 550 nm. Different concentrations of extracts were analyzed and then the halfminimal inhibitory concentration (IC50) was calculated by linear regression analysis. All tests were performed in triplicate, and the results were averaged. All chemicals used in these experiments were of analytical reagent grade and purchased from Sigma Chemicals USA, Merck Company and S.D Fine Chem Ltd.
Results Seventy seven species of the family Graphidaceae were extracted with aqueous solution of methanol containing dimethyl sulphoxide. Out of seventy seven, 30 species gave extract yields in the range of 7–77.5 µg and were found to show both superoxide scavenging activity (SSA) and inhibition of xanthine oxidase (IXO) activities (Table 1).
Fig. 1A. Effect of pH on the methanolic extracts of various species for the potential scavenging superoxide.
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Fig. 1B. Rate of xanthine oxidase inhibitory activity and superoxide scavenging activity by the methanolic extracts of the species.
Fig. 1C. Inhibition of xanthine oxidase and superoxide scavenging activity by the methanolic extracts incubated under various temperature.
Inhibitory activity of xanthine oxidase In order to confirm the precise superoxide-scavenging activity (SSA), both the cytochrome c (cyt.c) and NBT methods were used and compared. In these 30 species, SSA was found to be in the range of 11 to 84% by the cyt. c method and with NBT the activity was in the range of 8 to 77%. The SSA was found in the range of 30–45% in six species namely Graphina perstriatula, G. simulans, Graphis glauconigra, G. hossei, G. patwardhanii, and Phaeographopsis indica. Similarly 50 to 84% of SSA was found in five species namely Graphina multistriata, G. salacinilabiata, Graphis assamensis G. guimarana and G. sikkimensis. All other species showed SSA below 30%. In general the SSA detected by the cyt.c method was mostly higher than the SSA detected by the NBT method. 30 to 50% inhibition of xanthine oxidase (IXO) was found in 13 species namely Graphina acharii, G. glaucorufa, G. norlabiata, G. nylenderi, G. perstriatula,
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G. simulans, Graphis garoana, G. glauconigra, G. hossei, G. inquinata, G. sikkimensis, Phaeographis submaculata and Phaeographopsis indica. Similarly IXO was found in the range of 50 to 72% in 10 species namely Graphina multistriata, G. salacinilabiata, Graphis assamensis, G. exalbata, G. inamoena, G. patwardhanii, G. pyrrhocheloides, Phaeographina noralboradians, Phaeographis angulosa, and P. subtigrina. In all other species, the IXO was found to be below 30%. The species which showed 50% or greater than 50% IXO and SSA were selected for further physiological studies. Studies of the effect of pH on the extract for the ability of IXO and SSA have revealed no significant change in the IXO, when the reaction mixture was adjusted to an acidic or an alkaline pH. However, significant differences in SSA were observed at acidic or al-
Table 2. Inhibition of xanthine oxidase (IXO) and superoxide-scavenging activity (SSA) by the methanolic extracts of various species at day 0 and after 30 days incubation at room temperature. Name of the Species
Day 0
After 30 days
SSA(%)a
IXO(%)a
SSA(%)a
IXO(%)a
Graphina G. adscribens (Nyl.) Müll. Arg. G. glaucorufa (Vainio) Zahlbr. G. norlabiata Patw. & Kulk. G. nylenderi Patw. & Kulk. G. multistriata Müll. Arg. G. salacinilabiata Patw. & Kulk.
13.2 26.3 22.2 18.6 57.2 83.9
29.7 44.4 37.6 41.3 69.4 68.6
1.7 8.3 0.9 1.6 17.4 78.3
5.1 10.8 4.8 14.5 52.6 41.4
Graphis G.assamensis Nagarkar & Patw. G.exalbata Nyl. G.garoana Nagarkar & Patw. G.guimarana Vaino G. inamoena Zahlbr. G. inquinata (Kn. & Mitten) J.D. Hook G. nakanishiana Patw. & Kulk. G. patwardhanii Kulk. G. pyrrhocheiloides Zahlbr. G. sikkimensis Nagarkar & Patw.
61.2 27.4 10.8 38.5 23.4 29.5 17.5 44.2 14.7 63.3
54.3 56.7 33.4 28.6 58.3 44.8 28.5 59.8 72.4 49.5
58.5 7.3 2.1 18.1 1.5 2.8 3.5 13.2 3.2 17.4
33.5 34.3 21.3 21.2 44.7 12.8 12.5 43.5 56.2 18.1
Phaeographina P. noralboradians Patw. & Kulk.
27.3
66.3
5.6
47.3
Phaeographis P. angulosa Müll. Arg. P. submaculata Zahlbr. P. subtigrina (Vainio) Zahlbr.
12.4 13.8 22.9
53.7 49.1 51.2
0.3 2.3 3.2
17.4 22.2 33.2
Phaeographopsis P. indica (Patw. & Nagrkar) Sipman & Aptroot
41.2
33.5
39.6
22.5
a
Data are mean of three independent readings
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B. C. Behera et al.
kaline pH. The SSA was found to be below 10% at pH 3 and 5, while it was increased almost to double when the pH concentration of the buffer was increased to 6 and 9 (Fig. 1A). The rates of IXO and SSA by the active extract in different incubation periods have been presented in Fig. 1B. The results have indicated that on increase in the incubation period increased the rate of SSA and IXO. The extracts of all seven species namely Graphis assamensis G. guimarana, G. patwardhanii, G. sikkimensis, Graphina multistriata , G. salacinilabiata, and Phaeographopsis indica showed SSA over a range of 60 to 92% and IXO over a range of 53 to 72% after a period of one hour incubation. The effect of temperature on the extracts of species for the potential of SSA and IXO has been presented in Fig. 1C. The SSA shown by the active extracts of seven species was in the range from 53 to 84% at 4 °C, 33 to 63% at 40 °C, and it was as low as 2 to 22% at 100 °C. However, IXO was found to be in the range from 28 to 73% at 4 °C, 23 to 48% at 40 °C and 6 to 20% at 100 °C. As far as the extract concentration on the SSA and IXO is concerned, the SSA and IXO were increased with the increase in extract concentration in the reaction mixture. Stability of the extract to scavenge superoxide and to inhibit xanthine oxidase has been presented in Table 2. After a period of one month incubation, the maximum SSA was found in the samples of Graphina salacinilabiata (78%), Graphis assamensis (59%) and Phaeographopsis indica (40%). Extracts of other species showed 0.14 to 18% SSA. The maximum IXO was found in the samples of Graphis pyrrhocheiloides (56%), Graphina multistriata (53%), Graphina salacinilabiata (41%), Graphis assamensis (34%), G. exalbata (34%), G. inamoena (45%), G. patawardhanii (44%), Phaeographina noralboradians (47%) and Phaeographis subtigrina (33%). Comparision of absorption spectra of methanolic extracts of species before and after one month incubation has revealed that the maximum peaks were obtained in the UV region before incubation (starting of the expt.) and after one month incubation most of the peaks had disappeared and spectral changes were observed.
Discussion It is evident from the experiments that in both the assay systems (cyt. c and NBT) few species showed the similar scores for superoxide scavenging activity (SSA). However, extracts of many species showed variation in SSA in both the assay systems. This variation may occur when native xanthine oxidase does not reduce
cytochrome c directly, but does so only through the action of electron transfer mediators. When O2 is the only electron acceptor present, then O–2 mediates the reduction of cytochrome c. Quinones and certain dyes can also mediate electron transfer from xanthine oxidase to cytochrome c and such compounds may be present when assaying crude extracts (Bayer and Fridovich, 1987). We partially agree with the report of Cos et al. (1998) who reported that during the oxidation of xanthine to uric acid, an equivalent rate of superoxide radical is produced. Although, two different assay systems were used by us in these studies for the determination of SSA, however, only few species showed similar percentage of SSA in both cyt. c and NBT assay. This suggests that cytochrome c method didn’t measure only the SSA, but also measured the other biochemical properties. As far as the effect of pH on the extract, the scavenging of superoxide by the extract were found vary at different pH. The scavenging of superoxide was found to be higher in the reaction mixture with sample at alkaline pH than the acidic pH. However, there was no significant difference in IXO by the reaction mixture with extract at acidic or alkaline pH. These results have suggested that in case of scavenging of superoxide, in highly acidic condition probably the activation of the compound(s) relatively slow than the alkaline condition, thereby decreasing in SSA occurred in the highly acidic condition. The SSA and IXO were increased with the increasing incubation period suggesting that the time requirement for activation of the compound (s) of extracts of selected species is high enough to show maximum of SSA and IXO thereby showing higher SSA and IXO after 1 hour incubation. The samples incubated at 4 °C did not lose potential in scavenging of superoxide and IXO. The samples incubated at 40 °C showed 10 to 20% decrease in scavenging of superoxide and IXO. At 100 °C the samples lost their potential drastically in scavenging of superoxide and IXO. This might have been due to the decrease in the concentration of active compound(s) or to the decomposition of active compounds at higher temperatures (Behera and Makhija, 2002). Both the SSA and IXO activities linearly increased as the extract concentration increased in the reaction mixture suggesting that the activities were dose related. This may be probably due to the concentration and nature of bioactive compound(s) produced by the species. Further, lichen produces secondary metabolites which are mainly phenolic compounds. This may also be related to the high level of phenolic compounds as also have been reported earlier (Germano et al. 2002). Crude extracts may contain many compounds along with the various lichen substances. At this stage, how-
Inhibitory activity of xanthine oxidase ever, it is not possible to relate the biological activities with the lichen substances because both the activities found in the extracts of various species are with or without lichen substance(s). We can only conclude that both the biological activities reported here are species specific. The half–minimal inhibitory concentration (IC50) for 50% scavenging of superoxide was found to be 3.63 to 13.88 µg/ml and 2 to 5.26 µg/ml for 50% inhibition of xanthine oxidase by the extract of selected species of the family Graphidaceae. The IC50 value of the standard superoxide dismutase (from bovine erythrocyte; Sigma Chemical USA) was detected by us as 0.38 µg/ml. The IC 50 value of allopurinol (from Hi-Media, India) in clinical use as a xanthine oxidase inhibitory drug was detected by us as 6.37 µg/ml. On the effectiveness of naturally occuring compounds in the selected species of the family Graphidaceae having potential in scavenging of superoxide and inhibition of xanthine oxidase may provide protection against many chronic diseases and deserves further study for its importance in therapeutics and their cultivation should be encouraged to harvest large amounts of these naturally occuring compounds.
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Bangladeshi Indigenous Medicinal plants. Biosci Biotechnol Biochem 64(4): 837–840 Kong LD, Wolffonder JL, Christopher H, Cheng K, Hostottmann K, Tan RX (1999) Xanthine oxidase inhibitors from Brandisia hancei. Planta Medica 65: 744–746 Miao Z, Kayahara H, Tadasa K (1997) Superoxide-scavenging and tyrosinase-inhibitory activities of the extracts of some Chinese medicines. Biosci Biotechnol Biochem 61: 2106–2108 Niki E (1995) In Antioxidants – Free Radicals and Biological Defense (eds Niki E, Shimasaki H, Mino M): 3–5, Japan Scientific Press, Tokyo Noro T, Oda Y, Miyase T, Ueno A, Fukushima S (1983) Inhibitors of xanthine oxidase from the flowers and buds of Dafne genkwa. Chem Pharm Bull 31: 3984–3987 Schmeda Hirschmann G, Theodoluz C, Franco L, Ferro E, Rojas de Arias A (1987) Preliminary pharmacological studies on Eugenia uniflora leaves: xanthine oxidase inhibitory activity. J Ethnopharmacology 21: 183–186 Tiwari AK (2001) Imbalance in antioxidant defence and human diseases: Multiple approach of natural antioxidants therapy. Curr Sci 81(9): 1179–1187 Toda S, Kumura M, Ohnishi M (1991) Effects of phenolcarboxylic acids on superoxide anion and lipid peroxidation induced by superoxide anion. Planta Medica 57: 8–10 Tripathy YB, Upadhyay AK (2001) Antioxidant property of Mucuna pruriens L. Curr Sci 80(11): 1377–1378 Yamamoto Y, Miura Y, Higuchi M, Kinoshita Y (1993) Using lichen tissue cultures in modern biology. The Bryologist 96(3): 384–393 Yamamoto Y, Kinoshita Y, Matsubara H, Kinoshita K, Koyama K, Takahashi K, Kurokawa T, Yoshimura I (1998) Screening of biological activities and isolation of biological active compounds from lichens. Recent Res Devel Phytochem 2: 23–34 Zhang HY, Lu CS (1990) A study of the SOD-like activity of some copper (II)-small peptide and amino acid complexes. Acta Biochem Biophys Sinica (in Chinese) 22: 593–594
Address B. C. Behera, Plant Science Division, Agharkar Research Institute, G. G. Agarkar Road, Pune-411 004, India Fax: ++91-20-5651542 e-mail: [email protected]
Phytomedicine
Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae B. C. Behera, B. Adawadkar, and U. Makhija Plant Science Division, Agharkar Research Institute, Pune, India
Summary Results on the screening of species of the lichen family Graphidaceae for superoxide-scavenging activity (SSA) and xanthine-oxidase inhibitory (IXO) activity have been presented. The potential of the extracts for scavenging of superoxide and inhibition of xanthineoxidase under various physiological conditions has been evaluated. The methanolic extracts of the species of family Graphidaceae showed inhibitory properties of xanthine oxidase (IC50 = 2.0 to 5.26 µg/ml) with an additional superoxide scavenging capacity (IC50 = 3.63 to 13.88 µg/ml). The potential of the methanolic extracts for scavenging of superoxide and inhibition of xanthine oxidase remained stable at 4 °C. Thus the extracts can be maintained for longer periods for their therapeutic uses. Key words: Graphidaceae, inhibition of xanthine oxidase, Superoxide-scavenging activity, lichens, IC50
Introduction A majority of disease conditions like atherosclerosis, hypertension, ischemic diseases, Alzheimer’s disease, parkinsonism, cancer and inflammatory conditions are being considered to be primarily due to the imbalance between pro-oxidant and anti-oxidant homeostasis. Anti-oxidants from natural resources possess multifacetedness and magnitude of the activities provide enormous scope in correcting the imbalance. Therefore, much attention is being directed in harnessing and harvesting antioxidants from natural resources (Tiwari, 2001). Biological and chemical pro-oxidants are considered to be important for the provocation of free radical mediated diseases in an individual. Although free radicals are considered to be important for normal physiology, they cause cellular damage when produced in excess. The radicals initiate a chain reaction of lipid and protein peroxidation by attacking the double bonds of these molecules. About 40 diseases are now being
considered as free radical-mediated. Most of them are metabolic, nervous or other old age diseases (Tripathy and Upadhyaya, 2001). The involvement of free radicals and other oxidants in aging and in several diseases has been investigated in detail recently (Khanom et al. 2000, Niki, 1995, Toda et al. 1991). For example, active oxygen damages the skin directly and forms lipid peroxides which result in the formation of insoluble pigments such as eumelanins and phaeomelanins (Miao et al. 1997). Much physiological damage may be directly imputable to the hydroxyl radical, because it is highly reactive, and so any hydroxyl radical produced in vivo would react at or close to its site of formation. Eliminating superoxide would reduce H2O2 and thereby production of hydroxyl radicals. Xanthine-oxidase catalyses the oxidation of hypoxanthine and xanthine to uric acid, the excess of which triggers the symptomatology of gout in humans (Schmeda Hirschmann 0944-7113/03/10/06–07-536 $ 15.00/0
Inhibitory activity of xanthine oxidase et al. 1987). Furthermore, significant increments in the serum level of xanthine oxidase are clinically observed in patients who suffer from hepatitis, brain tumor, hyperuricemia and gout. Xanthine oxidase has been reported as a key enzyme associated with the incidence of hyperuricemia-related disorders (Kong et al. 1999). Therefore, it is important and urgent to search for superoxide scavengers and xanthine oxidase inhibitors from natural resources to enable the development of new drugs and the prevention of several diseases. Lichens have long been recognized as containing bioactive compounds, but little attempts have been made to screen them. Successful isolation and screening programs have been undertaken in Japan and UK (Hawksworth, 1994). Many natural lichens and cultured lichens have been screened for their biological activities and several novel compounds were also isolated and identified. There is little information available relating to their chemical constituents and biological properties like anti-oxidative activity and inhibition of xanthine oxidase (Yamamoto et al. 1993, 1998). We have recently reported the inhibition of reduction of nitro-blue tetrazolium and xanthine oxidase by the extract of natural thallus of a foliose lichen species Bulbothrix setschwanensis and of its cultures obtained in the laboratory (Behera and Makhija, 2002). There have been no comprehensive studies of a systematic group of lichens reporting the superoxide-scavenging activity (SSA) and the inhibition of xanthine oxidase (IXO). Therefore, the aim of this study was to search for superoxide scavenging compounds and inhibitors of xanthine oxidase in 77 species of different genera Graphina, Graphis, Phaeographina, Phaeographis and Phaeographopsis in the lichen family Graphidaceae which is one of the most abundantly and widely occuring groups of lichens in India to develop new drugs for the prevention of free radical-mediated diseases and hyperuricemia related disorders.
Materials and Methods Dried and identified herbarium specimens of the members (77 species) of the lichen family Graphidaceae collected from various parts of India and deposited in the Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune, Maharashtra, India have been used for the superoxide scavenging activity and inhibition of xanthine oxidase assays (Table 1). The specimens were soaked overnight in distilled water to ensure removal of other particles deposited on the thallus. Since all specimens were crustose and collected on barks of trees, care has also been taken to remove bark portions from the lichen thallus. After re-
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moving bark, 10 mg of wet lichen thallus of each specimen was crushed and suspended in a 10 ml screw cap bottle containing 5 ml of 10% dimethyl sulphoxide in distilled water (DMSO/DW) and kept in the freezer at 4 °C for 48 hours. The extract was then filtered and the filtrate was dried in vacuo for 8 hours at room temperature. The residue was weighed and then tested for its superoxide scavenging activity (SSA) and inhibition of xanthine oxidase activity (IXO). Samples showing SSA and IXO (10% or more) were further extracted with 10 ml of 10% aqueous solution of MeOH containing dimethyl sulphoxide (10%; w/v) and again tested for its IXO and SSA. The xanthine oxidase inhibitory activity was assayed spectrophotometrically at 290 nm (Noro et al. 1983). Test solutions were prepared by adding xanthine (final concentration 50 µM), hydroxylamine (final concentration 0.2 mM); EDTA (final concentration 0.1 mM) and extracts in various concentrations. The reaction was started by the addition of 0.6 ml of xanthine-oxidase (S.D. Fine-Chem Ltd.), 12.5 mU/ml in potassium phosphate buffer (0.1 M, pH 7.8). The mixture (total 3 ml) was incubated for 30 minutes at 37 °C prior to the measurement of uric acid production at 290 nm. The reaction was stopped by adding 0.3 ml of HCl (0.58 M). The uric acid production was calculated from the differential absorbance with a blank solution in which xanthine oxidase was replaced by buffer solution. Each test material initially dissolved in DMSO and then diluted with the same buffer was incorporated in the enzyme assay to assess the inhibitory activity. To measure accurately the superoxide-scavenging activity, assays were carried out using two different methods because certain redox-active compounds in the extracts may disturb the superoxide-generating reactions. The enzymatic method with cytochrome c (cyt. c); (Bayer and Fridovich, 1987) and non enzymatic method with nitro blue tetrazolium (NBT); ( Zhang and Lu, 1990) were therefore used for the evaluations. With the cyt. c method, superoxide anions were generated by a xanthine and xanthine oxidase system. The superoxide-scavenging activity was calculated by measuring the reduction rate of ferricytochrome c. Briefly the assay was performed in 3 ml of reaction mixture in a 1.0 cm cuvette maintained at 25 °C. The reaction mixture contained 0.3 ml of a sample solution, 0.5 ml of ferricytochrome c (1 × 10–5 M), 0.5 ml of xanthine (5 × 10–5 M) and 1.0 ml distilled water and 0.5 ml of a 0.1M potassium phosphate buffer (pH 7.8) containing EDTA (10–4 M). A solution of 0.2 ml of xanthine oxidase (0.5 × 10–8 M) was added to the reaction mixture to produce a rate of reduction of ferricytochrome c at 550 nm using 0.025 absorbance unit per 1.5 minutes, in a total volume of 3.0 ml reaction mixture. Data on the resulting straight line were taken for calculation by an
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established formula. With the NBT method, superoxide was generated by potassium peroxide. The inhibition rate was calculated by measuring the amount of the formazan which was reduced from NBT by superoxide. The same potassium phosphate buffer was used as that used in the cyt. c assay, and the total reaction mixture was again adjusted to 3 ml. The reaction mixture contained 0.5 ml of a potassium phosphate buffer, 0.3 ml
of a sample solution, 0.15 ml of NBT (0.5 × 10–4 M), 1.85 ml of water, and 0.2 ml of potassium peroxide (suspended in dimethyl sulfoxide; 0.33 × 10–3 M). After adding potassium peroxide, the optical density of the reaction mixture was measured at 550 nm, whereas, in the control sample, no potassium peroxide was used. The inhibitory rate was calculated using an established formula.
Table 1. Inhibition of xanthine oxidase (IXO) and superoxide-scavenging activities (SSA) showed by methanolic extracts of various species and the IC50 values of the extracts. Name of the species
Graphina G. acharii (Fée) Müll. Arg. G. adscribens (Nyl.) Müll. Arg. G. glaucorufa (Vainio) Zahlbr. G. multistriata Müll. Arg. G. norlabiata Patw. & Kulk. G. nylenderi Patw. & Kulk. G. perstriatula (Nyl.) Zahlbr. G. salacinilabiata Patw. & Kulk. G. simulans (Leight.) Müll. Arg.
Acc.no
Extract yield (µg)
IXO (%)a
SSA (%)a
IC50, µg/ml a
Cyt.c
NBT
SSA
IXO
75.167 77.1914 77.1715 77.538 76.295 74.2068 85.256 77.51 85.1124
13.2 17.2 21.5 21.6 11.4 8.9 25.6 65.3 17.3
37.2 29.7 44.4 69.4 37.6 41.3 38.5 68.6 34.1
25.7 13.2 26.3 56.9 22.2 18.6 36.3 83.9 33.2
14.4 14.1 8.1 38.2 14.7 26.4 12.7 76.5 41.5
– 11.4 5.7 4.8 6.8 8.1 – 2.9 –
– 5.1 3.4 2.2 4.0 3.6 – 2.2 –
Graphis G. assamensis Nagarkar & Patw. G. exalbata Nyl. G. garoana Nagarkar & Patw. G. glauconigra Vainio G. guimarana Vaino G. hossei Vaino G. inamoena Zahlbr. G. inquinata (Kn. & Mitten) J.D. Hook G. nakanishiana Patw. & Kulk. G. patwardhanii Kulk. G. persulcata Stirton G. persicina May & Flot. G. pyrrhocheiloides Zahlbr. G. sikkimensis Nagarkar & Patw. G. sorediosa Nagarkar & Patw.
77.1334 75.5 78.387 73.2479 74.543 75.175 77.1331 85.107
77.5 13.6 6.7 8.7 14.4 26.1 10.8 14.3
54.3 56.7 33.4 44.3 28.6 48.2 58.3 44.8
61.2 27.4 10.8 38.5 53.3 34.4 23.4 29.5
47.4 11.2 19.3 21.3 17.5 28.5 31.5 17.4
6.8 5.5 13.9 – 4.8 – 6.4 5.1
2.8 2.6 4.5 – 5.2 – 2.6 3.3
74.1646 73.3041 85.415 73.1208 77.1851 77.1806 78.364
16.3 61.6 7.1 13.7 11.3 9.4 21.9
28.5 59.8 13.5 19.4 72.4 49.5 21.2
17.5 44.2 27.6 27.4 14.7 63.3 14.2
21.3 27.3 34.4 31.6 21.4 48.7 13.9
8.6 5.6 – – 10.2 – –
5.3 2.5 – – 2.1 – –
Phaeographina P. caesioradians (Leight.) Redg. P. noralboradians Patw. & Kulk.
85.507 74.2044
27.1 12.4
18.2 66.3
24.2 27.3
16.5 31.2
– 5.5
– 2.3
Phaeographis P. angulosa Müll. Arg. P. submaculata Zahlbr. P. subtigrina (Vainio) Zahlbr.
73.3116 76.472 73.1984
15.9 13.7 8.8
53.7 49.1 51.2
12.4 13.8 22.9
8.2 16.3 9.6
12.1 10.9 6.6
2.8 3.1 2.9
77.127
24.8
33.5
41.2
58.4
3.6
4.5
Phaeographopsis P. indica (Patw. & Nagrkar) Sipman & Aptroot a
Data represent the mean of three independent measurements
Inhibitory activity of xanthine oxidase The superoxide-scavenging activity is expressed as the percentage inhibition (I%) of reduction of cyt. c or NBT in the presence of a sample, relative to that of the control (without the sample) using the following equation: Absorbance sample × 100 I% = 100 – –––––––––––––––––––––––– Absorbance control sample Physiological experiments
The methanolic extracts of seven species namely, Graphina multistriata, G. salacinilabiata, Graphis assamensis, G. guimarana, G. patwardhanii, G. sikkimensis, and Phaeographopsis indica, which showed 50% or more scavenging of superoxide and inhibition of xanthine oxidase, were selected for further physiological studies. In order to know the effect of pH on the activities the reaction mixtures were adjusted to pH 3, 5, 6, and 9 by adding 1 N NaOH or 1 N HCl before adding the extract. After addition of the extract absorbance was measured at the wavelengths 290 nm and 550 nm. To study the effect of temperature on the potential of scavenging of superoxide and IXO, the reaction mixture was incubated at three temperatures of 4°, 40° and 100 °C after adding the sample for one hour and then the absorbance was measured at 290 nm and 550 nm. Further, to find the stability of scavenging of superoxide and IXO activity, extracts were left in a screwed vial wrapped with aluminum foil for one month, and then the absorbance was measured at wavelengths 290 nm and 550 nm. They were also scanned in UV and
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visible regions to compare the absorption spectra of the extracts obtained before and after the incubation. In order to determine the rate of scavenging of superoxide and IXO by the methanolic extracts under various incubation periods, the reaction mixture with the extract was incubated for one hour. The absorbance was measured at 290 and 550 nm at different time intervals of 5, 20 and 60 minutes. The effect of concentration of extracts on the scavenging of superoxide and IXO was tested at concentrations of 2, 5 and 20 µg dried extract /ml of each sample added to the reaction mixture and then the absorbance was measured at 290 nm and 550 nm. Different concentrations of extracts were analyzed and then the halfminimal inhibitory concentration (IC50) was calculated by linear regression analysis. All tests were performed in triplicate, and the results were averaged. All chemicals used in these experiments were of analytical reagent grade and purchased from Sigma Chemicals USA, Merck Company and S.D Fine Chem Ltd.
Results Seventy seven species of the family Graphidaceae were extracted with aqueous solution of methanol containing dimethyl sulphoxide. Out of seventy seven, 30 species gave extract yields in the range of 7–77.5 µg and were found to show both superoxide scavenging activity (SSA) and inhibition of xanthine oxidase (IXO) activities (Table 1).
Fig. 1A. Effect of pH on the methanolic extracts of various species for the potential scavenging superoxide.
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Fig. 1B. Rate of xanthine oxidase inhibitory activity and superoxide scavenging activity by the methanolic extracts of the species.
Fig. 1C. Inhibition of xanthine oxidase and superoxide scavenging activity by the methanolic extracts incubated under various temperature.
Inhibitory activity of xanthine oxidase In order to confirm the precise superoxide-scavenging activity (SSA), both the cytochrome c (cyt.c) and NBT methods were used and compared. In these 30 species, SSA was found to be in the range of 11 to 84% by the cyt. c method and with NBT the activity was in the range of 8 to 77%. The SSA was found in the range of 30–45% in six species namely Graphina perstriatula, G. simulans, Graphis glauconigra, G. hossei, G. patwardhanii, and Phaeographopsis indica. Similarly 50 to 84% of SSA was found in five species namely Graphina multistriata, G. salacinilabiata, Graphis assamensis G. guimarana and G. sikkimensis. All other species showed SSA below 30%. In general the SSA detected by the cyt.c method was mostly higher than the SSA detected by the NBT method. 30 to 50% inhibition of xanthine oxidase (IXO) was found in 13 species namely Graphina acharii, G. glaucorufa, G. norlabiata, G. nylenderi, G. perstriatula,
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G. simulans, Graphis garoana, G. glauconigra, G. hossei, G. inquinata, G. sikkimensis, Phaeographis submaculata and Phaeographopsis indica. Similarly IXO was found in the range of 50 to 72% in 10 species namely Graphina multistriata, G. salacinilabiata, Graphis assamensis, G. exalbata, G. inamoena, G. patwardhanii, G. pyrrhocheloides, Phaeographina noralboradians, Phaeographis angulosa, and P. subtigrina. In all other species, the IXO was found to be below 30%. The species which showed 50% or greater than 50% IXO and SSA were selected for further physiological studies. Studies of the effect of pH on the extract for the ability of IXO and SSA have revealed no significant change in the IXO, when the reaction mixture was adjusted to an acidic or an alkaline pH. However, significant differences in SSA were observed at acidic or al-
Table 2. Inhibition of xanthine oxidase (IXO) and superoxide-scavenging activity (SSA) by the methanolic extracts of various species at day 0 and after 30 days incubation at room temperature. Name of the Species
Day 0
After 30 days
SSA(%)a
IXO(%)a
SSA(%)a
IXO(%)a
Graphina G. adscribens (Nyl.) Müll. Arg. G. glaucorufa (Vainio) Zahlbr. G. norlabiata Patw. & Kulk. G. nylenderi Patw. & Kulk. G. multistriata Müll. Arg. G. salacinilabiata Patw. & Kulk.
13.2 26.3 22.2 18.6 57.2 83.9
29.7 44.4 37.6 41.3 69.4 68.6
1.7 8.3 0.9 1.6 17.4 78.3
5.1 10.8 4.8 14.5 52.6 41.4
Graphis G.assamensis Nagarkar & Patw. G.exalbata Nyl. G.garoana Nagarkar & Patw. G.guimarana Vaino G. inamoena Zahlbr. G. inquinata (Kn. & Mitten) J.D. Hook G. nakanishiana Patw. & Kulk. G. patwardhanii Kulk. G. pyrrhocheiloides Zahlbr. G. sikkimensis Nagarkar & Patw.
61.2 27.4 10.8 38.5 23.4 29.5 17.5 44.2 14.7 63.3
54.3 56.7 33.4 28.6 58.3 44.8 28.5 59.8 72.4 49.5
58.5 7.3 2.1 18.1 1.5 2.8 3.5 13.2 3.2 17.4
33.5 34.3 21.3 21.2 44.7 12.8 12.5 43.5 56.2 18.1
Phaeographina P. noralboradians Patw. & Kulk.
27.3
66.3
5.6
47.3
Phaeographis P. angulosa Müll. Arg. P. submaculata Zahlbr. P. subtigrina (Vainio) Zahlbr.
12.4 13.8 22.9
53.7 49.1 51.2
0.3 2.3 3.2
17.4 22.2 33.2
Phaeographopsis P. indica (Patw. & Nagrkar) Sipman & Aptroot
41.2
33.5
39.6
22.5
a
Data are mean of three independent readings
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B. C. Behera et al.
kaline pH. The SSA was found to be below 10% at pH 3 and 5, while it was increased almost to double when the pH concentration of the buffer was increased to 6 and 9 (Fig. 1A). The rates of IXO and SSA by the active extract in different incubation periods have been presented in Fig. 1B. The results have indicated that on increase in the incubation period increased the rate of SSA and IXO. The extracts of all seven species namely Graphis assamensis G. guimarana, G. patwardhanii, G. sikkimensis, Graphina multistriata , G. salacinilabiata, and Phaeographopsis indica showed SSA over a range of 60 to 92% and IXO over a range of 53 to 72% after a period of one hour incubation. The effect of temperature on the extracts of species for the potential of SSA and IXO has been presented in Fig. 1C. The SSA shown by the active extracts of seven species was in the range from 53 to 84% at 4 °C, 33 to 63% at 40 °C, and it was as low as 2 to 22% at 100 °C. However, IXO was found to be in the range from 28 to 73% at 4 °C, 23 to 48% at 40 °C and 6 to 20% at 100 °C. As far as the extract concentration on the SSA and IXO is concerned, the SSA and IXO were increased with the increase in extract concentration in the reaction mixture. Stability of the extract to scavenge superoxide and to inhibit xanthine oxidase has been presented in Table 2. After a period of one month incubation, the maximum SSA was found in the samples of Graphina salacinilabiata (78%), Graphis assamensis (59%) and Phaeographopsis indica (40%). Extracts of other species showed 0.14 to 18% SSA. The maximum IXO was found in the samples of Graphis pyrrhocheiloides (56%), Graphina multistriata (53%), Graphina salacinilabiata (41%), Graphis assamensis (34%), G. exalbata (34%), G. inamoena (45%), G. patawardhanii (44%), Phaeographina noralboradians (47%) and Phaeographis subtigrina (33%). Comparision of absorption spectra of methanolic extracts of species before and after one month incubation has revealed that the maximum peaks were obtained in the UV region before incubation (starting of the expt.) and after one month incubation most of the peaks had disappeared and spectral changes were observed.
Discussion It is evident from the experiments that in both the assay systems (cyt. c and NBT) few species showed the similar scores for superoxide scavenging activity (SSA). However, extracts of many species showed variation in SSA in both the assay systems. This variation may occur when native xanthine oxidase does not reduce
cytochrome c directly, but does so only through the action of electron transfer mediators. When O2 is the only electron acceptor present, then O–2 mediates the reduction of cytochrome c. Quinones and certain dyes can also mediate electron transfer from xanthine oxidase to cytochrome c and such compounds may be present when assaying crude extracts (Bayer and Fridovich, 1987). We partially agree with the report of Cos et al. (1998) who reported that during the oxidation of xanthine to uric acid, an equivalent rate of superoxide radical is produced. Although, two different assay systems were used by us in these studies for the determination of SSA, however, only few species showed similar percentage of SSA in both cyt. c and NBT assay. This suggests that cytochrome c method didn’t measure only the SSA, but also measured the other biochemical properties. As far as the effect of pH on the extract, the scavenging of superoxide by the extract were found vary at different pH. The scavenging of superoxide was found to be higher in the reaction mixture with sample at alkaline pH than the acidic pH. However, there was no significant difference in IXO by the reaction mixture with extract at acidic or alkaline pH. These results have suggested that in case of scavenging of superoxide, in highly acidic condition probably the activation of the compound(s) relatively slow than the alkaline condition, thereby decreasing in SSA occurred in the highly acidic condition. The SSA and IXO were increased with the increasing incubation period suggesting that the time requirement for activation of the compound (s) of extracts of selected species is high enough to show maximum of SSA and IXO thereby showing higher SSA and IXO after 1 hour incubation. The samples incubated at 4 °C did not lose potential in scavenging of superoxide and IXO. The samples incubated at 40 °C showed 10 to 20% decrease in scavenging of superoxide and IXO. At 100 °C the samples lost their potential drastically in scavenging of superoxide and IXO. This might have been due to the decrease in the concentration of active compound(s) or to the decomposition of active compounds at higher temperatures (Behera and Makhija, 2002). Both the SSA and IXO activities linearly increased as the extract concentration increased in the reaction mixture suggesting that the activities were dose related. This may be probably due to the concentration and nature of bioactive compound(s) produced by the species. Further, lichen produces secondary metabolites which are mainly phenolic compounds. This may also be related to the high level of phenolic compounds as also have been reported earlier (Germano et al. 2002). Crude extracts may contain many compounds along with the various lichen substances. At this stage, how-
Inhibitory activity of xanthine oxidase ever, it is not possible to relate the biological activities with the lichen substances because both the activities found in the extracts of various species are with or without lichen substance(s). We can only conclude that both the biological activities reported here are species specific. The half–minimal inhibitory concentration (IC50) for 50% scavenging of superoxide was found to be 3.63 to 13.88 µg/ml and 2 to 5.26 µg/ml for 50% inhibition of xanthine oxidase by the extract of selected species of the family Graphidaceae. The IC50 value of the standard superoxide dismutase (from bovine erythrocyte; Sigma Chemical USA) was detected by us as 0.38 µg/ml. The IC 50 value of allopurinol (from Hi-Media, India) in clinical use as a xanthine oxidase inhibitory drug was detected by us as 6.37 µg/ml. On the effectiveness of naturally occuring compounds in the selected species of the family Graphidaceae having potential in scavenging of superoxide and inhibition of xanthine oxidase may provide protection against many chronic diseases and deserves further study for its importance in therapeutics and their cultivation should be encouraged to harvest large amounts of these naturally occuring compounds.
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Address B. C. Behera, Plant Science Division, Agharkar Research Institute, G. G. Agarkar Road, Pune-411 004, India Fax: ++91-20-5651542 e-mail: [email protected]