JOURNAL OF MEDICINAL FOOD J Med Food 9 (2) 2006, 270–275 © Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
Antioxidant Properties of Methanolic Extracts from Leaves of Rhazya stricta Shahid Iqbal,1 M.I. Bhanger,2 Mubeena Akhtar,2 Farooq Anwar,3 Khawaja Raees Ahmed,4 and Tabraz Anwer5 Departments of 1Chemistry and 4Biology, University of Sargodha, Sargodha; 2National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro; 3Department of Chemistry, University of Agriculture, Faisalabad; and 5Environmental Research Section, Medicinal Botanical Center, Peshawar, Pakistan ABSTRACT Because of increased safety concerns about synthetic antioxidants, exploitation of cheaper and safer sources of antioxidants based on natural origin is the focus of research nowadays. Rhazya stricta is a medicinally important plant native to South Asia. Extraction of antioxidants was carried out in different solvent systems, i.e., water, 80% methanol, 70% ethanol, and diethyl ether. The methanolic extract exhibited the highest total phenolic content among the extracts; therefore for further studies the methanolic extract was employed. Antioxidant activity measurement in the linoleic acid system, metal chelating activity, reducing power, scavenging effect on 1,1-diphenyl-2-picrylhydrazyl free radical, and superoxide anion radical scavenging activity were taken as the parameters for assessment of antioxidant potential of methanolic extracts. Results were compared with -tocopherol and the synthetic antioxidant butylated hydroxyanisole. The antioxidant potential of methanolic extracts of R. stricta leaves was comparable with previously exploited potent antioxidants and is strongly concentration dependent. KEY WORDS:
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antioxidant activity
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concentration effect
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INTRODUCTION
Rhazya stricta
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scavenging power
those of natural antioxidants with safer aspects.8 Several sources of natural antioxidants have been investigated, including plants and microorganisms.9,10 Natural antioxidants such as flavonoids, tannins, coumarins, curcuminoids, xanthones, phenolics, and terpenoids are found in various plant products such as fruits, leaves, seeds, and oils, and some of these are as effective as synthetic antioxidants in model systems,11,12 But with increasing demand from food and other relevant industries, there exists a need to exploit new sources of antioxidants. Pakistan is rich in medicinal flora. Many plants and herbs indigenous to Pakistan have been widely used in the treatment of various diseases with the traditional “Eastern system” of medication.13 Several genera of Apocyanaceae, including Rhazya, have been exploited as rich sources of indole alkaloids. The genus Rhazya comprises only two species, viz., Rhazya stricta Decaisne and Rhazya orientalis.13 R. stricta is abundantly distributed in various hilly areas of Pakistan. It is a small, glabrous, erect shrub, which grows in the northwest region of the subcontinent14,15 and has been previously used for the treatment of various ailments.16 Alkaloids of R. stricta has been reported to be used in the treatment of cancer and is cytotoxic against Eagles KB carcinoma of the nasopharynx in cell cultures,16 as a curative for chronic rheumatism, sore throat,17 and fever, has antineoplastic activity,18 and is active in antitumor treatment.19
A
NUMBER OF DISEASES,
including cancer, coronary heart disease, and atherosclerosis, in humans have been reported to be initiated because of oxidative stress.1,2 Diets rich in fruits and vegetables have been reported to be associated with lowering the risks of cancer and coronary heart disease.3 There is increasing evidence that changing one’s diet to an increased intake of food “relatively high” in selected natural antioxidants, such as plant polyphenols, vitamin C, or flavonoids, can reduce the incidence of chronic and degenerative diseases.4 On the basis of these studies, the search for food materials and dietary supplements having significant antioxidant potential is the focus of intense research nowadays.5 Many herbs and spices have been reported to be possess antioxidant properties.6 Their dual role in food preservation as well as disease prevention has highlighted their importance more appreciably as dietary factors. Previously, synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene have been in use, but because of concerns about their role in coronary heart disease and damage to DNA,7 scientists are keenly interested in looking to replace these synthetic sources with Manuscript received 1 February 2005. Revised manuscript accepted 25 November 2005. Address reprint requests to: Shahid Iqbal, Department of Chemistry, University of Sargodha, Sargodha-40100, Pakistan, E-mail:
[email protected]
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However, there are no reports describing the antioxidant activity of R. stricta available in the literature. Evidence that some alkaloids of this species are responsible for antioxidant activity is reported elsewhere.18 In the present work, the antioxidant potential of methanolic extracts of R. stricta leaves has been exploited.
MATERIALS AND METHODS Samples and reagents The leaves of R. stricta were collected from the periphery of the campus of the University of Sindh, Jamshoro, Pakistan, and authenticated in the Herbarium, Department of Botany, University of Sindh. BHA and -tocopherol were purchased from Fluka Chemical Co. (Buchs, Switzerland). All other reagents used were from E. Merck (Darmstadt, Germany) or Sigma Aldrich (St. Louis, MO) unless stated otherwise.
Extraction of total phenolics in different solvent systems Freeze-dried leaves of R. stricta (10 g) were extracted in different solvent systems, i.e., 300 mL of water, 80% methanol (150 mL), 70% ethanol (150 mL), and diethyl ether (150 mL), for 6 hours in a Soxhlet apparatus. The extracts were filtered, and residues were extracted again. This process was repeated three times to ensure complete extraction. Acid-catalyzed hydrolysis was carried out by adding 6–8 drops of 0.1 M HCl to the residue to convert bound phenolics to the free form.20 The combined filtrate was subjected to rotary evaporation under reduced pressure at 45°C, and finally the concentrated extract obtained was placed in a desiccator until analyses. Total phenolic content was measured for all the extracts. All experiments were conducted three times, and results were averaged. Data are reported as mean SD values.
Determination of total phenolics Total phenolic content, for all the extracts, was determined spectrophotometrically using the Folin-Ciocalteu reagent following a previously reported method.21 Briefly, the reaction mixture contained 1.0 mL of diluted extracts or standard solution (200 ppm), 1.0 mL of freshly prepared diluted Folin-Ciocalteu reagent (1:10), and 10 mL of 7.5% sodium carbonate. The resulting reaction mixture was diluted to 25 mL, mixed well, and subjected to incubation at room temperature for 2 hours to ensure the completion of reaction. Absorption was measured with a Lambda2 UV/VIS spectrophotometer (PerkinElmer Instruments, Norwalk, CT) at 760 nm. Gallic acid was used as the calibration standard, and results were expressed as g of gallic acid/100 g of extract. The experiment was conducted in triplicate, and results are reported as mean SD values.
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Antioxidant activity determination in the linoleic acid system The antioxidant activity of R. stricta extracts was determined following a reported method.22 Sample extracts (0.5 g) were added to a solution mixture of linoleic acid (0.13 mL), 99.8% ethanol (10 mL), and 0.2 M sodium phosphate buffer (pH 7.0, 10 mL). The total volume was adjusted to 25 mL with distilled water. The solution was incubated at 40°C, and the degree of oxidation was measured according to the thiocyanate method23 with 10 mL of ethanol (75%), 0.2 mL of an aqueous solution of ammonium thiocyanate (30%), 0.2 mL of sample solution, and 0.2 mL of ferrous chloride (FeCl2) solution (20 mM in 3.5% HCl) being added sequentially. After 3 minutes of stirring, the absorption values of mixtures measured at 500 nm were taken as peroxide contents. A control was performed with linoleic acid but without the extracts. One milliliter (200 ppm) of -tocopherol and synthetic antioxidants, i.e., BHA, was used as the positive control. The absorbance of the mixture was measured after every 8 hours up to 40 hours.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay The antioxidant activity of extracts, on the basis of the scavenging activity of the stable DPPH free radical (DPPH•), was determined following a previously described method24 with slight modifications. Appropriately diluted methanolic extract of R. stricta leaves, BHA, and -tocopherol (each 0.1 mL) were added to 5 mL of 0.004% methanolic solution of DPPH•. A control sample was placed under the same conditions. The resulting mixture was immediately placed in a Hitachi (Tokyo, Japan) U-2000 UV-visible spectrophotometer for measurement of absorbance at 517 nm, and the decrease in the absorbance was noted until the concentration of DPPH• reached 50%. The absorbance was noted at 0, 1, 2, 5, and 10 minutes. The remaining concentration of DPPH• in the reaction medium was calculated from a standard calibration curve.
Measurement of reducing power The reducing power of the extract was measured following the method of Yen and Chen.25 The methanolic extract and BHA (0–5.0 mg/ mL) were mixed individually with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was incubated at 50°C for 0.5 hour, followed by addition of 2.5 mL of trichloroacetic acid (10%) to the mixture, which was then centrifuged at 3,600 g for 10 minutes. The upper layer of the solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%). Absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated higher reducing power.
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Fe2-chelating activity was measured following a previously reported method.26 The reaction mixture contained extract (0.1–1.5 mg/mL), 2 mM FeCl2, and 5 mM ferrozine at the ratio of 10:1:2. The absorbance of the resulting ferrous ion–ferrozine complex was noted at 562 nm after the mixture was left at room temperature for 10 minutes. A lower absorbance indicates a higher chelating effect, and vice versa. The chelating effect of the ferrous ion was calculated using the following equation: Chelating effect (%) Absorbance of sample at 562 nm 1 100 Absorbance of control at 562 nm
Superoxide anion radical scavenging activity Superoxide anion radical scavenging activity was determined following a previously reported method.27 The phenazine methosulfate-NADH system was used for generation of superoxide radicals. The reaction mixture contained 0.25–2.0 mg/mL extract, 200 M nitro blue tetrazolium, 624 M NADH, and 80 M phenazine methosulfate in 0.1 M phosphate buffer (pH 7.4). After 2 minutes of incubation, absorbance was measured spectrophotometrically at 560 nm. The scavenging effect was calculated using the following formula: Scavenging effect (%) 100 1 Absorbance of control at 560 nm Absorbance of sample at 560 nm
RESULTS AND DISCUSSION Extraction of total antioxidants and measurement of total phenolic content Phenolics are aromatic secondary plant metabolites and are widely spread throughout the plant kingdom. Phenolics have been associated with color, sensory qualities, and nutritional and antioxidant properties of food.28 Water, 80% methanol, 70% ethanol, and diethyl ether were used for the extraction of phenolic compounds from leaves, total phenolic content was calculated by the Folin-Ciocalteu reagent method, and results were expressed as gallic acid equivalents (g/100 g). Although the Folin-Ciocalteu method is not considered authentically as an antioxidant test,29 this is the generally preferred method for measuring phenolics, because most plant-derived antioxidants contain large amounts of polyphenols.30 Total phenolic content was 2.64 0.17, 4.46 0.11, 3.86 0.23, and 3.42 0.14 g/100 g of leaves for water, 80% methanol, 70% ethanol, and diethyl ether, respectively. The highest phenolic content was found in the case of methanol, and these findings are in agreement with earlier reports31 demonstrating methanol as an effective solvent for extraction of antioxidants, and the values
are comparable to previously exploited natural sources of antioxidants.32
DPPH• scavenging activity DPPH• is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule.33 Measurement of radical scavenging activity using discoloration of DPPH has been widely used because of its stability, simplicity, and reproducibility.34 The free radical scavenging capacity of methanolic extracts of R. stricta was investigated, while BHA and -tocopherol served as standards, and a control was also assayed simultaneously. The scavenging activity, i.e., remaining amount (%) of DPPH•, was observed as a function of time. The scavenging activity gradually increased with increase in DPPH•-containing sample/ standard interaction time. At all times, BHA exhibited the highest scavenging activity, followed by R. stricta extracts, while -tocopherol exhibited the lowest activity among the series. A linear parallel relation in scavenging activity was observed for all the antioxidants. Initially, the increase in scavenging rate was sharper, but slowed with the increase in reaction time (Fig. 1). The results are in good agreement with other assays. Radical scavenging activity is considered as an authentic parameter for measurement of antioxidant activity and depends on the hydrogen donating ability of the sample; therefore extracts in polar solvents are suggested for maximum scavenging efficiency.35 The involvement of free radicals appears to be a feature of most, if not all, human disease, including cardiovascular diseases and cancer.36 Therefore, the free radical scavenging power of the natural antioxidant sources is important in fighting these diseases by conferring protection against free radical damage to cellular DNA, lipids, and proteins. On the basis of radical scavenging power, 1 g of peel and pulp of guava fruit has been reported to be equivalent to 104.1 and 54.0 mg of D,L--tocopherol, respectively.37
Reducing power Reducing power is assessed by the extent of conversion of the Fe3/ferricyanide complex to the Fe2/ferrous 120 % DPPH Remaining
Measurement of Fe2-chelating ability
100 80 60 40 20 0 0
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FIG. 1. Free radical scavenging activity of R. stricta leaves (), BHA (), and -tocopherol () compared with the control (). Data are mean SD values (n 3).
Abs. (700 nm)
ANTIOXIDANT PROPERTIES OF R. STRICTA EXTRACTS 3.0
Chelating activity
2.5
Chelating activity was measured against Fe2. Ferrous ion is commonly found in food systems and is known as an effective pro-oxidant.40 Phenolic compounds can chelate pro-oxidant metal ions, such as iron and copper, thus preventing free radical formation from these pro-oxidants and the ferrous ion-catalyzed oxidation.41 A direct correlation of chelating activity and total ferrous content was observed. The chelating effect increased with the increase in dose of extract up to 0.5 mg/mL, then the rate of chelation went on decreasing, and a very slow rise in chelating activity was observed from 0.5 to 1.5 mg/mL (Fig. 3). Activity against iron is significant in the sense that iron is essential for life because it is required for oxygen transport, respiration, and the activity of many enzymes. However, iron is an extremely reactive metal and will catalyze oxidative changes in lipids, proteins, and other cellular components.42
2.0 1.5 1.0 0.5 0.0 0.0
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1.0
1.5 2.0 2.5 3.0 3.5 Concentration (mg/mL)
4.0
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FIG. 2. Reducing power of methanolic extracts of R. stricta leaves () and BHA (). Data are mean SD values (n 3).
form. Some reports38 have shown that the reducing power of bioactive compounds was highly associated with antioxidant activity. Thus, it is necessary to determine the reducing power of phenolic constituents to elucidate the relationship between their antioxidant effect and their reducing power.39 The reducing power of the extract was observed at different concentrations, and results were compared with BHA (Fig. 2). A regular increase in reducing power, as a function of concentration, was observed up to a concentration of 1.5 mg/mL for the extract. Initially, the rate of increase in reducing power was high, but it gradually decreased, and finally became almost constant. BHA exhibited a very sharp increase in reducing power as a function of concentration, and observations could not be recorded above a concentration of 0.5 mg/mL. However, the reducing power is comparable to other previously exploited sources of antioxidants.22 Appreciable reducing power reveals the electron donor capacity of R. stricta extracts, leading to reaction with free radical and conversion to stable product and terminating the free radical chain reaction.25 The results for reducing power are in agreement with total antioxidant activity, as reducing power is a direct measure of antioxidant activity.23
Superoxide anion radical scavenging activity Superoxide anion radical is a precursor to active free radicals that have the potential for reacting with biological macromolecules and thereby inducing tissue damage.43 It has been reported to be involved in coronary, arteriosclerosis, and tumor diseases. Its elimination may lead to prevention of aging as well as many other diseases in vivo and in vitro; it may lead to increase in life span and living conditions of plants. Superoxide anion radical scavenging activity of R. stricta extracts was investigated as a function of concentration (Fig. 4). R. stricta extracts exhibited appreciable scavenging activity against the superoxide anion radical in a dosedependent manner. Initially, the scavenging effect rose sharply with the increase in concentration of extract, but the scavenging rate decreased gradually with the increase in dose and finally became almost constant. The results reveal appreciably the high medicinal value of R. stricta.
Antioxidant activity in the linoleic acid system The thiocyanate method was employed for measurement of antioxidant activity in the linoleic acid system. During
100 Scavenging Effects (%)
80 Chelating Effects (%)
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60 40 20 0
80 60 40 20 0
0.0
0.2
0.4
0.6 0.8 1.0 1.2 Concentration (mg/mL)
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FIG. 3. Chelating effect of R. stricta leaves on ferrous ion. Data are mean SD values (n 3).
0.0
0.5
1.5 1.0 Concentration (mg/mL)
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FIG. 4. Scavenging effects of crude methanolic extracts of R. stricta leaves on superoxide anion radical. Data are mean SD values (n 3).
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Absorbance (500 nm)
2.5 2.0 1.5 1.0 0.5 0.0 0
9
18
27
36
45
Time (h)
FIG. 5. Antioxidant activity of R. stricta leaves (), BHA (), and -tocopherol () in the linoleic acid system. Data are mean SD values (n 3).
the process, peroxides are formed in the emulsions. These peroxides are measured spectrophotometrically at 500 nm. A higher degree of absorbance is a measure of a higher concentration of peroxides formed and less antioxidant activity. Antioxidant activity of R. stricta extracts was determined as a function of time, while BHA and -tocopherol served as standards (Fig. 5). An increase in degree of oxidation/decrease in antioxidant activity was observed as a function of time. Initially the rate of increase in absorbance was slow and comparable among standards and extract. But after 8 hours, the absorbance increased sharply, and maximum absorbance was observed for -tocopherol followed by R. stricta extracts and BHA, respectively, at all times of analyses. After 32 hours of storage, a significant difference in absorbance was noted. The results reveal a strong antioxidant potential of R. stricta leaves comparable with those of BHA and -tocopherol.
CONCLUSION From the present study, it may be concluded that R. stricta is an important source of natural antioxidants with good free radical scavenging and anion radical scavenging capacities. It may be employed for treatment of various diseases like tumors, chronic rheumatism, arteriosclerosis, etc. Being abundantly available in the Asian continent and possessing antioxidant potential comparable to synthetic ones, it may be used in food systems and other industries.
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