Radical Scavenging Activity of Methanol Leaf Extracts of Orthosiphon stamineus.

Abstract

The antioxidant activity of methanol extracts of Orthosiphon stamineus. Benth (Lamiaceae), cultivated in different regions of Malaysia, was determined by measuring the scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and on superoxide anion. The results showed a variation in free-radical and superoxide-anion scavenging activities of the methanol extracts ranging from 62.82 to 92.34% and 53.29 to 75.88%, respectively. Methanol extract was active, and its antioxidative potency was comparable to that of pure quercetin and higher than that of the widely used synthetic antioxidant butylated hydroxylanisole (BHA). The UV and HPLC chromatographic fingerprints of the major phenolics were qualitatively similar for all the methanol leaf extracts of Orthosiphon stamineus. from various locations.

Keywords:

• Antioxidants
• free-radical scavenging
• Orthosiphon stamineus
• superoxide anion

Introduction

Natural antioxidant defenses are adequate to scavenge active radicals in healthy individuals, but the concentration of these antioxidants decreases with age, consequently increasing the amount of oxidation product with age. These have been implicated in various human diseases including the processes of aging, cancer, senile dementia, inflammation, and atherosclerosis (Sohal & Allen, 1985). There is increasing interest in the role of phenolics in prevention of such diseases and this has been the subject of extensive research. Phenolic compounds found in plants have been reported to have multiple biological effects including potent antioxidant activity that protect plants from oxidative damage, and they perform the same function for humans with their ability to inhibit oxidation of human low-density lipoprotein (LDL) (Decker, 1995; Evans & Miller, 1996; Robards et al., 1999). Phenolics have been shown to have the ability to block specific enzymes that cause inflammation and also modify the prostaglandin pathways and thereby protect platelets from clumping (Hertog, 1995). The antioxidant activity of natural extracts depends on the following; the quality of the original plant, climatic condition, geographic origin, harvesting period, storage, and technological factors (Cuvelier et al., 1996; Hagerman et al., 1998).

Malaysia has a tropical climate with high temperatures and rainfall all year, which have enabled Orthosiphon stamineus. Benth (Lamiaceae), known locally as misai kucing. or kumis kucing., to flourish extensively. In Malaysia, the plant is used for treatment of a wide range of diseases: eruptive fever, epilepsy, gallstone, hepatitis, rheumatism, hypertension, syphilis, gonorrhea, and renal calculus. Orthosiphon stamineus. (OS) is cultivated, and the leaves are used to prepare a diuretic tea, which has been reported to be active against kidney and bladder inflammation (Hegnauer, 1966; Wangner, 1982). The plant has several chemically active constituents, but one of the most important classes of compounds is the phenolics (Surmaryono et al., 1991). Natural antioxidant could be used for protecting living systems from oxidative damage by scavenging oxygen radicals. Studies on antioxidant acitivities of OS crude extracts are limited. This paper presents total phenolic content and free-radical and active oxygen scavenging activities of methanol leaf extracts of OS cultivated in different places in Malaysia.

Materials and Methods

Chemicals

Standard compounds of sinensetin, eupatorin, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, and rosmarinic acid were purchased from Indofine Chemical Co. (Hillsborough, NJ, USA). Solvents used for chromatography were tetrahydrofuran, methanol (HPLC grade), water (HPLC grade, the pH of water was adjusted to 3.0 using phosphoric acid), and ethanol (80%) obtained from Merck (Darmstadt, Germany). Folin-Ciocalteu reagent, Na₂CO₃, quercetin, butylated hydroxylanisole (BHA), caffeic acid, β-nicotinamide adenine dinucleotide (NADH, reduced form), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Membrane filters (0.45-µm pore size) from Millipore (Massachusetts, USA) were used for filtration of the mobile phase and the samples. All other chemicals were analytical grade or HPLC grade.

Plant materials

The plants were grown from cuttings using standard agronomic practices. The leaves were collected in the late afternoon, and were 30- to 45-day-old white-flowered plants of similar sizes and leaf area. Each specimen was labeled, numbered, and annotated with the date of collection and locality (Table 1). Voucher specimens of the plant material from different locations were deposited at Bilik Herba, School of Pharmaceutical Sciences, University Sains Malaysia.

Table 1.. Radical scavenging activity, total phenolic and soluble solid contents of methanolic extracts of Orthosiphon stamineus. leaf.^a.

				Antioxidant activity (%)
Location	State	Collection date^b.	Total phenolics (mg g^−1dry wt)	DPPH	NADH/PMS
Bohor Temak	Perlis	08/01/02	9.46	92.34 a	68.90 d
Kepala Batas	Pulau Pinang	26/05/02	7.61	88.68 b	72.81 bc
Bota Kanan	Perak	15/01/02	9.28	87.38 b	62.50 f
Kuala Nerang	Kedah	30/01/02	9.65	91.96 a	73.68 b
Kuching	Sarawak	20/09/01	9.59	83.75 d	71.27 c
Rawang	Selangor	26/12/02	8.86	88.49 bc	74.56 ab
Semonggok	Sarawak	05/03/02	9.77	90.31 b	72.80 b
Jengka	Pahang	09/04/02	7.36	62.82 e	72.59 b
Gua Musang	Kelantan	25/10/01	9.71	90.21 b	53.95 h
Parit	Perak	04/04/02	10.20	86.27 c	53.29 h
Pasir Puteh	Kelantan	25/07/02	6.69	88.83 bc	58.11 g
Bumbung Lima	Pulau Pinang	24/01/02	10.05	91.26 a	75.88 a
Quercetin				91.50 a	73.44 b
BHT				66.43 e	66.11 e

^a.The experiments were performed in January–February 2003. Leaves from white-flowered cultivars with similar fresh and dry weight (p > 0.05) were used for this experiment.

^b.Day/month/year.

Means within column with different letters indicate significantly different values (p < 0.05).

Sample preparation

One gram each of the dry leaf powder, from different places, was extracted with 100 ml of methanol for 4 h with continuous stirring with magnetic stirrer at 40°C on a water bath. The extracts were filtered through filter paper (Whatman no. 1) with a Buchner funnel under vacuum and cooled to room temperature. Aliquots of the extracts were kept in a refrigerator at –20°C until further use.

Determination of total phenolic and total soluble contents of methanol extracts

The concentration of total phenolics in extracts was determined by using Folin-Ciocalteu reagent and external calibration with caffeic acid. Briefly, 0.2 ml of extract solution in a test tube and 0.2 ml of Folin-Ciocalteu reagent was added and the contents mixed thoroughly. After 4 min, 1 ml of 15% Na₂CO₃ was added, then the mixture was allowed to stand for 2 h at room temperature. The absorbance was measured at 760 nm. The concentration of the total phenolics was determined as milligrams of caffeic acid equivalent by using an equation obtained from caffeic acid calibration curve, determined by linear regression. The total methanol soluble solids content of an aliquot (20 ml) of extract was determined in triplicate (Joubert, 1988).

Free-radical scavenging activity of extracts using the DPPH assay

The method for estimating the free-radical scavenging activity (RSA) of the methanol extracts of OS was adapted from that of Hatano et al. (1988) with some modifications. Two milliliters of methanol solution of DPPH (0.1 mM) was mixed with 200 µl of samples of OS extract (0.2 mg/ml) and made up with methanol to a final volume of 3 ml. After 60-min standing, the absorbance of the mixture was measured at 517 nm against methanol as blank using Perkin-Elmer Lambda 45 spectrophotometer. Quercetin and BHA were used as standards. The radical scavenging activity (%) of the tested samples was evaluated by comparison with a control (2 ml DPPH solution and 1 ml of methanol). Each sample was measured in triplicate and averaged. The radical scavenging activity was calculated using the formula: where A._c is the absorbance of the control and A._s is the absorbance of the tested sample after 60 min.

Scavenging effects on superoxide anion

Superoxide anion scavenging activity of the methanol extracts of OS was measured by using the method of Robak and Gryglewski (1988) with some modifications. Superoxide was generated in a nonenzymatic (phenazine methosulfate-NADH) system. The reaction mixture consisted of 100 µl of 60 µM PMS,1 ml of 468 µM NADH, 1 ml of 156 µM NBT solutions in 0.1 M phosphate buffer (pH 7.4), 100 µl of OS extracts (0.2 mg/ml), and 0.8 ml of 0.1 M phosphate buffer (pH 7.4). The reaction was initiated by 100 µl of PMS. After 5 min incubation at room temperature, the absorbance was measured at 560 nm against blank samples, which contain no PMS. The experiment was performed in triplicate and averaged. The percentage inhibition of the superoxide anion generation was calculated using the formula: where A._c is the absorbance of the control and A._s is the absorbance of the tested sample.

Spectrophotometric analysis of samples

UV analyses

The UV analyses were carried out using a Perkin-Elmer Lambda 45 UV spectrophotometer. A known concentration of the methanol leaf extracts from different locations (0.01 mg/ml) was scanned from a wavelength of 200.0 to 420 nm against methanol (HPLC grade) as blank.

High-performance liquid chromatography

HPLC was performed using an Agilent HPLC system (Agilent 1100 Series, Agilent Technologies, Waldbronn, Germany) with a 250 mm × 4.6 mm C-18 Lichrosorb column; the eluates were detected at 340 nm at 25°C. The flow rate was 1 ml/min. Samples were dissolved in methanol, and 20 µl of this solution was injected in the column. Rosamarinic acid (RA), 3′-hydroxy-5,6,7,4′-tetramethylflavone (TMF), eupatorin (EUP) and sinensetin (SEN) were used as reference markers (Fig. 1). The mobile phase was methanol:water at pH 3 (adjusted with phosphoric acid):tetrahydrofuran [4.5:5:0.5], and the total running time of analysis was 40 min. The HPLC method applied is a modification of that reported by Pietta et al. (1991) for the analysis of polymethoxylated flavones present in Orthosiphon. leaves.

Figure 1. Chemical structures of markers.

Figure 2. UV spectra of methanolic leaf extracts of Orthosiphon stamineus. from different locations in Malaysia.

Statistical analyses

Experimental results were mean ± SD of three parallel measurements and were analyzed by SPSS (version 10.0 for Windows 98, SPSS Inc.). Differences between means were determined using Tukey multiple comparisons. Correlations were obtained by Pearson correlation coefficient in bivariate correlations; p values < 0.05 were regarded as significant.

Results and Discussion

The UV and HPLC chromatographic profiles of flavonoids were qualitatively similar for all the methanol extracts of samples from different locations collected, giving reliable indication of the same identity; however, differences in relative concentration of the markers were observed (the quantitative data is not discussed in this article). The UV scan of the methanol extracts of the leaf showed UV spectra with peaks at 283 nm and 330 nm. In the HPLC analysis of the methanol extracts of the leaves, RA, TMF, SEN, and EUP (Fig. 1) used as markers were separated within a total time of 40 min by isocratic elution at 340 nm using a reverse-phase C-18 column (125 × 4 mm). All the markers were present in the chromatographic profiles of the samples from various locations. Their peaks were confirmed by comparison of the retention time with authentic standards, which were determined in a single run (Fig. 3) using the isocratic elution. The standards were resolved and eluted at 5.6, 12.5, 17.2, and 25.7 min with respect to RA, TMF, SEN, and EUP (Fig. 3). The RA peak was the prominent peak compared to the lipophilic flavones.

Figure 3. (a) HPLC chromatogram of reference markers, rosmarinic acid (RA), 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF), eupatorin (EUP), sinensetin (SEN). (b) HPLC fingerprint of methanol extract of Orthosiphon stamineus. leaf. Isocratic elution, methanol-water (pH 3)-tetrahydrofuran (4.5:5:0.5); flow rate, 1 ml/min; UV; 340 nm; temperature; 25°C; column; LiChrosorb RP-18.

The yield of extracts obtained from OS using methanol is shown in Figure 4. The yield of methanol extracts varied from 7.10% in leaf samples from Pasir Puteh (Kelantan) to 18.20% from Bohor Temak. The result of phenol analysis of the methanol extracts of OS from different locations is given in Table 1. The total phenolic content of the methanol extracts varied from 6.69 mg caffeic acid/g dry weight in the sample from Pasir Puteh (Kelantan) to 10.20 mg caffeic acid/g dry weight in the sample from Parit (Perak).

Figure 4. Methanol-soluble solid of Orthosiphon stamineus. leaf.

The results of free-radical scavenging activity of OS methanol extracts of samples from different locations by the DPPH method are shown in Table 1. A purple-colored DPPH is a stable free radical, which is reduced to α,α-diphenyl-β-picryl hydrazine (yellow colored) by reacting with an antioxidant. Antioxidants interrupt the free radical chain oxidation by donating hydrogen from hydroxyl groups to form a stable end product, which does not initiate or propagate further oxidation of lipids (Sherwin, 1978). The methanol extracts demonstrated a significant inhibitory activity against the DPPH radical at 0.2 mg/ml, and there was a similar radical scavenging activity for the reference compounds, quercetin and BHA (0.2 mg/ml). The data obtained showed that the methanol extracts of OS have a free-radical scavenging activity. The DPPH scavenging effect of the methanol extracts of OS from the locations and the standards decreased in the order Bohor Temak > Kuala Nerang > quercetin > Bunbung Lima > Semonggokk > Gua Musang > Pasir Puteh > Kepala Batas > Rawang > Bota Kanan > Parit > Kuching > BHT > Jengka. The results indicated that the methanol extracts of OS are free-radical inhibitors and primary antioxidants that react with free radicals.

Superoxide acts as an oxidizing or reducing agent, depending on the substrate oxidation potential (Ilan et al., 1976). Because superoxide and hydrogen peroxide serve as precursors of singlet oxygen and hydroxyl radicals, it indirectly initiates peroxidation of lipids (Kellogg & Fridovich, 1975; Aurand et al., 1977). It has also been reported by some researchers that superoxide can directly initiate lipid peroxidation (Lavelle et al., 1973; Goldstein & Weissmann, 1977). The results of the superoxide anion scavenging activity of the methanol extracts of samples from various locations in comparison with the same doses of quercetin and BHA are shown in Table 1. The addition of OS extracts (0.2 mg/ml) to the phenazine methosulfate-NADH (PMS-NADH) system significantly (p < 0.05) diminished production of superoxide. Quercetin and BHA (0.2 mg/ml) also inhibited superoxide production. The results revealed that the methanol extracts of OS exhibited superoxide radical activity comparable to quercetin and BHA. The variations in the free-radical and superoxide anion activity may be due to agronomic practices and environmental conditions. Though the plants selected for this experiment were of similar height, leaf area, fresh weight, and dry weight, the chemical composition of the leaves could be affected by the soil fertility levels (Schimdt, 1971; Taylor et al., 1983).

The antioxidant activity could be attributed to the high phenolic content of the extracts. OS contain several chemically active constituents, but one of the most important classes of compounds is the phenolics. Twenty phenolic compounds were isolated included nine lipophilic flavones, two flavonol glycosides, and nine caffeic acid derivatives such as rosmarinic acid and 2,3-dicaffeoyltartaric acid were identified from 80% aqueous methanol of OS and quantified by HPLC (Sumaryono et al., 1991). Plant phenolics are very effective free-radical scavengers and superoxide-anion scavengers and their antioxidant activities are well documented (Cuvelier et al., 1995; Evans & Miller, 1996).

To conclude, the methanol extracts of OS grown locally were potent with respect to free-radical scavenging activity and superoxide inhibition determined by the DPPH radical method and nonenzyme PMS/NADH-NBT systems. The samples from various locations showed a remarkable degree of variation of their antioxidant activity. The antioxidative potency of the extracts was comparable to that of pure quercetin and synthetic antioxidant BHA, thus presenting an alternative source for natural additives. In vivo. studies to assess the antioxidant effect in biological systems and xanthine oxidase inhibitory activity are proceeding in our laboratory.

Acknowledgment

The study was supported by Intensifying Research Priority Areas (IRPA) Grant from the Ministry of Science, Technology, and Environment, Malaysia.