In Vitro. Antioxidant Activity of Thirteen Medicinal Plants of India's Western Ghats

Abstract

Sixteen methanol extracts from thirteen medicinal plants collected from Western Ghats, India, were screened for in vitro. antioxidant activity using different models. The methanol extract of C. aromaticus. was found to be more active with low IC₅₀ value in most of the methods. The extracts of A. indica., C. arietinum., M. alba., O. dillenii., P. procumbens., S. androgynus., and S. oenopila. showed potent antioxidant activity against ABTS free radicals. Potent activity was observed for the extracts of B. calicynum. against DPPH free radicals and for the extracts of O. dillenii. and C. aromaticus. in the lipid peroxidation assay. Many of the extracts showed high total phenol content. The leaf extract of C. aromaticus. merits further investigation in animal models.

Keywords:

• Antioxidant
• flavonoids
• lipid peroxidation

Introduction

The high intake of plant products is associated with a reduced risk of a number of chronic diseases, such as atherosclerosis and cancer (Gosslau & Chen, 2004). In most cases, these beneficial effects have been partly attributed to compounds that possess antioxidant activity. Harmful free radicals and reactive oxygen species (ROS) have been found to play an important role in the causes of major chronic health problems, including diabetes, hypertension, cancer, malaria, etc. (Tsao & Deng, 2004). Antioxidants such as vitamin C and E are essential for protection against ROS. However, the majority of the antioxidant activity of plants may be from compounds such as phenolic acids and flavonoids, rather than from vitamin C, E, or β.-carotene (Guo et al., 1997). Hence, antioxidant phytochemicals such as flavonoids are the focus of many recent studies (Czeczot, 2000; Chu et al., 2000). In the present study, several plants collected from Western Ghats, Shimoga, India, were screened, using several standard methods, for their antioxidant activity based on their ethnomedical uses.

Materials and Methods

Plant material

Different parts of the plants Acalypha indica. L. (Euphorbiaceae), Bryophyllum calycinum. Salisb (Crassulaceae), Cicer arietinum. L. (Papilonaceae), Cissus quadrangularis. L. (Vitaceae), Coleus aromaticus. Benth (Lamiaceae), Cucumis trigonus. Roxb (Cucurbitaceae), Morus alba. L. (Moraceae), Opuntia dillenii. Haw (Cactaceae), Pavonia procumbens. Boiss (Malvaceae), Sauropus androgynus. L. Merr. (Euphorbiaceae), Spilanthes calva. DC (Compositeae), Vertivera zizanoides. L. Nash. (Gramineae,) and Zizuphus oenoplia. L. (Ramnaceae) were collected in the month of July 2004 from Shimoga, Karnataka, India. Authentication was done by Survey of Medicinal Plants and Collection Unit, Ootacamund, Tamilnadu, India. A voucher specimen is preserved in our laboratory.

Extraction

The plants were shade dried, powdered, and subjected to extraction (50 g each) by maceration in methanol (250 ml) at room temperature, with occasional shaking, for 7 days. The macerate was filtered, and the filtrate was dried at low temperature under vacuum in a rotary evaporator. The extracts were preserved in a refrigerator for further use. The yields of these extracts are given in Table 1.

Table 1.. In vitro. antioxidant activity of methanol extracts of thirteen medicinal plants by different methods.

		IC50 values ± SE (µg/ml) by method
Plants	Yield of extract (%)	ABTS	DPPH	Lipid peroxidation	H2O2	p.–NDA	Nitric oxide	Total phenol content (% w/w)	Total antioxidant capacity**
A. indica.^L	5.40	5.93 ± 0.06	230.00 ± 6.93	>1000	193.33 ± 3.00	>1000	>700	14.77 ± 0.22	2.86 ± 0.02
A. indica.^S	3.20	14.33 ± 0.30	212.83 ± 7.27	700.00 ± 10.00	380.00 ± 5.27	873.33 ± 17.64	490.00 ± 17.60	13.43 ± 0.19	2.27 ± 0.03
A. indica.^R	2.20	24.00 ± 1.50	208.5 ± 12.5	180.00 ± 8.90	205.00 ± 2.50	>1000	>700	5.89 ± 0.12	1.53 ± 0.03
B. calycinum.^L	1.50	96.66 ± 10.66	25.66 ± 0.84	145.00 ± 2.50	230.00 ± 3.81	>1000	>700	5.44 ± 0.40	0.57 ± 0.07
C. arietinum.^L	7.87	19.50 ± 0.28	170.83 ± 6.76	216.66 ± 5.06	199.14 ± 3.00	>1000	>700	17.01 ± 0.38	3.71 ± 0.02
C. quadrangularis.^L	3.80	118.00 ± 10.65	183.00 ± 4.94	160.00 ± 2.50	373.33 ± 1.66	>1000	>700	3.99 ± 0.19	2.10 ± 0.03
C. aromaticus.^L	2.20	7.50 ± 0.15	57.33 ± 0.87	83.00 ± 2.00	388.33 ± 1.66	956.6 ± 8.81	>700	10.06 ± 0.49	1.93 ± 0.03
C. trigonus.^F	1.79	99.66 ± 2.40	684.00 ± 24.41	160.00 ± 2.50	>1000	>1000	>700	4.99 ± 0.26	0.79 ± 0.03
M. alba.^L	9.40	23.00 ± 0.50	89.16 ± 5.23	487.50 ± 2.50	293.33 ± 3.33	270.00 ± 5.77	>700	8.51 ± 0.10	3.29 ± 0.03
O. dillenii.^F	3.00	45.5 ± 1.04	317.00 ± 28.94	352.50 ± 2.50	>1000	>1000	>700	8.40 ± 0.49	1.38 ± 0.04
O. dillenii.^L	2.80	27.00 ± 2.08	>1000	78.00 ± 4.16	>1000	>1000	>700	5.89 ± 0.49	0.38 ± 0.07
P. procumbens.^L	4.20	27.83 ± 0.66	370.00 ± 20.91	161.66 ± 12.44	175.00 ± 4.33	418.3 ± 10.13	>700	8.54 ± 0.44	2.52 ± 0.02
S. androgynus.^L	9.90	12.58 ± 1.08	341.00 ± 18.29	228.75 ± 1.25	370.00 ± 5.77	>1000	>700	9.82 ± 0.10	2.41 ± 0.04
S. calva.^L	4.55	46.33 ± 2.16	322.5 ± 11.23	766.66 ± 55.47	123.33 ± 2.20	>1000	>700	10.90 ± 0.12	2.63 ± 0.02
V. zizanoides.^Rh	2.79	56.83 ± 1.47	273.33 ± 10.54	312.50 ± 12.50	398.33 ± 6.00	>1000	>700	9.90 ± 0.16	0.72 ± 0.02
Z. oenoplia.^F.	23.10	24.00 ± 1.50	483.33 ± 18.95	370.00 ± 15.28	>1000	>1000	646.63 ± 12.05	12.07 ± 0.46	1.95 ± 0.04
Standards
Ascorbic acid	—	11.25 ± 0.49	2.69 ± 0.05	—	187.33 ± 3.93	>1000	—	—	—
Rutin	—	0.52 ± 0.01	3.91 ± 0.10	—	36.16 ± 0.16	205.83 ± 0.83	68.44 ± 2.56	—	—
BHA	—	—	—	95.00 ± 3.00	—	>1000	—	—	—

*Average of three determinations, L – Leaves, S – Stem, R – Root, F – Fruits, and Rh – Rhizome; in alkaline DMSO and deoxyribose methods, all the values were above 1000 µg/ml.

**Equivalent of ascorbic acid µM/g.

Chemicals

1,1-Diphenyl-2-picryl hydrazyl (DPPH) and 2,2′-azino-bis. (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Sigma Aldrich Co., St. Louis, USA. Rutin and p.-nitroso dimethyl aniline (p.-NDA) were obtained from Acros Organics, New Jersey, USA. naphthyl ethylene diamine dihydrochloride (NEDD) was obtained from Roch–Light Ltd., Suffolk, UK. All chemicals used were of analytical grade.

Preparation of test and standard solutions

Methanol extracts and the standard antioxidants (ascorbic acid, rutin, butylated hydroxy anisole, and α.-tocopherol) were dissolved in distilled dimethylsulphoxide (DMSO) separately and used for the in vitro. antioxidant assays using eight different methods, with the exception of the hydrogen peroxide method. For the hydrogen peroxide method, where DMSO interferes with the method, the extracts and standards were dissolved in distilled methanol and used. The stock solutions were serially diluted with the respective solvents to obtain the lower dilutions.

Total phenol estimation

The total phenol was determined using Folin-Ciocalteu reagent (Sadasivam & Manikam, 1992). In a series of test tubes, 0.4 ml of the methanol extract in methanol was taken and mixed with 2 ml of Folin-Ciocalteu reagent and 1.6 ml of sodium carbonate. After shaking, it was kept at 25°C for 2 h, and the absorbance was measured at 750 nm using a Shimadzu-UV-160 spectrophotometer. Using gallic acid monohydrate, a standard curve was prepared, and linearity was obtained in the range of 1–10 µ g/ml. Using the standard curve, the total phenol content was calculated and expressed as gallic acid equivalent in mg/g of extracts.

In vitro. antioxidant activity

The methanol extracts of thirteen plants were tested forin vitro. antioxidant activity using the following standard methods. In all these methods, a final concentration of 1000–0.45 µg/ml of the extract or standard was used. Absorbance was measured against a blank solution that contained extract or standard, but without the reagents. A control was performed without adding extracts or standards. Percentage scavenging and IC₅₀ value, which is the concentration of the sample required to scavenge 50% of the free radicals, was calculated.

Scavenging of ABTS radical cation

To 0.2 ml of various concentrations of the extract or standard, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution were added and incubated for 20 min. Absorbance of these solutions was measured spectrophotometrically at 734 nm (Re et al., 1999).

DPPH radical scavenging method

The extract or standard (10 µl) was added to DPPH in methanol solution (200 µl) in a 96-well microtitre plate (Tarsons, Kolkata, India). After incubation at 37°C for 30 min, the absorbance of each solution was determined at 490 nm using an ELISA microtitre plate reader (Bio Rad Laboratories Inc, California, USA, Model 550) (Hwang et al., 2001; Shreejayan & Rao, 1996).

Scavenging of hydroxyl radical by deoxyribose method

To the reaction mixture containing deoxyribose (3 mM, 0.2 ml), ferric chloride (0.1 mM, 0.2 ml), EDTA (0.1 mM, 0.2 ml), ascorbic acid (0.1 mM, 0.2 ml), and hydrogen peroxide (2 mM, 0.2 ml) in phosphate buffer (pH, 7.4, 20 mM) were added 0.2 ml of various concentrations of extract or standard in DMSO to give a total volume of 1.2 ml. The solutions were then incubated for 30 min at 37°C. After incubation, ice-cold trichloroacetic acid (0.2 ml, 15% w/v) and thiobarbituric acid (0.2 ml, 1% w/v) in 0.25 N HCl were added. The reaction mixture was kept in a boiling water bath for 30 min and then cooled, and the absorbance was measured at 532 nm (Barry et al., 1987; Elizabeth & Rao, 1990).

Scavenging of hydroxyl radical by p.-NDA method

To a solution mixture containing ferric chloride (0.1 mM, 0.5 ml), EDTA (0.1 mM, 0.5 ml), ascorbic acid (0.1 mM, 0.5 ml), hydrogen peroxide (2 mM, 0.5 ml), and p.-NDA (0.01 mM, 0.5 ml) in phosphate buffer (pH 7.4, 20 mM) was added various concentrations of extract or standard in distilled DMSO (0.5 ml) to produce a final volume of 3 ml. Absorbance was measured at 440 nm (Elizabeth & Rao, 1990).

Scavenging of hydrogen peroxide

A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer saline (PBS, pH 7.4). Various concentrations of the extract or standard in methanol (1 ml) were added to 2 ml of hydrogen peroxide solution in PBS. After 10 min the absorbance was measured at 230 nm (Jayaprakash et al., 2004).

Lipid peroxidation inhibitory activity

Lipid peroxidation was initiated by adding ferric chloride (100 µM, 0.25 ml) to a mixture containing rat brain homogenate (0.25 ml) and different concentrations of extract or standard (0.25 ml) in a total volume of 0.75 ml. The reaction mixture was incubated for 20 min at 37°C. After incubation, the reaction was stopped by adding 1 ml ice-cold 0.25 M HCl containing 15% trichloroacetic acid, 0.38% thiobarbituric acid, and 0.05% butylated hydroxytoluene. After heating at 80°C for 15 min, samples were cooled and then centrifuged at 1000 rpm for 10 min, and absorbance of the supernatant was measured at 532 nm (Sudheer Kumar et al., 2003).

Nitric oxide radical inhibition assay

The reaction mixture (6 ml) containing sodium nitroprusside (10 mM, 4 ml), phosphate buffer saline (1 ml), and extract or standard solution (1 ml) was incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture was removed, 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) was mixed and allowed to stand for 5 min for completion of diazotization reaction, and 1 ml of NEDD was added, mixed, and allowed to stand for 30 min in diffused light. The absorbance was measured at 540 nm against the corresponding blank solutions in a 96-well microtitre plate using an ELISA reader (Marcocci et al., 1994; Badami et al., 2003).

Scavenging of superoxide radical by alkaline DMSO method

To the reaction mixture containing 0.1 ml of NBT (1 mg/ml solution in DMSO) and 0.3 ml of extract or standard in DMSO was added 1 ml of alkaline DMSO (1 ml DMSO containing 5 mM NaOH in 0.1 ml water) to give a final volume of 1.4 ml, and the absorbance was measured at 560 nm (Elizabeth & Rao, 1990).

Evaluation of total antioxidant capacity

An aliquot of 0.1 ml of extract or standard solution in DMSO was combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) in an Eppendroff tube. The tubes were capped, incubated in a water bath at 95°C for 90 min, cooled to room temperature, and the absorbance of each solution was measured at 695 nm against a blank solution (Jayaprakash et al., 2004). The total antioxidant capacity was expressed as the equivalent of ascorbic acid per gram of the extract.

Results and Discussion

In the present study, 16 extracts belonging to 13 plants were screened for in vitro. antioxidant activity using several standard methods. Most of the plants showed potent antioxidant activity against ABTS free radicals (Table 1). These include A. indica. leaves, stem and root; C. arietinum. leaves; M. alba. leaves; O. dillenii. leaves and fruit; P. procumbens. leaves; S. androgynus. leaves; and S. oenoplia. fruits. The leaves of A. indica. and C. aromaticus. were found to be the most active, with IC₅₀ values of 5.93 ± 0.06 and 7.50 ± 0.15 µg/ml, respectively. These values were found to be lower than that of the standard ascorbic acid but higher than that of the standard rutin. The remaining plants, except B. calycinum., C. trigonus., and C. quadrangularis., showed moderate antioxidant activity. In the DPPH method, the extracts of B. calycinum. leaves exhibited potent antioxidant activity, and C. aromaticus. leaves and M. alba. leaves showed moderate antioxidant activity. In the lipid peroxidation assay, leaf extracts of O. dillenii. and C. aromaticus. showed potent antioxidant activity, higher than that of the standard BHA. In all other methods, all the extracts showed moderate or weak antioxidant activity.

In the evaluation of total antioxidant capacity by the phosphomolybdenum method, C. arietinum. leaves and M. alba. leaves were found to be more potent, with activity equivalent to 3.70 ± 0.02 and 3.29 ± 0.03 µM/g of ascorbic acid. The extracts of leaves of A. indica., P. procumbens., S. calva., and S. androgynus. also showed potent antioxidant activity in this method. The total phenol content of the extracts of C. arietinum. leaves, A. indica. leaves and stem, Z. oenoplia. fruits, S. calva. leaves, and C. aromaticus. leaves was found to be high. Hence, there was no correlation between the antioxidant activity of the extracts and their total phenol content.

In conclusion, among the 13 plants tested for in vitro. antioxidant activity using different methods, the methanol extract of C. aromaticus. leaves was found to be the most potent. Earlier studies indicated strong in vitro. antioxidant activity of aqueous extract of C. aromaticus. leaves in many models, including a few studied in the present work (Kumaran & Karunakaran, 2006). Our studies also confirmed this finding. Several phytoconstituents, including flavonoids quercetin, luteolin, and apigenin, were isolated from the leaves (Yoganarasimhan, 1996). Plant phenolics including flavonoids are known to possess strong antioxidant properties (Chu et al., 2000). Hence, the observed antioxidant activity may be due to the presence of flavonoids in the extract. C. aromaticus. leaves merit further investigation in animal models as well as to isolate its active constituents.

Acknowledgments

The authors are thankful to His Holiness Jagadguru Sri Sri Shivarathreeshwar Deshikendra Mahaswamigalavaru of Sri Suttur Mutt, Mysore, for providing the facilities. One of the authors, Mr. Channabasavaraj, would like to thank the All India Council for Technical Education, New Delhi, for providing financial support through its Quality Improvement Programme.