Free Radical Scavenging Activity of Extracts from Thai Plants in Guttiferae and Schisandraceae Families

ABSTRACT

Five Thai plants from the Guttiferae (Hypericum hookerianum. Wight & Arn, Garcinia speciosa. Wall, Garcinia xanthochymus. Hook f. ex. T. Anderson, Cratoxylum formosum. ssp. pruniflorum. (Kurz) Gogel, and Calophyllum polyanthum. Wall ex Choisy) and one from the Schisandraceae (Schisandra verruculosa.) were extracted by methanol and chloroform. The extracts were screened for free radical scavenging activity using the DPPH assay. All extracts showed a dose-dependent antioxidant activity. The most potent with the lowest IC₅₀ values were observed in the methanol extracts from the wood of G. speciosa., which were 2.5- and 5.3-fold more potent than the two standard antioxidants, ascorbic acid and α.-tocopherol, respectively. Free radical scavenging activities ranging from moderate to high were observed in both methanol and chloroform extracts from H. hookerianum., C. formosum. ssp. pruniflorum., G. xanthochymus., S. verruculosa. and C. polyanthum.. The information from this study can explain the traditional use and the further development of these extracts into new pharmaceuticals.

Keywords:

• DPPH
• free radical scavenging activity
• Guttiferae
• Schisandraceae

Introduction

Free radicals play an important role in the development of tissue damage and other pathological events such as cancer, aging, inflammation, and some degenerative diseases. The antioxidative properties of plant extracts and their isolated compounds which have free radical scavenging activity receive considerable attention for possible use in protection of cells and organs against oxidative damage (Siddhuraju et al., 2002; Velazquez et al., 2003). Natural antioxidants can be found in different plant tissues, including wood, bark, stem, leaf, root, flowers, and seeds. Many plant-derived antioxidants of free radical scavenging agents are phenolic or polyphenolic compounds (Zin et al., 2002). Several methods such as the ferric thiocyanate method and the thiobarbituric acid test are available for evaluating antioxidative activity. The free radical scavenging activity assay using DPPH (1,1-diphenyl-2-picrylhydracyl), a stable free radical, has been widely used to assess the free radical scavenging activity of antioxidants (Choi et al., 2002), as it is a simple and less time consuming method.

Compounds in plants from various parts of Southeast Asian have been screened and have exhibited significant biological activities. Garcinia. and Hypericum. species (Guttiferae) were found to be rich in secondary metabolites such as xanthonoids, biflavonoids, and triterpenoids (Xu et al., 1998). Some of these plants have been used in traditional medicines. For example, H. geminiflorum. (Hemsl), an endemic plant in Taiwan, is a Chinese folk medicine for the treatment of several bacterial infections, infectious hepatitis, gastrointestinal disorder, and tumor. Two new oxygenated xanthones and constituents with antiplatelet and anti-inflammatory activity have been found in these plants (Chung et al., 2002). For plants in the Schisandraceae family, more than 19 species are widely use in traditional Chinese medicine. These plants have proved to be rich in lignans and triterpenoids with various biological activities (Li et al., 2004). Some triterpenoids showed anti-HIV, antitumor, antihepatitis, and antioxidant activity (Hancke et al., 1999; Li et al., 2003). The antioxidative activity of polyphenols and phloroglucinol derivatives isolated from some Hypericum. species and dibenzocyclooctene lignans isolated from Schisandraceae have been studied (Lu & Liu, 1992; Couladis et al., 2002; Heilmann et al., 2003). The six selected plants in this study have never been previously investigated for antioxidative activity. The methanol and chloroform extracts from the wood, leaves, and fruit of six Thai plants (Hypericum hookerianum. Wight & Arn, Garcinia speciosa. Wall, G. xanthochymus. Hook. f. ex. T. Anderson, Crotoxylum formosum. ssp. pruniflorum. (Kurz) Gogel, Calophyllum polyanthum. Wall ex Choisy and Schisandra verruculosa. Gagnap) were screened for free radical scavenging activity and their potential to be developed into pharmaceutical products.

Materials and Methods

Chemicals

DPPH (1,1-diphenyl-2-picrylhydrazyl), ascorbic acid, and α-tocopherol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol and chloroform were obtained from Merck Ltd. (Darmstadt, Germany) and Labscan Asia Co., Ltd. (Bangkok, Thailand), respectively.

Plant samples

Six plants were collected from Chiang Mai Province, Thailand, in November and December 2002 (Table 1). The plant samples were authenticated by the Department of Biology, Faculty of Science and Faculty of Pharmacy, Chiang Mai University, Thailand, and the voucher specimens were deposited at the herbarium of the department.

Table 1. The percentage yield of methanol and chloroform extracts from various parts of the selected Thai plants in the Guttiferrae and Schisandraceae.

		Yield (%)
		Wood		Leaf		Fruit
Plant family	Local name	MeOH	CHCl3	MeOH	CHCl3	MeOH	CHCl3
Guttiferae
Hypericum hookerianum. Wight & Arn	Bua Thong	7.36	2.01	ND	ND	ND	ND
Garcinia speciosa. Wall	Phawa, Saraphi Pa	12.18	0.66	8.61	4.33	ND	ND
Garcinia xanthochymus. Hook. f. ex. T. Anderson	Mada Luang, Mada	19.04	1.02	12.45	1.67	23.60	9.69
Crotoxylum formosum. ssp. pruniflorum. (Kurz) Gogel	Tiew Khon, Tiew Leung	3.77	1.01	14.60	4.50	ND	ND
Calophyllum polyanthum. Wall ex Choisy	Pha Ong, Ma Nhae Doi	4.46	2.65	11.10	8.50	ND	ND
Schisandraceae
Schisandra verruculosa. Gagnap	—	1.77	1.22	4.14	3.94	ND	ND

MeOH, methanol extract; CHCl₃, chloroform extract; ND, not determined.

Preparation of the extracts

Wood, leaves, and fruits from the plants were separately reduced to small pieces, dried at 40°C in a hot air oven and comminuted to powder. The dried powder samples (100–300 g) were macerated in methanol for 48 h. The solvent was evaporated under reduced pressure by a rotary evaporator. The residues were re-extracted with chloroform and concentrated by partial evaporation under reduced pressure. Twenty-four extracts were obtained, and the percentage yields were calculated.

DPPH free radical scavenging assay

The free radical scavenging activities of all extracts and the standards (ascorbic acid and α-tocopherol) were determined by a modified DPPH assay of Tachibana et al. (2001). DPPH was used as a stable free radical. Briefly, 75 µl of the methanol extracts (6.25–1 mg/ml) and 75 µl of 200 µM ethanol solution of DPPH were put into each well of a 96-well microplate (Nalge Nunc International, NY, USA). The reaction mixtures were allowed to stand for 30 min at room temperature, and the absorbance was measured at 570 nm by a Well Reader (Seikagaku Corporation, Tokyo, Japan) against a blank (ethanol without DPPH). The experiments were done in triplicate. The DPPH free radical scavenging activity was calculated according to the following equation.

The scavenging activity was plotted against concentrations. The concentration that showed 50% DPPH scavenging activity (IC₅₀) was determined.

Results and Discussion

The percentage yields of the methanol and chloroform extracts from different parts of each plant are shown in Table 1. Methanol extracts of all plants showed higher percentage yield than the chloroform extracts. This might be due to the presence of more polar compounds in the plants, which are more soluble in methanol than in chloroform.

For scavenging activity, hydrogen donating ability of the extract to the free radical (DPPH) was determined. When DPPH is scavenged, the deep violet color turns to pale yellow, which can be determined spectrophotometrically. All extracts showed scavenging activity in a concentration-dependent pattern (Figs. 1-4). Table 2 demonstrates the IC₅₀ of the extract. All methanol extracts gave lower IC₅₀ values than the chloroform extracts. These results agree with the previous study of Moure et al. (2000) indicating the dependence of antioxidant activity of the plant extracts on the polarity of extracting solvents.

Figure 1 Comparison of free radical scavenging activity (%) of the methanol wood extracts of the six selected Thai plants and the standard antioxidants. Vertical bars represent the standard deviation of three replicates.

Figure 2 Comparison of free radical scavenging activity of the chloroform wood extracts of the six selected Thai plants and the standard antioxidants. Vertical bars represent the standard deviation of three replicates.

Figure 3 Comparison of free radical scavenging activity (%) of the methanol leaves extracts of the six selected Thai plants and the standard antioxidants. Vertical bars represent the standard deviation of three replicates.

Figure 4 Comparison of free radical scavenging activity (%) of the chloroform leaves extracts of the six selected Thai plants and the standard antioxidants. Vertical bars represent the standard deviation of three replicates.

Table 2. The IC₅₀ values of the selected Thai plant extracts.

	IC50 (µg/ml)
	Wood		Leaf		Fruit
Plant species	MeOH	CHCl3	MeOH	CHCl3	MeOH	CHCl3
Guttiferae
H. hookerianum.	19.08	65.42	—	—	—	—
G. speciosa.	9.75	142	65.13	168	—	—
G. xanthochymus.	32.10	89.56	58.69	59.83	25.58	26.68
C. formosum. ssp. pruniflorum.	23.96	91.04	93.28	162.34	—	—
C polyanthum.	44.29	242.25	51.88	69.41	—	—
Schisandraceae
S. verruculosa.	23.34	127.34	130	162.18	—	—

MeOH, methanol extract; CHCl₃, chloroform extract; IC₅₀, the concentration of extract that showed 50% DPPH scavenging activity. The IC₅₀ values of ascorbic acid and α.-tocopherol was 24.01 and 52.04 µg/ml, respectively.

In comparing the extract from wood and leaf of each plant, the scavenging activity of methanol wood extract of all plants exhibited higher scavenging activity than their leaves. This might be due to the higher content of the total polyphenolic compounds in the wood than in leaves. These polyphenolic compounds include flavonoids, anthraquinones, anthocyanidins, xanthones, and tannins. These compounds have been reported to scavenge free radicals, superoxide and hydroxyl radical by single electron transfer (Ho et al., 1999; Choi et al., 2002). The highest scavenging activity was found in the methanol wood extract of G. speciosa. with an IC₅₀ value of 9.75 µg/ml, which was 2.5- and 5.3-fold more potent than the standard antioxidants, ascorbic acid and α-tocopherol, respectively. H. hookerianum, S. verruculosa, C. formosum. ssp. pruniflorum, G. xanthochymus., and C. polyanthum. gave IC₅₀ values of 19.08, 23.34, 23.96, 32.10, and 44.29 µg/ml, respectively. In fact, some of these values were less than those obtained from the standard antioxidants ascorbic acid and α.-tocopherol (the IC₅₀ values of ascorbic acid and α.-tocopherol were found to be 24.01 and 52.04 µg/ml, respectively). For G. xanthochymus., the IC₅₀ values of methanol and chloroform extract were not significantly different in fruits (25.58 and 26.68 µg/ml) and leaves (58.69 and 59.83 µg/ml). The extracts from G. xanthochymus. using polar and nonpolar solvents appeared to give equipotency of the free radical scavenging activity. The results from this study suggest not only antioxidant activity use but also the potential of these selected plants to be developed into new pharmaceuticals. Further studies on other biological activities and elucidation of the active compounds of the extracts are warranted.

Acknowledgments

This work was partly supported by the Thailand Research Fund (TRF) under the RGJ-PhD program.