黄藤笋和4种常见茎菜抗氧化活性的比较研究

黄世能 黄雪梅 牛国才 张昭其

（1 中国林业科学研究院热带林业研究所 广州 510520 2 华南农业大学园艺学院 广州 510642）

Daemonorops margaritae Beccari belongs to the family Palmae. The genus Daemonorops consists of about 115 species[1]and D. margaritae Beccari is the only Daemonorops species native to China. It is widespread in tropical and south subtropical parts of southern China, occurring on Hainan Island,southern Guangdong and Guangxi Provinces, and in cultivation extending to southern Fujian and Yunnan Provinces. Its canes are used for all types of weaving and basket ware. Seeds are usually used for making necklaces. Its fresh shoots, the heart parts of young stems, have long been consumed by the natives in South China[1]. It is reported that shoots of D. margaritae can be severed as a natural vegetable as it contains high protein content, low fat content, rich in dietary fiber and all essential amino acids for humans as well as several minerals and victim C[2]. It is also reported that fruit of D.margaritae contained some elements common to sanguis draconis[3], a precious plant used in traditional Chinese medicine and described in the Chinese Pharmacopoeia. This implies that D.margaritae might have some medical values as sanguis draconis.

Despite the fact that its medical potential remains unknown, D. margaritae shoots have been proved to be a good forest vegetable. From the medical and epidemiological point of view, dietary fibers and secondary metabolites in fruits and vegetables usually have functions of antoxidation,anticancer, antiallergy, immune-enhancing, blood pressure regulation, cholesterol reducing, etc.[4-8]. Furthermore, vegetable and fruit consumption has been shown in epidemiological studies to be highly associated with the reduced risk of cancer,cardiovascular disease and aging[9-13].

Recent years have witnesses increased interest in the field of research on antioxidant activities of fruits and vegetables in China[14-16]. However, the antioxidant activity of D. margaritae shoots as a kind of nutrition-rich forest vegetable remains unknown.

The aim of the present study was threefold. The first was to investigate the antioxidant potency of D. margaritae shoots by 1,1-biphenyl-2-picrylhydrazyl (DPPH) radical scavenging method or DPPH essay, and compare its total antioxidant activity with those of four common stem vegetables, Asparagus officinalis L.,Zizania caduciflora Turcz, Lactuca sativa L. and Dendrocalamus latiflorus Munro. The second was to determine the radical scavenging effects of the fi ve test vegetables extracted with different extraction techniques and solvents. The third was to determine the radical scavenging effects of different concentrations of water extracts of the test vegetables.

1 MATERIALS AND METHODS

1.1 Plant materials

Fresh shoots of D. margaritae (Hance) Beccari were harvested from a 36-month old trial plantation for edible shoot production in Jiuwantan Forest Farm of Huadu District, Guangzhou City,Guangdong Province. Young plants with stem lengths of 60～80 cm were harvested and brought back to the lab for analysis. Fresh stems (shoots) of asparagus (Asparagus officinalis L.), water bamboo(Zizania caducifl ora Turcz), curled lettuce (Lactuca sativa L.) and sweet Ma bamboo (Dendrocalamus latifl orus Munro.) were purchased from a vegetable market in Tianhe District of Guangzhou, China.

1.2 Chemicals

1, 1-Diphenyl-2-picryl-hydrazyl (DPPH), was purchased from Sigma- Aldrich. All other chemicals used were of analytical grade.

1.3 Preparation of the plant extracts

Certain amount of the edible parts of the aforementioned plants was boiled in water for 3 min before sampling. Five grams of the material from each plant were sampled for one extraction.The samples were subjected to two extraction procedures: distilled water and 100% ethanol extraction. In all the cases, three 5.00 g samples of each plant material without water drops in the surface were used for extraction. The samples were pulped well with 20 ml of the above mentioned solvents. The pulped samples were made up to 40 ml by the addition of the same solvents and extracted for 30 min with employment of ultrasonic wave and 50 °C water bath extraction techniques.The aqueous extracts were left at room temperature over night, and then centrifugalized at 8 000 r/min for 15 min at 4 °C. After filtration, the residue was extracted again with 10 ml of the same solvent.The combined extract was stored at 4 °C for further test.

1.4 DPPH radical scavenging activity assay

The simplest method to determine the total antioxidant activity of any materials containing antioxidants is the utilization of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as it can be used for solid or liquid samples and is not specific to any particular antioxidant component,but applies to the overall antioxidant capacity of the sample[17]. As we have not known if the fresh shoots of D. margaritae would contain any antioxidant, this method was the only one that we could choose to use.

2.5 ml 6.5×10-5mol/L DPPH methanol solution was added to the test tube containing 0.5 ml aliquote of aqueous extract. The mixture was placed in a water bath for 25 min at 30 °C before measuring.The absorbance of all the sample solutions was measured at 517 nm. The percentage scavenging effect was calculated from the following equation.% DPPH scavenging =100×[1–(Ai–Aj) /A0] (1)Where A0=2.5 ml 6.5×10-5mol/L DPPH+0.5 ml of solvents (the blank), Ai=2.5 ml 6.5×10-5mol/L DPPH+0.5 ml of aqueous extracts (the sample) and Aj=2.5 ml of solvents+0.5 ml of aqueous extracts(the control).

In order to determine the radical scavenging effects of different concentrations of different plant extracts, the water extracts by using ultrasonic wave were diluted as many times as possible till the percentage scavenging effect could not be measured. All the measurements were carried out in triplicate.

2 RESULTS AND DISCUSSION

2.1 DPPH radical-scavenging activities in the extracts from fresh shoots of D. margaritae and other four stem vegetables

This study documented the first report of signifi cant results that showed fresh shoots of D. margaritae had the highest antioxidant activity, with DPPH radical-scavenging percentages of 82%～89%,among the five test stem vegetables both in water and ethanol extracts (Table 1). Statistical analysis showed that the antioxidant activity of water extracts of D. margaritae was signifi cantly higher than those of the rest test vegetables. In ethanol extracts, D. margaritae had significantly higher antioxidant activity than L. sativa, Z. caduciflora and D. latiflorus, but no significant differences in antioxidant activity were found between D. margaritae and A. officinalis. Generally D.latifl orus had the lowest the antioxidant activity.

Ethanol extracts of D. margaritae, A. officinalis and L. sativa had significantly higher antioxidant activity than water extracts. On the contrary,the antioxidant activities of water extracts of D.latiflorus and Z. caduciflora were significantly higher than ethanol extracts. It might be implied from the circumstances that the antioxidants of D.margaritae, A. officinalis and L. sativa were more water-extractable, while those of D. latifl orus and Z.caducifl ora more ethanol-extractable.

Comparison of DPPH radical-scavenging percentages of the same solvent and stem vegetable revealed that both extraction techniques, ultrasonic wave extraction and 50 °C water bath extraction,produced similar results. Only water extracts of D. latiflorus showed significant difference of radical-scavenging percentages between ultrasonic wave and 50 °C water bath extraction techniques.The ultrasonic wave extraction technique was characterized by unique extraction mechanism,better extraction effect than common technique,easy operation and energy saving, and continued to use in the follow-up test.

Table 1 DPPH radical-scavenging percentages of water- and ethanol-extracts of fresh shoots of D. margaritae and 4 common stem vegetables by using ultrasonic wave and 50 oC water bath extraction techniques

2.2 DPPH radical-scavenging percentages of water extracts of five stem vegetables extracted with ultrasonic wave at different dilutions

Table 2 showed D. margaritae had the highest DPPH radical-scavenging percentage at every dilution. The antioxidant activity of all the test vegetables decreased with increase in times of dilution. The radical-scavenging percentages of water extracts of D. latiflorus and Z. caduciflorareached zero (0) at 2 dilutions, while L. sativa and A. officinalis were zero (0) at 6 and 10 dilutions,respectively. The radical-scavenging percentage of D. margaritae decreased slowly before 100 fold dilution, and reached 0.33% while its water extracts were diluted 3 200 times, indicating that D.margaritae has not only high antioxidant activity but also its antioxidant has a high dilution endpoint.This can be interpreted as that only 1/320 dose of extracts of D. margaritae shoots is needed while one dose of extracts of A. officinalis, a well-known vegetable having the highest antioxidant activity so far, is needed in the utilization of these two extracts as additives in the production of healthcare products.

Table 2 DPPH radical-scavenging percentages of water extracts of different test vegetables by using ultrasonic wave extraction technique at different dilutions

3 CONCLUSION

The current study provides some useful insight into the antioxidant potency of fresh shoots of D. margaritae as a new and remarkable forest vegetable. In all the cases, extracts of fresh D.margaritae shoots had the highest antioxidant activity among the fi ve test vegetables, even higher than A. officinalis which is well known vegetable with the highest antioxidant activity so far. The high DPPH radical- scavenging ability of D.margaritae shoots indicates that this species has a promising potential in the production of healthcare foods, drinks as well as medicines. However,the constituents present in the extract, which are responsible for this activity, remain unknown.Therefore, further studies are needed to investigate the constituents and their functions involved in the antioxidant activity of D. margaritae.

ACKNOWLEDGEMENTS

Financial support for this work was provided,in part, by the International Tropical Timber Organization (ITTO) (PD 100/01 Rev. 3 (I)), the Ministry of Science and Technology of China(Grant no. 2006BAD19B0903) and the Guangzhou Municipal Forestry Administration (Grant no.2004-2). The authors would like to thank Ms.Zheng Yi and Ms. Zhao Xia of the Research Institute of Tropical Forestry, Chinese Academy of Forestry and Ms. Chen Yunjiao of the College of Horticulture, South China Agricultural University for their assistance in field sample collection and laboratory work.

REFERENCE

[1] Dransfield J. & Manokaran N., eds. Rattans. Plant Resources of South-East Asia No. 6. Indonesia,PROSEA. 1994.

[2] Zhao Xia, Huang Shineng, Xian Guangyong, et al.Analysis and evaluation of nutritional components in Daemonorops margaritae shoots. Nonwood Forest Research, 2007, 25(1): 46-48.

[3] Yin Guangtian, Yang Jinchang, Sun B, et al.Identification and extracting of Dragon Blood's elements from fruits of Daemonorops margaritae(Hance) Becc. Journal of Central South University of Forestry & Technology, 2005, 25(6): 74-76.

[4] Sun Jie, Chu Yifang, Wu Xianzhong, et al. Antioxidant and antiproliferative activities of common fruits. J Agric Food Chem, 2002(50): 7449-7454.

[5] Gardner PT, White T A C, Mcphail D B, et al. The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices.Food Chem, 2000(68): 471-474.

[6] Gardner P T, Mcphail D B, Crozier A, et al. Electron spin resonance (ESR) spectroscopic assessment of the contribution of quercetin and other flavonols to the antioxidant capacity of red wines. J Sci Food Agric, 1999(79): 1011-1014.

[7] Gardner P T, Mcphail D B, Duthie G G. Electron spin resonance spectroscopic assessment of the antioxidant potential of teas in aqueous and organic media. J Sci Food Agric, 1997(76): 257-262.

[8] Rice-Evans C A, Miller N J, Paganga G. Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med, 1996(20): 933-956.

[9] Bocco A, Cuvelier M E, Richard H, et al. Antioxidant activity and phenolic composition of citrus peel and seed extracts. J Agric Food Chem, 1998(46): 2123-2129.

[10] Wang Hong, Cao Guohua, Prior R L. Total antioxidant capacity of fruits. J Agric Food Chem, 1996(44):701-705.

[11] Velioglu Y S, Mazza G, Gao L, et al. Antioxidant activity and total phenolics in selected fruit, vegetable and grain products. J Agric Food Chem, 1998(46):4113-4117.

[12] Doll R. An overview of the epidemiological evidence linking diet and caner. Proc. Nutr. Soc. 1990(49):119-131.

[13] Ames B N. Identifying environmental chemicals causing mutations and cancer. Science, 1979(204):587-593.

[14] Fang Min, Wang Yaofeng, Gong Zhiyong. Study on antioxidant activities of fifty kinds of fruits and thirty-three kinds of vegetables. Food Science, 2008,29 (10): 97-100.

[15] Li Anping, Xie Bixia, Tao Junkui, et al. Evaluation of the antioxidant activity of extracts from bamboo shoots. Acta Nutrimenta Sinica, 2008, 30 (3): 321-323.

[16] Yang Dongmei, Jin Yueting, Ke Leqin, et al.Antioxidant activities of 12 common vegetables.Journal of Chinese Institute of Food Science and Technology, 2007, 7(5): 24-29.

[17] Blois M S. Antioxidant Determinations by the Use of a Stable Free Radical. Nature, 1958(181): 1199 – 1200.