Chemical Constituents and Antioxidant Properties of Phyllostachys prominens Gramineae (W Y Xiong ) Leaf Extracts

Purpose: To isolate and identify chemical components of Phyllostachys prominens (Poaceae) leaf extracts, and measure their antioxidant activities. Methods: Ethanol extracts of P. prominens leaves were subjected to different chromatographic methods: macroporous resin column chromatography, Sephadex LH-20 column chromatography, and semi-preparative, reversed-phase (RP) high performance liquid chromatography (HPLC). Plant extract components were identified by ultraviolet spectroscopy (UV), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to measure the radical scavenging activity of the compounds . Results: We isolated fourteen compounds including six flavonoids, two lignans, two phenolic glycosides, a phenolic acid, a phenylpropanoid, a monoterpene glycoside, and amarusine from the leaves of Phyllostachys prominens. The DPPH assay showed that eleven compounds (compounds 1, 4, 6, 7, 8, 9, 10, 11, 12, 13, and 14) exhibited radical scavenging activity. (The half maximal inhibitory concentration ranged from 33.52 to 100.58 μg/mL). The half maximal inhibitory concentration (IC 50 ) values of compounds 1, 4, 6 and 7 were 33.52, 40.61, 47.10, and 3 5.84 μg/mL respectively, while the IC 50 of the positive control, butylated hydroxytoluene, was 46.32 μg/mL. Conclusion: Fourteen compounds have been successfully all isolated from Phyllostachys prominens for the first time. Eleven of the compounds have radical scavenging activity.


INTRODUCTION
Bamboo is a perennial plant of the Gramineae family that grows in China, Korea, Japan, and other parts of Southeast Asia, and represents an important commodity.It is used as a building material, handicraft component, food ingredient, and component of traditional medicines.Bamboo leaves have a long medicinal utilization history in China [1].Bamboo leaf extracts have been reported to contain flavonoids, coumarins, lignans, polysaccharides, and anthraquinones [2].In the past few years, reports have described the beneficial effects of bamboo leaf extracts on human health, which include antioxidant, antiaging, antibacterial, lipid regulating, and antitumor activities [3][4][5][6][7].
Trop J Pharm Res, March 2016; 15(3): 570 Phyllostachys prominens, which belongs to the tribe Bambuseae, is an important bamboo species that is widely distributed in the south of China [8].Phyllostachys prominens is not only used as a building material, but also as a source of high quality edible bamboo shoots.However, huge leaves of Phyllostachys prominens are generally disposed of as waste; thus, how to utilize such large amounts of bamboo leaves is an urgent problem.Exploring the medical value of bamboo leaves could potentially uncover a solution to this problem.Synthetic antioxidants have many risks because of their carcinogenicity and toxicity to the liver.Therefore, the development and utilization of more effective antioxidants of natural origins is desired.However, at present, little work has been done on the chemical composition of leaf extracts from Phyllostachys prominens.Therefore, in this research, we isolated and identified the chemical components of Phyllostachys prominens leaf extracts, and evaluated the antioxidant activity of the isolated compounds.

EXPERIMENTAL Materials and equipment
Column chromatography for fractionation of leaf extracts was carried out using a semi-preparative reversed-phase (RP) column packed with macroporous resin (AB-8, 10 × 80 mm), Sephadex LH-20 (GE Healthcare), and semipreparative RP high performance liquid chromatography (HPLC) (Shimadzu) with an YMC-Pack ODS-A column (250 × 10 mm, 5 μm, particles).UV spectra were determined using a Waters 2695 HPLC with a photodiode array detector (PAD).NMR spectra were scanned using a Bruker instrument operating at 300 MHz.Mass spectroscopy was performed on an Agilent 6540 high-resolution quadruple time-of-flight mass spectrometer.

Plant materials
Phyllostachys prominens leaves were collected from Hangzhou City, Zhejiang Province, China in September 2013.The plant identity was confirmed by Professor Chen Shuang-Lin from the Research Institute of Subtropical Forest, Chinese Academy of Forestry.A voucher specimen (No. 201310-00) has been deposited in the state forestry administration Key Open Laboratory, International Center for Bamboo and Rattan in Beijing, China.

Phyllostachys prominens leaves
Five kilograms of dried Phyllostachys prominens leaves were ground to a powder and extracted three times with 95 % ethanol at room temperature.The extracts were combined and evaporated under reduced pressure at 318 K on a rotary evaporator to yield a solid residue.The residue (451.0 g) was resuspended in water, followed by successive partition with ethyl acetate (111.0 g) and n-butanol (55.0 g).
During the following fractionation steps, HPLC analysis of the fractions was performed.Briefly, 1.0 ml of each fraction was filtered through a 0.45 μm membrane filter before 10 μL was injected into the UPLC system for analysis.The mobile phase was composed of solutions A (MeOH) and B (Water) with a gradient elution, and the flow rate was 1.0 mL/min.For semipreparative HPLC steps, the fraction was filtered through a 0.45 μm membrane filter before 100 μL was injected into the semi-preparative UPLC system.The mobile phase was composed of solutions A (MeOH) and B (Water) and the flow rate of the mobile phase was 6.0 mL/min.

Measurement of radical scavenging activity by DPPH assay
These 14 compounds were evaluated for radical scavenging activity using a modified DPPH assay (Sigma, St. Louis, MO, USA) [9].Briefly, each compound was dissolved in 400 μL of DMSO and serially diluted to 200, 100, 50, 20, 10, and 5 µg/mL.The reaction mixtures consisted of 200 μL of each serial dilution and 200 µg/mL DPPH in triplicate.After 30 min of incubation in the dark, the absorbance of each reaction was read at 517 nm.The positive control was butylated hydroxytoluene (BHT).The half maximal inhibitory concentration (IC 50 ) values represent the concentration of sample at which 50 % of the DPPH was scavenged.Data were calculated as mean absorbance values.

RESULTS
Fourteen compounds were isolated from leaf extracts of Phyllostachys prominens, and were identified according to the HRMS, 1 H-NMR, and       ).These data are in good agreement with those of 5-O-caffeoylquinic acid [13].
Compounds 1-14 were tested for antioxidant activity using the DPPH method.The results are shown in Table 1

DISCUSSION
In the present study, 14 compounds, including six flavonoids, two lignans, two phenolic glycosides, a phenolic acid, a phenylpropanoid, a monoterpene glycoside, and amarusine, were isolated from the leaves of Phyllostachys prominens.To our knowledge, these were the first compounds isolated from leaf extracts of Phyllostachys prominens that subsequently were shown to have anti-oxidant activities.
Bamboo leaves are rich in flavonoids [3],which were considered to have many functions, such as removing active oxygen, preventing hemal sclerosis, improving nutrition for tissue, antiaging and preventing aging dementia [6].In our study, we also six flavonoids exhibting antioxidant activity.In addition to flavonoids, we isolated two lignans from the bamboo leaf extracts.Lignans, which are a type of phytoestrogen, have a variety of biological activities including antioxidant activity [19].In the future research on the chemical constituents of bamboo leaves, more attention should be paid on lignans.
The DPPH assay results showed that, in addition to flavonoids, the two lignans and phenolic acid, which are important compounds in bamboo leaves, also had antioxidant activity.These findings suggest that the antioxidant activity of the bamboo leaf extracts could be attributed to several different compounds.