Phytochemical Compounds in Arundo donax L. Rhizome and Antimicrobial Activities

Arundo donax L. (Poaceae) is a giant reed like grass with versatile economic (biofuel) and biomedical uses. The rhizome of this plant grows horizontally underground and has a storage function which has been the source material for pharmaceutical and biomedical utilizations. Acidic and neutral/ alkaline fractions have been reported to inhibit Prymnesium parvum, ichthyotoxic golden alga.1 In addition, traditional medicine has used A. donax L. as an emollient and a diuretic agent.2,3 Furthermore, basic biomedical research has used the rhizome from which purified n-acetyl-D-glucosamine was obtained. This phytochemical compound has been reported to exhibit anti-proliferation activity against a human cancer cell line.4


INTRODUCTION
Arundo donax L. (Poaceae) is a giant reed like grass with versatile economic (biofuel) and biomedical uses. The rhizome of this plant grows horizontally underground and has a storage function which has been the source material for pharmaceutical and biomedical utilizations. Acidic and neutral/ alkaline fractions have been reported to inhibit Prymnesium parvum, ichthyotoxic golden alga. 1 In addition, traditional medicine has used A. donax L. as an emollient and a diuretic agent. 2,3 Furthermore, basic biomedical research has used the rhizome from which purified n-acetyl-D-glucosamine was obtained. This phytochemical compound has been reported to exhibit anti-proliferation activity against a human cancer cell line. 4 In view of these biomedical uses, investigation of phytochemical compounds in A. donax L. is a warranted procedure in order to establish the knowledge needed to further exploit this plant for pharmaceutical uses. A number of phytochemical compounds from A. donax L. have been extracted using various procedures as well as isolated/ identified using chromatographic techniques. For example, n-acetyl-D-glucosamine, a specific lectin, has been isolated from A. donax L. rhizomes using affinity chromatography. 4 The phytochemical compounds of A. donax stems were extracted with acetone and re-dissolved in chloroform before fractionation. 5 The analysis of stem extract by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) revealed compounds such as long-chain n-fatty acids, n-alkanes, n-aldehydes, n-alcohols, monoglycerides, free and esterified sterols, and terpenols. Recently, the non-structural carbohydrates in A. donax L. were identified and the results revealed that the contents affected the dry matter and water extracts, which further caused the differences in their biomass. 6 However, the identification of phytochemical compounds in A. donax L. is still unclear with concerning to their types and contents. Therefore, in this study, the A. donax L. rhizomes extracted with various solvents were phytochemically characterized including the antimicrobial activities of their metabolites.
rhizomes were dried at 50°C before pulverizing. Sequential extraction based on the polarity index of various solvents used hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and methanol (MeOH), respectively. The rhizome was initially extracted by immersing 1-g powder with 10 ml HEX, followed by 15 min of sonication, then overnight maceration. Consequently, the residues were removed from the HEX extract by filtration through a membrane paper (Whatman® no. 1). The residue was consecutively extracted with DCM, EA, and MeOH, respectively. Then, the solvents were removed from all the extracts by vacuum evaporation, and the extracts were stored at -20°C for subsequent antimicrobial activity test and phytochemical analysis by GC-MS.

GC-MS analysis
GC-MS was used to characterize the phytochemical compounds which were dissolved in their extracted solvents for a final concentration of 10 mg/ml. Methyl heptadecanoate was used as an internal standard. The extracts were analyzed using an HP5-MS capillary-fused silica column (30 m length, 0.25 mm I.D., and 0.25 m film thickness), helium as a carrier gas at a flow rate of 1 ml/min, and 250°C injector temperature in splitless mode. The oven temperature condition was as follow; 40°C initiation, increasing 4°C/min to 300°C/min and increasing 5°C/min to 320°C/min for 10 min. The mass range of analyzed compounds detected at m/z 30-500 amu. 7 Screening against the WILEY07 library enabled characterization of the phytochemical compounds from A. donax L., with similarity of at least 90% warranting further analysis.

Antimicrobial activity assay
The antimicrobial activities assay was performed by the disc diffusion method 8 against yeast C. albicans and two types of bacteria: 1) Grampositive (S. aureus ATCC 25923, B. cereus ATCC11778, and B. subtilis ATCC6633) and 2) Gram-negative (E. coli ATCC25922). Bacteria were cultured in Luria-Bertani broth at 37°C for 16-18 h; the broth was replaced with fresh Luria-Bertani broth and the culture continued for 2-3 h. C. albicans was cultured in yeast extract peptone dextrose (YPD) liquid medium at 30°C for 16-18 h; the medium was replaced with fresh YPD medium and the culture continued for 2-3 h. Each microbial inoculum was diluted in Mueller-Hinton (MH) broth to obtain 0.1 OD 600 . Then, the pour plate technique was performed by mixing 1 ml of all microbial inocula with 9 ml of MH agar. All extracts were prepared by dissolving in 100% dimethyl sulfoxide at a concentration 100 µg/ µl. The extracts (calculated as 2 mg/disc) were dropped on paper discs sized 6 mm in diameter and allowed to dry in a laminar flow cabinet; 100% dimethyl sulfoxide was used as a control. The discs with extracts were placed on microbial plates before overnight co-incubation at 37°C for all tested bacteria, and 30°C for C. albicans. Growth inhibition was detected measuring the clear zone.

Hierarchical clustering analysis (HCA)
The extracted phytochemical compounds with similarity values ≥ 90% and antimicrobial activity (only inhibited microbial strains) were grouped using HCA as previously reported by Sumner et al. (2003). 9 The overall dataset was visually analyzed and defined by multiexperiment viewer (MeV), as Pearson correlation, using average linkage clustering. 10 The relationship between detected compounds and antimicrobial activity was presented as a heat map pattern with the degree of difference indicated in a representative color.

RESULTS AND DISCUSSION
Phytochemical compounds in A. donax L. extract detected by GC-MS Dried rhizomes were sequentially extracted with HEX, DCM, EA, and MeOH, respectively, as described in the experimental section.
All the four extracts were stored at -20°C before to analysis of the phytochemical compounds by GC-MS. The compounds were screened against the WILEY07 library; 84 phytochemicals were identified and the compounds with a matching-similarity ≥ 95% were presented in Table 1.
Methyl heptadecanoate was used as the internal standard for comparing the phytochemical contents in different extracts. Nineteen identical compounds were detected in the HEX extract, such as 2-benzoxazolone and heptadecane, which were not found in the other extracts. Five, 11, and seven specific compounds were detected only in DCM, EA, and MeOH extracts, respectively. Interestingly, two compounds, phenol, 3,5-bis(1,1-dimethylethyl)-and hexadecanoic acid (or palmitic acid), were detected in all the extracts. The most diverse identified compounds (49 compounds) were detected in the HEX extract, whereas fewer compounds were detected in the DCM (38 compounds), EA (34 compounds), and MeOH extracts (18 compounds). Use of various solvents for extraction resulted in different contents and types of compounds.
All detected phytochemicals in each extract were grouped, as shown in Figure 1, based on their functional groups as follows 1) lipids, fatty acid and conjugates 2) hydrocarbons 3) phenolic compounds 4) sterols 5) terpenoids 6) xanthones and xanthenes, and 7) other compounds.
The phytochemical compounds in A. donax L. stems analyzed by GC-MS revealed that long-chain fatty acids, alkanes, aldehydes, alcohols, ketones, sterols and conjugates, terpenoids, monoglycerides, and glucosides were the major constituents in stem fibres. 5 However, in this study, sterols were found to be the major constituents of the HEX, DCM, and EA extracts from A. donax L. with 52%, 42%, and 30% relative content, respectively. Although sterol was found in methanolic   Hydrocarbons were detected in all extracts, except the MeOH extract.
Since the electrons of hydrocarbons are equally shared by the adjacent atoms between the bonds, this compound group is better extracted by a non-polar solvent or a solvent with low polar index. Hydrocarbon compounds were detected in extracts derived from solvents with lower polarity index (P′), such as HEX (P′=0.1), DCM (P′=3.1), and EA (P′=4.4), but not in the MeOH (P′=5.5) extract. 14 A low proportion of the terpenoid squalene was detected in the HEX (1%) and EA (0.1%) extracts. Squalene, a precursor of sterol production, was reported to have antioxidant and immune-boosting, 15 and protective activities against ultraviolet-induced skin cancer. 16 In the root part of several plants, genes involved in secondary metabolite production might be expressed at a low level, as revealed by the low content of artemisinin, a terpenoid with antimalarial properties, in the root part of Artemisia annua. 17 Xanthone and xanthene were detected in the DCM (0.3%), EA (1 %), and MeOH (2%) extracts. Xanthone could be found in various plant organs, such as fruit peel (e.g. mangosteen pericarp), bark, or root. It exhibited anticancer, [18][19] immune modulation, 20 antioxidant, 21 and antimicrobial activity. 22 The analysis of A. donax L. extracts in this study revealed that rhizomes are an important source of valuable phytochemical metabolites, which could have medicinal applications. All the four extracts were further analyzed for their effects on micro-organism growth, and the relationship between the detected phytochemicals and their anti-microbial activities was evaluated using HCA.

HCA of detected metabolites in A. donax L. and their antimicrobial activities
Antimicrobial activities of the four A. donax L. extracts including Hex, DCM, EA, and MeOH in crude forms were examined against the bacteria and yeast. Growth inhibition was determined based on clear zone after overnight co-incubation of the disc containing 2 mg of each A. donax L. extract with bacteria and yeast. The results showed that all four extracts inhibited the growth of B. subtilis as presented in Table  2; the DCM extract showed the highest activity (18 mm of inhibition zone). The HEX, EA, and MeOH extracts showed no significant activity against B. subtilis (p≥ 0.01). The growth of B. cereus, a human pathogenic bacterium, was inhibited by the DCM and EA extracts with 17.8 mm and 7.8 mm inhibition zone, respectively.
However, the HEX and MeOH extracts could not suppress its growth at this concentration. These results indicated that the DCM extract had the best antibacterial activity against the tested strains. Nevertheless, all four extracts could not inhibit E. coli, S. aureus, and C. albicans growth in this study. This first report proved that extraction of A. donax L. using different solvents resulted in differences in antibacterial activities. Moreover, A. donax L. has potential activities against B. subtilis and B. cereus. Since B. cereus is a Gram-positive human-pathogenic bacterium that causes foodborne diseases and some severe symptoms, such as vomiting and diarrhea, 23 the antibacterial activity of A. donax L. extracts, especially DCM extract, indicates that A. donax L. is a potential source of phytochemical compounds for antibiotic production.
Heat map visualization of antibacterial activities and relative phytochemical compounds was performed by HCA ( Figure 2).
The results revealed that hexadecanoic acid had an important role in anti-B. subtilis activity as presented in the same cluster and pattern of heat map. Hexadecanoic acid, or palmitic acid, is a saturated fatty acid found in many organisms including plants. It was detected in all the extracts, with the DCM extract presenting the highest relative content (108.97%), while the MeOH extracts showed the lowest at 27.65%. This indicates that a higher hexadecanoic acid content would result in a higher anti-B. subtilis activity. In a further study, the anti-B. subtilis activity of A. donax L. extract could be predicted from this fatty acid content. Isolated n-hexadecanoic acid from Canthium parviflorum leaves also showed antimicrobial activities against E. coli, S. aureus, B. subtilis, and C. albicans at dose 100 and 150 µg. 24 According to the same pattern of expression in heat map, xanthone is the main compound which inhibited the growth of B. cereus. The DCM and EA extracts showed anti-B. cereus activities; xanthone was detected and identified in these two rhizome extracts with a content of 2.5-4.3%. Xanthone is a tricyclic aromatic compound, which could be found in the A. donax L. stem. 5 In addition to anticancer, immune modulation, and antioxidant activities, xanthones, the major active component derived from Calophyllum brasiliense Cambess. and Mammea americana L., showed antibacterial activities against S. aureus and E. coli. 25 The dose used in this study (2 mg extract/disc) was in the range similar to that used in previous studies, 1-2 mg/disc for Andrographis paniculata, Morinda citrifolia, Piper sarmentosum, and Mitracarpus hirtus L. extracts; 8, 26, 27 and 5 mg/disc for A. annua extract. 28 Further studies on the investigation of bioactivity of A. donax L. rhizomes should focus on these two active compounds.