Chemical Constituents and Antibacterial Properties of Indocalamus latifolius McClure Leaves, the Packaging Material for “Zongzi”

The glutinous rice dumpling named “Zongzi” in Chinese is a type of traditional food that is popular in East Asian countries. “Zongzi” is made of glutinous rice and wrapped in the leaves of Indocalamus latifolius McClure as the packaging material. Four new compounds, latifoliusine A (2), (7S,8R) syringylglycerol-8-O-4′-sinapyl ether 4-O-β-d-glucopyranoside (7), (7S,8S) syringylglycerol-8-O-4′-sinapyl ether 7-O-β-d-glucopyranoside (8), and (7R,8S) syringylglycerol-8-O-4′-sinapyl ether 7-O-β-d-glucopyranoside (10), along with six known compounds (1, 3–6 and 9) were isolated from I. latifolius McClure leaves. The structures and relative configurations of the compounds were determined by detailed spectroscopic analysis, high-resolution electrospray ionization mass spectroscopy (HRESIMS), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), nuclear overhauser enhancement (NOE) and circular dichroism (CD). All of the isolated compounds were screened for their antibacterial activities in vitro. The results indicated that apigenin 6-C-α-l-arabinopyranosyl-8-C-β-d-glucopyranoside (5) and apigenin 7-O,8-C-di-glucopyranoside (6) have antibacterial activities against four bacterial strains (Staphylococcus aureus, Bacillus thuringiensis, Escherichia coli and Pseudomonas solanacearum).


Introduction
"Zongzi", which is believed to have a history of more than 2000 years, is a type of famous Chinese food that is also popular in many Asian countries [1].It is made of glutinous rice and wrapped in the large flat leaves of Indocalamus latifolius McClure."Zongzi" has been characterized by a long shelf life since ancient times.
In our previous research, several new compounds have been identified from the leaves of different bamboo species including the following: Three novel lignans were isolated from Bambusa tuldoides Munro [10]; a new polyketide derivative named Amarusine A was isolated from the leaves of Pleioblastus amarus [11]; two new compounds, xylitol 1-O-(6′-O-p-hydroxylbenzoyl)-glucopyranoside and bambulignan B, were isolated from the leaves of Pleioblastus amarus (Keng) keng f [12]; and four diastereoisomeric oxyneolignans were isolated and characterized from Bambusa tuldoides Munro [13].In the present research, on the basis of our continuing research interest in the phytochemistry of bamboo, we examined the phytoconstituents of I. latifolius McClure leaves in detail and their antibacterial activities against two Gram-positive and two Gram-negative bacterial strains for the first time.
New compound 10 was also obtained as a yellow powder ([α]D = +4.55°;c = 1.0, methanol).The molecular formula of 10, C28H38O14, was confirmed by negative HRESIMS (m/z 633.1953 [M + Cl] − , calculated 633.1950) and coincided with that of 8.The IR and UV spectra of 8 and 10 showed similar absorption patterns.The 1 H-and 13 C-NMR spectra of 10 were very similar to those of 8, suggesting that the overall structure of 10 was the same as that of 8.Moreover, the HSQC and HMBC correlations of 10 corroborated the aforementioned deduction.

Antibacterial Activities of the Isolated Compounds
The agar-disk diffusion method is a traditional method for measuring the antibacterial activities of compounds, and their antibacterial effects can be visually observed [31][32][33].
The results of the antibacterial activity tests indicated that the 10 compounds had selective antibacterial properties.Figure 4 shows the zones of inhibition for each compound against the four test strains.All 10 compounds showed inhibition zones, which varied from 0.13 to 1.69 mm.
Compounds 5 and 6 had antibacterial activities against all four bacterial strains and, more notably, these two compounds showed strong antibacterial activities against S. aureus and E. coli, which are food-contaminating bacteria.Of the remaining two test strains, B. thuringiensis was most sensitive to compound 9, and P. solanacearum was most sensitive to compound 6.

Discussion
Consumers, nowadays, have a strong demand for greener food preservation techniques; hence there is great potential for developing naturally-derived antimicrobial agents.Extensive research has documented that compounds isolated from plants contain a large number of secondary metabolites and possess the capacity to inhibit the growth of bacteria and fungi [34].The antimicrobial compounds in plants are a part of the self-defense mechanisms for combating harmful microbes in a natural environment [35].Many of these compounds are under investigation and are not yet exploited commercially.Hao et al. [36] found the alcohol extracts of angelica root, banana purée, bay, caraway seed, carrot root, clove (eugenol), marjoram, pimento leaf, and thyme showed inhibition of A. hydrophila and L. monocytogenes in refrigerated poultry.Ahn et al. [37] also found grape seed extract and pine bark extract could control the growth of microorganisms in cooked beef.Kotzekidou et al. [38] tested plant extracts and essential oils with potent antimicrobial activities in chocolate at different temperatures and in dry or humidified environment, the most inhibitory action was observed by lemon flavor applied on chocolate inoculated with E. coli cocktail culture after storage at 20 °C for 9 days.Martinez-Romero et al. [39] reported that the vapor atmosphere of carvacrol could reduce the fungal growth in grape berries.
Another application of natural derived antimicrobials is in the bioactive packaging technologies for food preservation.Seydim [40] found the antimicrobial activity of some spice extracts could be expressed in a whey protein isolate (WPI)-based edible film; hence, they may act as releasable antimicrobial constituents in food packaging.Oussalah [41] studied milk protein-based edible films containing plant essential oils mix on beef muscle slices for controlling the growth of pathogenic bacteria during storage at 4 °C; the film containing oregano showed the most effective against two test bacteria.Nicholson [42] suggested naturally-occurring bio-preservatives could be applied in the food packaging system as part of a multiple hurdle technique, and should lead to increases in both the food safety and shelf-life of perishable foods.
Naturally-derived preservatives for food have been investigated for practical applications in the last 10 years; however, there are also challenges.Plant extracts, especially the EOs, always have strong odor/flavor and may transfer into the food.In this research, we investigated the compounds from a traditional natural packaging material, the leaves of Indocalamus latifolius McClure.In addition to the antibacterial capacity, we also found compound (2) has a pleasant smell.Thus, the isolated compounds in our research could act as an antimicrobial agent or as a component in antimicrobial packages, and also as an odor/flavor enhancer for packaged foods.
Whereas, the results and data obtained from laboratory in vitro experiments may not be applied to food products as foods are complex, the natural antimicrobial agents may offer exclusive advantages for food preservation, and the applications of naturally-derived antimicrobial agents in food will rise steadily in the future.

Analytical Methods
HPLC analysis utilized a binary elution system consisting of solvent A (MeOH) and solvent B (water containing 0.2% acetic acid) with a YMC-PACK ODS-AQ C18 column.The flow rate was 1 mL/min, the column temperature was 30 °C and the injection volume was 10 μL.The PAD detection wavelength monitoring range was 210 to 400 nm.LC-MS analysis was performed to confirm the molecular weights of the compounds using an Rp-18 column and ESI-MS.The mobile phases were solvent A (MeOH) and solvent C (water containing 0.1% formic acid).The flow rate was 0.3 mL/min, the column temperature was 30 °C, and the effluent was monitored at either 220 or 270 nm.The 1 H-, 13 C-NMR, and 2D (HSQC, HMBC, and NOE) NMR spectra were recorded on 500 MHz Bruker spectrometers using DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal standard.The chemical shifts were expressed in δ (ppm), and the coupling constants were reported in Hertz.The concentration of the compound was 12 mg/mL, the NMR acquisition duration was 2 min for 1 H-NMR and 5 h for 13 C-NMR, and the widths of the NMR spectra were 0-14 ppm for 1 H-NMR and 0-220 ppm for 13 C-NMR.

Microbial Strains
Two food contaminating bacteria Staphylococcus aureus (Gram (+)) and Escherichia coli (Gram (−)) were selected as test strains, another Gram -ositive bacteria (Bacillus thuringiensis) along with another Gram-negative bacteria (Pseudomonas solanacearum) were selected for testing the antibacterial selectiveness of isolated compounds, all four bacteria strains were obtained from the Agricultural Product Key Laboratory of Anhui Agriculture University, Hefei City, Anhui, China.

Figure 3 .
Figure 3.The relative configuration of compound 2.

Figure 4 .
Figure 4. Antibacterial activities of the compounds isolated from the leaves of Indocalamus latifolius McClure.

3. 1 .
Plant Material I. latifolius McClure leaves were collected from the Century Garden of Bamboos in Yibin city, Sichuan, China.A voucher specimen was deposited in the State Forestry Administration Key Open Laboratory at the International Centre for Bamboo and Rattan in Beijing 100102, China.

Table 1 .
NMR spectroscopic data (measured at 500 MHz) of the isolated compounds 2, 7, 8, and 10 in DMSO from the leaves of Indocalamus latifolius McClure.