UPLC-Q-Orbitrap-MS2 analysis of Moringa oleifera leaf extract and its antioxidant, antibacterial and anti-inflammatory activities

Abstract Moringa oleifera leaf acetone extract (MLE) was prepared. Phytochemicals of MLE and their antioxidant, antibacterial, and anti-inflammatory activities were evaluated. Results showed that MLE contained total phenolic content of 20.16 mg gallic acid equivalents/g dry weight. A total of 39 compounds were identified by mass spectrometry. The contents of acetyl-glucomoringin, caffeoylquinic acid, feruloylquinic acid, and coumarylquinic acid were high. MLE had high DPPH· and ABTS•+ scavenging activities and reducing powder. In addition, MLE could effectively inhibit S. aureus and B. subtilis, but little effect on E. coli was found. The anti-inflammatory effect of MLE was evaluated using a lipopolysaccharide (LPS) -induced RAW 264.7 cell model. MLE significantly inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA levels in LPS-induced RAW 264.7 cells. The inhibitory activity increased in a dose-dependent manner. The bioactivities of MLE were related to its phenolic content and phenolic profiles.


Introduction
Moringa oleifera is widely cultivated and M. oleifera leaves (ML) have been used in traditional medicine to alleviate pain mainly (Mart ınez-Gonz alez et al. 2017;Manguro and Lemmen 2007). In China, ML is used as a new food resource. Some studies have focused on the phenolic compounds of M. oleifera and their bioactivities (Wang et al. 2017;Coppin et al. 2013;Vongsak et al. 2013;Rodr ıguez-P erez et al. 2016;Pontual et al. 2017). Previous phytochemical analyses of ML from different countries have been performed, and the profiles and contents of phytochemical compounds are distinct (Moyo et al. 2011;Rodr ıguez-P erez et al. 2015;Nouman et al. 2016;Makita et al. 2017). However, studies on M. oleifera cultivated in China are few. In this study, ML was collected in autumn from Yunnan province, China. ML acetone extract (MLE) was prepared and the phytochemical compounds were quantitatively and qualitatively determined. Furthermore, the bioactivities of MLE in vitro and their underlying mechanisms were evaluated.

Total phenolic content and phytochemical compounds of MLE
Total phenolic content of MLE was 20.16 mg GAE/g DW. Its UPLC-Q-Orbitrap-MS 2 profile is shown in Figure S1. A total of 39 compounds were tentatively characterized, including 8 organic acids, 2 amino acids, 2 hydroxybenzoic acids, 6 hydroxycinnamic acids, 14 flavonols, 2 flavones, and 5 thioglycosides. Their MS and MS 2 were further analyzed. Table S1 shows the overall results.

Organic acids
Citric acid (compound 1) and malic acid (compound 2) were tentatively identified with 199.71 and 658.86 mg/g DW. The peak of compound 15 was identified as benzoic acid. Compound 33, 35, 37, 38 and 39 were tentatively characterized as 5 low-polar organic acids. Our results showed the contents of linolenic acid and linoleic acid were high and the contents of trihydroxyoctadecadienoic acid, hydroperoxyoctadecatrienoic acid and 9-hydroxyoctadeca-10, 12, 15-trienoic acid were low.

Amino acids
Two amino acids of L-phenylalanine (compound 6) and tryptophan (compound 8) with 182.46 and 143.99 mg/g DW were characterized from MLE.

Hydroxybenzoic acid derivatives
Compound 4 was proposed as protocatechuic acid hexoside with four produced ions at 108, 152, 109, and 153. Salicylic acid O-glycoside was proposed for compound 5 with the fragments at m/z 137.
The content of coumaroylquinic acid (compound 10) was 878.30 mg/gDW. Feruloylquinic acid (compound 11) was also abundant compound in MLE, being 900.98 mg/g DW. It presented two main fragments at m/z 134 and 193.

Flavones
Two different peaks of flavones were tentatively characterized. Compound 16 was characterized as multiflorin B, and compound 18 was tentatively identified as apigenin glucoside.

Thioglycosides
Glucomoringin (compound 3) was identified with content of 242.42 mg/g DW. Compound 14 was characterized as acetyl-glucomoringin with the ions at m/z 97(HSO 4 ) and 259; its content was abundant in MLE, attaining 2293.08 mg/g DW.
Three isothiocyanate derivatives were tentatively identified, being compound 32, 34, and 36. They showed identical fragments at m/z 58 (CNS), and obtained a total content of 1152.81 mg/g DW.

Antioxidant activities of MLE
Antioxidant activities of MLE were determined. As shown in Table 1, the IC 50 values of MLE, vitamin C (V c ), and butylated hydroxytoluene (BHT) for scavenging DPPHÁwere 5.18, 3.31, and 26.45 mg/mL, respectively. The DPPHÁ scavenging activity of MLE was slightly lower than that of V c and higher than that of BHT. FRAP capacity was in the order of V c > MLE > BHT, and the IC 50 value of MLE was 2.66 mg/mL. MLE showed significantly stronger ABTS þ -scavenging activity than BHT and Trolox. Our results demonstrated that ML is a good antioxidant source. The previous studies showed that quercetin and its derivatives had high antioxidant abilities (Rodr ıguez-P erez et al. 2015). In this study, MLE were rich in quercetin and its glycosides, coumaroylquinic acid, and chlorogenic acids. The high antioxidant activities of MLE were related to its phenolic profiles.

Antibacterial activity of MLE
The inhibitory effects of MLE against three bacteria, including E. coli, S. aureus and B. subtilis were determined, compared with kanamycin and vancomycin. As shown in Table S2, MLE showed significant inhibitory activity against S. aureus and B. subtilis. However, our results indicated MLE had little inhibitory activity against E. coli in the concentrations. Previous studies found that gram-negative bacteria were more resistant to polyphenol-based plant extracts than gram-positive bacteria, which could be mainly associated with the cell wall structures difference.

Protective effects of MLE on inflammation in LPS-stimulated RAW 264.7 cells
As shown in Figure S2A, nitric oxide (NO) production of the LPS-induced group significantly increased compared with the control (p < 0.05). MLE significantly inhibited NO secretion (p < 0.05) compared with the LPS-induced group. The inhibitory activity increased in a dose-dependent manner. MLE at 80 mg/mL could decrease the NO content by 70.29%. As shown in Figure S2B, MLE effectively inhibited iNOS mRNA expression levels of LPS-induced RAW 264.7 macrophages (p < 0.05), and the inhibitory activity was similar to the activity of NO production. MLE decreased the expression of iNOS in a dose-dependent manner. In general, phenolic compounds possess antiinflammatory activity by reducing the production of inflammatory mediators or through free radical scavenging capacity. Chlorogenic acid, quercetin-3-O-glucuronide, kaempferol-3-O-galactoside, isothiocyanates, kaempferol with high anti-inflammatory activity were previously reported (Moldovan et al. 2016;Waterman et al. 2014). Our results indicated the anti-inflammatory activities of MLE might contribute to the main phenolic compounds, such as quercetin rutinoside, chlorogenic acid, kaempferol, isothiocyanates, quercetin-3-O-glucoside, and kaempferol-3-O-glucoside.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Natural Science Foundation of China (Grant number 31360381)