LC/MS characterization of phenolic antioxidants of Brindle berry (Garcinia gummi-gutta (L.) Robson)

Abstract Characterisation of antioxidant fraction of the fruit of Garcinia gummi-gutta was done using liquid chromatography mass spectroscopic (LC/MS) analyses. Total poly phenolics and radical scavenging activity of various extracts such as acetone, methanol and hydroalcohol were estimated spectrophotometrically. The active extract was analysed by LC/MS in order to identify the molecular mass and tentative structures of major compounds. Phenolic compounds such as luteolin 7-O-glucuronide, kaempferol 3-O-(6-O-acetyl) glycoside, dicaffeoylquinic acid, apigenein-6-C-pentosyl-8-C-hexoside and p-coumarylquinic acid were identified from hydro alcoholic extract of G. gummi-gutta.


Introduction
Phenolic compounds have received much attention due to their effective antioxidant properties, and their beneficial effects are attributed to their donating electrons and scavenging free radicals. In addition, natural antioxidants have the capacity to improve food quality and stability, and can also act as nutraceuticals to terminate free radical chain reactions in biological systems, and thus may provide additional health benefits to consumers (Zhao et al. 2014;Sulaiman et al. 2014).
Brindle berry (Garcinia gummi-gutta) is an ever green medium-sized tree occurring in Southern India and Sri Lanka. The fruit is a fleshy, globose, sub-globose to ovoid berry, green turning yellow, orangey or reddish when ripe (Lim 2012). It has been used traditionally to treat respiratory infections like sore throat and cough. The fruit contains hydroxy citric acid and it has been found to suppress fatty acid synthesis, lipogenesis and hence to promote weight loss (Antony et al. 1998;Jena et al. 2002). Phenolics such as caffeoyl glucose, quercetin 3-glucronide and quercetin 3-rhamnoside have been reported from the fruits (Sulaiman et al. 2013). This species was not much explored phytochemicaly; however, chemical and biological studies have been reported in allied species such as Garcinia travancorica (Anu Aravind et al. 2016), Gracinia dulcis (Saelee et al. 2015;Mahabusarakam et al. 2016), Garcinia nobilis (Fouotsa et al. 2014), Garcinia mangostana (Zhou et al. 2015;Tran et al. 2016) and Garcinia hombroniana (Jamila et al. 2016). Generally, fruits have been considered as excellent sources of antioxidants, polyphenols, vitamins C and E and carotenoid (Vinson et al. 2001). In the current study, antioxidants of hydro alcoholic extract of G. gummi-gutta have been characterised by Mass spectroscopic analysis. Structural elucidation was done by mass fragmentation on collision-induced dissociation.

Results and discussion
The total phenolic content and radical scavenging activity of different extracts are presented in Table S1. The highest TPC was showed by hydro alcoholic extract (18.36 mg GAE) followed by acetone extract (12.80 mg GAE/g extract). dPPH radical scavenging capacity was found to be in correlation with total poly phenolics. Hydro alcoholic extract showed highest scavenging activity against dPPH radicals with least EC 50 value (6.38 μg/mL). Comparing with the standard catechin, hydro alcoholic extract showed significant radical scavenging activity.
Various extracts analysed such as acetone, methanol and hydro alcohol showed potent scavenging activity against dPPH radical as they contain significant amount of phenolics. The similar correlation between phenolic contents and radical scavenging activity has been reported in various Brazilian fruits (Almeida et al. 2011). Chemically, phenolics are free radical scavengers due to the presence of hydroxyl groups in ortho-and para-positions (Lapornik et al. 2005). The interrelation between total phenolics and antioxidant activity is well established (Fernandes de Oliveira et al. 2012;Terpinc et al. 2012).
Liquid chromatography profile was recorded at 265 nm on diaode array detection. The HPLC anlaysis of hydro alcoholic extract showed major peaks at Rt 2.54, 2.72, 3.10, 3.38, 3.64 and 4.59 ( Figure S1). Online UV spectrum of each peak was analysed separately. The UV λ max revealed that most of the peaks were phenolic in nature.
The hydro alcoholic extract was further characterised by LC-MS/MS analysis. Total ion chromatogram was extracted into base peak chromatogram using Agilent Mass Hunter software ( Figure S2). The MS spectra generated by negative ESI analyses yielded deprotoneated [M-H] molecules. Fragmentation was performed by auto MS/MS analysis by varying collision voltage. Most of the compounds were identified by comparing with the previously reported fragmentation pattern. The tentative identification of compounds corresponding to the MS/MS fragmentation is given in Table S2. The peak with m/z 461.07 presented two major fragments with m/z 285.04 and it was identified as luteolin 7-O-glucronide.The fragmentation pattern [M-176-H]-was in consistent with its previously reported spectra (Lin & Harnly 2010;Ibrahim et al. 2015). The molecular ion with m/z 489.10 yielded a fragment at m/z 285 corresponding to kaempferol 3-O-(6-O-acetyl) glycoside which has been previously reported in other fruit extracts (Tolonen et al. 2002;Kazuma et al. 2003;Apáti et al. 2004). The peak with m/z 515.12 was identified as dicaffeoylquinic acid by comparing the previously reported mass fragmentation pattern (Plazonić et al. 2009;Simirgiotis et al. 2013). At 2.25 min, a peak with m/z 563.13 was observed. On collisioninduced dissociation, it was fragmented as 503.10 and 473.10. It was consistent with mass fragmentation of apigenein-6-C-pentosyl-8-C-hexoside (Rodríguez-Medina et al. 2009). The peak at 2.80, m/z 337.08, yielded a fragment with m/z 173. It was consistent with mass fragmentation of p-coumarylquinic acid. The fragment 173 was due to the loss of water from deprotonated quinic acid group (Rodríguez-Medina et al. 2009). All these compounds were identified for the first time from this species. Total polyphenolic content, radical scavenging activity together with the identified polyphenolic compounds indicate that Brindle berry fruit can be considered as a potential source of natural antioxidants for food and medicinal applications.

Conclusion
The polyphenolic-rich diet has become an increasingly important area of human nutrition research. The characterisation of antioxidant fraction of G. gummi-gutta was carried out using multiple chromatographic analyses. The data obtained by LC-MS/MS spectra provided complimentary information about the structure of compounds which might be responsible for the antioxidant activity. Phenolic compounds like luteolin 7-O-glucuronide, kaempferol 3-O-(6-O-acetyl) glycoside, dicaffeoylquinic acid, apigenein-6-C-pentosyl-8-C-hexoside and p-coumarylquinic acid were identified from the active fraction. The current study concluded that the fruit of G. gummi-gutta is a natural source of polyphenolics with potent radical scavenging activity. It is beneficial to add the same with dietary sources due to the evidence of their defensive impact on human health.

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

Funding
This work was supported by TATA Trust.