Studies related to the chemical composition, biological activities and toxicity of methanolic extracts of noni (Morinda citrifolia) fruits and leaves

Abstract Morinda citrifolia is a plant that grows in Brazilian northeast and presented a wide range of therapeutic, industrial and technological applications. Based on this, the aim of this work was to study the chemical composition, main biological activities and potential toxicity of its extracts, aiming their industrial application. Important compounds were identified in the methanolic extracts obtained by ultrasonic and Soxhlet extractions from leaves and fruits. GC × GC allowed for the identification of phytosterols, fatty acids and methyl esters, besides others (scopoletin, hydrocarbons, alcohols, terpenes). By HPLC-DAD, compounds like catechin, rutin, quercetin could be also identified and quantified. Their content of polyphenols and flavonoids was considered between the international standards. The extracts showed high antioxidant activities (EC50 ∼ 300 μg mL−1, using DPPH assay) compared with those from the literature. The extracts did not show toxicity or mutagenicity, but presented cytotoxicity, which can indicate their use safely in phytotherapic or nutritional applications. Graphical Abstract


Introduction
Morinda citrifolia Linn (Noni) is a perennial fruitful plant that reaches 6 m in height (Chan-Blanco et al. 2007;Nerurkar et al. 2015) and belongs to the Rubiaceae family, being traditionally used as therapeutic treatment of diseases such as arthritis, tuberculosis, diabetes and hypertension (Ali et al. 2016). Due to its adaptability to edaphoclimatic conditions and its value in the market, the cultivation of noni tends to grow in Brazil, mainly aiming its medicinal use and functional foods production (Vasconcelos et al., 2014). Its juice has antioxidant and anti-inflammatory properties (Motshakeri and Ghazali 2015). Its commercialization is not allowed in Brazil due to the lack of studies proving their action and nontoxicity (Deng et al. 2010). Almeida et al. (2019) carried out an excellent review involving updated and comprehensive information on M. citrifolia (mainly fruits), such as its traditional use, biochemical, herbal and toxicological properties, processes and standardizations of derived products. In view of the wide range of therapeutic, industrial and technological applications of the different components of M. citrifolia, this work aims to study the chemical composition and main biological activities of M. citrifolia fruits and leaves extracts from the Northeast region of Brazil (Sergipe) with a view to their industrial, nutritional and pharmacological application.

Results and discussion
The extractions by sonication revealed higher yields (fruits 14.7% and leaves 15.95%) if compared to Soxhlet (fruits 5.66% and leaves 5.24%) for both samples. To facilitate the data interpretation, it was used the following codes: Soxhlet extract of fruits (SEF), Soxhlet extract of leaves (SEL), ultrasonic extract of fruits (UEF) and ultrasonic extract do leaves (UEL).
The HPLC analysis of the extracts revealed the presence of nine different polyphenols ( Figure S2 and Table S3, supplementary material). Sonication extracts showed higher concentrations for all the nine compounds than Soxhlet extracts; regarding advantages such as lower solvent consumption and shorter extraction time. The sonication technique allowed for the increase the yield of bioactive compounds such as rutin and quercetin by five and six times, reducing the extraction time by 3.2 times. In the fruit extracts, quercetin and catechin were the major constituents (15.6 and 15.0 mg g À1 , respectively). These compounds are reported as the main constituents in Noni fruit extracts, however in lower concentration than that obtained in the present study (Huang et al. 2016;Meinhart et al. 2019). The content of rutin in Brazilian Noni was higher than those obtained by other authors.
In the leave extracts, the concentration of epigallocatechin, gallocatechin and quercetin can be highlighted. The concentration of these compounds was higher than those reported in the literature for Noni leaves extracts obtained by conventional techniques (Hui et al. 2020). Rutin, cathechin and quercetin, flavonoids identified in these extracts, exhibit significant biological activities, such as antioxidant, antimicrobial, among others (Prakash et al. 2019;Ara ujo et al. 2021).
The results of cytotoxic and mutagenic activities, evaluated by A. cepa bioassay, are summarized in Table S3. All extracts showed cytotoxic activity represented by a decrease in germination index (GI) and an increase in mitotic index (MI), when compared to the negative control (p < 0.05) (Francisco et al. 2018). These effects increased with the increasing of the concentration (0.1 to 1.0 mg mL À1 ). No significant differences were observed for mutagenicity index (MTI), thus no mutagenic effects were observed, at any concentration, when compared to the negative control. Boontha et al. (2018) reported cytotoxic activity against human breast cancer cells for fruit extracts of Noni. Extracts from leaves and some isolated compounds (damnacanthal, rutin, and scopoletin) also showed cytotoxic effect against cancer cell according to Thani et al. (2010) and crude extract showed higher activity than the isolated compounds, indicating synergic effect. Thus, the presence of rutin and scopoletin in the extracts obtained in the present study could explain the cytotoxicity reported.

Conclusions
The ultrasonic extracts showed higher yields than those obtained by Soxhlet, with the reduction of the extraction time and solvent consumption. GC Â GC allowed for the identification of important compounds, with activities recognized in the literature, such as fatty acids, scopoletin, phytosterols and terpenes. HPLC-DAD allowed for the identification of nine different compounds in leaves fruit extracts, noteworthy: catechin, quercetin, gallic acid, gallocatechin and epigallocatechin. The extracts were nontoxic in A. salina assay and showed some cytotoxicity, but did not show mutagenicity, in A. cepa bioassay. These results indicated the potential use of the extracts from Brazilian Morinda citrifolia in anticancer therapies, as the literature already has been reported.

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

Funding
The authors thank CNPq, CAPES and FAPITEC for the financial support for this study.