Nutritional and Biological Evaluation of Leaves of Mangifera indica from Mauritius S .

Mango trees are evergreen plants that are present all around Mauritius. In this study, mango leaves, Mangifera indica grown in Mauritius were investigated for their nutritional values involving proximate composition, total flavonoid (TFC), total phenolic (TPC), and mineral content, and phytochemicals as well as its antioxidant and antibacterial properties. +e ash, crude fat, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) of the mango leaves were found to be 12.61, 3.92, 35.32, 34.98, and 12.86%, respectively. +e calcium content (2.15%) was above the normal required range, while the phosphorus content (0.12%) and crude protein content (13.60%) were within the normal required range of common fodders. +e phytochemical results showed the presence of saponins, alkaloids, phenols, tannins, and flavonoids in the crude, EtOAC, and MeOH extracts.+e values of TPC and TFC were higher for the EtOAC extract compared to theMeOH extract. Several secondary metabolites were identified from the leaves of the Mangifera indica which include 11 phenols, 4 xanthones, 9 flavanols, 10 benzophenones, 7 terpenoids, and 4 derivatives of gallotannins using UPLC-MS/MS. +e presence of these metabolites is responsible for good antioxidant and antibacterial properties. Hence, mango leaves can be exploited for its potential use as a supplementary fodder for ruminants.


Introduction
Mauritius relies heavily on importation of meat and dairy products.Local production of cattle and goat is limited and not well organized.Being a small island, pasture fields are limited and are on decline.Small livestock breeders need to look for alternative feed sources for their animals especially during the dry months of the year.
Tree leaves play an important role in the nutrition of grazing animals in areas where few or no alternatives are available [1].Fodder trees are an important source of supplementary protein, vitamins, and minerals in developing countries and are an alternative source of livestock feeding which have the potential for alleviating some of the feed shortages and nutritional deficiencies for small ruminants, goats, and sheep diets [2].Mango trees are evergreen plants which withstand dry periods very efficiently, and they can be used as a food supplement.Although a number of studies have been conducted for the different uses of mango fruits, peels, juice, and stem bark, there are limited reports on the importance of Mangifera indica leaves and its suitability to be used as fodder.Mango leaves fed at high percentage may cause poisoning for cattle [3]; however, it can form an important part of feed given to ruminants [4].According to the study conducted in Nigeria, it was shown that when goats were fed on mango leaves together with Guinea grass (Panicum maximum), the weight gain recorded was higher compared to when the mango leaves were replaced by Ficus (Ficus thonningii) or Gliricidia (Gliricidia sepium).e study also pointed out that mango leaves were accepted by the goats and are therefore palatable to the animals [5].
Mangifera indica have been the focus of intense research in search of a variety of biomolecules from different parts of the plants such as stems, leaves, fruits, and seed kernels.Its medicinal value is well established and has been used for centuries for the treatment of different kinds of diseases [6].It is found to possess different pharmacological properties including the antibacterial property [7].
Previous research on the phytochemical screening of the leaves of Mangifera indica revealed the presence of phenols, saponins, tannins, steroids, flavonoids, anthraquinone, and glycosides [7][8][9].erefore, the purpose of this work was to investigate the nutritional values in terms of proximate analysis, TPC, TFC, antioxidant, and antibacterial studies and to identify the major secondary metabolites present in the leaves of Mangifera indica from Mauritius.

Sample Preparation.
e fresh leaves of Mangifera indica (dauphiné, Mauritian variety) collected from the central region of Mauritius were washed with water and dried in a drying cabinet.e dried leaves were then reduced to a particle size of 1 mm using an electric grinder and stored in a well-closed plastic container for further use.

Proximate Analysis.
e different chemical parameters for proximate analysis were determined based on the Association of Official Analytical Chemists (AOAC), 2005, Official Method [10].

Determination of Crude Protein.
e total nitrogen content was obtained from a Eurovector EA 3000 elemental analyzer, and the crude protein content was calculated by multiplying the N% by a factor of 6.25.

Crude Fat Content.
e fat content was extracted from the ground mango leaves (2 g) using 140 mL of petroleum ether in a Soxhlet extractor for 6 h.After extraction, the solvent was distilled off, the residue was weighed, and the percentage of fat was calculated as a percentage of the sample used.

Ash Content.
Two grams of powdered mango leaves was heated on a hot plate in a 50 mL silica crucible which was further heated for 3 h at 525 °C in a muffle furnace.e residual ash was weighed and was expressed as percentage of mass of ash with respect to mass of the original sample.

Determination of Fiber Content.
e neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using the Fibertec method according to the method described in Foss Application Note AN 304 (2005) and Foss Application Note AN 3429 (2005), respectively [11].
Determination of NDF involved boiling 1 g of dried powdered leaves (W 1 ) for 1 h in 100 mL of neutral detergent solution (disodium hydrogen phosphate, sodium tetraborate decahydrate, EDTA disodium salt dehydrate, sodium dodecyl ethoxyethanol, and anhydrous sodium sulphite), in which 1 drop of octan-2-ol and 1 or 2 drops of α-amylase were added.e resulting residue was dried at 105 °C in an oven for 3 h and weighed (W 2 ).
e residue was further ashed for 3 h in a muffle furnace, cooled, and weighed (W 3 ).e percentage of NDF was calculated according to the following equation: whereW 1 � mass of the sample, W 2 � mass of crucible + residue, and W 3 � mass of the crucible and ash.
For ADF content, 100 mL of acid detergent solution (conc.sulphuric acid and N-cetyltrimethyl ammonium bromide) were added to 1 g of powdered leaves (W 1 ) in a fritted predried Foss crucible followed by adding 1 drop of octan-2-ol, filtered, dried for 3 h at 105 °C in the ovenm, and weighed (W 3 ).e percentage of ADF was calculated according to the following equation: where W 1 � mass of the sample, W 2 � mass of the crucible, and W 3 � mass of the crucible and residue.

Determination of Acid Detergent Lignin (ADL).
Acid detergent lignin (ADL) was determined according to the method described in Foss Application Note AN 3430 (2005) [10].e ADF residue was soaked in conc.sulphuric acid (72%) for 3 h, ashed for 3 h at 525 °C, and weighed.e percentage of ADL was calculated according to the following equation: where W 1 � mass of the ADF residue, W 2 � mass of crucible + ADF residue, and W 3 � mass of crucible + ash.

Determination of Calcium and Phosphorus Content.
e ash obtained as mentioned in Section 2.3.3 was double ashed using a mixture of 2 : 1 conc.HCl/HNO 3 (3 mL) in a muffle furnace at 525 °C for 3 h.After the double ashing, the residue was dissolved in conc.HCl : HNO 3 (3 : 1) and the solution was made up to 200 mL.e obtained filtrate was used for the determination of calcium content using atomic absorption spectrophotometer (AAS) and phosphorus content using UV-Vis spectrophotometer at a wavelength of 650 nm.

Preparation of Extracts.
Ten grams of powdered leaves was extracted in the Soxhlet extractor using hexane, EtOAc, and MeOH, successively.
e extracts were concentrated and stored at 4 °C until further analysis.0.5 g of each extract was dissolved in 100 mL of methanol, and these diluted extracts were qualitatively tested for the presence of different phytochemicals using standard procedures [8,13,14] and antioxidant activity.

Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC).
e total phenolic content (TPC) of the extracts was determined using the reported procedure [15].0.2 mL of the extract was mixed with 0.2 mL of the Folin-Ciocalteu reagent, and 0.8 mL of sodium carbonate (2%) was added after 5 min.e samples were then incubated at room temperature.e TFC was determined using the aluminium chloride colorimetric method [16].0.25 mL of extract in 1.25 mL of distilled water was mixed with a solution containing sodium nitrite (0.075 mL, 5%), aluminium chloride (0.15 mL, 10%), and sodium hydroxide (0.5 mL, 1 M), and the mixture was made up to 2.5 mL with distilled water.e absorbance of solution was read at 650 nm for TPC and 510 nm for TFC using the Biochrom Libra S22 UV-Vis spectrometer.e TPC/TFC extract solutions were expressed as gallic acid/quercetin equivalent (mg/g) of ground leaves.

Antioxidant Activity.
e free radical scavenging capacity of the extracts was determined using the DPPH assay [17]. 2 mL of the methanolic sample with different concentrations, ranging from 100 to 5000 ppm, was added to 2 mL of methanolic DPPH solution (4.5 g/100 mL).e absorbance was measured at 492 nm using a Labsystems Multiskan Ms. EIA reader ( ermo Fisher Scientific/lab system, California, USA) after 30 min of incubation at 37 °C.Ascorbic acid and methanol were used as positive and negative controls, respectively.e ability to scavenge the DPPH was calculated using (6), where A 0 and A s are the absorbances of the control and sample, respectively.e concentration of the sample required to scavenge 50% of DPPH was determined as follows:

Results and Discussion
Availability of proper animal feed and efficient feeding are the foundation of successful livestock production.Proper animal feeding is the supply of a diet balance in all nutrients which increases the animal productivity, quality of product, and animal welfare.e availability of accurate reliable and reproducible analytical data is imperative for proper feed formulation.

Proximate Analysis.
Proximate analysis gives valuable information to access the quality of the plant material as fodder.Proximate analyses were carried out on the leaves of Mangifera indica grown locally to evaluate the nutritional value and to investigate whether the leaves can be used as fodder for ruminants.e proximate composition of Mangifera indica leaves in terms of ash, crude fat (CF), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) is given as percentage of dry matter in Table 1.e % ash content of major fodder trees has been reported to be in the range of 5-17% [19], and the ash content of locally grown Mangifera indica leaves was found to be 12.61% which indicates that locally grown mango leaves have a relatively high mineral content.e CF content was 3.92% which was lower than the value (8%) which is considered to be harmful to ruminants [20].
e CP content was within the range of CP values (12-14%) of top fodder trees which is regarded as the amount required to support the growth of ruminants [19].Detergent fiber (NDF, ADF, ADL, HC, and CEL) composition of the local mango leaves together with Nigerian [5] and Laos [21] varieties are summarized in Table 1.e range of values of different parameters for the major fodder trees from Nepal are also given in Table 1 [19].e values of ADF and ADL obtained for local mango leaves were within the range as reported for major fodder trees and therefore considered as acceptable and digestible by the ruminants.In the locally grown mango leaves, the cellulose (CEL) and hemicellulose (HC) levels were quite low.Relatively low CEL and HC have been reported in various fodder tree leaves found elsewhere [19].

Calcium and Phosphorus Content.
Calcium and phosphorus are considered as macrominerals and are needed in gram quantities.In animals a Ca : P ratio above 2 helps to increase the absorption of calcium in the intestine [22].Fodder with Ca : P ratio of 1.5-1.3 is considered to be good.However, it is reported that fodder which is too rich in calcium may hamper the absorption and assimilation of phosphorus, magnesium, zinc, copper, and other microelements.
e normal requirement for calcium and phosphorus contents for ruminants is in the range of 0.19-0.82and 0.12-0.48%,respectively [23].
e Ca : P ratio in the local mango leaves was 17.9 : 1.However, a few major fodder trees have been reported with higher calcium than that obtained for the mango leaves.High Ca : P ratio can be dangerous for ruminants, and this could be corrected by adding cereal by-products to the fodder which are low in calcium and high in phosphorus.

Phytochemical Analysis.
e phytochemical screening of the crude, hexane, EtOAC, and MeOH extracts indicated the presence of an array of phytochemicals including saponins, alkaloids, phenols, tannins, flavonoids, glycosides, diterpenes and coumarins in the Mangifera indica leaves, and these findings were in line with data reported elsewhere [8,9] (Table 2).Phenols, flavonoids, alkaloids, and tannins were identified mainly in the EtOAc and MeOH extracts.
erefore, in this study, the EtOAc and MeOH extracts of the Mangifera indica leaves were analyzed using UPLC-MS/MS and different metabolites were identified by comparison with reference compounds/literature data/their mass spectral fragments from both the positive-and negative-ion modes.

Phenolic Compounds.
A number of polyphenolic compounds characterized as benzophenone derivatives, flavonols, xanthones, and gallotannins by their molecular weight and mass spectrometric data, and comparison with reported data are given in Tables 3 and 4 ( e mass spectra used to support the findings of this study are included within the supplementary information files (available here)).
Seven phenolic compounds were identified in the protonated form, [ Journal of Chemistry Journal of Chemistry   3).

Benzophenone Derivatives and Related Compounds.
Ten benzophenone derivatives were identified from the EtOAC and MeOH extracts.e compound with a [M+H] + ion at m/z 263 with a major fragment at m/z 124 was identified as maclurin.Moreover, a number of iriflophenone galloyl glucosides or maclurin glucoside derivatives were identified at m/z 409, 545, 713, and 865 (entry 23-26, Table 3) in the positive-ion mode and 423, 543, 575, and 727 (entry 8-12, Table 4) in the negative-ion mode.e compound having m/z 405 (entry 22, Table 3) in the positive-ion mode is most probably a derivative of iriflophenone glucoside based on its mass fragments.4.

Terpenoids.
In the positive mode, the peak at m/z 427 corresponded to the terpenoid lupeol.Six cycloartane terpenoids derivatives (Tables 3 and 4) were identified from the leaves of locally grown mango leaves.

Biological Properties
4.1.Antibacterial.It has been reported that the phytochemicals present in the leaves of Mangifera indica are responsible for antibacterial, antioxidant, and anti-inflammatory activities.Previous research conducted by Doughari and Manzara [7] reported that the extracts of Mangifera indica showed a potent antibacterial activity against Gram-positive and Gram-negative bacteria with MIC values ranging from 12.5 to 175 mg/mL.e results of the present study revealed that the crude extract of the leaves exhibited moderate to good antibacterial properties against Gram-positive bacteria with zone of inhibition of 8.6 ± 0.6 mm (P.aeruginosa), 10.3 ± 1.5 mm (B.cereus), and 11 ± 1.0 mm (S. aureus), and against Gram-negative bacteria with zone of inhibition of 10 ± 1.0 mm (S. epiderdimis), 16 ± 0.2 mm (K.pneumoniae), and 20.3 ± 0.6 mm (E.coli) at the conc. of 1 mg/mL.e MIC values were found to be 0.062 for S. aureus, B. cereus, E. coli, S. epidermis, and P. aeroginosa while for K. pneumonia, an MIC value of 0.031 mg/mL was obtained.
Values of TPC and TFC of the crude, EtOAc, and MeOH extracts of the locally grown Mangifera indica are given in Table 5. e crude extract exhibited antioxidant potential which can be related to its high TPC and TFC. e EtOAc extract showed higher antioxidant activity attributed to a more elevated concentration of phenolic and flavonoid contents as compared to the MeOH extract.

Conclusion
is study was conducted in a view to understand the nutritional characteristics of leaves from Mangifera indica trees grown in Mauritius for their use as fodder for ruminants.It is observed that mango leaves is a good source of mineral elements since it has a high percentage of ash content.e crude fat was low while crude protein was within the range required to support the growth of ruminants.e parameters (NDF, ADF, and ADL) were comparable to values reported for major fodder trees apart from HC which was found to be below the range.
e phytochemical studies indicated the presence of various phenolic compounds including xanthones, flavanols, benzophenones, terpenoids, and tannins which can be the source of natural antioxidant and antibacterial agents.ese scientific findings would be environmentally and economically sustainable for considering mango leaves as good fodder for feeding ruminants.
ions and were attributed to mangiferin, mangiferin-methyl ether, and mangiferin-Ogallate, respectively.e other peak at m/z 421 ([M−H] − ion) corresponded to isomangiferin.e fragment ions at m/z 331 and 301 corresponded to the loss of C-glycoside phenolic compounds.3.3.3.Flavonols.In the literature, a number of flavanol and flavanol glycosides have been detected in mango peels, bark, and seed kernels.In the EtOAc and MeOH extracts, nine flavanols have been detected in the protonated or deprotonated form.e peak at [M−H] − with m/z 285 corresponded to kaemferol while that at [M−H] − with m/z 463 was attributed to quercetin 3-O-glucoside.e other quercetin derivatives were identified in the positive-ion mode [M+H] + at m/z 303, 317, 347, 435, 449, 459, and 479 based on their mass fragmentation and by comparison with literature data (Table

Table 2 :
Summary of the phytochemical screening test performed on the different extracts.

Table 3 :
Compounds determined by UPLC-MS/MS in the EtOAc and MeOH extracts of Mangifera indica in the positive-ion mode.

Table 4 :
Compounds determined by UPLC-MS/MS in the EtOAC and MeOH extracts of Mangifera indica in the negative-ion mode.

Table 5 :
Total phenolic content and total flavonoid content in crude, EtOAc, and MeOH extracts of Mangifera indica.