# Nutritional and Biological Evaluation of Leaves ofMangifera indica from Mauritius

Authors: S. Jhaumeer Laulloo; M. G. Bhowon; S. Soyfoo; L. S. Chua
Journal: Journal of Chemistry
 (2018)

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## Abstract

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. The 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. The 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. The phytochemical results showed the presence of saponins, alkaloids, phenols, tannins, and flavonoids in the crude, EtOAC, and MeOH extracts. The values of TPC and TFC were higher for the EtOAC extract compared to the MeOH 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. The 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.


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## Body

## 1. 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 ofMangifera 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). The 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 ofMangifera indica revealed the presence of phenols, saponins, tannins, steroids, flavonoids, anthraquinone, and glycosides [7–9]. Therefore, 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.


## 2. Materials and Methods

### 2.1. General

Ethylenediamine tetraacetic acid disodium salt, ascorbic acid, antimony potassium (+) tartrate, and disodium tetraborate were purchased from BDH (England). Gallic acid monohydrate, sodium dodecyl sulphate, copper acetate, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, and Folin–Ciocalteu reagent were obtained from Sigma-Aldrich.N-Cetyltrimethyl ammonium bromide (CTAB) and ammonium molybdate were purchased from Techno Pharmachem, India. All other common chemicals and solvents used were of analytical grade.


### 2.2. Sample Preparation

The fresh leaves ofMangifera indica (dauphiné, Mauritian variety) collected from the central region of Mauritius were washed with water and dried in a drying cabinet. The 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.


### 2.3. Proximate Analysis

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


#### 2.3.1. Determination of Crude Protein

The total nitrogen content was obtained from a Eurovector EA 3000 elemental analyzer, and the crude protein content was calculated by multiplying theN% by a factor of 6.25.


#### 2.3.2. Crude Fat Content

The 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.


#### 2.3.3. 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. The residual ash was weighed and was expressed as percentage of mass of ash with respect to mass of the original sample.


#### 2.3.4. Determination of Fiber Content

The 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 (W1) 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. The resulting residue was dried at 105°C in an oven for 3 h and weighed (W2). The residue was further ashed for 3 h in a muffle furnace, cooled, and weighed (W3). The percentage of NDF was calculated according to the following equation:(1)%NDF=W2−W3W1×100,where W1 = mass of the sample, W2 = mass of crucible + residue, and W3 = mass of the crucible and ash.For ADF content, 100 mL of acid detergent solution (conc. sulphuric acid andN-cetyltrimethyl ammonium bromide) were added to 1 g of powdered leaves (W1) 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 (W3). The percentage of ADF was calculated according to the following equation:(2)%ADF=W3−W2W1×100,where W1 = mass of the sample, W2 = mass of the crucible, and W3 = mass of the crucible and residue.


#### 2.3.5. 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]. The ADF residue was soaked in conc. sulphuric acid (72%) for 3 h, ashed for 3 h at 525°C, and weighed. The percentage of ADL was calculated according to the following equation:(3)%ADL=W3−W2W1×100,where W1=mass of the ADF residue, W2=mass of crucible+ADF residue, and W3=mass of crucible+ash.


#### 2.3.6. Determination of Hemicelluloses, Cellulose, and Lignin in the Fiber and Total Digestible Nutrient

The percentage of hemicelluloses, cellulose, and lignin in the fiber was calculated as follows [12]:(4)Hemicellulose=%NDF−ADF=35.32−34.98=0.34%,Cellulose=%ADF−ADL=34.98−12.86=22.12%,Lignin=%ADL=12.86%.Total digestible nutrient (TDN) was computed from the following formula:(5)TDN=88.9−ADF%×0.7=88.9−34.98×0.7=64.41%.


#### 2.3.7. Determination of Calcium and Phosphorus Content

The ash obtained as mentioned in Section2.3.3 was double ashed using a mixture of 2 : 1 conc. HCl/HNO3 (3 mL) in a muffle furnace at 525°C for 3 h. After the double ashing, the residue was dissolved in conc. HCl : HNO3 (3 : 1) and the solution was made up to 200 mL. The 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.


### 2.4. Phytochemicals Screening

#### 2.4.1. Preparation of Extracts

Ten grams of powdered leaves was extracted in the Soxhlet extractor using hexane, EtOAc, and MeOH, successively. The 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.


#### 2.4.2. Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The 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. The samples were then incubated at room temperature. The 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. The absorbance of solution was read at 650 nm for TPC and 510 nm for TFC using the Biochrom Libra S22 UV-Vis spectrometer. The TPC/TFC extract solutions were expressed as gallic acid/quercetin equivalent (mg/g) of ground leaves.


### 2.5. Antioxidant Activity

The 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). The absorbance was measured at 492 nm using a Labsystems Multiskan Ms. EIA reader (Thermo 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. The ability to scavenge the DPPH was calculated using (6), where A0 and As are the absorbances of the control and sample, respectively. The concentration of the sample required to scavenge 50% of DPPH was determined as follows:(6)%  scavengingactivity=A0−AsA0×100.


### 2.6. Antibacterial Activity

Antimicrobial tests were performed against three Gram-positive bacteriaStaphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), and Bacillus cereus (ATCC 10876) and three gram negative bacteria Escherichia coli (ATCC 22922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 13883) using agar disk diffusion method [18] at varying concentrations (1, 0.5, 0.25, 0.125, 0.062, and 0.031 mg/mL). The diameter of the zone of inhibition was recorded in mm.


## 2.1. General

Ethylenediamine tetraacetic acid disodium salt, ascorbic acid, antimony potassium (+) tartrate, and disodium tetraborate were purchased from BDH (England). Gallic acid monohydrate, sodium dodecyl sulphate, copper acetate, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, and Folin–Ciocalteu reagent were obtained from Sigma-Aldrich.N-Cetyltrimethyl ammonium bromide (CTAB) and ammonium molybdate were purchased from Techno Pharmachem, India. All other common chemicals and solvents used were of analytical grade.


## 2.2. Sample Preparation

The fresh leaves ofMangifera indica (dauphiné, Mauritian variety) collected from the central region of Mauritius were washed with water and dried in a drying cabinet. The 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.


## 2.3. Proximate Analysis

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


### 2.3.1. Determination of Crude Protein

The total nitrogen content was obtained from a Eurovector EA 3000 elemental analyzer, and the crude protein content was calculated by multiplying theN% by a factor of 6.25.


### 2.3.2. Crude Fat Content

The 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.


### 2.3.3. 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. The residual ash was weighed and was expressed as percentage of mass of ash with respect to mass of the original sample.


### 2.3.4. Determination of Fiber Content

The 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 (W1) 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. The resulting residue was dried at 105°C in an oven for 3 h and weighed (W2). The residue was further ashed for 3 h in a muffle furnace, cooled, and weighed (W3). The percentage of NDF was calculated according to the following equation:(1)%NDF=W2−W3W1×100,where W1 = mass of the sample, W2 = mass of crucible + residue, and W3 = mass of the crucible and ash.For ADF content, 100 mL of acid detergent solution (conc. sulphuric acid andN-cetyltrimethyl ammonium bromide) were added to 1 g of powdered leaves (W1) 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 (W3). The percentage of ADF was calculated according to the following equation:(2)%ADF=W3−W2W1×100,where W1 = mass of the sample, W2 = mass of the crucible, and W3 = mass of the crucible and residue.


### 2.3.5. 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]. The ADF residue was soaked in conc. sulphuric acid (72%) for 3 h, ashed for 3 h at 525°C, and weighed. The percentage of ADL was calculated according to the following equation:(3)%ADL=W3−W2W1×100,where W1=mass of the ADF residue, W2=mass of crucible+ADF residue, and W3=mass of crucible+ash.


### 2.3.6. Determination of Hemicelluloses, Cellulose, and Lignin in the Fiber and Total Digestible Nutrient

The percentage of hemicelluloses, cellulose, and lignin in the fiber was calculated as follows [12]:(4)Hemicellulose=%NDF−ADF=35.32−34.98=0.34%,Cellulose=%ADF−ADL=34.98−12.86=22.12%,Lignin=%ADL=12.86%.Total digestible nutrient (TDN) was computed from the following formula:(5)TDN=88.9−ADF%×0.7=88.9−34.98×0.7=64.41%.


### 2.3.7. Determination of Calcium and Phosphorus Content

The ash obtained as mentioned in Section2.3.3 was double ashed using a mixture of 2 : 1 conc. HCl/HNO3 (3 mL) in a muffle furnace at 525°C for 3 h. After the double ashing, the residue was dissolved in conc. HCl : HNO3 (3 : 1) and the solution was made up to 200 mL. The 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.


## 2.3.1. Determination of Crude Protein

The total nitrogen content was obtained from a Eurovector EA 3000 elemental analyzer, and the crude protein content was calculated by multiplying theN% by a factor of 6.25.


## 2.3.2. Crude Fat Content

The 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.


## 2.3.3. 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. The residual ash was weighed and was expressed as percentage of mass of ash with respect to mass of the original sample.


## 2.3.4. Determination of Fiber Content

The 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 (W1) 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. The resulting residue was dried at 105°C in an oven for 3 h and weighed (W2). The residue was further ashed for 3 h in a muffle furnace, cooled, and weighed (W3). The percentage of NDF was calculated according to the following equation:(1)%NDF=W2−W3W1×100,where W1 = mass of the sample, W2 = mass of crucible + residue, and W3 = mass of the crucible and ash.For ADF content, 100 mL of acid detergent solution (conc. sulphuric acid andN-cetyltrimethyl ammonium bromide) were added to 1 g of powdered leaves (W1) 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 (W3). The percentage of ADF was calculated according to the following equation:(2)%ADF=W3−W2W1×100,where W1 = mass of the sample, W2 = mass of the crucible, and W3 = mass of the crucible and residue.


## 2.3.5. 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]. The ADF residue was soaked in conc. sulphuric acid (72%) for 3 h, ashed for 3 h at 525°C, and weighed. The percentage of ADL was calculated according to the following equation:(3)%ADL=W3−W2W1×100,where W1=mass of the ADF residue, W2=mass of crucible+ADF residue, and W3=mass of crucible+ash.


## 2.3.6. Determination of Hemicelluloses, Cellulose, and Lignin in the Fiber and Total Digestible Nutrient

The percentage of hemicelluloses, cellulose, and lignin in the fiber was calculated as follows [12]:(4)Hemicellulose=%NDF−ADF=35.32−34.98=0.34%,Cellulose=%ADF−ADL=34.98−12.86=22.12%,Lignin=%ADL=12.86%.Total digestible nutrient (TDN) was computed from the following formula:(5)TDN=88.9−ADF%×0.7=88.9−34.98×0.7=64.41%.


## 2.3.7. Determination of Calcium and Phosphorus Content

The ash obtained as mentioned in Section2.3.3 was double ashed using a mixture of 2 : 1 conc. HCl/HNO3 (3 mL) in a muffle furnace at 525°C for 3 h. After the double ashing, the residue was dissolved in conc. HCl : HNO3 (3 : 1) and the solution was made up to 200 mL. The 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.


## 2.4. Phytochemicals Screening

### 2.4.1. Preparation of Extracts

Ten grams of powdered leaves was extracted in the Soxhlet extractor using hexane, EtOAc, and MeOH, successively. The 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.


### 2.4.2. Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The 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. The samples were then incubated at room temperature. The 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. The absorbance of solution was read at 650 nm for TPC and 510 nm for TFC using the Biochrom Libra S22 UV-Vis spectrometer. The TPC/TFC extract solutions were expressed as gallic acid/quercetin equivalent (mg/g) of ground leaves.


## 2.4.1. Preparation of Extracts

Ten grams of powdered leaves was extracted in the Soxhlet extractor using hexane, EtOAc, and MeOH, successively. The 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.


## 2.4.2. Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The 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. The samples were then incubated at room temperature. The 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. The absorbance of solution was read at 650 nm for TPC and 510 nm for TFC using the Biochrom Libra S22 UV-Vis spectrometer. The TPC/TFC extract solutions were expressed as gallic acid/quercetin equivalent (mg/g) of ground leaves.


## 2.5. Antioxidant Activity

The 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). The absorbance was measured at 492 nm using a Labsystems Multiskan Ms. EIA reader (Thermo 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. The ability to scavenge the DPPH was calculated using (6), where A0 and As are the absorbances of the control and sample, respectively. The concentration of the sample required to scavenge 50% of DPPH was determined as follows:(6)%  scavengingactivity=A0−AsA0×100.


## 2.6. Antibacterial Activity

Antimicrobial tests were performed against three Gram-positive bacteriaStaphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), and Bacillus cereus (ATCC 10876) and three gram negative bacteria Escherichia coli (ATCC 22922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 13883) using agar disk diffusion method [18] at varying concentrations (1, 0.5, 0.25, 0.125, 0.062, and 0.031 mg/mL). The diameter of the zone of inhibition was recorded in mm.


## 3. 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. The availability of accurate reliable and reproducible analytical data is imperative for proper feed formulation.


### 3.1. 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 ofMangifera indica grown locally to evaluate the nutritional value and to investigate whether the leaves can be used as fodder for ruminants. The 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. The % 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. The CF content was 3.92% which was lower than the value (8%) which is considered to be harmful to ruminants [20].Table 1
Proximal composition expressed in % (dry basis) ofMangifera indica.
Constituents (%)
Mangifera indica (dauphiné Mauritian variety)
Mangifera indica (Nigerian variety)
Mangifera indica (Laos variety)
Major fodder trees (Nepal)
Ash
12.61
10
—
(5 to 17)
CF
3.92
0.48
—
—
CP
13.60
20.38
6.90
(11 to 22)
NDF
35.32
43.14
50.10
(27 to 75)
ADF
34.98
51.48
—
(20 to 54)
ADL
12.86
2.47
—
(6 to 35)
HC
0.34
8.34
—
(1 to 31)
CEL
22.12
49.01
—
(13 to 33)
Calcium
2.15
—
—
>1
Phosphorus
0.12
—
—
(1.16 to 0.40)
— = not reported.The 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. The range of values of different parameters for the major fodder trees from Nepal are also given in Table 1 [19]. The 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].


### 3.2. 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. The normal requirement for calcium and phosphorus contents for ruminants is in the range of 0.19–0.82 and 0.12–0.48%, respectively [23]. The 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.


### 3.3. Phytochemical Analysis

The 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 theMangifera 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.Table 2
Summary of the phytochemical screening test performed on the different extracts.
Phytochemicals
Crude
Hexane
EtOAc
MeOH
Saponin
+
−
+
+
Alkaloids
+
−
+
+
Phenols
+
−
+
+
Tannins
+
−
+
+
Flavonoids
+
−
+
+
Steroids
+
−
−
+
Starch
−
−
−
−
Glycosides
+
+
−
−
Diterpenes
+
+
+
−
Anthocyanins
−
−
−
−
Amino acids
−
−
−
−
Coumarins
−
−
−
−Therefore, in this study, the EtOAc and MeOH extracts of theMangifera 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.


#### 3.3.1. 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 Tables3 and 4 (The mass spectra used to support the findings of this study are included within the supplementary information files (available here)).Table 3
Compounds determined by UPLC-MS/MS in the EtOAc and MeOH extracts ofMangifera indica in the positive-ion mode.
Identified compounds (name, formula)
Retention time
[M+H]+
Mol. wt.
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Protacatechuic, C7H6O4
4.570
155
154
153, 125, 79
√
√
[24, 25]
2
Gallic acid, C7H6O5
2.174
171
170
153, 127, 109, 107, 97, 81, 79, 69
√
[25, 26]
3
Methyl gallate, C8H8O5
5.612
185
184
153, 125
√
[27]
4
2,5-Di-tert-butylphenol, C14H22O
7.148
207
206
189, 174, 159, 147, 119, 105, 91, 95, 79, 77
√
√
[28]
5
Tetrahydroxy sodium benzoate, C7H5O6Na
4.211
209
208
191, 173, 167, 163/151/121, 107, 93, 91, 79, 77
√
√
6
Derivative of gallic acid
18.951
235
234
194, 171, 153, 137, 118, 84, 73
√
√
7
Derivative of gallic acid
2.022
276
275
258, 230, 212, 194, 153, 112, 97
√
8
Ellagic acid, C14H6O8
6.876
303
302
257, 229
√
[29]
9
Derivative of theogallin with one OH missing, C14H16O9
8.121
329
328
271, 249, 235, 225, 192, 176, 153, 107
√
10
Theogallin, C14H16O10
2.257
345
344
327, 299, 209, 153, 137, 125
√
√
[30]
Xanthones
11
Mangiferin, C19H18O11
8.928
423
422
√
√
[31]
12
Mangiferin 3-methyl ether, C20H20O11
8.299
437
436
√
[32]
13
Mangiferin-6’-O-gallate, C26H22O15
8.875
575
574
465, 455, 439, 423, 327, 313, 303, 285, 261, 193
√
[31]
Flavonols
14
Quercetin, C15H10O7
9.284
303
302
√
√
[33]
15
Rhamnetin, C16H12O7
12.036
317
316
262, 167, 156, 154, 126
√
[34]
16
Quercetin carboxylic acid, C16H10O9
10.643
347
346
302, 284, 255, 227
√
17
Quercetin pentoside, C20H18O11
8.394
435
434
354, 311, 259, 188, 162
√
√
[31]
18
Quercetin 3-O-rhamnoside C21H20O11
9.640
449
448
285, 284, 255
√
√
[31, 34]
19
Epicatechin gallate hexamalonate, C22H18O11
13.983
459
458
√
√
[24]
20
Rhamnetin hexoside, C22H22O12
14.067
479
478
315, 169
√
√
[30]
Benzophenones
21
Maclurin, C13H10O6
10.549
263
262
167, 124, 121, 111, 99, 65
√
√
[31]
22
Iriflophenone glucoside derivative
7.860
405
404
369, 351, 327, 313, 298, 273, 253, 99, 81
√
√
23
Iriflophenone 3-C-β-D-glucopyranoside, C19H20O10
7.232
409
408
391, 355, 325, 313, 231, 195, 177, 165, 121
√
√
[35]
24
Maclurin 3-C-(6’’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.011
545
544
√
[36]
25
Iriflophenone-di-O-galloyl-glucoside, C33H28O18
9.302
713
712
√
[31, 36]
26
Iriflophenone tri-O-galloyl-glucoside, C40H32O22
9.902
865
864
√
[36]
Terpenoids
27
Lupeol, C30H50O
7.819
427
426
√
√
[37, 38]
28
Mangiferonic acid, C30H46O3
9.575
455
454
√
[39, 40]
29
Manglanostenoic acid, C30H48O3
14.444
457
456
√
[40]
30
Cycloart-25-ene-3,24,27-triol, C30H50O3
13.910
459
458
√
√
[38]
31
Cycloartane-3,24,25-triol, C30H52O3
7.013
461
460
√
√
[38]
Other compounds
32
Unknown
1.216
224
223
219, 201, 183, 155, 127, 99, 81
√
33
Unknown
1.844
229
230
142, 70, 65
√
34
Unknown
6.930
240
240
180, 174, 67
√
37
Unknown
3.502
367
366
349, 332, 258, 229, 202, 188, 156, 144, 118, 91
√
40
Unknown
5.884
391
392
373, 325, 249, 211, 191, 167, 149, 109, 95
√
√
42
Unknown
9.041
567
566
549, 436, 322, 209, 114
√Table 4
Compounds determined by UPLC-MS/MS in the EtOAC and MeOH extracts ofMangifera indica in the negative-ion mode.
Entry
Identified compounds (name, formula)
Retention time
[M−H]− (m/z)
Mol. wt. (m/z)
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Sodium gallate, C7H5NaO5
1.567
191
192
173, 109, 93, 85
√
√
[27]
Xanthones
2
Mangiferin, C19H18O11
7.715
421
422
331, 301, 285, 272, 259, 243, 227, 183, 171, 163
√
√
[34]
3
Isomangiferin, C19H18O11
6.999
421
422
331, 301
√
√
[34]
4
Mangiferin, 3-methyl ether C20H20O11
9.575
435
436
313, 255, 169, 140, 124
√
[32]
5
Mangiferin, 6-O-gallate C26H22O15
3.994
573
574
403, 331, 301
√
[34]
Flavonol
6
Kaemferol, C15H10O6
11.150
284
286
269, 240, 224, 212
√
[25]
7
Quercetin 3-O-glucoside, C21H19O12
10.863
462
463
445, 419, 401, 383, 355, 301, 291, 247, 229, 218, 171, 164
√
[33]
Benzophenones
8
Maclurin 3-C-β-D-glucoside, C19H20O11
8.859
423
424
332, 302, 286, 272, 259, 227, 216, 165
√
√
[41]
9
3-Glucosyl-2,3',4,4',6-pentahydroxybenzophenone, C19H20O11
4.423
423
424
314, 169, 109
√
[36]
10
Maclurin 3-C-(6’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.862
543
544
271, 258, 211, 193, 190, 175, 169, 163, 150
√
√
[36]
11
Maclurin mono-O-galloyl-glucoside, C26H24O15
8.574
575
576
231, 283
[31, 42]
12
Maclurin di-O-galloyl-glucoside, C33H28O19
9.718
727
728
287, 270, 245, 211, 193, 169
√
[31, 42]
Terpenoids
Cycloartane-3,29-diol; 3β-form, C30H52O2
3.242
443
444
237, 189, 95, 59
√
[38]
3,27-Dihydroxycycloart-24-en-26-oic acid, C30H48O4
12.009
472
472
256
√
[38]
Gallotannins
Digalloyl glucoside, C20H20O14
11.865
483
484
254, 245, 153
√
Tri-O-galloyl glucoside, C27H24O18
7.285
635
636
211, 169
√
[27, 43]
Tetra-O-galloyl glucoside, C34H28O22
9.146
787
788
618, 466, 313, 295, 211, 193, 169
√
√
[26, 43]
Penta-O-gallose-glucose, C41H32O26
9.577
939
940
√
[25, 44]
Other compounds
Ferulic acid hexoside, C16H20O9
12.439
355
356
183
√
[24]
Unknown
13.582
666
667
311, 282, 267, 226, 159
√
Unknown
10.005
681
682
529, 331, 288, 269, 211, 169
√
√
Unknown
12.725
815
816
410, 309, 355, 323
√
√
Unknown
13.725
817
818
622, 283, 271, 255, 243
√
Unknown
13.867
960
961
914, 415, 397, 277, 255, 233
√Seven phenolic compounds were identified in the protonated form, [M+H]+ ions: protocatechuic acid [C7H7O4]+ of m/z 155 (entry 1), gallic acid [C7H7O5]+ of m/z 171 (entry 2), methyl gallate [C8H9O5]+ of m/z 185 (entry 3), di-tert-butylphenol [C14H22O]+ of m/z 207 (entry 4), and tetrahydroxy sodium benzoate of m/z ion 209 (entry 5); based on its fragmentation pattern, ellagic acid [C14H7O8]+ (entry 8) and theogallin [C14H17O10]+ (entry 10) were detected at m/z 302 and 345. The entry 6 and 7 with an m/z value of 235 and 276 were detected in both the MeOH and EtOAc extracts, and based on their mass spectral fragmentations at m/z 194 (sodium gallate), 171 (gallic acid), and 153 (procatechuic acid) are most probably derivatives of gallic acid. The compound at m/z 329 (entry 9) is most probably a derivative of theogallin (Table 3). Another phenolic compound, sodium gallate (entry 1) was identified as a [M−H]− deprotonated molecule m/z 191 and yielded ion fragments at m/z 173, 109, 93, and 85 (Table 4).


#### 3.3.2. Xanthones

Four xanthone derivatives were detected in the MeOH and EtOAc extracts of the leaves ofMangifera indica. The peaks at m/z 421/423, 435/437, and 573/575 corresponded to [M−H]−/[M+H]+ ions and were attributed to mangiferin, mangiferin-methyl ether, and mangiferin-O-gallate, respectively. The other peak at m/z 421 ([M−H]− ion) corresponded to isomangiferin. The 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. The 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. The 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 3).


#### 3.3.4. Benzophenone Derivatives and Related Compounds

Ten benzophenone derivatives were identified from the EtOAC and MeOH extracts. The 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. The 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.


#### 3.3.5. Gallotannins

In the MeOH extract, a number of gallotannins were identified atm/z 483, 635, 787, and 939 as [M−H]− ions corresponding to di-O-galloyl, tri-O-galloyl, tetra-O-galloyl, and penta-O-galloyl glucosides as given in Table 4.


#### 3.3.6. Terpenoids

In the positive mode, the peak atm/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.


## 3.1. 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 ofMangifera indica grown locally to evaluate the nutritional value and to investigate whether the leaves can be used as fodder for ruminants. The 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. The % 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. The CF content was 3.92% which was lower than the value (8%) which is considered to be harmful to ruminants [20].Table 1
Proximal composition expressed in % (dry basis) ofMangifera indica.
Constituents (%)
Mangifera indica (dauphiné Mauritian variety)
Mangifera indica (Nigerian variety)
Mangifera indica (Laos variety)
Major fodder trees (Nepal)
Ash
12.61
10
—
(5 to 17)
CF
3.92
0.48
—
—
CP
13.60
20.38
6.90
(11 to 22)
NDF
35.32
43.14
50.10
(27 to 75)
ADF
34.98
51.48
—
(20 to 54)
ADL
12.86
2.47
—
(6 to 35)
HC
0.34
8.34
—
(1 to 31)
CEL
22.12
49.01
—
(13 to 33)
Calcium
2.15
—
—
>1
Phosphorus
0.12
—
—
(1.16 to 0.40)
— = not reported.The 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. The range of values of different parameters for the major fodder trees from Nepal are also given in Table 1 [19]. The 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].


## 3.2. 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. The normal requirement for calcium and phosphorus contents for ruminants is in the range of 0.19–0.82 and 0.12–0.48%, respectively [23]. The 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.


## 3.3. Phytochemical Analysis

The 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 theMangifera 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.Table 2
Summary of the phytochemical screening test performed on the different extracts.
Phytochemicals
Crude
Hexane
EtOAc
MeOH
Saponin
+
−
+
+
Alkaloids
+
−
+
+
Phenols
+
−
+
+
Tannins
+
−
+
+
Flavonoids
+
−
+
+
Steroids
+
−
−
+
Starch
−
−
−
−
Glycosides
+
+
−
−
Diterpenes
+
+
+
−
Anthocyanins
−
−
−
−
Amino acids
−
−
−
−
Coumarins
−
−
−
−Therefore, in this study, the EtOAc and MeOH extracts of theMangifera 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.


### 3.3.1. 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 Tables3 and 4 (The mass spectra used to support the findings of this study are included within the supplementary information files (available here)).Table 3
Compounds determined by UPLC-MS/MS in the EtOAc and MeOH extracts ofMangifera indica in the positive-ion mode.
Identified compounds (name, formula)
Retention time
[M+H]+
Mol. wt.
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Protacatechuic, C7H6O4
4.570
155
154
153, 125, 79
√
√
[24, 25]
2
Gallic acid, C7H6O5
2.174
171
170
153, 127, 109, 107, 97, 81, 79, 69
√
[25, 26]
3
Methyl gallate, C8H8O5
5.612
185
184
153, 125
√
[27]
4
2,5-Di-tert-butylphenol, C14H22O
7.148
207
206
189, 174, 159, 147, 119, 105, 91, 95, 79, 77
√
√
[28]
5
Tetrahydroxy sodium benzoate, C7H5O6Na
4.211
209
208
191, 173, 167, 163/151/121, 107, 93, 91, 79, 77
√
√
6
Derivative of gallic acid
18.951
235
234
194, 171, 153, 137, 118, 84, 73
√
√
7
Derivative of gallic acid
2.022
276
275
258, 230, 212, 194, 153, 112, 97
√
8
Ellagic acid, C14H6O8
6.876
303
302
257, 229
√
[29]
9
Derivative of theogallin with one OH missing, C14H16O9
8.121
329
328
271, 249, 235, 225, 192, 176, 153, 107
√
10
Theogallin, C14H16O10
2.257
345
344
327, 299, 209, 153, 137, 125
√
√
[30]
Xanthones
11
Mangiferin, C19H18O11
8.928
423
422
√
√
[31]
12
Mangiferin 3-methyl ether, C20H20O11
8.299
437
436
√
[32]
13
Mangiferin-6’-O-gallate, C26H22O15
8.875
575
574
465, 455, 439, 423, 327, 313, 303, 285, 261, 193
√
[31]
Flavonols
14
Quercetin, C15H10O7
9.284
303
302
√
√
[33]
15
Rhamnetin, C16H12O7
12.036
317
316
262, 167, 156, 154, 126
√
[34]
16
Quercetin carboxylic acid, C16H10O9
10.643
347
346
302, 284, 255, 227
√
17
Quercetin pentoside, C20H18O11
8.394
435
434
354, 311, 259, 188, 162
√
√
[31]
18
Quercetin 3-O-rhamnoside C21H20O11
9.640
449
448
285, 284, 255
√
√
[31, 34]
19
Epicatechin gallate hexamalonate, C22H18O11
13.983
459
458
√
√
[24]
20
Rhamnetin hexoside, C22H22O12
14.067
479
478
315, 169
√
√
[30]
Benzophenones
21
Maclurin, C13H10O6
10.549
263
262
167, 124, 121, 111, 99, 65
√
√
[31]
22
Iriflophenone glucoside derivative
7.860
405
404
369, 351, 327, 313, 298, 273, 253, 99, 81
√
√
23
Iriflophenone 3-C-β-D-glucopyranoside, C19H20O10
7.232
409
408
391, 355, 325, 313, 231, 195, 177, 165, 121
√
√
[35]
24
Maclurin 3-C-(6’’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.011
545
544
√
[36]
25
Iriflophenone-di-O-galloyl-glucoside, C33H28O18
9.302
713
712
√
[31, 36]
26
Iriflophenone tri-O-galloyl-glucoside, C40H32O22
9.902
865
864
√
[36]
Terpenoids
27
Lupeol, C30H50O
7.819
427
426
√
√
[37, 38]
28
Mangiferonic acid, C30H46O3
9.575
455
454
√
[39, 40]
29
Manglanostenoic acid, C30H48O3
14.444
457
456
√
[40]
30
Cycloart-25-ene-3,24,27-triol, C30H50O3
13.910
459
458
√
√
[38]
31
Cycloartane-3,24,25-triol, C30H52O3
7.013
461
460
√
√
[38]
Other compounds
32
Unknown
1.216
224
223
219, 201, 183, 155, 127, 99, 81
√
33
Unknown
1.844
229
230
142, 70, 65
√
34
Unknown
6.930
240
240
180, 174, 67
√
37
Unknown
3.502
367
366
349, 332, 258, 229, 202, 188, 156, 144, 118, 91
√
40
Unknown
5.884
391
392
373, 325, 249, 211, 191, 167, 149, 109, 95
√
√
42
Unknown
9.041
567
566
549, 436, 322, 209, 114
√Table 4
Compounds determined by UPLC-MS/MS in the EtOAC and MeOH extracts ofMangifera indica in the negative-ion mode.
Entry
Identified compounds (name, formula)
Retention time
[M−H]− (m/z)
Mol. wt. (m/z)
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Sodium gallate, C7H5NaO5
1.567
191
192
173, 109, 93, 85
√
√
[27]
Xanthones
2
Mangiferin, C19H18O11
7.715
421
422
331, 301, 285, 272, 259, 243, 227, 183, 171, 163
√
√
[34]
3
Isomangiferin, C19H18O11
6.999
421
422
331, 301
√
√
[34]
4
Mangiferin, 3-methyl ether C20H20O11
9.575
435
436
313, 255, 169, 140, 124
√
[32]
5
Mangiferin, 6-O-gallate C26H22O15
3.994
573
574
403, 331, 301
√
[34]
Flavonol
6
Kaemferol, C15H10O6
11.150
284
286
269, 240, 224, 212
√
[25]
7
Quercetin 3-O-glucoside, C21H19O12
10.863
462
463
445, 419, 401, 383, 355, 301, 291, 247, 229, 218, 171, 164
√
[33]
Benzophenones
8
Maclurin 3-C-β-D-glucoside, C19H20O11
8.859
423
424
332, 302, 286, 272, 259, 227, 216, 165
√
√
[41]
9
3-Glucosyl-2,3',4,4',6-pentahydroxybenzophenone, C19H20O11
4.423
423
424
314, 169, 109
√
[36]
10
Maclurin 3-C-(6’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.862
543
544
271, 258, 211, 193, 190, 175, 169, 163, 150
√
√
[36]
11
Maclurin mono-O-galloyl-glucoside, C26H24O15
8.574
575
576
231, 283
[31, 42]
12
Maclurin di-O-galloyl-glucoside, C33H28O19
9.718
727
728
287, 270, 245, 211, 193, 169
√
[31, 42]
Terpenoids
Cycloartane-3,29-diol; 3β-form, C30H52O2
3.242
443
444
237, 189, 95, 59
√
[38]
3,27-Dihydroxycycloart-24-en-26-oic acid, C30H48O4
12.009
472
472
256
√
[38]
Gallotannins
Digalloyl glucoside, C20H20O14
11.865
483
484
254, 245, 153
√
Tri-O-galloyl glucoside, C27H24O18
7.285
635
636
211, 169
√
[27, 43]
Tetra-O-galloyl glucoside, C34H28O22
9.146
787
788
618, 466, 313, 295, 211, 193, 169
√
√
[26, 43]
Penta-O-gallose-glucose, C41H32O26
9.577
939
940
√
[25, 44]
Other compounds
Ferulic acid hexoside, C16H20O9
12.439
355
356
183
√
[24]
Unknown
13.582
666
667
311, 282, 267, 226, 159
√
Unknown
10.005
681
682
529, 331, 288, 269, 211, 169
√
√
Unknown
12.725
815
816
410, 309, 355, 323
√
√
Unknown
13.725
817
818
622, 283, 271, 255, 243
√
Unknown
13.867
960
961
914, 415, 397, 277, 255, 233
√Seven phenolic compounds were identified in the protonated form, [M+H]+ ions: protocatechuic acid [C7H7O4]+ of m/z 155 (entry 1), gallic acid [C7H7O5]+ of m/z 171 (entry 2), methyl gallate [C8H9O5]+ of m/z 185 (entry 3), di-tert-butylphenol [C14H22O]+ of m/z 207 (entry 4), and tetrahydroxy sodium benzoate of m/z ion 209 (entry 5); based on its fragmentation pattern, ellagic acid [C14H7O8]+ (entry 8) and theogallin [C14H17O10]+ (entry 10) were detected at m/z 302 and 345. The entry 6 and 7 with an m/z value of 235 and 276 were detected in both the MeOH and EtOAc extracts, and based on their mass spectral fragmentations at m/z 194 (sodium gallate), 171 (gallic acid), and 153 (procatechuic acid) are most probably derivatives of gallic acid. The compound at m/z 329 (entry 9) is most probably a derivative of theogallin (Table 3). Another phenolic compound, sodium gallate (entry 1) was identified as a [M−H]− deprotonated molecule m/z 191 and yielded ion fragments at m/z 173, 109, 93, and 85 (Table 4).


### 3.3.2. Xanthones

Four xanthone derivatives were detected in the MeOH and EtOAc extracts of the leaves ofMangifera indica. The peaks at m/z 421/423, 435/437, and 573/575 corresponded to [M−H]−/[M+H]+ ions and were attributed to mangiferin, mangiferin-methyl ether, and mangiferin-O-gallate, respectively. The other peak at m/z 421 ([M−H]− ion) corresponded to isomangiferin. The 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. The 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. The 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 3).


### 3.3.4. Benzophenone Derivatives and Related Compounds

Ten benzophenone derivatives were identified from the EtOAC and MeOH extracts. The 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. The 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.


### 3.3.5. Gallotannins

In the MeOH extract, a number of gallotannins were identified atm/z 483, 635, 787, and 939 as [M−H]− ions corresponding to di-O-galloyl, tri-O-galloyl, tetra-O-galloyl, and penta-O-galloyl glucosides as given in Table 4.


### 3.3.6. Terpenoids

In the positive mode, the peak atm/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.


## 3.3.1. 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 Tables3 and 4 (The mass spectra used to support the findings of this study are included within the supplementary information files (available here)).Table 3
Compounds determined by UPLC-MS/MS in the EtOAc and MeOH extracts ofMangifera indica in the positive-ion mode.
Identified compounds (name, formula)
Retention time
[M+H]+
Mol. wt.
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Protacatechuic, C7H6O4
4.570
155
154
153, 125, 79
√
√
[24, 25]
2
Gallic acid, C7H6O5
2.174
171
170
153, 127, 109, 107, 97, 81, 79, 69
√
[25, 26]
3
Methyl gallate, C8H8O5
5.612
185
184
153, 125
√
[27]
4
2,5-Di-tert-butylphenol, C14H22O
7.148
207
206
189, 174, 159, 147, 119, 105, 91, 95, 79, 77
√
√
[28]
5
Tetrahydroxy sodium benzoate, C7H5O6Na
4.211
209
208
191, 173, 167, 163/151/121, 107, 93, 91, 79, 77
√
√
6
Derivative of gallic acid
18.951
235
234
194, 171, 153, 137, 118, 84, 73
√
√
7
Derivative of gallic acid
2.022
276
275
258, 230, 212, 194, 153, 112, 97
√
8
Ellagic acid, C14H6O8
6.876
303
302
257, 229
√
[29]
9
Derivative of theogallin with one OH missing, C14H16O9
8.121
329
328
271, 249, 235, 225, 192, 176, 153, 107
√
10
Theogallin, C14H16O10
2.257
345
344
327, 299, 209, 153, 137, 125
√
√
[30]
Xanthones
11
Mangiferin, C19H18O11
8.928
423
422
√
√
[31]
12
Mangiferin 3-methyl ether, C20H20O11
8.299
437
436
√
[32]
13
Mangiferin-6’-O-gallate, C26H22O15
8.875
575
574
465, 455, 439, 423, 327, 313, 303, 285, 261, 193
√
[31]
Flavonols
14
Quercetin, C15H10O7
9.284
303
302
√
√
[33]
15
Rhamnetin, C16H12O7
12.036
317
316
262, 167, 156, 154, 126
√
[34]
16
Quercetin carboxylic acid, C16H10O9
10.643
347
346
302, 284, 255, 227
√
17
Quercetin pentoside, C20H18O11
8.394
435
434
354, 311, 259, 188, 162
√
√
[31]
18
Quercetin 3-O-rhamnoside C21H20O11
9.640
449
448
285, 284, 255
√
√
[31, 34]
19
Epicatechin gallate hexamalonate, C22H18O11
13.983
459
458
√
√
[24]
20
Rhamnetin hexoside, C22H22O12
14.067
479
478
315, 169
√
√
[30]
Benzophenones
21
Maclurin, C13H10O6
10.549
263
262
167, 124, 121, 111, 99, 65
√
√
[31]
22
Iriflophenone glucoside derivative
7.860
405
404
369, 351, 327, 313, 298, 273, 253, 99, 81
√
√
23
Iriflophenone 3-C-β-D-glucopyranoside, C19H20O10
7.232
409
408
391, 355, 325, 313, 231, 195, 177, 165, 121
√
√
[35]
24
Maclurin 3-C-(6’’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.011
545
544
√
[36]
25
Iriflophenone-di-O-galloyl-glucoside, C33H28O18
9.302
713
712
√
[31, 36]
26
Iriflophenone tri-O-galloyl-glucoside, C40H32O22
9.902
865
864
√
[36]
Terpenoids
27
Lupeol, C30H50O
7.819
427
426
√
√
[37, 38]
28
Mangiferonic acid, C30H46O3
9.575
455
454
√
[39, 40]
29
Manglanostenoic acid, C30H48O3
14.444
457
456
√
[40]
30
Cycloart-25-ene-3,24,27-triol, C30H50O3
13.910
459
458
√
√
[38]
31
Cycloartane-3,24,25-triol, C30H52O3
7.013
461
460
√
√
[38]
Other compounds
32
Unknown
1.216
224
223
219, 201, 183, 155, 127, 99, 81
√
33
Unknown
1.844
229
230
142, 70, 65
√
34
Unknown
6.930
240
240
180, 174, 67
√
37
Unknown
3.502
367
366
349, 332, 258, 229, 202, 188, 156, 144, 118, 91
√
40
Unknown
5.884
391
392
373, 325, 249, 211, 191, 167, 149, 109, 95
√
√
42
Unknown
9.041
567
566
549, 436, 322, 209, 114
√Table 4
Compounds determined by UPLC-MS/MS in the EtOAC and MeOH extracts ofMangifera indica in the negative-ion mode.
Entry
Identified compounds (name, formula)
Retention time
[M−H]− (m/z)
Mol. wt. (m/z)
Fragment ions
EtOAC
MeOH
Reference
Phenolic compounds
1
Sodium gallate, C7H5NaO5
1.567
191
192
173, 109, 93, 85
√
√
[27]
Xanthones
2
Mangiferin, C19H18O11
7.715
421
422
331, 301, 285, 272, 259, 243, 227, 183, 171, 163
√
√
[34]
3
Isomangiferin, C19H18O11
6.999
421
422
331, 301
√
√
[34]
4
Mangiferin, 3-methyl ether C20H20O11
9.575
435
436
313, 255, 169, 140, 124
√
[32]
5
Mangiferin, 6-O-gallate C26H22O15
3.994
573
574
403, 331, 301
√
[34]
Flavonol
6
Kaemferol, C15H10O6
11.150
284
286
269, 240, 224, 212
√
[25]
7
Quercetin 3-O-glucoside, C21H19O12
10.863
462
463
445, 419, 401, 383, 355, 301, 291, 247, 229, 218, 171, 164
√
[33]
Benzophenones
8
Maclurin 3-C-β-D-glucoside, C19H20O11
8.859
423
424
332, 302, 286, 272, 259, 227, 216, 165
√
√
[41]
9
3-Glucosyl-2,3',4,4',6-pentahydroxybenzophenone, C19H20O11
4.423
423
424
314, 169, 109
√
[36]
10
Maclurin 3-C-(6’-O-p-hydroxybenzoyl)β-D-glucoside, C26H24O13
9.862
543
544
271, 258, 211, 193, 190, 175, 169, 163, 150
√
√
[36]
11
Maclurin mono-O-galloyl-glucoside, C26H24O15
8.574
575
576
231, 283
[31, 42]
12
Maclurin di-O-galloyl-glucoside, C33H28O19
9.718
727
728
287, 270, 245, 211, 193, 169
√
[31, 42]
Terpenoids
Cycloartane-3,29-diol; 3β-form, C30H52O2
3.242
443
444
237, 189, 95, 59
√
[38]
3,27-Dihydroxycycloart-24-en-26-oic acid, C30H48O4
12.009
472
472
256
√
[38]
Gallotannins
Digalloyl glucoside, C20H20O14
11.865
483
484
254, 245, 153
√
Tri-O-galloyl glucoside, C27H24O18
7.285
635
636
211, 169
√
[27, 43]
Tetra-O-galloyl glucoside, C34H28O22
9.146
787
788
618, 466, 313, 295, 211, 193, 169
√
√
[26, 43]
Penta-O-gallose-glucose, C41H32O26
9.577
939
940
√
[25, 44]
Other compounds
Ferulic acid hexoside, C16H20O9
12.439
355
356
183
√
[24]
Unknown
13.582
666
667
311, 282, 267, 226, 159
√
Unknown
10.005
681
682
529, 331, 288, 269, 211, 169
√
√
Unknown
12.725
815
816
410, 309, 355, 323
√
√
Unknown
13.725
817
818
622, 283, 271, 255, 243
√
Unknown
13.867
960
961
914, 415, 397, 277, 255, 233
√Seven phenolic compounds were identified in the protonated form, [M+H]+ ions: protocatechuic acid [C7H7O4]+ of m/z 155 (entry 1), gallic acid [C7H7O5]+ of m/z 171 (entry 2), methyl gallate [C8H9O5]+ of m/z 185 (entry 3), di-tert-butylphenol [C14H22O]+ of m/z 207 (entry 4), and tetrahydroxy sodium benzoate of m/z ion 209 (entry 5); based on its fragmentation pattern, ellagic acid [C14H7O8]+ (entry 8) and theogallin [C14H17O10]+ (entry 10) were detected at m/z 302 and 345. The entry 6 and 7 with an m/z value of 235 and 276 were detected in both the MeOH and EtOAc extracts, and based on their mass spectral fragmentations at m/z 194 (sodium gallate), 171 (gallic acid), and 153 (procatechuic acid) are most probably derivatives of gallic acid. The compound at m/z 329 (entry 9) is most probably a derivative of theogallin (Table 3). Another phenolic compound, sodium gallate (entry 1) was identified as a [M−H]− deprotonated molecule m/z 191 and yielded ion fragments at m/z 173, 109, 93, and 85 (Table 4).


## 3.3.2. Xanthones

Four xanthone derivatives were detected in the MeOH and EtOAc extracts of the leaves ofMangifera indica. The peaks at m/z 421/423, 435/437, and 573/575 corresponded to [M−H]−/[M+H]+ ions and were attributed to mangiferin, mangiferin-methyl ether, and mangiferin-O-gallate, respectively. The other peak at m/z 421 ([M−H]− ion) corresponded to isomangiferin. The 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. The 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. The 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 3).


## 3.3.4. Benzophenone Derivatives and Related Compounds

Ten benzophenone derivatives were identified from the EtOAC and MeOH extracts. The 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. The 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.


## 3.3.5. Gallotannins

In the MeOH extract, a number of gallotannins were identified atm/z 483, 635, 787, and 939 as [M−H]− ions corresponding to di-O-galloyl, tri-O-galloyl, tetra-O-galloyl, and penta-O-galloyl glucosides as given in Table 4.


## 3.3.6. Terpenoids

In the positive mode, the peak atm/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.


## 4. Biological Properties

### 4.1. Antibacterial

It has been reported that the phytochemicals present in the leaves ofMangifera 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. The 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. The 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 grownMangifera indica are given in Table 5. The crude extract exhibited antioxidant potential which can be related to its high TPC and TFC. The EtOAc extract showed higher antioxidant activity attributed to a more elevated concentration of phenolic and flavonoid contents as compared to the MeOH extract.Table 5
Total phenolic content and total flavonoid content in crude, EtOAc, and MeOH extracts ofMangifera indica.
Extract
TPC (mg/g)
TFC (mg/g)
SC50 (µg/mL)
Crude
230 ± 2
131 ± 1
64
EtOAc
186 ± 1
191 ± 2
80
MeOH
99 ± 1
46 ± 1.0
313


## 4.1. Antibacterial

It has been reported that the phytochemicals present in the leaves ofMangifera 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. The 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. The 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 grownMangifera indica are given in Table 5. The crude extract exhibited antioxidant potential which can be related to its high TPC and TFC. The EtOAc extract showed higher antioxidant activity attributed to a more elevated concentration of phenolic and flavonoid contents as compared to the MeOH extract.Table 5
Total phenolic content and total flavonoid content in crude, EtOAc, and MeOH extracts ofMangifera indica.
Extract
TPC (mg/g)
TFC (mg/g)
SC50 (µg/mL)
Crude
230 ± 2
131 ± 1
64
EtOAc
186 ± 1
191 ± 2
80
MeOH
99 ± 1
46 ± 1.0
313


## 5. Conclusion

This study was conducted in a view to understand the nutritional characteristics of leaves fromMangifera 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. The crude fat was low while crude protein was within the range required to support the growth of ruminants. The 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. The 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. These scientific findings would be environmentally and economically sustainable for considering mango leaves as good fodder for feeding ruminants.



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*Source: 6869294-2018-08-01.xml*