Antidiabetic and Anticancer Potentials of Mangifera indica L. from Different Geographical Origins

Mango fruit is well known for its nutritional and health benefits due to the presence of a plethora of phytochemical classes. The quality of mango fruit and its biological activities may change depending upon the variation in geographical factors. For the first time, this study comprehensively screened the biological activities of all four parts of the mango fruit from twelve different origins. Various cell lines (MCF7, HCT116, HepG2, MRC5) were used to screen the extracts for their cytotoxicity, glucose uptake, glutathione peroxidase activity, and α-amylase inhibition. MTT assays were carried out to calculate the IC50 values for the most effective extracts. The seed part from Kenya and Sri Lanka origins exhibited an IC50 value of 14.44 ± 3.61 (HCT116) and 17.19 ± 1.60 (MCF7). The seed part for Yemen Badami (119 ± 0.08) and epicarp part of Thailand (119 ± 0.11) mango fruit showed a significant increase in glucose utilization (50 μg/mL) as compared to the standard drug metformin (123 ± 0.07). The seed extracts of Yemen Taimoor seed (0.46 ± 0.05) and Yemen Badami (0.62 ± 0.13) produced a significant reduction in GPx activity (50 μg/mL) compared to the control cells (100 μg/mL). For α-amylase inhibition, the lowest IC50 value was observed for the endocarp part of Yemen Kalabathoor (108.8 ± 0.70 μg/mL). PCA, ANOVA, and Pearson’s statistical models revealed a significant correlation for the fruit part vs. biological activities, and seed part vs. cytotoxicity and α-amylase activity (p = 0.05). The seed of mango fruit exhibited significant biological activities; hence, further in-depth metabolomic and in vivo studies are essential to effectively utilize the seed part for the treatment of various diseases.


Introduction
Mangifera indica (mango) is one of the most prevalent tropical fruits belonging to the genus Mangifera, which involves about 30 species of fruit trees in the Anacardiaceae family [1]. Although mango is known to be native to India, it is now widely cultivated in several countries, Mainly China, Thailand, Indonesia, Mexico, and Pakistan. Several parts of mango, including fruits, leaves, flowers, bark, and roots, have been traditionally used to treat numerous diseases. For instance, an aqueous extract of mango stem bark obtained by decoction has been used for the treatment of diarrhea, menorrhagia, anemia, cutaneous infection, and diabetes [2]. Additionally, it is commonly used in Cuba to enhance the quality of life of cancer patients [3]. Numerous pharmacological studies on mango fruits have been conducted to validate the traditional uses of Mangifera indica in the management of diverse diseases. These studies demonstrated that mango fruits have antioxidant, anticancer, and antidiabetic activities [4]. For example, an in vitro study of five Indian mango cultivars showed that different solvent extracts of mango peel have antioxidant, antimicrobial, and anti-inflammatory activities [5]. Regarding the anticancer effect, a recent study revealed that the ethanolic extract of mango peel has antioxidant and cytotoxic effects on lung cancer cell lines [6]. Another study illustrated the antiproliferation effect of the acetone extracts of peel and pulp of different mango cultivars in hepatocellular carcinoma (HepG2) [7]. Furthermore, a group of researchers observed oxidative stress mediated apoptosis induced by ethanolic extract of mango kernels in cultured estrogen receptor-positive breast cancer (MCF-7) [8]. For the antidiabetic potential, mango peel extract exhibited antidiabetic properties via improving antioxidant enzymes in diabetic rats [9]. Additionally, another study found that the methanolic extracts of mango fruits (pulp) offered antidiabetic effects by inhibiting α-amylase and α-glucosidase activity [10]. Moreover, an in vivo study found that long-term administration (21 days) of aqueous and methanolic extract of mango seed effectively reduces blood glucose levels in diabetic rats [11]. Collectively, a considerable amount of the literature proposed the potential application of mango fruit extract in the management of diabetes and cancer. The medicinal benefits of mango are attributed to many bioactive compounds, including polyphenols, terpenoids, carotenoids, and phytosterols [12]. Mangiferin is the prominent potent active constituent of mango with multiple pharmacological properties involving anti-inflammatory, antioxidant, antidiabetic, and anticancer [13]. Mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside) is a polyphenol and is mainly isolated from all parts of the Mangifera indica tree (fruits, leaves, stem bark, and roots). Different mango cultivars exhibit a wide diversity in mango fruit characteristics (shape, size, color, texture, taste, and aroma) and their phytochemical profiles. Consequently, the bioactivity of each cultivar might be varied. Similarly, various parts of an individual mango fruit (peel, flesh, seed) possess many biological effects due to the variation in the type and quantity of chemical compounds [14]. Indeed, only Nam Doc Mai peel extract showed anti-proliferative effects when a study compared the methanolic extract of peel and pulp from three mango varieties (Irwin, Nam Doc Mai, and Kensington Pride) for their growth-inhibitory potentials on MCF-7 human breast cancer cells. Overall, fruit parts and habitat differences are essential factors to consider when evaluating medicinal plants' bioactivity. To the best of our knowledge, this is a first-time study to evaluate the anticancer and antidiabetic activities of an aqueous extract of four separate parts (epi-, meso-, endocarp, and seed) of eight mango fruits from different geographical origins (Indian, Egyptian, Pakistani, Yemen, Thailand, Sri Lanka, Kenya, and Vietnam). In addition, it assesses the correlation between the Mangiferin amount and the observed biological activities. To illustrate, the cytotoxicity potential of the mango fruit extract was tested in selected cancer cell lines (Human breast adenocarcinoma, Human colorectal carcinoma, and Hepatocellular carcinoma) and normal human fetal lung fibroblast. Also, the effect of mango extracts on the glutathione peroxidase enzyme was assessed. Lastly, the antidiabetic activity was investigated by the inhibition of α-amylase, as well as the glucose uptake capacity in hepatocellular carcinoma.

The Effect of Extracts on Glutathione Peroxidase Activity (GPx) Using HepG2
For relative GPx activity, MTT-assisted cell viability was evaluated as described earlier.

Geographical Origin IC 50
Yemen

Statistical Analysis
The results are expressed with a mean (±standard deviation (SD)) from at least three independent experiments. The data were analyzed for statistical significance between the treated and control group with the help of GraphPad Prism V-9.  (Table 1).

Correlation Analysis Pearson's Correlation
The statistical analysis for Pearson's analysis showed a significant correlation between geographical origin and MG amount (−0.603, p = 0.000). With regard to biological activities, a significant correlation was observed for the fruit part vs. HT116 (−0.550, p = 0.000), MCF7 (−0.442, p = 0.002), and α-amylase activity (0.315, p = 0.029). Glucose uptake and GPx activities do not correlate with the geographical origin, fruit part, MG amount, or biological activities (Table 4).

Component Analysis for Variance
The principal component analysis (PCA), an eigenvalue-based statistical tool, is employed to reduce the dimensions of a large dataset into principal components based on %variability. The PCA maximizes the variance for uncorrelated variables based on an individual and cumulative variance, with an impactful display of the relevant correlated variables in the same component. Herein, three components, PC1 (33.09%), PC2 (20.48%), and PC3 (14.65%), with a cumulative % variability of 68.23, were observed. For PC1 with major % variability, the variables presented were fruit part (−0.70), HCT116 (0.92), MCF7 (0.87), and α-amylase activity (−0.60), geographical origin (−0.91) and MG amount (0.83) were observed in PC2, whereas glucose uptake (0.62) and GPx activity (0.79) were observed in PC3, i.e., with the least % variability. The KMO-Bartlett's test of sphericity showed a high X 2 -value of 129.78 with a significance value of 0.00 (p = 0.05) the data for PCA are shown in Table 5. A graphical presentation of the components observed with their corresponding eigenvalues is shown in Figure 1.
Variables Correlation with ANOVA ANOVA with inter-and intra-correlation for the data variables also confirmed the outcomes from Pearson's correlation and PCA analysis. The correlation of geographical origin with MG amount (−0.603), as well as fruit part with HCT116 (−0.550), MCF7 (−0.442), and α-amylase activity (0.315), was observed to be significant (0.00) with a high F-value of 627.81. The correlation for within and between groups, along with the sum of squares and mean square values, is shown in Table 6 (p = 0.05). Variables Correlation with ANOVA ANOVA with inter-and intra-correlation for the data variables also confirmed the outcomes from Pearson's correlation and PCA analysis. The correlation of geographical origin with MG amount (−0.603), as well as fruit part with HCT116 (−0.550), MCF7 (−0.442), and α-amylase activity (0.315), was observed to be significant (0.00) with a high F-value of 627.81. The correlation for within and between groups, along with the sum of squares and mean square values, is shown in Table 6 (p = 0.05).

Discussion
It is a well-known fact that variations in the geographical origin of a plant or fruit significantly affect its quality in terms of phytochemical profile and biological activities. The main aim of this study was to compare the quality variation among mango fruits obtained from different geographical origins. Mango fruits from eight different cultivars were collected and processed for green extraction and characterization of phytochemical profile based on MG amount in four parts of each mango fruit (epi-, endo-, mesocarp, and seed). The details regarding the novel green extraction and LCMSMS characterization for MG amount in these four parts of eight cultivars are described in our previous study [15]. The extracts from these mango fruit samples were screened for biological activities consisting of cytotoxicity (HCT116, MCF7, MRC5 cell lines), glucose uptake assay and GPx activity in HepG2 cells, and α-amylase activity. Statistical models were created to establish a correlation between the phytochemicals present in the mango fruit with the biological activities tested.
For cytotoxicity (HCT116, MCF7, MRC5), a general followed by an in-depth screening was performed to determine the IC 50 values. The extract samples with <50% of cell viability were studied further at six different concentrations using an additional cell line of MRC5. In general, the seed parts of these mango cultivars were observed with considerable cytotoxic potential. The seed extract from Kenya mango (large and round) was seen to be more cytotoxic towards the tested cell lines (HCT116, MCF7) in general screening, whereas the MTT assay for selectivity against the three cell lines (HCT116, MCF7, and MRC5) suggested Sri Lanka (large yellowish green) seed extract with the lowest IC 50 value followed by Thailand (large green) seed extract. Though various cytotoxic studies have been reported for mango fruit extract including the following: aqueous flesh extract [16], peel and flesh for Australian cultivars [14], peel extract from six Brazilian cultivars [17,18], seed extract [8,19,20] as well as skin and flesh extract [21], this is a first-time study to report a comprehensive characterization for the whole mango fruit parts from eight different cultivars. With regard to phytochemicals in mango fruit, a good amount of MG was observed in all the fruit parts of these mango cultivars. Though seeds were reported with an average amount of MG, the epi-and mesocarp parts of the mango fruit presented the highest MG amount. MG and MG gallate [22], along with phenols and oleanolic acid, have been reported to possess significant cytotoxic potential [6]. The 48 extracted samples from the eight cultivars exhibited a significant (p < 0.05) increase in glucose uptake and utilization in HepG2 cell lines. The epicarps (Thailand (large green), Yemen Taimoor (large yellowish green), Yemen Badami (large yellow), Kenya (large round red)), endocarps (Yemen Badami (large yellow), Kenya (large round red), Thailand (large green)), and mesocarps (Kenya (large round red), Indian Alphonso (small round green), Thailand (large green)) parts were more effective as compared to the seeds of the mango fruits (100 µg/mL). In addition, the extracts of Yemen Badami (large yellow) seed and Thailand (large green) epicarp revealed a significant increase in glucose uptake at 50 µg/mL. As mentioned earlier, the MG amount was found more in the mentioned parts of these different cultivars fruit samples, epicarp, and mesocarp in particular. Mangiferin has been reported to promote glucose utilization and metabolism in a dose-dependent manner [23], promote PPARα-induced free fatty acids metabolism in HepG2 cells [24], enhance the utilization of peripheral glucose [23], and decrease β-cells apoptosis [25]. The GPx (glutathione peroxidase) enzyme prevents cell damage via the reduction of free radicals [26,27]; however, with an increase in free radicals in various disease conditions, an increase in GPx level is usually witnessed, which returns to its normal level with the use of specific therapeutic agents [28,29]. An elevated level of GPx prevents oxidative damage and inflammation, but this may block apoptotic cell death, resulting in a higher survival rate for the altered cells. This shows a complex role for GPx in the development and progression of cancer due, in part, to its role in the modulation of intracellular ROS [29]. The extracts were tested for a potential role in GPx enzyme inhibition. Herein again, the epicarps (Yemen Taimoor (large yellowish green), Sri Lanka (large yellowish green)), mesocarps (Thailand (large green), Yemen Kalabathoor (large round red)), and endocarps (Egypt (reddish green), Thailand (large green), Yemen Taimoor (large yellowish green)) parts of these mango fruits samples showed considerable reduction of GPx activity in HepG2 cells. A significant activity reduction (as compared to the positive control, i.e., tannic acid) was recorded for the extracts of Egypt (reddish green) endocarp and Thailand (large green) mesocarp; however, in vivo assays are required to further investigate and confirm the effect of these extracts on GPx level. The more MG amount in the mango fruits epicarp and mesocarp may be suggested to play a role in GPx restoration and its normalization to the normal level, as reported previously [30,31]. One of the investigative targets for diabetes management is the control of postprandial glucose levels via inhibition of the carbohydrate-digesting enzyme, i.e., α-amylase in the intestine [32,33]. In order to find the antidiabetic potential for the mango fruit parts, α-amylase inhibition activity was investigated. The general screening results suggested considerable activity for the extracts of Yemen Kalabathoor (large round red) endocarp, Vietnam (large yellowish green) seed, India Badami (large green) seed, and Yemen Taimoor (small reddish-green) seed. The extracts from these cultivars were studied further for selectivity (i.e., being selectively cytotoxic to cancer cells and less cytotoxic to normal cells. Extracts with lower cytotoxicity to normal cells are considered a selective extract against cancer cell lines) and IC 50 value determination, where the endocarp from Yemen Kalabathoor (large round red) exhibited considerably low IC 50 values when compared to the standard drug acarbose. The amount of MG in the endocarp is reported to be higher compared to seeds; hence, MG, along with some other constituents, may be responsible for the α-amylase activity. Previous studies have confirmed the α-amylase inhibitory role of mango peel-extracted MG [34][35][36]. Furthermore, in vivo studies on mango peel extract also supported the antidiabetic potential for MG [9,34,37]. However, phenolic compounds may also play a vital role as suppressors of postprandial hyperglycemia at different concentrations [38].
The outcomes of the biological activities suggest the presence of more than one bioactive phytochemical in mango fruit and its different parts. Albeit MG was observed with an average amount in all the 48 samples extracted for mango fruit, higher in epicarp and mesocarp parts, a systemic in-depth metabolomic analysis may be more useful to obtain a broader profile of the phytochemicals present in different varieties of the mango fruit (prone to variation based on the change in geography and other factors) vs. the effectiveness in biological activities of these samples. Moreover, the active phytochemicals (based on biological assays) need to be isolated and tested further via in vivo studies in different concentrations. The isolated compounds might show lower activity compared to the mixture of compounds in the extract.
The statistical models were applied to show the correlation among the biological activities of the different parts of the mango fruit from different cultivars. Pearson's test revealed a significant correlation between the geographical origin vs. MG amount and fruit part vs. cytotoxicity (HCT116, MCF7) and α-amylase activity. Glucose uptake assay and GPx inhibition showed no correlation with the mentioned biological activities. For the fruit part, it was the seed part of the fruit that appeared as the most effective in cytotoxicity and α-amylase inhibitory activity. Likewise, the PCA analysis showed more %variability (intra-correlation) for PC1 with components of fruit part, HCT116, MCF7, and α-amylase. Geographical origin and MG amount were placed in PC2, whereas glucose uptake and GPx activities were grouped in PC3 with the least % variability, suggesting a more significant correlation for the seed fruit part with cytotoxicity and α-amylase activity. The ANOVA with inter-and intra-correlation for the dataset further confirmed an alike significant correlation with a high F-value as reported for Pearson's and PCA analysis. The outcomes of the statistical model suggest a prominent role for the seed part of the mango fruit in various biological activities. It is noteworthy to mention that it is the first time to report the mangiferin-based characterization of the different mango fruit parts (epi-, endo-, mesocarp, and seed) from eight different cultivars (48 samples) with an extensive biological screening of cytotoxicity (HCT116, MCF7, MRC5), glucose utilization (HepG2), GPx inhibition (HepG2), and α-amylase inhibition. None of the literature is available to be compared with the outcomes herein. The authors suggest a detailed metabolomic or phytochemical analysis to establish a prominent role in a series of biological activities for the seed part of the mango fruit. These in vitro studies may be followed by appropriate in vivo pharmacological experiments in order to confirm the role of mango seed, which may become a good source of phytochemicals, food products, and nutraceuticals to be utilized for the management and cure of various ailments.

Collection and Preparation of Samples
Fresh mango fruit varieties from different geographical origins (Indian, Egyptian, Pakistani, Yemen, Thailand, Sri Lanka, Kenya, and Vietnam) were collected from local markets at Khobar, Eastern Province, Kingdom of Saudi Arabia. A green ultrasonic-assisted extraction (using water as a solvent at a temperature of 40 • C) was performed for four different parts (epi-, meso-, endocarp, and seed) of every individual mango fruit, as reported in our previous study [15].

Characterization and Standardization of the Extracts
The extracts were quantified for Mangiferin amount (MG amount) in all the mango fruit samples. UPLCMS/MS was applied to develop and validate a green, efficient, and fast analytical method for MG quantification [15]. Though reported in our previous study, the MG amount found in these samples is presented in the table below for ease of understanding of the correlation.

Cell Culture
All cell lines were cultured and maintained according to the procedure reported by Ahmad et al. [39].

Determination of Cytotoxicity and Selectivity
The cytotoxicity of the extracts was evaluated by MTT assay, as previously reported [40] and the already reported method of Ahmad et al. [39].

Glucose Uptake Assay
Odeyemi et al. modified method was used for glucose uptake assay [39,41].

Determination of Glutathione Peroxidase Activity
The assay was performed according to the manufacturer's protocol, and our group reported procedures [26,28,39].

α-Amylase Inhibition Activity
The inhibitory activity of α-amylase was ascertained as described by Quan et al. [42]. The extracts were initially tested for 500 µg/mL, extracts that inhibited the enzyme were further evaluated for 1000, 500, 100, 50, 25, and 10 µg/mL, with acarbose as a positive control [39]. Absorbance was measured using a multi-plate reader at 550 nm for each well, calculating the percentage of inhibition utilizing the following equation: where A = the absorbance of the reaction mixture in the presence of the extract, B = the absorbance of the mixture without the enzyme, and C = the absorbance of the reaction mixture in the absence of any extract.

Conclusions
This study investigated the quality of eight different cultivars of mango fruit in terms of the biological activities of the fruit parts. All parts of the mango fruit showed cytotoxicity, glucose uptake, GPx, and α-amylase inhibition. The statistical model suggested a significant correlation with most activities attributable to the seed part of the mango fruit. MG was correlated to the screened biological activities; however, a comprehensive phytochemical characterization with advanced in vivo studies and clinical trials may further confirm the role of mango fruit for the cure of diseases in the form of food products, nutraceuticals, or isolated medicinal compounds. The study assessed quality variation among different mango cultivars and established a correlation between phytochemistry and the biological activity of mango fruit and its parts from different geographical origins.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement:
All the authors showed the consent to publish the work.

Data Availability Statement:
The data used to generate the outcomes is completely presented in this document.