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ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 2  |  Page : 186-193  

Genetic, chemical, and biological diversity in Mangifera indica L. cultivars


1 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
3 Department of Pharmacology, National Research Center, Giza, Egypt

Date of Web Publication18-May-2020

Correspondence Address:
Dr. Rehab M. S. Ashour
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Ainy, Cairo 11562
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_99_19

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   Abstract 


Context: Mango is a valuable plant with vital economic importance; the leaves of its cultivars show several morphological similarities. Aims: Full differentiation of the leaves of eight Mangifera indica L. cultivars depending on genetic, chemical, and biological bases. Settings and Design: Chemometric analysis was applied to fully distinguish the diversity among cultivars; also, their gastroprotective activity was studied. Subjects and Methods: DNA fingerprinting of eight mango cultivars using random amplified polymorphic DNA–polymerase chain reaction technique and high-performance liquid chromatography (HPLC) analysis of phenolic compounds and flavonoids were compared using chemometric analysis. Furthermore, estimation of total polyphenolics and flavonoids and gastroprotective activity was studied.Statistical Analysis Used: One-way analysis of variance was used, followed by Tukey's post hoc test. Results: Primers OPA-O7 and OPA-O8 showed 100% polymorphism. Total polyphenolics and flavonoids concentrations varied greatly (14.58 in Tommy atkins to 29.54 in Fagrklan g gallic acid equivalent/100 g extract and 22.49 in Tommy atkins to 93.40 in Fagrklan g rutin equivalent/100 g extract, respectively). HPLC quantification revealed that Kent had relatively high mangiferin content (732.446 mg/kg), and caffeic acid was recorded in the tested cultivars (2266.66 in Keitt to 1106.94 mg/kg in Naaomy). Pylorus ligation model in rats was used to assess gastroprotective potential at a dose of 200 mg/kg using standard ranitidine. High percentage protection was observed in Kent (65.62%), whereas Keitt showed the lowest percentage protection (45.31%). No direct correlation could be deduced between concentration of detected metabolites and the gastroprotective effect, so this activity might be attributed to synergistic effect between all secondary metabolites. Conclusions: This study spots the light on the great variation among the tested extracts; in addition, it provides effective techniques that pave the way for complete discrimination of these mango cultivars.

Keywords: Chemometric analysis, gastroprotection, high-performance liquid chromatography, Mangifera indica L., mangiferin, random amplified polymorphic DNA


How to cite this article:
El-Hawary SS, Ashour RM, El-Gayed SH, Gad HA, Jaleel GA. Genetic, chemical, and biological diversity in Mangifera indica L. cultivars. Phcog Res 2020;12:186-93

How to cite this URL:
El-Hawary SS, Ashour RM, El-Gayed SH, Gad HA, Jaleel GA. Genetic, chemical, and biological diversity in Mangifera indica L. cultivars. Phcog Res [serial online] 2020 [cited 2020 Jun 5];12:186-93. Available from: http://www.phcogres.com/text.asp?2020/12/2/186/284427

Authors Seham S. El.Hawary and Rehab M. S. Ashour contributed equally to this work.




SUMMARY

  • Eight mango cultivars were fully differentiated using RAPD-PCR. Chemical diversity was evidenced by Folin Ciocalteau and Aluminium chloride methods. Great variation was detected in different metabolites quantified using HPLC, and also in gastroprotective activity evaluated using Pylorus ligation model.




Abbreviations Used: RAPD: Random amplified polymorphic DNA; HPLC: High performance liquid chromatography; UV: Ultraviolet; PCA: Principle component analysis; PCR: Polymerase chain reaction.


   Introduction Top


Mangoes are members of genus Mangifera; it consists of about 70 genera, family Anacardiaceae. Historical records showed that its cultivation started in India more than 4000 years ago.[1] Over one thousand mango cultivars are found around the world.[2] It is usually cultivated for its fruit, which is considered to be as the “king of fruits” or “superfruit.”[3]Mangifera indica L. is an important medicinal plant not only the fruit but also different parts of mango tree had various reported biological activities.[4] Mangoes possess hypolipidemic, anticancer, antiparasitic, anti-HIV, antispasmodic, antidiarrheal, gastroprotective immunomodulation, antimicrobial, antifungal, antipyretic, anthelmintic and hepatoprotective activities.[5] In Egypt, the immunostimulant, anticancer, and antimicrobial activities of the volatile oil of the peel of three mango cultivars (Zebdeya, Hindi, and Cobaneya) were investigated.[6] They are considered a rich source of polyphenolics mainly mangiferin, phenolic acids, and flavonoids, found in all parts (pulp, peel, seed, bark, leaf, and flower) in various concentrations. The importance of polyphenolics arises primarily from their antioxidant capabilities, thus protection against many diseases.[7]

Peptic ulcer developed due to the imbalance among aggressive factors (acid, pepsin, and bile salts) and defensive factors (mucus, bicarbonate, prostaglandins, epithelial cell restoration, and blood flow).[8] However, still the mechanism of the gastric ulcer is not well understood.[9],[10] Different therapeutic agents including proton-pump inhibitors, antihistaminic, and antacids are available for the treatment of this disorder, but the incidence of relapses, drug interactions, and side effects were reported. Thus, search for herbal drugs that decrease relapse and offer better protection is deemed of interest.[10] Different models can be employed to induce peptic ulcer, for example, induction with ethanol, with nonsteroidal anti-inflammatory drugs, using stress, and by pylorus ligation.[11]

A previous study reported the potential gastroprotective effect of the aqueous decoction of mango leaves and stated that it may be attributed to the bioactive phenolic compounds present, representing 57.3% of the total extract.[12] The aqueous decoction of mango flowers revealed to have significant gastroprotective and ulcer healing properties; meanwhile, using pylorus ligation, it significantly decreased the acid output, which proves its antisecretory effect leading to gastroprotection.[13] Moreover, the stem bark methanolic extract of mango demonstrated significant dose-dependent ulceration inhibition.[14]

The eight mango cultivars under investigation, namely, Naaomy, Haidy, Fagrklan, Palmer, Keitt, Maya, Tommy atkins, and Kent, were misleading to be identified depending on their leaves' morphology.

The use of the appropriate cultivar is believed to be extremely important in herbal medicine to get the desired pharmacological action. This study aims to provide useful tools for the precise discrimination of these eight mango cultivars. The use of random amplified polymorphic DNA (RAPD) was reported to be an effective tool for the identification of plant cultivars,[15] so it was the technique of choice to assess their genetic variability. In addition, quantification of the total polyphenolics and flavonoids was performed using Folin–Ciocalteu and aluminum chloride reagents, respectively. Further identification and quantification of different metabolites (mangiferin, polyphenolics, and flavonoids) in the tested extracts were achieved using high-performance liquid chromatography (HPLC)/ultraviolet (UV) detector.

Pylorus ligation-induced peptic ulcer or Shay's method was mainly employed to investigate and compare the effect of the tested extracts on gastric secretions and subsequently their possible gastroprotective potential. This model has the advantage of being capable of assessing the antisecretory and cytoprotective potential of drugs.[11] Finally, application of principal component analysis (PCA), utilizing data obtained from both RAPD and HPLC, was employed to fully discriminate the mango cultivars under study.


   Subjects and Methods Top


Standards and chemicals

Ranitidine, aluminum chloride, and rutin were obtained from E-Merck, Darmstadt, Germany, whereas gallic acid from Sigma-Aldrich, USA. Folin–Ciocalteu was obtained from Loba-Chemie, India. All solvents were of the analytical grade and water was distilled. Standards of flavonoid aglycones and phenolic compounds, used in HPLC analysis, were obtained from different manufacturers and were of HPLC purity grade.

Plant material

The leaves of M. indica L. cultivars, namely, Naaomy, Haidy, Fagrklan, Palmer, Keitt, Maya, Tommy atkins, and Kent, were collected in July 2015 from the Ministry of Agriculture and Land Reclamation (Egypt). The plant was authenticated by Professor Dr. Gamal Haseeb, Fruit Department Faculty of Agriculture, Cairo University. Voucher specimens numbered (2.4.2017 I-VIII) were placed at the Herbarium of the Faculty of Pharmacy (Pharmacognosy Department), Cairo University. DNA analysis was conducted in National Research Center, Dokki, Giza. HPLC analysis was performed at the Food Technology of Agriculture and Land Reclamation, Giza, Egypt.

Genetic profiling (DNA fingerprint)

Material for DNA

0.5 g of freeze-dried leaves[16] of each of the eight mango cultivars was powdered in liquid nitrogen. Isolation of the DNA from the frozen plants was done using cetyltrimethylammonium bromide method.[17] Ice-cold isopropanol was used to precipitate the nucleic acid.

Polymerase chain reaction

Amplifications were performed using 10 random arbitrary primers (OPA-01-10), synthesized by Operon biotechnologies Inc., Alameda, California, USA.[18] Sequences of the primers are as follows: (5'-CAGGCCCTTC-3'), (5'-TGCCGAGGTG-3'), (5'-AGTCA GCCAC-3'), (5'-AATCGGGCTG-3'), (5'-AGGGG TCTTG-3'), (5'-GGTCCCTGAC-3'), (5'-GAAACGGGTG-3'), (5'-GTGAC GTAGG-3'), (5'-GTGACGTAGG-3'), and (5'-GTGATCGCAG-3'), respectively.

Amplification was performed in 25 μl reaction volume with the following reagents: 0.5 μl of dNTPs (10 mM), 1.5 μlMgCl2 (25 mM), 5 μl of 10× reaction buffer, 2.0 μl of primer (5 pmol), 2.5 μl of total genomic DNA (20.4 ng/μl), 0.25 μl of Taq polymerase (10/μl), and 14.75 μl of sterile double-distilled H2O.

Polymerase chain reaction program and temperature profile

DNA amplification was carried out in a Perkin Elmer 2400 thermal cycler, using the following program: for 3 min, one cycle at 95°C (separation of initial strand), followed by 2 min, 45 cycles at 92°C (for denaturation), 1 min at 37°C (for annealing), 2 min at 72°C (for elongation), 10 min, 1 cycle at 72°C (for final extension), and finally 4°C (infinitive).

Electrophoresis of polymerase chain reaction products

Separation of amplified DNA fragments was done on 2% agarose gel plate. 10 μl of each polymerase chain reaction (PCR) product was loaded onto the wells of the gels after being mixed with 2 μl loading buffer. The gels were run at 100 volts for about 30 min.

Visualization, scoring, and photography

After electrophoresis, visualization was performed by staining with 0.2 μg/ml ethidium bromide solution and photographed using a gel documentation system under UV light. RAPD markers were scored as DNA fragments present in some lanes and absent in others.

Spectrophotometric quantitative estimation of total polyphenolics

Total polyphenols were determined colorimetry by Folin–Ciocalteu reagent. 0.5 g of dried leaves of each cultivar was homogenized, separately, in methanol using mortar and pestle, and the homogenate was centrifuged at 10,000 cycles/min for 20 min. The supernatant was used for the estimation of total polyphenols. 2.5 ml of Folin–Ciocalteu reagent was added to 0.5 ml of each of methanolic extract and then 2.5 ml of 7.5% sodium carbonate was added. The contents were incubated for 45 min at room temperature. The absorbance was measured at 710 nm. Samples were prepared in triplicates and the mean value of absorbance was obtained. Blank was concurrently prepared. The same procedure was repeated for gallic acid as standard. Total polyphenolic content was calculated from the regression equation of the standard plot (Y = 0.001X + 0.0154, r2 = 0.9993), where Y = absorbance, X = concentration, expressed as g gallic acid equivalent/100 g dried extract.[19]

Spectrophotometric quantitative estimation of total flavonoids

Aluminum chloride colorimetric method was used to determine flavonoid content. 1 ml of sample extract was mixed with 3 ml of methanol and 0.2 ml of 10% aluminum chloride. 0.2 ml of 1M potassium acetate and 5.6 ml of distilled water and remains at room temperature for 30 min. The absorbance was measured at 420 nm. Rutin was used as standard (1 mg/ml). Flavonoid content was calculated from the regression equation of the standard plot (Y = 0.001X + 0.0286, r2 = 0.991) expressed as g rutin equivalent/100 g of dried extract.[20]

Material for high-performance liquid chromatography

Preparation of plant extracts for high-performance liquid chromatography and biological study

500 g of dried leaves of each of the eight cultivars, namely, Naaomy, Haidy, Fagrklan, Palmer, Keitt, Maya, Tommy atkins, and Kent, was macerated in 70% alcohol at room temperature. The resulting extracts were concentrated under vacuum, to yield 25 g, 40 g, 50 g, 32 g, 46 g, 30 g, 41 g, and 22 g, respectively.

Sample preparation for high-performance liquid chromatography

Extraction, hydrolysis, and identification of flavonoids and polyphenolic compounds were performed according to Mattila et al. and Goupyet al.[21],[22]

Standards for phenolic components of the samples were prepared in methanol as 50–600 μg/ml solutions. Quantification was based on retention times comparison and measuring the peak areas of both samples and standards using the external standard method. All experiments were made in triplicates and the average was taken.

Chromatographic conditions for high-performance liquid chromatography analysis of phenolic compounds

Detailed conditions are attached in Supplementary File S-A.[Additional file 1]

Chromatographic conditions for high-performance liquid chromatography analysis of flavonoids and mangiferin

Detailed conditions are attached in Supplementary File S-B.

Gastroprotective activity

Animals

Adult male Swiss albino mice (30–40 g) and male Wistar albino rats, weighing 150–170 g, were obtained from the National Research Centre animal house in Dokki, Giza, Egypt. The animals were housed in an air-conditioned room at 22°C ± 3°C and 55% ± 5% humidity, in metal cages. Standard laboratory diet was provided and water ad libitum under standard conditions of 12 h dark/12 h light. Experiments were conducted in the period between 9:00 and 15:00 h. Procedures of all experiments were performed according to the laboratory animals care and use guide and approved by the National Research Centre ethics committee, registration number (Mp2536). They also followed the recommendations provided by the Health Guide of National Institutes for Care and Use of Laboratory Animals (Publication No. 85–23, revised 1985).

Acute toxicity (LD50) study

The median lethal dose (LD50) for each mango cultivar extract was determined orally in mice adopting Lorke's method[23] with modifications. Detailed procedures are attached in Supplementary File S-C.

Experimental procedure

Pylorus ligation-induced ulceration

Pylorus ligation was done as described by Shay[24] with slight modifications. Detailed procedures are attached in Supplementary File S-D1.

Determination of gastric wall mucus content

The mucus of gastric wall was estimated according to Corne et al. (1974).[25] Detailed procedures are attached in Supplementary File S-D2.

Determination of peptic activity

Detailed procedures are attached in Supplementary File S-D3.

Determination of gastric mucin content

This was achieved as described by Winzler.[26] Detailed procedures are attached in Supplementary File S-D4.

Histopathology

The samples of the stomach from different groups were preserved using 10% buffered formalin. They were processed for paraffin block preparation. Sections of approximately 5 mm thickness were cut. Hematoxylin and eosin was used for staining. Examination under a microscope for histopathological changes such as degeneration, erosion, edematous appearance, hemorrhage, and necrosis was performed.

Statistical and chemometric analysis

One-way analysis of variance was used for results analysis, followed by Tukey's post hoc test and expressed as mean ± standard error of the mean. The statistical software used to analyze the data was SPSS version 15 (IBM corp., Armonk, N.Y., USA). The obtained results were considered significant when P < 0.05. PCA was performed employing Unscrambler® 9.7 (CAMO SA, Oslo, Norway).


   Results Top


Genetic profiling (DNA fingerprint)

RAPD analysis of the eight mango cultivars was performed using ten decamer primers, from OPA-01 to OPA-10, respectively. The banding profiles produced are recorded in [Table 1] and [Table 2]. The ten DNA primers generated a total of 479 fragments in all eight species, where 94 fragments were generated in cultivar Haidy and 40 fragments in cultivar Palmer.
Table 1: Total number of random amplified polymorphic DNA-polymerase chain reaction fragments

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Table 2: Monomorphic and polymorphic bands generated by 10 primers

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Monomorphic bands (common in all species) were 12 bands, whereas 375 fragments were polymorphic (present in at least one species and absent in others), representing a total level of polymorphism of 72.28%. The highest percentage of polymorphism 100% was observed with primers A-O7 and A-O8, whereas the least percentage 37.25% was obtained with primer A-O4.

Spectrophotometric estimation of total polyphenolics

Relatively high phenolic content was observed in Fagrklan, Palmer, and Haidy cultivars (29.54, 28.66, and 27.25 g gallic acid equivalent/100 g extract, respectively), whereas Kent, Keitt, Naaomy, and Maya cultivars showed lower phenolic content (24.63–22.68 g gallic acid equivalent/100 g extract). Meanwhile, Tommy atkins had the lowest phenolic content. The results are shown in [Table 3].
Table 3: Spectrophotometric quantitative estimation of polyphenolics and flavonoids

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Spectrophotometric estimation of total flavonoids

Great variation in flavonoid content was observed in the tested cultivars. The highest concentration was recorded in Fagrklan (93.40), whereas the lowest was in Tommy atkins (22.49) calculated as g rutin equivalent/100 g extract. The results are shown in [Table 3].

High-performance liquid chromatography quantification of mangiferin

Mangiferin concentration was high in Kent (732.446 mg/kg), followed by Keitt and Naaomy (673.801 and 641.261 mg/kg, respectively) and then by Fagrklan and Haidy (575.921 and 531 mg/kg, respectively), whereas relatively lower mangiferin concentrations were observed in Maya, Tommy, and Palmer (488.114, 420.968, and 341.077 mg/kg, respectively).

High-performance liquid chromatography quantification of polyphenolics

HPLC analysis led to the identification of 18 phenolic compounds in the leaves of the eight mango cultivars under study. The results are shown in [Table 4].
Table 4: High-pressure liquid chromatography quantification of polyphenolics

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Maya showed high content of ethyl vanillic acid (5742.22 mg/kg), followed by Palmer and Tommy (3233.38 and 2306.37 mg/kg, respectively). Caffeic acid was detected in considerably high amounts relative to other phenolic compounds in all tested mango cultivars with concentration ranging from 2266.66 mg/kg in Keitt to 1106.94 mg/kg in Naaomy. Meanwhile, vanillic acid concentration was 1342.15 and 1224.55 mg/kg in Tommy and Naaomy, respectively. Catechol was detected in its highest concentration in Kent, followed by Haidy (1788.34 and 588.55 mg/kg, respectively).

High-performance liquid chromatography quantification of flavonoids

A total of nine flavonoids were identified and quantified in the tested mango cultivars. The results are shown in [Table 5].
Table 5: High-pressure liquid chromatography quantification of flavonoids

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Hesperidin was the main flavonoid detected in all cultivars with concentration ranging from 20.482 mg/kg in Tommy to 3.068 mg/kg in Fagrklan. Meanwhile, rutin was observed at concentration of 6.997, 4.610, and 3.042 mg/kg in Palmer, Haidy, and Kent, respectively.

Pharmacological assessment

Acute toxicity tests

Both phases ( first and second) of acute toxicity study showed no notable toxicity signs in mice.

Antisecretory gastroprotective activity

Macroscopic examination (ulcer number, ulcer index, and percentage protection)

Stomachs of ulcer control rats (rats with pyloric ligation) appeared with clear ulceration in their glandular area in comparison with normal control rats. Significant reduction in ulcer index with 67% protection was established upon pretreatment with ranitidine. Pretreatment with tested extracts significantly reduced ulcer index. Kent showed the highest protection (65%), followed by Haidy and Fagrklan (64%), Naaomy (62%), Tommy atkins (53%), Palmer (51%), Maya (50%), and Keitt (45%) [Table 6].
Table 6: Ulcer index and percentage protection of the eight tested mango cultivars

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Effects of extracts on gastric juice parameters and on gastric wall mucus content

Pretreatment with all tested extracts significantly decreased total acidity, acid output, and peptic activity as compared to ulcer control group (P < 0.05) and also significantly increased gastric wall mucus production and mucin content (P < 0.05) as compared to the control group. Fagrklan showed the most potent effect mimic to ranitidine standard [Table 7].
Table 7: Gastroprotective activity on pylorus ligation induced ulcer in rats

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Histopathological study

Photomicrography of stomach subjected to pylorus ligation revealed the lack of secreting lining of the epithelium as well as congested vascular spaces and moderate edema [Figure 1]a. On the other hand, ranitidine-treated rats showed that the secreting layer of epithelium was restoring its activity and continuity, along with decreased edemas and congestion at the submucosal level [Figure 1]b.
Figure 1: Photomicrographs of stomach sections of different treatment groups stained by H and E. (a) Pylorus ligated, (b) Ranitidine, (c) Naamoy, (d) Haidy, (e) Fagrklan, (f) Keitt, (g) Palmers, (h) Maya, (i) Tommy atkins, (j) Kent

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The highest degree of healing and least remaining pathology was shown in a descending order starting from Fagrklan, Haidy, Maya, and Kent cultivars as shown in [Figure 1]d, [Figure 1]e, [Figure 1]h and [Figure 1]j, respectively. The covering mucosa was intact in the four groups, whereas tissue edema, areas of hemorrhage, and inflammatory cellular infiltrate were increasing from Fagrklan, to Haidy, and Maya cultivars, being mostly expressed in Kent cultivar. On the other hand, Naaomy, Keitt, Palmer, and Tommy atkins cultivars had a similar presentation of slugged surface epithelium with massive tissue edema, inflammatory cellular infiltrate, and showing mucosal ulceration with submucosal edema and hemorrhage as represented in [Figure 1]c, [Figure 1]f, [Figure 1]g and [Figure 1]i, respectively.

Chemometric analysis

This was done by applying PCA, utilizing the ten primers in the eight cultivars studied, as shown in [Figure 2]. PCA score plot could successfully discriminate and segregate different mango cultivars, where the score plot explained about 92% of the variance in 180-dimensional space using only the first two components (the first PC accounts for 86% of the total variance followed by the second PC with 6%). As obvious, samples Haidy, Kent, and Fagrklan were positioned on the right side of the plot (positive PC1) and they were completely segregated confirming their genetic diversity. However, all other samples were placed on the left side (negative PC1) with sample Tommy on the lower quadrant away from all other samples. Samples Keitt, Palmer, Naaomy, and Maya were very close to each other, indicating their genetic similarity. In addition, the primers having the greatest influence on the scores plot were detected from the loading plot, as shown in [Figure 3], where primers A-O4, A-O7, and A-O8 were the main markers responsible for the segregation of samples Fragklan, Haidy, and Kent, respectively.
Figure 2: Principal component analysis score plot utilizing ten primers of eight mango cultivars

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Figure 3: Principal component analysis loading plot utilizing ten primers of eight mango cultivars

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To estimate the discriminative ability of the identified compounds by HPLC, PCA analysis was employed as a data reduction technique using the relative peak areas of the identified components as input data, to generate a visual plot for qualitative assessment on the similarity and dissimilarity of the tested samples. PCA score plot [Figure 4] resulted in two orthogonal PCs, which explained about 93% of the variance in 180-dimensional space using only the first two components (the first PC accounts for 81% of the total variance followed by the second PC with 12%). From the scatter points, different mango cultivars could be completely discriminated. On the right side of the plot, Tommy and Palmer are positioned (positive PC1 values). However, samples (Haidy, Keitt, Naaomy, and Fagrklan) were placed on the far left side (negative PC1 values) without any overlap among samples. Haidy and Keitt samples were separated from Naaomy and Fagrklan in relation to their position regarding PC2. Two samples Maya and Kent were detected as outliers, which investigated their clear compositional differences among all tested samples. The specific peaks, which had the most influence on the separation among different mango cultivars, were found out with the help of PCA loading plot. The loading plot of PCA [Figure 5] indicated that catechol, caffeic acid, vanillic acid, and ethyl vanillic may have more influence on the discrimination of different cultivars. These variables could be used as chemical markers in HPLC quality control of different mango cultivars in the future.
Figure 4: Principal component analysis score plot of relative peak areas of total compounds identified by high-performance liquid chromatography in eight mango cultivars (average of 3 replicates)

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Figure 5: Principal component analysis loading plot of relative peak areas of total compounds identified by high-performance liquid chromatography in eight mango cultivars (average of 3 replicates)

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   Discussion Top


The current study aimed to fully discriminate the leaves of eight tested mango cultivars based on genetic, chemical, and biological features. Furthermore, chemometric analysis was applied to provide strict evidences about relationships between the studied species. This discrimination is believed to be extremely valuable to prevent unfortunate misleading use of one cultivar instead of another, which might lead to altered pharmacological effect than expected. The choice of the leaves was decided considering the beneficial use of mango by-products in health and industry.

DNA-based tools are an evolving measure for authentication and identification of medicinal plants.[27] The results of RAPD-PCR indicated high diversity among the cultivars under study. Primers A-O7 and A-O8 (100% polymorphism) can be effectively used to differentiate between the eight mango cultivars. Moreover, chemometric analysis was able to discriminate the different cultivars based on the primers used, where primers A-O4, A-O7, and A-O8 are the main markers responsible for the segregation of samples Fragklan, Haidy, and Kent, respectively.

Polyphenolics, flavonoids, and mangiferin have been frequently reported not only in the edible part of mango fruits but also in the seed, skin, and leaves.[28] Upon estimation of the total phenolic content, Fagrklan showed almost double the phenolic content (29.54 g gallic acid equivalent/100 g extract) compared to Tommy atkins (14.58 g gallic acid equivalent/100 g extract). A previous study from India indicated the phenolic content of mango leaf to be 49.76 g/100 g gallic acid equivalent.[29] Furthermore, the total flavonoid content was highest in Fagrklan (93.40 g rutin equivalent/100 g extract), whereas Tommy atkins showed remarkable almost 4-fold decrease (22.49 g rutin equivalent/100 g extract). These findings with profound diversity in the quantitative analysis drove our interest to deeply explore the chemical composition of the eight mango cultivars, to get a more comprehensive insight about their chemical constituents and their relation to the gastroprotective and antisecretory effect using pylorus ligation model.

HPLC quantification of different metabolites (mangiferin, polyphenolics, and flavonoids) generally revealed great variation in the concentration of these metabolites among the tested cultivars. This compositional difference may be attributed to environmental and biological factors.[30]

Mangiferin concentration was 732.446 mg/kg in Kent, whereas in Palmer, it dropped more than 2-fold to 341.077 mg/kg. Mangiferin (C-glycosyl xanthone) is reported to be the main phenolic constituent in mango;[31] it can be obtained from leaves, fruits, bark, and roots.[28] This variation detected in mangiferin concentration is in accordance with that previously reported on 11 mango pulp cultivars, in which it was only detected in five of them with variable concentrations (0.032–3.20 mg/100 g).[28]

In spite of the fact that Kent showed the highest mangiferin concentration as well as the highest percentage of gastric protection, the rest of the results showed no direct correlation between this constituent concentration and the activity under study. This was clearly evidenced by the percentage protection of Keitt cultivar that took the second place in mangiferin concentration among the tested samples, yet it revealed the lowest percentage protection (45.31%). Polyphenolics, for example, caffeic acid and catechol as well as flavonoids, play an important role as protective agents against ulcer through their cytoprotective, antisecretory, and antioxidant effects.[32]

Although, in all eight tested cultivars, caffeic acid, ethyl vanillic acid, and vanillic acid were the most abundant phenolic acids identified. caffeic acid was found in highest concentration in Keitt (2266.66 mg/kg), while maximum concentration of ethyl vanillic acid was observed in Maya (5742.22 mg/kg), and vanillic acid concentration was optimum in Tommy (1342.15 mg/kg).

It was remarkable that Kent showed relatively high concentrations of catechol (1788.34 mg/kg), followed by Haidy (588.55 mg/kg), compared to other cultivars under study. This great variation in different constituents' concentration is also seen concerning the detected flavonoids, for example, hesperidin concentration, which varied from 20.48 in Tommy atkins to 3.07 in Fagrklan. No direct relation was observed between any of the detected phenolics or flavonoid concentrations and the obtained protection against ulcer.

Finally, it could be concluded that the gastroprotective effect for the tested cultivars might be due to synergistic effect of all secondary metabolites present in the leaves of these cultivars rather than to a single component.

By utilizing the data obtained from HPLC in combination with chemometrics, the results showed the successful application of PCA in the segregation of different mango cultivars based on the identified peak areas, which confirmed the diversity in their composition quantitatively. PCA loading plot investigated the main chemical markers responsible for cultivar discrimination, which are identified as catechol, caffeic acid, vanillic acid, and ethyl vanillic acid.

This great variation spots the importance of the precise recognition, of which cultivar can be used medicinally to prevent any health hazards. Strict identification of the cultivar used should be adopted to get the desired pharmacological action.


   Conclusions Top


All tested M. indica cultivars showed great variation in secondary metabolites. No correlation was observed between a specific metabolite and the gastroprotective activity. Full differentiation using DNA fingerprinting, chemical analysis, and PCA was successfully achieved. This study provided the most precise information to set strict boundaries between the eight mango cultivars under study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.


   Supplementary Files Top


Supplementary Data

S-A: Chromatographic conditions for HPLC analysis of phenolic compounds

A reversed phase column Alltima C18(150 mm × 4.6 mm i.d., 5 μm) was used, with 30 ° C column temperature, and the solvent system was A (acetic acid 2.5%), B (Acetic acid 8%) and C (acetonitrile). The best separation obtained was using 5% B at 0 min, 10% B at 20 min, 30% B at 50 min, 50% B at 55 min, 100% B at 60 min, 50% B and 50% C at 100 min, 100% C at 110 min. The flow rate was 1 ml/min, and the injection volumes were 10 μL, ultraviolet wave length was 280 nm.

S-B: Chromatographic conditions for high-performance liquid chromatography analysis of flavonoids

Analysis was done on Inertsil (GL Sciences, Inc., Japan) with ODS-3 column (4.0 mm × 150 mm, 3 μm) with a guard column C-18. Column temperature was 35°C. Gradient elution using mobile phase: 50 mM H3 PO4(pH 2.5) as solution A and acetonitrile as solution B was adopted as follows: 0–5 min, isocratic elution 95% A/5% B, 5–55 min linear gradient from. The flow rate was 0.7 mL/min, and. Major flavonoids were identified by their retention times comparison to those of the standards. Peak areas of both standards and samples were used for quantification employing the external standard method. Calibration curves of standard flavonoids were established by dilution of stock standards in methanol to give 2–20 μg/mL. All experiments were made in triplicates and the average was taken.

Standards in high-performance liquid chromatography purity grade were used for flavonoid aglycones. Stock solutions (5 mg/50 mL in methanol) were prepared for all standards, except for apigenin and luteolin (5 mg/50 mL in DMF/MeOH, 1:6, v/v), and rhamnetin and isorhamnetin (5 mg/50 mL in DMF/MeOH, 1:10, v/v).

Biological Study

S-C: Acute toxicity (LD50) study

The median lethal dose (LD50) for each extract was determined orally in mice adopting Lorke's method[1] with modifications. Concerning the first phase, nine mice were randomized into 3 groups (each of 3 mice). They were treated with the extract at doses of 500, 1000 and 2000 mg/kg, p.o., to evaluate the range of LD50. The same conditions were provided to all mice. They were monitored for signs of toxicity, which include but not limited to stretching, paw-licking, respiratory distress and mortality for 24 h. During the second phase of the study doses of 3000, 4000 and 5000 mg extract/kg body weight, respectively were administered orally to another fresh set of 3 groups (three mice each). Signs of toxicity were also observed as well as mortality for 24 h. The geometric mean of doses that caused 0 and 100% mortality (the oral median lethal dose) was calculated. In each group, the number of deaths within 24 h was recorded; also the final LD50 values were measured as the geometric mean of the highest nonlethal dose (with no deaths) and the lowest lethal dose (where deaths occurred).

S-D: Experimental procedure

S-D1: Pylorus ligation-induced ulceration

Pylorus ligation was done as described by[2] with slight modifications. Sixty-three rats were randomly divided into 9 groups (number of rats in each group = 6). Group I was treated with vehicle (10% DMSO) as negative control, Group II was treated with 100 mg/kg ranitidine (p.o.), as positive control. The rest of groups were treated orally with 200 mg/kg of each of the eight mango cultivars; Naaomy, Haidy, Fajrklan, Palmer, Keitt, Maya, Tommy atkins and Kent, for 7 days. 1 h after the administration of the last dose of tested extracts, pylorus ligation was done on 48 h fasted rats Carbajal et al. 2000.[3] 4 h after ligation, the animals were sacrificed by cervical dislocation. The stomach was removed and the gastric juice was drained. The stomach was opened along the greater curvature. The following scoring system was used: 0 for normal mucosa; 0.5 for blushing; 1 for spot ulcers; 1.5 for hemorrhage streaks; 2 for ulcers less than 3mm; and 3 for ulcers more than 5 mm.[4] The ulcer index was calculated according to the method described by[5] by dividing the sum of the total length of long ulcers and hemorrhagic spots in each group of rats by the number of animals. The percentage protection was calculated using the equation described by.[6]

Centrifugation of gastric juice for 10 min at 2000 rpm was done, followed by collection of the supernatant. It was used for the determination of gastric juice volume, as well as free and total acidity. The volume was expressed as mL/100 g/4 h. Estimation of both free and total acidity of gastric juice was performed as described by.[7] This was done by titrating 1ml 0.01 N sodium hydroxide using phenolphthalein as indicator.[8] The acidity was expressed as mEq/L/100g while the acid output as mEq/100 g/4 h.[9]

S-D2: Determination of gastric wall mucus content

The mucus of gastric wall was estimated following the method of.[10] Removal and weighing of the glandular segment of the stomach was done. This was followed by incubation for 2 h, in tubes containing solution of 1% Alcian blue which consists of 0.16 M sucrose in 0.05 M sodium acetate, with pH 5.8. Centrifugation of Alcian blue binding extract was done, followed by measuring the supernatant absorbance at 498 nm. Calculation of the amount of Alcian blue extracted (mg/g of glandular tissue) was then done. From a standard curve, the quantity of Alcian Blue extract per gram wet stomach was calculated.

S-D3: Determination of peptic activity

The main proteolytic activity of gastric secretion was estimated in terms of the amount of proteases produced after incubation of the substrate with pepsin for half an hour. The pepsin proteolytic activity in gastric juice was measured spectrophotometrically at 280 nm.[11]

S-D4: Determination of gastric mucin content

This was achieved as described.[12] Briefly, orcinol (1.6%) and sulphuric acid (60%) were added to diluted samples, which were then vortexed and boiled for 10 min. Cooling of mixtures in ice-cold water was done in order to cease the reaction. The absorbance was determined spectrophotometrically at 425 nm.


   References Top


  1. Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87.
  2. Shay H. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 1945;5:43-61.
  3. Carbajal D, Molina V, Noa M, Valdés S, Arruzazabala ML, Aguilar C, et al. Effect of D-002 on gastric mucus composition in ethanol-induced ulcer. Pharmacol Res 2000;42:329-32.
  4. Kulkarni S. New drug discovery process. In: Handbook of Experimental Pharmacology. 3rd ed. New Delhi: Vallabh Prakashan; 1999. p. 135-7.
  5. Cho CH, Ogle CW. Cholinergic-mediated gastric mast cell degranulation with subsequent histamine H1-and H2-receptor activation in stress ulceration in rats. Eur J Pharmacol 1979;55:23-33.
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  8. Grossman MI, Kirsner JB, Gillespie IE. Basal and histalog-stimulated gastric secretion in control subjects and in patients with peptic ulcer or gastric cancer. Gastroenterology 1963;45:14-26.
  9. Brodie DA, Hooke KF. The effect of vasoactive agents on stress-induced gastric hemorrhage in the rat. Digestion 1971;4:193-204.
  10. Corne SJ, Morrissey SM, Woods RJ. Proceedings: A method for the quantitative estimation of gastric barrier mucus. J Physiol 1974;242:116P-7P.
  11. Sanyla A, Debnath P, Bhattacharya S, Gode K. The effect of cyproheptadine on gastric activity: An experimental study. In: Pfeiffer CJ, editor. Peptic Ulcer. Copenhagen: Munksgaard; 1971. p. 312-8.
  12. Winzler R. Determination of serum glycoproteins. Methods Biochem Anal 1955;2:279-311.




 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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