Study of physicochemical parameters of different cultivars of Mangifera indica L. leaves for their use as a source of Mangiferin.
Jacqueline A. Romero1*, Roberto Vandama1, Marilin López1, Maricela Capote2 Carlos Ferradá1, Caridad Carballo1, René Delgado1, Wim Vanden Berghe3, Sandra Apers4
1Laboratory of Analytical Chemistry. Center of Development and Drug Research, Ave 26 # 1605, La Habana, Cuba
2Institute of Tropical Fruit Research, Ave. 7ma. No. 3005 entre 30 y 32. Miramar, Playa. La Habana. Cuba.
3Laboratory of Protein Science, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University Antwerp, Campus Drie Eiken, Universiteitsplein 1, Wilrijk, Belgium.
4Natural Products & Food – Research and Analysis, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium
Available Online: 25th May, 2015
ABSTRACT
The leaves of M. indica are used as a source of mangiferin for the pharmaceutical industry. The present study deals with the phytochemical and physicochemical evaluation of different varieties in order to identify the most suitable one. The parameters included in this comparative study are extractable matter, mangiferin content, water content and total ash. ANOVA revealed a significant difference between the physicochemical parameters of the different cultivars. The Hierarchical Ascendant Classification divided the 29 cultivars into 4 clusters. Eventhough the Filipino and Biscochuelo cultivars showed a higher content of Mangiferin, the cluster of cultivars including the cultivar Corazón is the most suitable one taking the results of the extractable matter into account. In addition, the seasonal variation was investigated for the latter cluster, which revealed that optimal collection period includes the flowering and green fruit stages. Furthermore, based on the investigation of the influence of the drying and storage conditions, plant material for industrial isolation of mangiferin, can be dried using a sun dryer and stored in glass or polypropylene containers for one year.
Keywords: Mangifera indica L., leaves, cultivars, mangiferin
INTRODUCTION
Mango (Mangifera indica L.) is one of the most important tropical fruits worldwide in terms of production and consumer acceptance1. Extensive plant breeding has generated hundreds of cultivars, the fruits of which show a pronounced diversity in size, shape, color, flavor, seed size, and composition2. M. indica contains various constituents such as carbohydrates, tannins, proteins, saponins, mucilage, terpenoids, flavonoids and glycosides. Mangiferin (MgF), a xanthone glycoside is a main constituent of M. indica present in the leaves, fruits, stem bark and root. MgF has a wide panel of pharmacological activities such as antipyretic, antioxidant, antitumour, immunomodulatory and neuroprotective3-7. It is traditionally known to be useful for the treatment of a wide range of diseases like throat infection, burns, scalds, and is applied in antidiabetic, antioxidant, antimicrobial, antiviral and antibacterial treatments8-14. A careful survey of literature revealed that little work has been done on the phytochemical comparison, and more specifically the mangiferin content, of the leaves of M. indica from different cultivars grown in Cuba.The aim of this work is a preliminary phytochemical characterization of M. indica leaves in order to use them as a source for obtaining MgF as a raw material for the pharmaceutical industry.
MATERIALS AND METHODS
Plant material
The cultivars studied were: ʻLancetillaʼ,ʻDeliciaʼ, ʻSeñoraʼ, ʻEstero del Pinarʼ, ʻKeittʼ, ʻSanta Cruzʼ, ʻBombay tardíoʼ, ʻTommy Atkinsʼ, ʻMininʼ, ʻMameyzónʼ, ʻSan Felipeʼ, ʻPedroʼ, ʻBiscocheloʼ, ʻChino Amarilloʼ, ʻChino Rojoʼ, ʻFilipinoʼ, ʻChino Esperónʼ, ʻCorazónʼ, ʻLa Pazʼ, ʻSan Diegoʼ, ʻJulieʼ, ʻSuper Hadenʼ, ʻDeliciosoʼ, ʻDomʼ, ʻRenʼ, ʻReina de Méxicoʼ, ʻHadenʼ, ʻSpringfieldʼ, ʻMario’, ʻMacho’, ʻFlorida’ and ʻSmithʼ. Plant material was donated by the genebank of the Tropical Fruit Research in Artemisa, Cuba and the samples were authenticated by Dr. Victor Fuentes. Also leaves at different phenological stages, including. vegetative, flowering, green and ripe fruit were collected from July to October, 2012 and 2013. Voucher specimens (ROIG 4776) were deposited at the herbarium Medicinal Plant Experimental Station, ̏Juan Tomás Roig˝ San Antonio de los Baños, Mayabeque, Cuba. Leaves were washed in running water and dried using a sun dryer. They were homogenized to a fine powder (2-3mm) and stored in air tight packets.
Reagents
Mangiferin standard was donated by the Center of Pharmaceutical Chemistry and certified by the State Center for Drug Control (CECMED), Cuba. The HPLC purity of the reference material was 93.8%. The reagents used were of analytical grade (MERCK, Germany).
Physicochemical parameters
Physicochemical studies including total ash, loss on drying at 105 °C and extractive values (water soluble and ethanol soluble) were carried out according to the WHO guidelines15.
Mangiferin content
Procedure
Mangiferin was quantified by HPLC. The separation was performed using a Knauer Smartline HPLC system (Knauer, Germany) equipped with a Smarline 1000 pump, a Smartline 2500UV detector, a Smarline 3900 Thermoautosampler, a Manager 5050 degasser and ClarityChrom software. A Luna C-18 reverse-phase analytical column (150 mm x 4,6 mm i.d., 5µm particle size), Phenomenex, USA was used. The following gradient was applied with mobile phase A being acetic acid 0.2% and acetonitrile (85:15) and B acetonitrile: 18 min, 100% A; 20 min, 20 % A; 22 min, 100% A. The flow rate was 1 mL min-1. Absorbance was monitored at 254 nm.
Preparation of standard
To construct the calibration curve, a stock solution of 100 ppm of reference compound MgF was prepared in a 50% dioxane solution. A series of dilutions were subsequently carried out to obtain 1/2, 1/4, 1/8, 1/16 and 1/32, of the original concentration for the preparation of standard solutions.
Sample Preparation
About 1 g of dried leaves were accurately weighed and processed by continuous extraction for 4 hours in a Soxhlet apparatus, using dioxane as a solvent. The extract was transferred to a 500 mL volumetric flask and made up to volume with a mixture of dioxane: water (1:1) v/v. A 5,0 mL aliquot was transferred to a 50 mL volumetric flask and made up to volume with the same mixture. All the samples were analyzed in triplicate.
Statistical analysis
The statistical analysis was performed using Stargraphic software version 5.1. Results were considered statistically significant at P<0.05. The means and standard deviations for the physicochemical parameters were calculated. Hierarchical Ascendant Classification (HAC) was used to divide the M. indica cultivars in different clusters based on their extractable matter and MgF content.
Drying study
Four drying methods were used: oven drying at 40°C ± 1°C, under shade, sun and in a sun drier. The plant material of 5 cultivars (Macho, Haden, Springfield, Flipino and Florida) was dried until constant weight. Dried leaves were stored in an airtight amber bottle at temperature of 4° ± 1 °C, until the analyses were performed.
Storage study
The storage study was conducted at 25°C ± 2°C/ 70 % RH ± 5 %, using 4 kinds of containers: polypropylene, polyethylene, glass and paper. The dried leaves of Super Haden cultivar were stored for one year and were checked for residual humidity, MgF content and organoleptic characteristics at time points 0, 3, 6and 12months.
RESULTS AND DISCUSION
Comparison of different cultivars
The results for mangiferin content and the physicochemical parameters of 29 cultivars grown in Cuba are shown in table 1. The total ash ranged from 7.4±0.15 to 14.6±0.21 and the water from 4.4±0.12- 13.2±0.15 percent. The contents of water soluble and ethanol soluble fractions ranged from 16.2±0.12 to 23.7±0.35 g/100g, and 16.3±0.20 to 32.5±0.15 g/100g respectively and the mangiferin content ranged from 1.24±0.01 to 5.00±0.05 g/100g of dry leaves. The quality of the plant material is determined by its mangiferin content, while the total ash and water content are parameters that are indicative for the purity. In addition, the values of the determination of the extractable matter in ethanol 70% (v/v) and water allow us to predict the amount of other material extracted simultaneously with MgF, as these solvents are used as the first step in the industrial production of MgF as active pharmaceutical ingredient in our plant production site. Statistically significant differences between all parameters for the different cultivars were observed (Table 1.).
A significant positive correlation among the majority of the physicochemical parameters evaluated was observed except for the total ash. All the characters, were negatively correlated with total ash. Substances soluble in water and in ethanol 70% show correlation between them (0.6404) and with the total ash (-0.6908 and -0.5014 respectively) and water content (0.4281 and 0.6965 respectively), all these correlations with statistical significance for a confidence level of 95%. The mangiferin content showed no statistically significant correlation with any of the parameters analyzed. The biosynthesis and the accumulation of MgF varies depending on the variety, but is independent from the effect of the physicochemical parameters analyzed in this study. The Hierarchical Ascendant Analysis of the results led to four clusters with variable characteristics (Table 2). Cluster 1 was formed by cultivars Haden, Bombay tardio, Keitt, Springfield, Señora, Chino esperón, Mario, Lancetilla, Delicia, Delicioso, Tommy Atkins, Minin, Pedro, Eldon; cluster 2: Smith, Estero del Pinar, Super Haden, Chino rojo, Corazón, Santa Cruz, Reina de Mexico, cluster 3: Julie, La Paz, Filipino, Chino Amarillo, Bizcochuelo, cluster 4: Kent, San Felipe, San Diego, Mameyzon. Cluster 3 groups cultivars that combine a high content of MgF with the lowest content of extractable substances in water and ethanol 70%. Although a higher MgF content was found in Filipino and Biscochuelo cultivars, less compounds are extracted together with the metabolite of interest, both in water and in 70% Ethanol in the case of the Corazón cultivar. So we conclude that the group of cultivars that are part of the cluster number 2 would be the most suitable for use in obtaining MgF. Seasonal variation of Mangiferin content
The content of MgF in the leaves of different phenological states of seven cultivars were determined .From these results, i.e. for Super Haden cultivar during the stages of flowering, green fruit, ripe fruit and vegetative state was: 4.28 ± 0.86, 3.25 ± 0.03, 1.63 ± 0.11, 1.56 ± 0.12 respectively, it could be concluded that the highest content was found in the leaves collected during flowering and green fruit. The highest content of mangiferin was found in the leaves collected during flowering , i.e. the phenological stage that leads to fruiting which could be related to the role of this metabolite in the plant as an important secondary metabolite to protect the plants against various forms of biotic and abiotic stress16-18.
Drying study
In order to obtain a high quality of plant material to be used as a herbal drug it is important to determine which drying method is the best, i.e. the method leading in the shortest time to the starting material with the lowest amount of water. In our study all drying methods show differences in the residual moisture for the five cultivars studied while the MgF content remained unchanged for each cultivar studied. In general, the best results were obtained using artificial drying at 40 ⁰C, except for Haden cultivar for which the residual moisture values were lower in the case of the sun dryer. For all methods this parameter remained below 12% w/w. (Table 3). For improved energy efficiency in the production of medicinal plants technology, conventional hot air drying using fuel or electricity can be replaced by the use of solar dryers which has the advantage of energy savings with obtaining a dehydrated product quality and hygienic conditions where sanitation compared to the natural drying is higher.
Storage
In order to avoid possible damage and contamination by moulds, insects and other animal contamination, medicinal plant materials must be processed and packaged immediately after harvest. Proper long term storage of the drug should ensure product stability and conservation of physical, chemical, organoleptic and therapeutic properties. Therefore it is important to carefully analyze the influence of the type of packaging used and the conditions of storage. Table 4 shows the results for the storage study during a year in four kinds of containers. The results showed no statistically significant differences in mangiferin content between the different types of containers or between the different time points while organoleptic characters changed, i.e. loss of color and odor, for packaging paper and nylon features after ninth months of storage. For samples stored in glass and polypropylene containers these features remained unchanged. The residual water content is maintained in a range between 7.2 and 9.8. So the storage for one year in glass or polypropylene containers guarantees the retaining of all the physicochemical characteristics.
ACKNOWLEDGEMENTS
We thank VLIR-UOS for support of this work.
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