Comparative Analysis on aucubin content in different ecotyes of plantain plant

An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 9 COMPARATIVE ANALYSIS ON AUCUBIN CONTENT IN DIFFERENT ECOTYES OF PLANTAIN PLANT (Plantago sp.) Nguyen Van Ay1, Mai Vu Duy1, Do Tan Khang1, Bui Nhi Binh1, Khajidusren Altantsetseg2, Oyungerel Baatartsogt2, Vanjidorj Enkhchimeg2 1Can Tho University 2Mongolian University of Life Sciences Information: Received: 28/11/2016 Accepted: 19/01/2017 Published: 06/2017 Keywords: Plantain, vegetative growth stage, geo-ecological condition, aucubin content ABSTRACT Evaluation on aucubin accumulation in different ecotypes of plantain (Plantago sp.)” was carried out on on different ecotypes of plantain collected from Mongolia and Vietnam. The results showed that: (i) the amount of aucubin had been increased together with the growth stages. Minimum level of aucubin of P. depressa was found in vegetative stage (1.66 mg/g), maximum in fruiting stage (12.46 mg/g); (ii) all of ecotypes of Plantago sp. varied in their aucubin content. In addition, aucubin content of Mongolian ecotypes was higher than those of Vietnamese ecotypes’; and (iii) the geo-ecological conditions (Vietnam and Mongolia) had affected significantly on the aucubin content in different ecotypes. P. depressa contains higher amount of aucubin than P. major. The highest aucubin constituent is on P. depressa ecotype which collected in Middle Khalkh dry steppe, Mongolia (21.87 mg/g) and the lowest on P. major in Thanh Hoa-Vietnam (2.08 mg/g). 1. INTRODUCTION Plantain (Plantago sp.), an important medicinal plant belonging to the Plantaginaceae family, is a genus of about 200 species. It is a native plant in Europe and Asia, but it is now widely distributed around the world, including temperate climate areas and some tropical regions (Clapham et al., 1989). Plantain is quite rich in bioactive substances, minerals and vitamins, which have been utilized in Vietnam, Lao, China, Mongolia and some other countries for traditional and industrial production in term of health, food product and medicine (Ho, 2000). Among bioactive compounds, plantain highly contains of aucubin belonging to iridoid glucoside group which is the typical substance for the whole Plantago genus (Andrzejewska-Golec, 1995, Taskova et al., 2002, Ronsted et al., 2000 and Jurisic et al., 2003). It has shown pharmacological properties, such as hepatoprotective, anti-toxic, anti-inflammatory, anti-oxidant, anti-aging, wound healing activity, anti-osteoporosis, neurotrophic, removal uric acid and diuretic (Kolak et al. 2011). Environmental factors such as light intensity, temperature, water availability, type and composition of soil and several other have a substantial influence on the quality and productivity of medicinal plants. They are not stable in habitats and the consequences of their variation are difficult to predict (Jolita et al. 2012). Plants of the same species occurring in different environments may differ significantly in their content of particular secondary metabolites (Szakiel et al., 2010). The evaluation of secondary An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 10 products accumulation under modified environment reveals more precise information about influences of environment factors on metabolic pathways. Considerable research work in the last period addresses to various aspects of modelling these factors using new numerical methods (Mihailovic & Eitzinger, 2007). The factors that can be easy to control in greenhouse are light intensity and temperature. On the other hand, they are known as the crucial environment factors strongly related to the phenological development and secondary metabolism of medicinal plants (Hornok, 1992; Zobayed et al., 2005). The stimulating of biosynthesis on secondary metabolites in medicinal crops by control and optimization of external and internal factors may be applied to develop the biotechnologies of high quality drug production (Poutaraud & Girardin, 2005). On the other hand, research on Plantago sp. which originated from Mongolia and Vietnam have been still missing, especially bioactive substance aspects. Thus, this study was conducted to comparatively analyze the amount of bio-active constituent aucubin in different ecotypes of Plantago sp. which are cultivated in Mongolia and Vietnam. 2. MATERIALS AND METHODS 2.1 Materials Seed and whole plant samples of 10 ecotypes of Plantago sp. were collected in 10 different experimental sites in Mongolia and Vietnam (Table 1). Voucher specimens of all samples, morphologically authenticated by Professor Altantsetseg, have been deposited at School of Animal Sciences and Biotechnology, Mongolian University of Life Sciences, Mongolia. Afterward, all of ecotypes of Plantago sp. were cultivated in the greenhouse of Can Tho University (83% relative humidity, 35oC, light intensity about 5000 lux) and/or Mongolian University of Life Sciences (51% relative humidity, 20oC, light intensity 2000 lux). Table 1. Geographical locations of Plantago sp. collected from different locations in Mongolia and Vietnam Location Country Scientific name Location (Latitude, Longitude) Time of Collection Middle Khalkh dry steppe Mongolia Plantago depressa 47.8864° N 106.9057° E July, 2015 Dzungarian Gobi desert P. depressa 45.4511° N 95.8506° E July, 2015 Mongolian Altai Mountain steppe Plantago major 48.3983° N 89.6626° E July, 2015 East Mongolia steppe P. depressa 47.4658° N 115.3927° E July, 2015 Alashan Gobi desert P. major 43.5000° N 104.2861° E July, 2015 Can Tho Vietnam P. major 10.0452° N 105.7469° E August, 2015 Ca Mau P. major 9.1527° N 105.1961° E August, 2015 Kien Giang P. major 9.8250° N 105.1259° E August, 2015 Nam Dinh P. major 20.4388° N 106.1621° E August, 2015 Thanh Hoa P. major 20.1291° N 105.3131° E August, 2015 An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 11 2.2 Preparation of samples Whole plants of the samples were harvested in 4 different growth phases: vegetative (2 months old), budding (3 months old), flowering (4 months old) and fruiting stage (5 months old), from 10 randomly selected pots for each phase. All of selected plants were uprooted and washed under tap water. Next, the flowers, petioles and roots were separated from the leaves and dried in a convectional dryer and mechanically ground to obtain the homogenous powder. Aucubin (standard chemical, purity > 98%), methanol, acetonitrile and other solvents were purchased from Sigma-Aldrich, USA. 2.3 Extraction of samples We applied high performance liquid chromatography (HPLC) to measure iridoid content in Plantago ecotypes followed by the modified method (described by Hogedal and Molgaard, 2000) and Kartini & Azminah (2012). Fifty mg of dried plant material was added to 10 mL of 100% methanol solution. Samples were sonicated at 55 °C in a Sonorex Super model RK 52H ultrasonic bath for 60 min and then the extract mixture was centrifuged at 18,000 rpm in 15 min to remove particulates. All extract were stored at 4oC until analyzed. Prior to the analysis, all standards and sample solutions were filtered through Waters sterile filters with 0.20 µm pores. 2.4 Effect of ecological conditions and vegetative periods on aucubin content Observation on P. depressa ecotype collected at Middle Khalkh dry steppe, Mongolia. This ecotype was cultivated in 2 different conditions, Mongolia and Vietnam. To compare the aucubin amount, the experiment was arranged in a Randomized Complete Bock Design (RCBD) with 2 factors including 8 treatments, 5 replications per each treatment. All treatments were arranged in various vegetative stages (2, 3, 4 and 5 months old; vegetative, budding, flowering and fruiting stages, respectively) and (ii) two ecological conditions. 2.5 Effect of types of ecotypes on aucubin content All ecotypes were cultivated in the same conditions in Vietnam (humidity, temperature, water availability, fertilizers). After fruiting stage, they were collected to comparative analyze of aucubin constituent. The experiment was arranged in a RCBD with 10 treatments (ecotypes), 5 replications per each treatment. 2.6 Determination of aucubin content by high performance liquid chromatography Each 10 μl volume of centrifuged sample was automatically injected into HPLC system for analysis. All experiments included in 5 replications per each ecotype. A Waters 2847 Dual Wavelength Absorbance Detector instrument was set to read samples at 202 nm, and flow rate was set to 0.3 μL/min. The column used in the analysis was a Nova-Pak reversed-phase C18 column (2 mm x 150 mm, 5 μm particle size; Phenomenex) and the solvent system consisted of isocratic (non-changing) of 7% acetonitrile solution (Sigma-Aldrich) in Milli-Q water. Sample runtime was 25 minutes. Chromatogram peaks were quantified by integrating the peak areas. Major peaks other than the internal standard were assumed to be aucubin, based on authentic aucubin standard (Fig.1B). 2.7 Building up the standard curve The stock solutions of reference standards were prepared by dissolving in methanol. The concentrations of aucubin reference standard used for calibration were at 2.5, 5, 10 and 50 mgL-1. The standard curve was calibrated using the linear regression equation (Y= 19730X - 54075, R2=0.9994) derived from the peak areas (Fig 1A). An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 12 Figure 1. The spectrum of aucubin of reference standard (A) and sample (B) 2.8 Statistical analysis All the results were performed for the statistical package for social sciences (Version 16.0 for windows, SPSS Inc.). The data were subjected to the analysis of variance (ANOVA). Post-hoc comparisons of mean quantities were made using the Duncan Multiple Range Test. A value of p < 0.01 was considered to be significant. 3. RESULTS AND DISCUSSION 3.1 Effect of growth conditions and vegetative stages on aucubin accumulation A large number of internal and external factors effect on the chemical composition of plants and it is difficult to determine which factor affected particular changes in chemical compounds. The factors that can be easy to control in greenhouse are light intensity and temperature. On the other hand, they are known as the crucial environment factors strongly related to the phenological development and secondary metabolism of medicinal plants (Hornok, 1992; Zobayed et al., 2005). It is possible to achieve high production of secondary metabolites within a very short period of cultivation under optimized conditions in a controlled environment system (Afreen, 2005). In present study, we evaluated the influences of external factors, as temperature and light intensity in Vietnam and Mongolia, together with internal factor – phenological development stages on accumulation of amount of aucubin in P. depressa ecotype which originated at Middle Khalkh dry steppe of Mongolia (Table 2). The present study revealed that high temperature and light intensity positively influence the accumulation of aucubin. The data revealed that the amount of aucubin (15.83 mg/g) in P. depressa cultivated in Vietnam is much higher than those in Mongolia (2.04 mg/g), significantly different at P < 0.01 level. Table 2. Effect of growth conditions and vegetative stages on the aucubin content (mg/g, dried weight) in plantain (P. depressa) Vegetative stages (months old) Growing place (location) Aucubin content (mg/g, dried weight) Mongolia Vietnam 2 (Vegetative) 1.00(±0.03) e 2.31(±0.41) cd 1.66(±0.74) d 3 (Budding) 1.60(±0.17) de 19.43(±0.93) b 10.51(±0.94) c 4 (Flowering) 2.51(±0.10) c 19.71(±1.06) b 11.11(±0.90) b 5 (Fruiting) 3.06(±0.19) c 21.87(±0.61) a 12.46 (±0.61) a 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 uV(x100,000) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 uV(x1,000,000) A B An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 13 Vegetative stages (months old) Growing place (location) Aucubin content (mg/g, dried weight) Mongolia Vietnam Aucubin content on ecological conditions (B) 2.04(±0.82) b 15.83(±0.90) a P(A) ** P(B) ** P(A*B) ** CV (%) 6.43 The data were observated on P. depressa ecotype collected at Middle Khalkh dry steppe, Mongolia; Means with same letters indicate no significant difference by Duncan’s multiple-range test; ** = significant at 0.01 level. Medical chemicals are synthesized and accumulated based on some genes induced by environmental conditions. It is well known that light is a physical factor which can affect the metabolite production. In addition, temperature is one of the most important environmental factors controlling plant secondary metabolite production (Kaplan et al., 2004; Ramakrishna and Ravishankar, 2011). Temperature significant influences photosynthetic processes, ontogenetic development, the carbon balance of plants, and the influence can be positive or negative (Morison and Lawlor, 1999). Higher light intensity and temperature stimulate the photosynthetic efficiency and accumulation of aucubin in P. depressa and an increase of aucubin under higher light intensity and temperature linked to higher abundance of dark glands, which are considered as synthesis sites of corresponding compounds (Briskin and Gawienowski, 2001; Zobayed et al., 2006). Indeed, according to Anasori and Asghari (2008) light is a physical factor which can affect the metabolite production. The production of secondary metabolites is often low and depends greatly on the physiological and developmental stage of the plant (Rao and Ravishankar, 2002). In Table 11, the result also shows that significantly different aucubin content on 4 different vegetative stages. The tendency of aucubin increases gradually from vegetative to fruiting stage. Furthermore, the content of aucubin at fruiting stage were the highest (12.46 mg/g) among the 4 different vegetative stages while lowest at vegetative stage (1.66 mg/g), significant at 0.01 level. It should be noted that various parts of the plants may differ quantitatively and qualitatively as far as the content of iridoids is concerned. Noro et al. (1990) observed that the overground parts of Plantago asiatica contained more aucubin than the underground ones, and young leaves were more than the older ones. According to Bowers and Stamp (1992), the contents of aucubin and catalpol in Plantago lanceolata was determined genetically and it also depended on the age of leaves. Phylogenetically, the secondary bioactive compounds in plants appear to be randomly synthesised – but they are not useless junk. Several of them are found to hold important functions in the living plants (Bernhoft, 2010). Thus, according to Jensen (1991) and Damtoft et al. (1993), in P. major and Scrophularia racemosa, aucubin is sythesized from a precursor which is 8-epi-deoxyloganic acid or 8-epi- iridodial. Afterward, because of being an intermediate, aucubin can be used to forming capatal finally or others under specific circumstances. Moreover, the interaction of ecological conditions and vegetative stages on aucubin content of P. depressa, significant at 0.01 level (Table 2). The maximum is at fruiting stage (21.87 mg/g) cultivated in Vietnam, the minimum is at vegetative stage (1 mg/g) in Mongolia. The present result shows that P. depressa cultivated in different An Giang University Journal of Science – 2017, Vol. 5 (2), 9 – 18 14 environments could differ significantly in aucubin content. Jolita et al. (2012) concluded that the quality and productivity of medicinal plants can be affected by environmental factors such as light intensity, temperature, water availability, type and composition of soil. Bioactive compounds also are not stabile in habitats and the consequences of their variation are difficult to predict. On the other hand, even the same species growing in different environments may vary significantly in their content of particular biosubstance compounds (Szakiel et al., 2010). Iridoids belong to terpenoids, which are a class of secondary metablites found in a wide variety of plants. Aucubin is one of the most common iridoids in the plant kingdom. Terpenoids represent the largest and most diversified class of chemicals among the innumerable compounds produced by plants. Plants require terpenoids metabolites for a variety of basic functions in growth and development as photosynthetic pigments (carotenoids), electron carriers (side-chains of ubiquinone and plastoquinone), regulators of growth and development (gibberellins, abscisic acid, strigolactones, brassinosteroids, cytokinins), in protein glycosylation (dolichols), or as elements of membrane structure and function (phytosterols), specialized terpenoid metabolites, in particular, have been recognized for an array of biological roles. Moreover, using of terpenoids for more specialized chemical interactions and protection in the abiotic and biotic environment (Tholl, 2015; Loreto et al., 2014; Behnke et al., 2007; Velikova et al., 2014). On the other hand, the typical defense compounds of Plantaginaceae are the iridoid glycosides. In plants, glycosidic defense compounds and hydrolytic enzymes often form a dual defense system, in which the glycosides are activated by the enzymes to exert biological effects. The concentration of iridoid glycosides as well as the β-glucosidase activity in leaves of different ages. The concentrations of defense compounds as well as the β-glucosidase activity, were highest in younger leaves and decrease with increasing leaf age (Buschmann and Muller, 2013). Thus, all previous research strongly supported for our results that the aucubin content do differ in vegetative stages and even ecological conditions. 3.2 Effect of ecotypes on aucubin content According to Andrzejewska-Golec (1995), among species of the genus Plantago, there are 38 investigated species, which contain aucubin and differ in their aucubin content. Beside the accumulation of secondary metabolites highly depends on external factors, they also strongly depend on their gene expression through environmental interaction. The data in Table 12 indicates that the aucubin content of P. depressa (21.87, 14.37 and 14.17 mg/g at experimental sites of Middle Khalkh dry steppe, Dzungarian Gobi desert and East Mongolia steppe respectively) is higher than in P. major (12.78, 13.41, 7.43, 4.83, 3.12, 13.67 and 2.08 mg/g; Mongolian Altai Mountain steppe, Alashan Gobi desert, Can Tho, Ca Mau, Kien Giang, Nam Dinh and Thanh Hoa sites, respectively), significant at 0.01 level. The highest is at Middle Khalkh dry steppe site and the lowest at Thanh Hoa site. Furthermore, the aucubin content of plantains originated in Mongolia are much higher than those in Vietnam (Table 3). Although in our study we set up all ecotypes in the same conditions, aucubin content of those differed. It could be due to some reason: Aucubin is an iridoid glycoside, which belongs to Terpenoids. In pathway of biosynthesis of iridoids in plant, aucubin is an intermediate chemical (Damtoft et al., 1993). I