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Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth.

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Waqas Khan Kayani a, Rehana Rani a, Ihsan-ul-Haq b, Bushra Mirza a,⇑ a b

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

a r t i c l e

i n f o

Article history: Received 28 October 2013 Received in revised form 6 March 2014 Accepted 29 April 2014 Available online xxxx Keywords: Ajuga bracteosa HPLC Morphology 20-Hydroxyecdysone Phytogeography

a b s t r a c t Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metabolism and morphology of a plant, the present study was aimed at evaluating the impact of phytogeography, season and tissue type on morphology and 20-HE content of A. bracteosa. The results revealed large morphological variations in various ecotypes of A. bracteosa. However, plants from the same altitude, regardless of their phytogeography, represented similar morphology. Effect of habitat on 20-HE content remained non-significant except for Karot (1608 lg/g) and Kahuta (728 lg/g). Effect of tissue types was significant (p value summer (617 lg/g). The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amount radically rose in flowers (l = 2814 lg/g). The aerial portion of Karot ecotype harvested in winter offers a valuable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracteosa raised in vitro at different temperature regime was carried out. On the basis of these results we hypothesize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as a defense response. Ó 2014 Published by Elsevier Inc.

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1. Introduction


Ajuga bracteosa Wall. ex Benth. (Labiatae) is a valuable aromatic, medicinal, soft, villous and decumbent herb of 10–30 cm height [1,2]. It is found on exposed slopes, grasslands and open fields in subtropical and temperate regions of the world [3] at an altitude ranging from 1300 to 2400 m [4]. Large morphological variations have been reported in its ecotypes [2]. It is used as a medicine since ancient times and has a variety of applications. In ethnomedicine, its use is reported as an astringent, hypoglycemic, anthelmintic, antifungal, antibacterial, anti-inflammatory and it also remediates gastrointestinal disorders [5]. A. bracteosa is traditionally used to treat fever and phlegm in China [6]. It is recommended in Ayurveda to treat gout, palsy, amenorrhea and rheumatism [7,8]. Leaves of A. bracteosa are stimulant, diuretic and locally used to treat malaria [7,9], hence regarded as an alternate of cinchona [10].

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⇑ Corresponding author. Tel.: +92 51 90643007; fax: +92 51 90644050.

A. bracteosa contains a variety of important categories of compounds including neo-clerodane diterpenoids, iridoid glycosides, withanolides and phytoecdysteroids [5]. Phytoecdysteroids are polyhydroxysteroids which are usually present in plants in small amounts [11], while animals contain even lesser ecdysteroids than plants [12]. In plants, they act as growth regulators generally [13], and in some species actively defend them against insect predation [14]. 20-Hydroxyecdysone (20-HE) is one of the naturally occurring phytoecdysteroids (Fig. 1). In plants, 20-HE imitates the indigenous hormones which induce a lethal precocious ecdysis of arthropods upon ingestion [15]. In insects, 20-HE gradually reduces feeding and induces starvation, resulting in fat body lipolysis [16]. Studies conducted on mice models, in which body fat, plasma insulin levels and glucose tolerance decreased, proved the anti-obesitic and anti-diabetic potentials of 20-HE [17]. Human trials exhibited that 20-HE declines body fat, increases muscle mass and improves athletes’ performance [18]. A few reports reveal biosynthesis and accumulation of 20-HE in plant kingdom. Its content depends on climatic conditions [11] and

E-mail address: [email protected] (B. Mirza). 0039-128X/Ó 2014 Published by Elsevier Inc.

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Fig. 1. Structural formula of 20-hydroxyecdysone.

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varies during plant development [19]. Annual plants accumulate maximum ecdysteroids in their apical regions while perennials recycle them in their deciduous organs and perennial tissues. In a study conducted on Achyranthes japonica, 20-HE was found increasing in first leaf pair stage followed by a decrease with vegetative growth of the plant [14]. It was also estimated in the aerial portion of some species of asteraceae and caryophyllaceae [20]. 20HE has been detected in more than 100 plant families [21] and in some members of Chenopodiaceae, ecdysteroids content differs largely among different organs of the same plant [22]. However its spatial and temporal tissue type-based estimation was conducted only in Pfaffia glomerata [23]. The effect of transformation on 20-HE content has also been studied in some plants. For example 20-HE production was found in higher amounts in regenerants derived from hairy roots of Ajuga reptans [24] while a tenfold increase was noted in transformed Ajuga multiflora hairy roots as compared to the wild type [25]. To the best of our knowledge, though various tissue types have been assessed for 20-HE estimation in a few plant species, yet not a single comprehensive report exists explaining the accumulative effect of key factors i.e. season, habitat and climate. A. bracteosa can be a potential source of 20-HE [5] but it has not been subjected to any of such estimation yet. In this study, 20-HE content is evaluated in naturally growing A. bracteosa at various altitudes and in different seasons to identify the right tissue, geographical location and season for its maximum harvest. Moreover, various morphological characteristics have been studied in selected chemotypes.

University campus; HMP-460), Kahuta (Rawalpindi; HMP-461), Karot (Eastern Rawalpindi; HMP-462), Sehnsa (District Kotli, AJK; HMP-463), Sarsawa (District Kotli, AJK; HMP-464) and Neelum Valley (District Neelum, AJK; HMP-465) and abbreviated as IS, KH, KR, SE, SA and NV respectively. The plants were identified by Prof. Dr. Rizwana Aleem Qureshi (taxonomist) in Plant Sciences Department Quaid-i-Azam University (QAU). A voucher specimen of each location (numbering HMP-460–465) was deposited in the ‘‘Herbarium of medicinal Plants of Pakistan’’ in QAU Islamabad, Pakistan. The plant material was collected in three consecutive seasons i.e. summer and winter of 2011, and spring of 2012.


2.2.2. Morphological study For morphological analysis, several parameters were studied including plant height, stem branching, stem color, number of leaves, leaf color, flower color, number of flowers per plant, flowering time, root branching, presence of hairs and nodules.


2.2.3. Surface sterilization and tissue culture conditions Healthy plants of A. bracteosa were collected from the lawns of Department of Biotechnology Quaid-i-Azam University. Nodal sections of these plants were taken as explants. They were washed under running tap water for 1 h. It was followed by washing with sodium hypochlorite (15% v/v) for 15 min and four times washing with distilled autoclaved water in laminar flow hood. Afterwards, washing of these explants was performed with 70% ethanol (v/v) for 30 s followed by rinsing in 0.1% mercuric chloride and repeated washing with distilled autoclaved water. Finally, these explants were blotted on sterile filter paper and cultured on MS [26] basal medium containing 3% sucrose and solidified with 0.8% agar.


2.2.4. Temperature treatments Nodal explants of 1 month old tissue cultured A. bracteosa were chosen for the treatment to a range of temperature [10 °C,15 °C,20 °C,25 °C (controlplant) and 30 °C]. These explants were inoculated on MS medium solidified with 0.8% agar in sterilized magenta jars. Five groups of 30 explants were assigned to each temperature range and incubated in identical growth chambers for a period of one month. The cultures were maintained at 50–60% relative humidity and 16 h illumination (2000 lux). The medium was changed after 2 weeks. After one month, fully grown plantlets were removed, washed thoroughly with distilled water and their fresh biomass was calculated. The plantlets were rinsed gently with distilled water to remove media and further processed as described in Section 2.2.3.


2.2.5. Plant processing for 20-hydroxyecdysone analysis After collection (20 adult plants as mentioned in Section 2.2.1), the plants were rinsed with distilled water to remove soil/mud from roots and dust from aerial parts. It was followed by gentle separation of four parts (leaves, flowers, stem and roots) and subsequent air drying under shade. Fully dried plant material (both from wild type and temperature treated in vitro grown shoots mentioned in Section 2.2.4) was subjected to vacuum drying in vacucell under 0.1 bar pressure to ensure that it is completely moisture free. These parts were separately homogenized to fine powder in lab-scale grinder with short intervals under controlled temperature of milling. The ground powder was sealed in air tight bags and stored at 20 °C till further processing.


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2. Experimental


2.1. General



GPS (Giko 301, Garmin) was used to record geographical data. Vacucell (Vacucell 55, MMM, Germany) was used to dry the plant material. Lab-scale blender was used for grinding plant material. HPLC grade solvents (Sigma Aldrich, GmbH Buchs Switzerland) were used for extraction of 20-HE with the help of Elmasonic Sonicator (E30-H Germany). Centrifugation was performed in eppendorf 5417C (Germany) and filtration with Sartolong Polyamide filter paper (Germany). Samples were stored at 70 °C (Thermo Electron Corporation, 5702, USA). HPLC analysis was performed with a Discovery C18 HPLC column (Agilent 1200 series, SUPELCO USA) using a Diode Array Detector (G1315B-DAD). 2.2. Plant material

2.3. Extraction solvent for 20-hydroxyecdysone and sample preparation


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2.2.1. Collection A. bracteosa Wall. ex Benth. (Labiatae) was collected from six different locations of Pakistan, viz. Islamabad (Quaid-i-Azam

20-HE was extracted according to the standard procedure [27] with some modifications. Briefly, powdered plant material (200 mg each of leaves, roots, flowers and stems of all seasons


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W.K. Kayani et al. / Steroids xxx (2014) xxx–xxx Table 1 Folklore name of A. bracteosa from collected habitats with geographical parameters.

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S. No.


Vern. name


Elev. (m)



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Campus, QAU (Islamabad) Kahuta (Rawalpindi) Karot (Eastern Rawalpindi) Sehnsa (District Kotli, AJK) Sarsawa (District Kotli, AJK) Neelum Valley (AJK)

Booti Kora Kora Kori booti Kora booti Jan-e-Adam


600 723 462 644 966 1817

33.74949° 33.59634° 33.59904° 33.51127° 33.53311° 34.430743°

73.14905° 73.53209° 73.60848° 73.74409° 73.78472° 74.35156°

and locations) was soaked in 1 ml of methanol and ethyl acetate (1:1 v/v) and sonicated for 5 min at 25 °C under 50/60 Hz. It was followed by occasional shaking for 20 min. Following sonication, shaking and re-sonication (as described earlier), the mixture was centrifuged for 5 min at 13,000 rpm and supernatant was filtered with 0.2 lm pore size (25 mm) filter paper.


2.4. HPLC analysis


For HPLC analyses of 20-HE, already optimized method [28] was followed with slight modifications according to the system’s suitability. Mobile phase A (acetonitrile:methanol:water:acetic acid: :5:10:85:1 v/v) and B (acetonitrile:methanol:acetic acid::40:60:1 v/v) were prepared and filtered through 47 mm pore size filter paper followed by sonication for 5 min to degas the solvents. The

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gradient program started with 0% B, followed by a gradual increase to 50% B at 20 min, which in turn was followed by 100% B in 25 min and finally a linear gradient to 0% B during 30–40 min. Injection volume was kept 20 ll. Analysis was performed with a C18 HPLC column (250  4.6 mm and 5 lm particle size). Flow rate of mobile phase was optimized to 1 ml/min and constant pressure of 350 bar. 20-HE was detected by using a Diode Array Detector at an absorbance of 245 nm with retention time12–13 min.


2.5. Statistical analysis


Statistical analysis including descriptive statistics and ANOVA was performed on XlStat and Origin 7.5Ò SR6 Software. Multivariate analysis such as Principle Component Analysis (PCA) was performed by SPSS Statistical Package (version 16.0).


Fig. 2. Map showing the collection sites.

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Fig. 3. Pictorial presentation of the representative samples for morphological analysis of A. bracteosa.


3. Results


3.1. Morphological analysis


3.1.1. Effect of different seasons and geographical locations on the morphology The plant samples were collected from quite diverse areas with respect to altitude (Table 1) and climate. The areas of collection

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ranged from temperate to sub-tropical (Fig. 2). Summer is generally hot here and winter very cold. High fluctuation is recorded in temperature (Supplementary Fig. 1). Snowfall occurs only in NV habitat. Multiple parameters of morphology were studied (Supplementary Table 1). A. bracteosa is short lived, glandular and hairy with leaves nearly alternate and simple ex-stipulate, and flowers in verticillaster inflorescence. Results reveal that this endangered herb possesses remarkable diversity as shown by its different

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Fig. 4. Root of (a) SE ecotype, (b) SA ecotype and (c) IS ecotype.


ecotypes. Vegetative growth was found directly proportional to the plant length. IS ecotype represented maximum height during summer followed by SE. Minimum plant height was recorded in NV ecotype in all seasons and in KH during winter. Maximum number of stem branches was recorded in KH ecotype while in SE, IS and SA, branching is rather rare. KH ecotype exhibited supreme vegetative growth in comparison with other ecotypes (Fig. 3). Studied ecotypes also differed considerably in leaf color. Green or light green leaf color was commonly found in majority of ecotypes. On the contrary, KH ecotype showed purplish indigo color, especially on lower side of the lamina, whereas NV ecotype had dark green leaves, specifically during winter. Normally, petals of the plants were found white or white with blue lines, but NV ecotype has shown seasonal variation in petal color i.e. blue petals in winter, light blue in spring and blue with white lines during summer. Regardless of petal color, all the studied ecotypes contained white corolla tube. Moreover, ecotypes of IS, SE and KH contained maximum number of flowers/plant (70). Roots of the studied ecotypes were without nodules except for SE ecotype which displayed prominent nodules throughout the year (Fig. 4). Aerial portions, especially leaf and stem of KH and KR ecotypes did not contain white hairs while the plant from the rest of the habitats contained white hair in all seasons. Besides, least root branching was recorded in KH ecotype. These branches were dense and thin during winter.


3.2. 20-Hydroxyecdysone analysis


3.2.1. Effect of different seasons and geographical locations on 20-HE biosynthesis in different tissues 20-HE content was estimated keeping in view the effect of season, tissue type and habitat of collection. All the possible combinations were analyzed to scrutinize the significant parameters affecting 20-HE content. Individual effect of each parameter on 20-HE content represented KR as the best habitat (Fig. 5a). Habitats remained non-significant with reference to each other and followed the descending order: KR (1608 lg/g) > SE (1456 lg/g) > NV (1238 lg/g) > SA (1116 lg/g) > IS (1032 lg/g) > KH (728 lg/g). Seasons contributed significantly (p value < 0.001) to the 20-HE content with the descending order: winter (1902 lg/g) > spring (1071 lg/g) > summer (617 lg/g) (Fig. 5b). Tissue types also remained significant with reference to each other for 20-HE content (p value leaf (1376 lg/g) > stem (1098 lg/g) > root (692 lg/g). Based on the interaction of tissue type to the habitat, the overall trend pointed out that 20-HE content follows the ascending order: root < stem < leaf < flower. High 20-HE yielding ecotypes (KR and SE) followed the same pattern, while in IS and SA ecotypes, leaf displayed maximum amount of 20-HE. In general, maximum amount of 20-HE was detected in the aerial parts of the plants of all studied habitats (Fig. 6a). Tissue type versus season interaction exhibited that 20-HE content followed the general pattern: leaf > flower >

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stem > root, but during winter its amount was raised in flowers to the maximum (2814 lg/g). The amount of 20-HE is minimum in summer and gradually increases in spring and reaches its maximum level in winter (p value

Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth.

Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As ...
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