seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different...

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Steroids xxx (2014) xxx–xxx

STE 7560 No. of Pages 9, Model 5G

14 May 2014

Contents lists available at ScienceDirect

Steroids

journal homepage: www.elsevier .com/locate /s teroids

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

http://dx.doi.org/10.1016/j.steroids.2014.04.0170039-128X/� 2014 Published by Elsevier Inc.

⇑ Corresponding author. Tel.: +92 51 90643007; fax: +92 51 90644050.E-mail address: [email protected] (B. Mirza).

Please cite this article in press as: Kayani WK et al. Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in dtissue types of wild Ajuga bracteosa Wall. ex Benth. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.017

Waqas Khan Kayani a, Rehana Rani a, Ihsan-ul-Haq b, Bushra Mirza a,⇑a Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistanb 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 a b s t r a c t

293031323334353637383940

Article history:Received 28 October 2013Received in revised form 6 March 2014Accepted 29 April 2014Available online xxxx

Keywords:Ajuga bracteosaHPLCMorphology20-HydroxyecdysonePhytogeography

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Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeuticimportance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metab-olism 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 largemorphological 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 contentremained non-significant except for Karot (1608 lg/g) and Kahuta (728 lg/g). Effect of tissue types wassignificant (p value <0.016) for 20-HE content and followed ascending order: root < stem < leaf < flower,representing the tender aerial tissues’ hormonal supremacy. Seasons showed a significant impact (p value<0.001) on 20-HE content with the pattern: winter (1902 lg/g) > spring (1071 lg/g) > summer (617 lg/g).The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amountradically rose in flowers (l = 2814 lg/g). The aerial portion of Karot ecotype harvested in winter offers avaluable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracte-osa raised in vitro at different temperature regime was carried out. On the basis of these results we hypoth-esize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as adefense response.

� 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 insubtropical and temperate regions of the world [3] at an altituderanging from 1300 to 2400 m [4]. Large morphological variationshave been reported in its ecotypes [2]. It is used as a medicine sinceancient 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 remediatesgastrointestinal disorders [5]. A. bracteosa is traditionally used totreat fever and phlegm in China [6]. It is recommended in Ayurvedato 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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A. bracteosa contains a variety of important categories of com-pounds including neo-clerodane diterpenoids, iridoid glycosides,withanolides and phytoecdysteroids [5]. Phytoecdysteroids arepolyhydroxysteroids which are usually present in plants in smallamounts [11], while animals contain even lesser ecdysteroids thanplants [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 occur-ring phytoecdysteroids (Fig. 1). In plants, 20-HE imitates the indig-enous hormones which induce a lethal precocious ecdysis ofarthropods upon ingestion [15]. In insects, 20-HE graduallyreduces feeding and induces starvation, resulting in fat body lipol-ysis [16]. Studies conducted on mice models, in which body fat,plasma insulin levels and glucose tolerance decreased, proved theanti-obesitic and anti-diabetic potentials of 20-HE [17]. Human tri-als exhibited that 20-HE declines body fat, increases muscle massand improves athletes’ performance [18].

A few reports reveal biosynthesis and accumulation of 20-HE inplant kingdom. Its content depends on climatic conditions [11] and

ifferent

http://dx.doi.org/10.1016/j.steroids.2014.04.017

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http://www.elsevier.com/locate/steroids


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Fig. 1. Structural formula of 20-hydroxyecdysone.

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varies during plant development [19]. Annual plants accumulatemaximum ecdysteroids in their apical regions while perennialsrecycle them in their deciduous organs and perennial tissues. Ina study conducted on Achyranthes japonica, 20-HE was foundincreasing in first leaf pair stage followed by a decrease with veg-etative growth of the plant [14]. It was also estimated in the aerialportion of some species of asteraceae and caryophyllaceae [20]. 20-HE has been detected in more than 100 plant families [21] and insome members of Chenopodiaceae, ecdysteroids content differslargely among different organs of the same plant [22]. Howeverits spatial and temporal tissue type-based estimation was con-ducted only in Pfaffia glomerata [23]. The effect of transformationon 20-HE content has also been studied in some plants. For exam-ple 20-HE production was found in higher amounts in regenerantsderived from hairy roots of Ajuga reptans [24] while a tenfoldincrease was noted in transformed Ajuga multiflora hairy roots ascompared to the wild type [25].

To the best of our knowledge, though various tissue types havebeen assessed for 20-HE estimation in a few plant species, yet not asingle comprehensive report exists explaining the accumulativeeffect of key factors i.e. season, habitat and climate. A. bracteosacan be a potential source of 20-HE [5] but it has not been subjectedto any of such estimation yet. In this study, 20-HE content is eval-uated in naturally growing A. bracteosa at various altitudes and indifferent seasons to identify the right tissue, geographical locationand season for its maximum harvest. Moreover, various morpho-logical characteristics have been studied in selected chemotypes.

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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 plantmaterial. 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 Soni-cator (E30-H Germany). Centrifugation was performed in eppendorf5417C (Germany) and filtration with Sartolong Polyamide filterpaper (Germany). Samples were stored at �70 �C (Thermo ElectronCorporation, 5702, USA). HPLC analysis was performed with a Dis-covery C18 HPLC column (Agilent 1200 series, SUPELCO USA) usinga Diode Array Detector (G1315B-DAD).

2.2. Plant material

2.2.1. CollectionA. bracteosa Wall. ex Benth. (Labiatae) was collected from six

different locations of Pakistan, viz. Islamabad (Quaid-i-Azam

Please cite this article in press as: Kayani WK et al. Seasonal and geographicaltissue types of wild Ajuga bracteosa Wall. ex Benth. Steroids (2014), http://dx.

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 NeelumValley (District Neelum, AJK; HMP-465) and abbreviated as IS,KH, KR, SE, SA and NV respectively. The plants were identified byProf. Dr. Rizwana Aleem Qureshi (taxonomist) in Plant SciencesDepartment Quaid-i-Azam University (QAU). A voucher specimenof 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 consecutiveseasons i.e. summer and winter of 2011, and spring of 2012.

2.2.2. Morphological studyFor morphological analysis, several parameters were studied

including plant height, stem branching, stem color, number ofleaves, leaf color, flower color, number of flowers per plant, flower-ing time, root branching, presence of hairs and nodules.

2.2.3. Surface sterilization and tissue culture conditionsHealthy plants of A. bracteosa were collected from the lawns of

Department of Biotechnology Quaid-i-Azam University. Nodal sec-tions of these plants were taken as explants. They were washedunder running tap water for 1 h. It was followed by washing withsodium hypochlorite (15% v/v) for 15 min and four times washingwith 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 repeatedwashing with distilled autoclaved water. Finally, these explantswere blotted on sterile filter paper and cultured on MS [26] basalmedium containing 3% sucrose and solidified with 0.8% agar.

2.2.4. Temperature treatmentsNodal 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 explantswere inoculated on MS medium solidified with 0.8% agar in steril-ized magenta jars. Five groups of 30 explants were assigned to eachtemperature range and incubated in identical growth chambers for aperiod of one month. The cultures were maintained at 50–60% rela-tive humidity and 16 h illumination (2000 lux). The medium waschanged after 2 weeks. After one month, fully grown plantlets wereremoved, washed thoroughly with distilled water and their freshbiomass was calculated. The plantlets were rinsed gently with dis-tilled water to remove media and further processed as describedin Section 2.2.3.

2.2.5. Plant processing for 20-hydroxyecdysone analysisAfter collection (20 adult plants as mentioned in Section 2.2.1),

the plants were rinsed with distilled water to remove soil/mudfrom roots and dust from aerial parts. It was followed by gentleseparation of four parts (leaves, flowers, stem and roots) and sub-sequent air drying under shade. Fully dried plant material (bothfrom wild type and temperature treated in vitro grown shootsmentioned in Section 2.2.4) was subjected to vacuum drying invacucell under 0.1 bar pressure to ensure that it is completelymoisture free. These parts were separately homogenized to finepowder in lab-scale grinder with short intervals under controlledtemperature of milling. The ground powder was sealed in air tightbags and stored at �20 �C till further processing.

2.3. Extraction solvent for 20-hydroxyecdysone and samplepreparation

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

impact on the morphology and 20-hydroxyecdysone content in differentdoi.org/10.1016/j.steroids.2014.04.017


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Table 1Folklore name of A. bracteosa from collected habitats with geographical parameters.

S. No. Habitat Vern. name Abbrv. Elev. (m) �N �E

1 Campus, QAU (Islamabad) Booti IS 600 33.74949� 73.14905�2 Kahuta (Rawalpindi) Kora KH 723 33.59634� 73.53209�3 Karot (Eastern Rawalpindi) Kora KR 462 33.59904� 73.60848�4 Sehnsa (District Kotli, AJK) Kori booti SE 644 33.51127� 73.74409�5 Sarsawa (District Kotli, AJK) Kora booti SA 966 33.53311� 73.78472�6 Neelum Valley (AJK) Jan-e-Adam NV 1817 34.430743� 74.35156�

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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 wasfollowed by occasional shaking for 20 min. Following sonication,shaking and re-sonication (as described earlier), the mixture wascentrifuged for 5 min at 13,000 rpm and supernatant was filteredwith 0.2 lm pore size (25 mm) filter paper.

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2.4. HPLC analysis

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

Fig. 2. Map showing th


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

2.5. Statistical analysis

Statistical analysis including descriptive statistics and ANOVAwas performed on XlStat and Origin 7.5� SR6 Software. Multivari-ate analysis such as Principle Component Analysis (PCA) was per-formed by SPSS Statistical Package (version 16.0).

e collection sites.



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



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3. Results

3.1. Morphological analysis

3.1.1. Effect of different seasons and geographical locations on themorphology

The plant samples were collected from quite diverse areas withrespect to altitude (Table 1) and climate. The areas of collection


ranged from temperate to sub-tropical (Fig. 2). Summer is gener-ally hot here and winter very cold. High fluctuation is recordedin temperature (Supplementary Fig. 1). Snowfall occurs only inNV habitat. Multiple parameters of morphology were studied (Sup-plementary Table 1). A. bracteosa is short lived, glandular and hairywith leaves nearly alternate and simple ex-stipulate, and flowers inverticillaster inflorescence. Results reveal that this endangeredherb 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.



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ecotypes. Vegetative growth was found directly proportional to theplant length. IS ecotype represented maximum height during sum-mer followed by SE. Minimum plant height was recorded in NVecotype in all seasons and in KH during winter. Maximum numberof stem branches was recorded in KH ecotype while in SE, IS andSA, branching is rather rare. KH ecotype exhibited supreme vegeta-tive growth in comparison with other ecotypes (Fig. 3).

Studied ecotypes also differed considerably in leaf color. Greenor light green leaf color was commonly found in majority of eco-types. On the contrary, KH ecotype showed purplish indigo color,especially on lower side of the lamina, whereas NV ecotype haddark green leaves, specifically during winter. Normally, petals ofthe plants were found white or white with blue lines, but NV eco-type has shown seasonal variation in petal color i.e. blue petals inwinter, light blue in spring and blue with white lines during sum-mer. Regardless of petal color, all the studied ecotypes containedwhite corolla tube. Moreover, ecotypes of IS, SE and KH containedmaximum number of flowers/plant (�70). Roots of the studiedecotypes were without nodules except for SE ecotype which dis-played prominent nodules throughout the year (Fig. 4). Aerial por-tions, especially leaf and stem of KH and KR ecotypes did notcontain white hairs while the plant from the rest of the habitatscontained white hair in all seasons. Besides, least root branchingwas recorded in KH ecotype. These branches were dense and thinduring winter.

3.2. 20-Hydroxyecdysone analysis

3.2.1. Effect of different seasons and geographical locations on 20-HEbiosynthesis in different tissues

20-HE content was estimated keeping in view the effect of sea-son, tissue type and habitat of collection. All the possible combina-tions were analyzed to scrutinize the significant parametersaffecting 20-HE content.

Individual effect of each parameter on 20-HE content repre-sented KR as the best habitat (Fig. 5a). Habitats remained non-sig-nificant with reference to each other and followed the descendingorder: 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 contributedsignificantly (p value < 0.001) to the 20-HE content with thedescending order: winter (1902 lg/g) > spring (1071 lg/g) > sum-mer (617 lg/g) (Fig. 5b). Tissue types also remained significantwith reference to each other for 20-HE content (p value <0.016)(Fig. 5c) with the descending order: flower (1619 lg/g) > leaf(1376 lg/g) > stem (1098 lg/g) > root (692 lg/g).

Based on the interaction of tissue type to the habitat, the overalltrend pointed out that 20-HE content follows the ascending order:root < stem < leaf < flower. High 20-HE yielding ecotypes (KR andSE) followed the same pattern, while in IS and SA ecotypes, leaf dis-played maximum amount of 20-HE. In general, maximum amountof 20-HE was detected in the aerial parts of the plants of all studiedhabitats (Fig. 6a). Tissue type versus season interaction exhibitedthat 20-HE content followed the general pattern: leaf > flower >


stem > root, but during winter its amount was raised in flowersto the maximum (2814 lg/g). The amount of 20-HE is minimumin summer and gradually increases in spring and reaches its max-imum level in winter (p value <0.002) suggesting that low temper-ature is favorable for its accumulation (Fig. 6b).When seasons wereplotted against habitats for 20-HE content, the same trend wasfound, winter being the best season (p value <0.005) followed byspring and summer. However, the ecotypes of KH and IS deviatedfrom this pattern and remained almost consistent in 20-HE distri-bution throughout the year in all tissue types (Fig. 6c).

3.2.2. Effect of different temperature treatments on 20-HE biosynthesisin in vitro grown plants

To confirm the effect of low temperature we executed 20-HEprofiling of A. bracteosa raised in vitro at different temperatureregimes. Qualitative analysis of in vitro raised plants at varioustemperature conditions was carried out by using reverse phaseHPLC. Results revealed that temperature treatment significantlyaffected the production of 20-HE (p < 0.05) (Table 2) with the max-imum amount in plants raised at 15 ± 1 �C. Other temperaturetreatments did not induce appreciable biosynthesis of 20-HE.

3.3. Principal component analysis (PCA)

Principal Component Analysis (PCA) was conducted to elucidatethe effect of studied factors on the 20-HE content of the plant. PCAinitially extracted two components (PC-I and PC-II), collectivelyaccounting for 99.84% variance of the data (Fig. 7).

The clusters of items in PC-I accounted for a large proportion ofthe variance (91.37%) and represented 80% of the total data (G-1and G-2). G-1 revealed loadings (39% of the samples) which repre-sented maximum amount of 20-HE yielding parameters rangingfrom 453 to 3719 lg/g dry weight (DW) of the plant. Aerial por-tions of the plants collected in winter and spring dominated G-1(83%). G-2 is the largest group of this plot exhibiting 41% of thetotal data. The plants in this group exhibited moderate 20-HE con-tent ranging from 322 to 2517 lg/g DW of the plant. It comprises50% of the plant samples collected during spring season. All thethree aerial portions have almost the same number in G-2, con-trary to relatively low representation of root tissue (16%). Overalltrend of PC-I specifies dominancy of aerial tissue types to possess20-HE and winter season as significant contributors stimulating20-HE accumulation (progressive effect), and decreased localiza-tion during the summer season in root tissue type (regressiveeffect).

PC-II accounted for a minor proportion of the variance (8.47%)representing 20% of the data. G-1 of the PC-II represented a smallamount of 20-HE contents ranging from 116 to 665 lg/g DW ofthe plant. This component represented small amount of 20-HEunder the negative influence of summer season especially in theroot. It contains 75% of the plants collected during summer, 25%during spring, and also 50% root tissues. Contrary to this, G-2explains 14% of the data and harbors factors responsible for least



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Fig. 5. Effect of (a) habitats, (b) seasons and (c) tissue types on 20-HE content (lg/g)at 0.05a and values are presented as mean ± SD of at least three independent

Q2 experiments.

Fig. 6. Effect of (a) habitats versus tissue types (b) seasons versus tissue types and(c) habitats versus in seasons on 20-HE content (lg/g) at 0.05a and values arepresented as mean ± SD of at least three independent experiments.



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20-HE contents (119–576 lg/g DW of the plant). G-2 contains 50%root tissues and consists exclusively of 100% of the plants collectedduring summer.

4. Discussion

A. bracteosa is a widely distributed medicinal plant with severalchemotypes. In this study, these chemotypes were assessed on


morphological basis and screened for 20-HE distribution in differ-ent tissue types collected during different seasons from variousgeographical locations of Pakistan. Large morphological variationswere observed in different chemotypes of A. bracteosa. Plants fromKH habitat possessed maximum vegetative growth which is prob-ably due to environmental feasibility i.e. temperature and rainfall.However, plants collected from the same altitude regardless of



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Table 2Fresh biomass and 20-HE biosynthesis in in vitro cultured A. bracteosa under differenttemperature regimes.

Temperature (�C) Fresh biomass (g) 20-HE (lg/g DW)

10 ± 1 3.06 ± 0.93 670 ± 5.715 ± 1 7.1 ± 0.9 1980 ± 22.9120 ± 1 13.99 ± 1.1 1002 ± 12.530 ± 1 17.4 ± 1.2 852 ± 11.525 ± 1 (Control) 19.3 ± 2.6 788 ± 11.9



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their geographical location, represented similar vegetative growthpattern.

A. bracteosa is a non-leguminous plant, but interestingly SE eco-type possesses enormous number of root nodules throughout theyear. This suggests that it can be an actinorhizal host, being capa-ble of nodules formation when infected with a nitrogen-fixing acti-nomycete Frankia [29]. We found enormous color shift in differenttissue types. Stem and leaf color was green with purple or indigotinge during spring, light green in summer and dark green withpurple to pink or indigo shade in winter. In mature leaves, rise incarbohydrate concentration triggers plants to use surplus amountsto produce anthocyanin. The flowers of A. bracteosa are white orwhite with blue lines but NV ecotype contains blue flowers duringwinter. Ontogenetic color changes in flowers are widespreadthroughout the angiosperms [30]. Anthocyanin pigments are con-sidered to be responsible for blue colors of various tissue typesin majority of plants [31]. Anthocyanin undergoes acylation toenhance and stabilize color intensity and can induce blue color[32].

HPLC analysis of collected plant samples was performed toassess 20-HE concentration. Earlier it has been suggested thatdevelopmental stage, habitat and season of the plant collection

Fig. 7. Principal Component Analysis of tissue type and season based localization of 20-Hthird letter representing tissue type (L = leaf, F = flower, S = stem and R = root) and last t


affect the concentration of the phytoecdysteroids [5,19,33–35].Distribution of phytoecdysteroids has been reported and their fluc-tuation at different growth stages has been scrutinized in manyplant species including A. reptans [14,35]. In the present study,20-HE was found in all tissue types of A. bracteosa which is inaccordance with a previous study [36]. Maximum amount of 20-HE was found in flowers i.e. 1619 lg/g (0.16%) followed by leaf(1377 lg/g) and stem (1098 lg/g) while root contained the leastamount of all. Compared to our observation, exploration of P. glom-erata for 20-HE revealed its maximum amount in flowers (0.82%)followed by roots (0.66%), leaves (0.60%), and stems (0.24%) [23].

We found that flowers of A. bracteosa from KR and SE habitatsdisplayed the highest contents of 20-HE (1608 lg/g and 1456 lg/g respectively). Both KR and SE habitats are located in North Westof Pakistan. These locations have minimum altitude and a hilly ter-rain. Although IS location is also at the same altitude, its geograph-ical location is different as it is situated near the hot plains ofPunjab. Moreover, the ecotype of SE contains the second highestplant height and maximum number of flowers (�70) followed bythat of KR which contains maximum branches. Based upon thesefacts, it seems that the aerial portion of the said plants from hillyzones can be ideal for 20-HE harvest.

Seasons contributed significantly to the 20-HEcontent of theplant representing maximum amount during winter (1902 lg/g),followed with a significantly low value in spring (1071 lg/g) andthe least in summer (616 lg/g). Although the summer season pre-sents maximum vegetative growth of plants, probably due to enor-mous rainfall at all the studied habitats, it exhibits the leastamount of 20-HE. 20-HE content is reported to decrease duringmaximum vegetative growth [37]. Principal Component Analysissignified that aerial tissue types represented highest 20-HE con-tents (up to 0.37%) under the influence of winter season (G-1 of

E in studied habitats. Each case is named as; first two letters representing habitat,wo letters representing seasons (Sum = summer, Sp = spring and Win = winter).



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PC-I) while summer season has negative impact on 20-HE contentespecially in root (0.06%) (G-1 of PC-II).Varying amount of 20-HEover seasons can be related to defense response of A. bracteosatowards low temperatures. During spring, when average tempera-ture is below the optimum temperature for plant growth, 20-HEwas found in moderate amount and its quantity gradually fell dur-ing summer. However, when the low temperature stress hamperedthe growth of this plant during winter, maximum quantity of 20-HE was observed which indicates its accumulation and defensefunction in response to cold stress. Earlier it has been observed thatecdysteroids increase in amount on mechanical damage [38],which indicates their involvement in defense mechanism [39].20-HE has tissue specific functions in plants and may have ecolog-ical significance too [23]. When phytophagus insects feed on theplants with higher phytoecdysteroid content, they undergo lethaleffects e.g. premature molting, weight loss and metabolic defects[40,41]. Our study has revealed that soft tissues contain higheramount of 20-HE as compared to harder parts. As tissue hardnessincreases, 20-HE concentration decreases, suggesting that juveniletissues are more prone to the attack of grazers, herbivores andenvironmental constraints supporting the idea of stress inducedaccumulation of 20-HE in tender aerial parts.

Aerial portions of A. bracteosa (flower, stem and leaf) containmaximum 20-HE quantity as previously found in spinach [35].20-HE was found distributed in apical leaves and stems of spinach,suggesting its biosynthesis in older leaves (source) and transloca-tion to younger leaves (sink) [35] where it may be accumulated[42]. 20-HE content increases on tissue injury [38], methyl jasmo-nate application and insect predations [43] which supports 20-HEinvolvement in plant protection and defense [44]. It is a well-known fact that in defense, plants produce jasmonic acid, whichtriggers defense responses [45]. Jasmonic acid (JA) also induces risein 20-HE content, which suggests that JA pathway is involved insignaling the damage-induced accumulation of 20-HE [38,43].

Based on the results of the experiments conducted on wild typeplants, it was hypothesized that low temperatures during winterswould act as an elicitor in situ and would be crucial in profoundproduction of 20-HE in A. bracteosa. The temperature treatmentexperiment performed in control conditions confirmed maximum20-HE biosynthesis procurement at low temperature (15 ± 1 �C).This suggests that ontogeny or developmental stages could notbe the only cause for increased production of 20-HE in A. bracteosa.Concentrations of plant’s defensive compounds could be influ-enced by environmental conditions [46].

It is widely known that plants induce the production of second-ary metabolites during low temperature as part of their defense[47]. Enhancement of secondary metabolites during low tempera-ture season is of great physiological significance, as it provides atool to ascertain harvesting times based on secondary metaboliteyield. The absolute values of withanolides were higher in the stressexperiment than in situ Withania somnifera plants [48]. In an ex situexperiment, a plant is subjected to low temperature and thereforemetabolic energy is reconfigured in a different way. Plants alwaystend to follow the less energy consuming path even when encoun-tering stress. This may or may not involve increased production ofsecondary metabolites as seen during salt stress in Swertia chirata[49]. Temperature stress is known to cause many physiological,biochemical and molecular changes in plant metabolism and pos-sibly alter the secondary metabolite production in plants [50].Results revealed that relatively high or low temperatures reducedthe photosynthetic efficiency of the leaves of St. John’s wort plantsand resulted in low CO2 assimilation. Indeed, biotic and abioticstresses exert a considerable influence on the levels of secondarymetabolites in plants [51]. We suggest temperature stress asanother factor for induction of 20-HE accumulation.


5. Conclusion

A. bracteosa is a morphologically diverse, endangered and highlymedicinal species with several ecotypes. Large morphological vari-ations were observed in different ecotypes of A. bracteosa. The hab-itats with an altitude of nearly 600 m represented almost similarvegetative growth pattern regardless of their geographical loca-tion. The plant offers a valuable source of 20-HE and its aerial por-tion can be used as a substitute of other sources. Winter is the bestseason for collection of A. bracteosa from all the said locations.However the chemotype of KR habitat (Western Kahuta) revealedmaximum 20-HE content. Tender aerial portions are prone toattack by grazers and herbivores and contain substantial 20-HEcontent. As the tissue rigidity increases, 20-HE content decreasessupporting the idea of its defense/stress induced production. Wefound low temperature (15 ± 1 �C) inducing considerably higherlevel of 20-HE content in in vitro grown plants. We also speculatethat biosynthesis of 20-HE in this plant is not only a direct conse-quence of plants phenology, but induced in response to low tem-perature stress.

Acknowledgements

Our acknowledgements are due to Dr. Yoshinori Fujimoto,Department of Chemistry and Material Sciences, Tokyo Instituteof Technology, Meguro, Japan for the provision of standard. Weare grateful to Higher Education Commission of Pakistan for grant-ing us funds to conduct this research. We are also grateful to Mr.Majid Mehmood and Mr. Atif Kamal for their assistance in statisti-cal analysis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.steroids.2014.04.017.

References

[1] Hedge I, Nasir Y, Ali S. Flora of Pakistan. Karachi: University of Karachi,Department of Botany; 1990. p. 192.

[2] Kirtikar K, Basu B. Indian medicinal plants. 2nd ed. Dehradhun: InternationalBook Distributors; 1935.

[3] Gupta AK, Tandon N. Reviews on Indian medicinal plants. New Delhi: IndianCouncil of Medical Research; 2004.

[4] Chandel S, Bagai U. Screening of antiplasmodial efficacy of Ajuga bracteosa Wallex Benth. Parasitol Res 2011;108:801–5.

[5] Israili ZH, Lyoussi B. Ethnopharmacology of the plants of genus Ajuga. Pak JPharm Sci 2009;22:425–62.

[6] Shen X, Isogai A, Furihata K, Sun H, Suzuki A. Two neo-clerodane diterpenoidsfrom Ajuga macrosperma. Phytochemistry 1993;33:887–9.

[7] Al-Musayeib NM, Mothana RA, Matheeussen A, Cos P, Maes L. In vitroantiplasmodial, antileishmanial and antitrypanosomal activities of selectedmedicinal plants used in the traditional Arabian Peninsular region. BMCComplement Altern Med 2012;12:49.

[8] Kaithwas G, Gautam R, Jachak SM, Saklani A. Antiarthritic effects of Ajugabracteosa Wall ex Benth. in acute and chronic models of arthritis in albino rats.Asian Pac J Trop Biomed 2012;2:185–8.

[9] Pavela R. Larvicidal effects of various Euro-Asiatic plants against Culexquinquefasciatus Say larvae (Diptera: Culicidae). Parasitol Res 2008;102:555–9.

[10] Pal A, Pawar R. A Study on Ajuga bracteosa Wall ex. Benth for analgesic activity.Int J Curr Biol Med Sci 2011;1:12–4.

[11] Saatov Z, Gorovits M, Abubakirov N. Phytoecdysteroids of plants of the genusSilene. Chem Nat Compd 1993;29:551–7.

[12] Lafont R, Connat J. Pathways of ecdysone metabolism. Stuttgart: EcdysoneGeorg Thieme-Verlag; 1989. p. 167–73.

[13] Hendrix SD, Jones RL. The activity of b-ecdysone in four gibberellin bioassays.Plant Physiol 1972;50:199.

[14] Boo KH, Lee D, Jeon GL, Ko SH, Cho SK, Kim JH, et al. Distribution andbiosynthesis of 20-hydroxyecdysone in plants of Achyranthes japonica Nakai.Biosci Biotechnol Biochem 2010;74:2226–31.





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14 May 2014

[15] Browning C, Martin E, Loch C, Wurtz J-M, Moras D, Stote RH, et al. Critical roleof desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor.J Biol Chem 2007;282:32924–34.

[16] Wang S, Liu S, Liu H, Wang J, Zhou S, Jiang R-J, et al. 20-Hydroxyecdysonereduces insect food consumption resulting in fat body lipolysis during moltingand pupation. J Mol Cell Biol 2010;2:128–38.

[17] Kizelsztein P, Govorko D, Komarnytsky S, Evans A, Wang Z, Cefalu WT, et al.20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-inducedobesity mice model. Am J Physiol Endocrinol Metab 2009;296:E433–9.

[18] Gadzhieva RM, Portugalov SN, Paniushkin VV, Kondrat’eva II. A comparativestudy of the anabolic action of ecdysten, leveton and Prime Plus, preparationsof plant origin. Eksp Klin Farmakol 1995;58:46–8.

[19] Ramazanov NS. Phytoecdysteroids and other biologically active compoundsfrom plants of the genus Ajuga. Chem Nat Compd 2005;41:361–9.

[20] Tuleuov BI. 20-Hydroxyecdysone content of several representativesof the families Asteraceae and Caryophyllaceae. Chem Nat Compd2009;45:762–3.

[21] Adler JH, Grebenok RJ. Biosynthesis and distribution of insect-moltinghormones in plants—a review. Lipids 1995;30:257–62.

[22] Dinan L. Distribution and levels of phytoecdysteroids within individual plantsof species of the Chenopodiaceae. Eur J Entomol 1995;92:295.

[23] Festucci-Buselli RA, Contim LA, Barbosa LCA, Stuart JJ, Vieira RF, Otoni WC.Level and distribution of 20-hydroxyecdysone during Pfaffia glomeratadevelopment. Braz J Plant Physiol 2008;20:305–11.

[24] Tanaka N, Matsumoto T. Regenerants from Ajuga hairy roots with highproductivity of 20-hydroxyecdysone. Plant Cell Rep 1993;13:87–90.

[25] Kim OT, Manickavasagm M, Kim YJ, Jin MR, Kim KS, Seong NS, et al. Genetictransformation of Ajuga multiflora Bunge with Agrobacterium rhizogenesand 20-hydroxyecdysone production in hairy roots. J Plant Biol2005;48:258–62.

[26] Murashige T, Skoog F. A revised medium for rapid growth and bio assays withtobacco tissue cultures. Physiol Plant 1962;15:473–97.

[27] Ghosh D, Laddha K. Extraction and monitoring of phytoecdysteroids throughHPLC. J Chromatogr Sci 2006;44:22–6.

[28] Wu JJ, Cheng KW, Wang H, Ye WC, Li ET, Wang M. Simultaneous determinationof three phytoecdysteroids in the roots of four medicinal plants from the genusAsparagus by HPLC. Phytochem Anal 2009;20:58–63.

[29] Obertello M, Oureye M, Laplaze L, Santi C, Svistoonoff S, Auguy F, et al.Actinorhizal nitrogen fixing nodules: infection process, molecular biology andgenomics. Afr J Biotechnol 2004;2:528–38.

[30] Weiss MR. Floral color change: a widespread functional convergence. Am J Bot1995:167–85.

[31] Grotewold E. The genetics and biochemistry of floral pigments. Annu Rev PlantBiol 2006;57:761–80.

[32] Yonekura-Sakakibara K, Nakayama T, Yamazaki M, Saito K. Modification andstabilization of anthocyanins. Anthocyanins: Springer; 2009. p. 169–90.

[33] Dinan L. The analysis of phytoecdysteroids in single (preflowering stage)specimens of fat hen Chenopodium album. Phytochem Anal 1992;3:132–8.

620


[34] Dinan L. The association of phytoecdysteroids with flowering in fat hen,Chenopodium album, and other members of the Chenopodiaceae. Experientia1992;48:305–8.

[35] Grebenok RJ, Adler JH. Ecdysteroid distribution during development ofspinach. Phytochemistry 1991;30:2905–10.

[36] Adler JH, Grebenok RJ. Occurrence, biosynthesis, and putative role ofecdysteroids in plants. Crit Rev Biochem Mol Biol 1999;34:253–64.

[37] Lafont R, Ho R, Raharivelomanana P, Dinan L. Ecdysteroids in ferns:distribution, diversity, biosynthesis, and functions. Springer, Working withFerns; 2010. p. 305–19.

[38] Schmelz EA, Grebenok RJ, Galbraith DW, Bowers WS. Damage-inducedaccumulation of phytoecdysteroids in spinach: a rapid root responseinvolving the octadecanoic acid pathway. J Chem Ecol 1998;24:339–60.

[39] Hunter MD. Out of sight, out of mind: the impacts of root-feeding insects innatural and managed systems. Agric For Entomol 2001;3:3–9.

[40] Blackford MJ, Dinan L. The effects of ingested 20-hydroxyecdysone on thelarvae of Aglais urticae, Inachis io, Cynthia cardui (Lepidoptera: Nymphalidae)and Tyria jacobaeae (Lepidoptera: Arctiidae). J Insect Physiol 1997;43:315–27.

[41] Soriano IR, Riley IT, Potter MJ, Bowers WS. Phytoecdysteroids: a novel defenseagainst plant–parasitic nematodes. J Chem Ecol 2004;30:1885–99.

[42] Bakrim A, Maria A, Sayah F, Lafont R, Takvorian N. Ecdysteroids in spinach(Spinacia oleracea L.): biosynthesis, transport and regulation of levels. PlantPhysiol Biochem 2008;46:844–54.

[43] Schmelz EA, Grebenok RJ, Galbraith DW, Bowers WS. Insect-induced synthesisof phytoecdysteroids in spinach, Spinacia oleracea. J Chem Ecol1999;25:1739–57.

[44] Schmelz EA, Grebenok RJ, Ohnmeiss TE, Bowers WS. Interactions betweenSpinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids. ArchInsect Biochem Physiol 2002;51:204–21.

[45] Howe GA, Jander G. Plant immunity to insect herbivores. Annu Rev Plant Biol2008;59:41–66.

[46] Gambarana C, Ghiglieri O, Tolu P, De Montis MG, Giachetti D, Bombardelli E,et al. Efficacy of an Hypericum perforatum (St. John’s wort) extract inpreventing and reverting a condition of escape deficit in rats.Neuropsychopharmacology 1999;21:247–57.

[47] Janská A, Maršík P, Zelenková S, Ovesná J. Cold stress and acclimation – what isimportant for metabolic adjustment? Plant Biol 2010;12:395–405.

[48] Kumar A, Abrol E, Koul S, Vyas D. Seasonal low temperature plays an importantrole in increasing metabolic content of secondary metabolites in Withaniasomnifera (L.) Dunal and affects the time of harvesting. Acta Physiol Plant2012;34:2027–31.

[49] Abrol E, Vyas D, Koul S. Metabolic shift from secondary metabolite productionto induction of anti-oxidative enzymes during NaCl stress in Swertia chirataBuch.-Ham. Acta Physiol Plant 2012;34:541–6.

[50] Levitt J. Responses of plants to environmental stresses. Water, radiation, salt,and other stresses, vol. II. Academic Press; 1980.

[51] Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism. Plant Cell1995;7:1085.



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