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Original article
12 (
8
); 2644-2654
doi:
10.1016/j.arabjc.2015.04.020

Transgenic Artemisia dubia WALL showed altered phytochemistry and pharmacology

, , ,
a Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan
c Department of Pharmacy, Quaid-i-Azam University, Islamabad 45320, Pakistan

⁎Corresponding author. Tel.: +92 51 90643007. dr.bushramirza@gmail.com (Bushra Mirza)

Received: , Accepted: ,
© The Author(s) 2015
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

The rol genes have been shown to enhance the production of secondary metabolites in plants. This report examines the effect of trans-genes (rol ABC) on possible high production of biologically important phytochemicals and enhanced pharmacological activities. Three transgenic lines (1, 2 and 3) of Artemisia dubia WALL (transformed with Agrobacterium tumefaciens harboring rol ABC genes) were subjected to phytochemical analysis and pharmacological studies. A great variation in phytochemistry and the pharmacological activities was observed not only between the transgenic and non-transgenic control plants but also among the transgenic lines itself. Comparative chemical profile obtained via HPLC, TLC and spectrophotometry showed high degree of variations in the quantity of phytochemicals. An increased production of total flavonoids (71.1% in transgenic line 2) and total phenolics (110.8% in transgenic line 1), increase in caffeic acid and catechin and a decrease in gallic acid content in the extracts of transformed plants compared to the untransformed control plants was decreased. In case of pharmacological activities, moderate to high level increase in antimicrobial (antibacterial and antifungal) activities, cytotoxicity (14.1%), antitumor (29%) and antioxidant activities (23.9%) was observed (in transgenic line 2). In general all the three transgenic lines under study showed improvement in their pharmacological activities in the order of transgenic line 2 > 1 > 3 > control. The implication of these findings will help to meet the increasing demand of pharmacologically important compounds.

Keywords

Artemisia dubia
Pharmacology
Phytochemistry
rol ABC genes
Transformation
Transgenic lines
1

1 Introduction

Genetic engineering has allowed the production of plants with altered content of secondary metabolites (Marion et al., 1996; Muhammad et al., 2008). As the secondary metabolites play important role in plant defense mechanism, therefore it is quite possible that such engineered plants may show changes (alteration) in different pharmacological activities. For the last 2 decades scientists are trying to introduce trans-genes into agriculturally and medicinally important plants in order to get desirable outcomes. Although it is rare (Gómez-Galera et al., 2007; Pandey et al., 2010), however examples could be found where the transgenes have been introduced to medicinally important plants to get the enhanced level of phytochemical/s (secondary metabolites) which otherwise are not produced or produced in a very little quantity (Ray et al., 1996; Ray and Jha, 1999; Bandyopadhyay et al., 2007; Baldi et al., 2008; Chaudhuri et al., 2009 and Kiani et al., 2012). For example in one study the transformed root cultures of Withania somnifera (through Agrobacteirum rhizogenes) showed increased production of secondary metabolites, with increase in biomass production (Chaudhuri et al., 2009). The same strategy has also been used for the enhancement of important pharmacological activities like a significant enhancement of the antioxidant activity was reported in transformed roots of W. somnifera by Kumar et al. (2005) and increase in resistance against pathogens by Hain et al. (1993).

Plant secondary metabolites are useful in the long term, often for defense purposes, and give plants specific characteristics such as color. Secondary plant metabolites are also used in signaling and regulation of primary metabolic pathways. Moreover, several plant secondary metabolites are used for the production of medicines, dyes, insecticides, flavors and fragrances. Numerous investigations have shown that the Agrobacterium rol genes can induce high levels of secondary metabolites in hairy root cultures of most transformed plant species (Giri and Narasu, 2000; Sevon and Oksman-Caldentey, 2002). Rol genes have a large impact on diverse biochemical processes and effect broad range of genes in transformed plant cells, which leads to the enhanced production of secondary metabolites (Bulgakov, 2008). We hypothesized that transformation with the rol ABC genes could induce increased secondary metabolites production through stimulation of synthesis pathway.

Over the past 2 decade, novel features of plants transformed with the A. rhizogenes harboring rol genes have been revealed, such as increased production of secondary metabolites. Several groups have shown that transformation with rol genes can increase secondary metabolite production through modification of its biosynthetic pathway. Among various rol genes, the rol A gene has emerged as a stimulator of growth and secondary metabolism (Altvorst et al., 1992; Schmulling et al., 1993). The rol B protein on the other hand, has been shown to have a tyrosine phosphatase activity and therefore play a possible role in the auxin signal transduction pathway (Filippini et al., 1996). While, Estruch et al. (1991) have demonstrated that rol C can be involved in the release of active cytokinins from their inactive glucosides due to its cytokinin glucosidase activity. Collectively, these genes play a major role in the pathway that leads to high level production of secondary metabolites. Although now it is well known that the rol genes act via transcriptional activation of defense genes, the mechanism of activation is unclear (Bulgakov, 2008). However, production of transgenic plants by utilizing these genes seems to be an appropriate choice to improve production of secondary metabolites of any plant and hence to increase the pharmacological activities.

In continuation to our previous studies Kiani et al. (2012) the aim of the investigation reported here was to evaluate the possible impact of Agrobacterium mediated transformation (with rol ABC genes) on phytochemistry and pharmacological properties (like antimicrobial, antitumor, antioxidant and cytotoxicity activities) of Artemisia dubia.

2

2 Materials and methods

2.1

2.1 Plant materials and extract preparation

Three different transgenic lines (1, 2 and 3) of A. dubia obtained through transformation with Agrobacterium tumefaciens strain LBA4404 containing pRT99 harboring rol ABC genes as reported in our previous study (Kiani et al., 2012) were used in this study. These transgenic plants along with control plants were grown to maturity in green house and used for pharmacological evaluation. When the age of plants was five months, shoots (T) and roots (RT) were separated, dried under shade and crushed using laboratory grinder.

The ground plant material was soaked in methanol (100 g/500 ml) for 7 days at room temperature. The methanol was filtered and the filtrate was concentrated in rotary evaporator (Rotavapor R-200, Buchi) at 45 °C and finally dried to a constant weight at the same temperature in vacuum oven (Vacucell, Einrichtungen GmbH) to get crude extract.

2.2

2.2 Phytochemical analysis

2.2.1

2.2.1 Determination of total flavonoid content

For total flavonoids determination, aluminum chloride colorimetric method was used (Chang et al., 2002). The extracts/samples (0.5 ml of 1 mg/ml of methanol) were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. The resulting mixture was kept at room temperature for 30 min and absorbance was measured at 415 nm using spectrophotometer (Agilent, 8453). The standard curve for calculating the total flavonoid contents in equivalence to quercetin was plotted using quercetin solutions at concentrations of 0.0–8.0 μg/ml in methanol.

2.2.2

2.2.2 Determination of total phenolic content

The total phenolic content was determined according to Velioglu et al. (1998) using Folin–Ciocalteu reagent. An aliquot of the solutions of extracts/samples was prepared at a concentration of 1 mg/ml. An aliquot of 200 μl was transferred into a test tube and 1.5 ml of Folin–Ciocalteu reagent (previously diluted 10-fold with deionized water) was added and mixed. The resulting mixture was kept at room temperature for 5 min and then 1.5 ml of 6% (w/v) solution of sodium carbonate was added to the mixture and stirred thoroughly. The resulting reaction mixture was kept at room temperature for 90 min, followed by the measurement of absorbance at 725 nm using spectrophotometer (Agilent, 8453). The standard calibration curve was plotted using gallic acid (0.0–25 μg/ml). The total phenolic content was expressed as gallic acid equivalents in percentage weight by weight.

2.2.3

2.2.3 Analysis of important phytochemicals using HPLC

Stock solutions of rutin, kaempferol, myricetin, gallic acid, catechins, caffeic acid and quercetin were prepared in methanol, at concentration of 1 mg/ml and then further diluted with methanol to get 10, 20, 50, 100, 150 and 200 μg/ml for the preparation of standard calibration curve. All the solutions were filtered through 0.2 μm Sartolon polyamide membrane filter (Sartorius).

The extracts/samples for HPLC analysis were prepared at concentration of 10 mg/ml in methanol. The samples were dissolved in methanol via ultra-sonication and were filtered through 0.2 μm Sartolon Polyamide membrane filter. All the samples were freshly prepared and used for analysis immediately or stored at 4 °C if not tested for more than 1 h.

Chromatographic analysis was carried out using HPLC-DAD attached with Discovery C-18 analytical column. Method followed was as described by Zu et al. (2006) with slight modification according to the system suitability. Briefly, mobile phase A was methanol–acetonitrile–water–acetic acid (10:5:85:1) and mobile phase B was methanol–acetonitrile–acetic acid (60:40:1). A gradient of time 0–20 min for 0–50% B, 20–25 min for 50–100% B and then isocratic 100% B till 30 min was used. Flow rate was 1 ml/min and injection volume was 20 μl. Rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kaempferol were analyzed at 368 nm. Different wavelengths were selected according to method described by Zu et al. (2006). Every time column was reconditioned for 10 min before the next analysis.

2.2.4

2.2.4 Thin layer chromatography (TLC) fingerprinting of A. dubia phytochemistry

For TLC fingerprinting 10 mg of each extract/sample was dissolved in 1 ml of methanol (HPLC grade). Four silica gel 60 F254 TLC plates (4 × 6.66 cm, Merck, Germany) were used. Four different mobile phases (MP) (a, b, c and d) were used to separate out different phytochemicals. Mobile phases composition was such that a = Chloroform:Ethylacetate; 20:1, b = Chloroform:Ethylacetate; 3:1, c = 100% ethyl acetate and d = Ethylacetate:Methanol; 3:1.

A 15 ml of mobile phase was poured in TLC tank and covered with lid for vapors saturation. After 20 min, plates were placed in TLC tank carefully and allowed to develop. When the mobile phase reached at upper end, the plates were taken out. Plates were air-dried and visualized after dipping into phosphomolybdic acid (10% W/V ethanol) and heating on hot plate at 250 °C.

2.3

2.3 Biological activities

2.3.1

2.3.1 Antibacterial assay

Antibacterial assay was performed by disk diffusion method as described by Bibi et al. (2011). Antibacterial activity was studied against five bacterial strains (three gram positive i.e. Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Micrococcus luteus (ATCC 10240) and two gram negative i.e. Escherichia coli (ATCC 15224), and Enterobacter aerogenes (ATCC 13048)). Briefly, 100 μl of 24 h old bacterial cultures in nutrient broth was spreaded over petriplates (7 cm) containing 25 ml solidified nutrient agar. Sterile filter paper disks (diameter 6 mm) impregnated with 10 μl of extract dilutions (1, 5 and 10 mg/ml DMSO) were placed over each of the culture plates with the help of sterilized forceps. The plates were then incubated at 37 °C for 24 h. The experiment was performed in triplicate whereas Cefaxime and pure DMSO were used as positive and negative control respectively. Antibacterial activity was determined by measuring zones of inhibition around the disks in each plate. Minimum inhibitory concentration (MIC) was noted.

2.3.2

2.3.2 Antifungal assay

Antifungal assay was performed as described by Jagessar et al. (2008). Antifungal activity was studied against five fungal strains namely Mucor species (FFBP 0300), Aspergillus niger (FFBP 0198), Aspergillus flavis (FFBP 0064), Aspergillus fumigatus (FFBP 66) and Fusarium solani (FFBP 0291). All fungal strains were grown on 6.5% SDA (Sabouraud dextrose agar) at 28 °C and preserved at 4 °C until used. Terbinafine was used as standard drug while DMSO was used as negative control. All extract/samples were checked at 4 different concentrations. Plates containing 25 ml SDA were inoculated with 100 μl fungal spores suspensions, prepared by harvesting fungal spores in 2% w/v tween-20 solution (in distilled water) and turbidity adjusted to 0.5 McFarland turbidity standards. Sterile filter paper disks (diameter 6 mm) impregnated with 10 μl of extract/sample (1, 5, 10 and 50 mg/ml in DMSO) were placed over each of the culture plates followed by the incubation of plates at 28 °C for 24 h. Clear zones of inhibition around the disks were measured and MIC values were determined.

2.3.3

2.3.3 Brine shrimp cytotoxicity assay

Brine shrimp (Artemia salina) cytotoxicity assay was performed following Haq et al. (2012a,b). Brine shrimp eggs were hatched at 37 °C in artificial sea water. In a 20 ml glass vial, cytotoxicity activity of extracts/samples was checked at final concentration of 1000, 100 and 10 μg/ml (in 5 ml of sea water). Ten shrimps were transferred into each vial and kept under florescence light at 37 °C for 24 h. Test was performed in triplicate, percentage death was calculated by Abbott’s formula and LD50 values were determined by Finney computer program (Finney, 1971).

2.3.4

2.3.4 Antitumor assay

Potato disk antitumor assay was performed by following the standard procedure as described by Bibi et al. (2011). A 48 h old culture of Agrobacterium tumefaciens strain At 10 was used to generate tumors in potato disks. An inoculum of 1.5 ml containing plant extracts (10,000, 1000, 100, 10 and 1 μg/ml), bacterial cultures and distilled water was prepared. DMSO was used as negative control and vinblastine sulfate was used as positive control. Red skinned potatoes were surface sterilized with 0.1% HgCl2 solution followed by washing with distilled water. Using sterile borer and scalpel potato disks (4 mm diameter) were prepared. Autoclaved agar solution (1.5% w/v) was poured in petriplates (20 ml) and allowed to solidify. Ten disks were placed on agar surface of each petriplate and 50 μl of already prepared inoculum was poured on the top of each disk. The plates were sealed with parafilm to avoid moisture loss and incubated at 28 °C in dark. After 21 days of incubation, potato disks were stained with Lugol’s solution (10% KI, 5% I2), and tumors were counted under dissecting microscope. More than 20% tumor inhibition was considered significant (Ferrigini et al., 1982). Tumor inhibition was calculated using following formula: % of tumor inhibition = ( 1 - Ts / Tc ) × 100 where “Ts” is number of tumors in the sample and “Tc” is number of tumors in control.

2.3.5

2.3.5 Free radical scavenging activity

Free radical scavenging activity of the extracts/samples was measured using 2,2, diphenyl-1-picrylhydrazyl (DPPH) assay. The assay was performed according to Haq et al. (2012a,b). A 2800 μl volume of DPPH solution (3.92 mg/100 ml methanol) was added into glass vials followed by the addition of 200 μl of plant extract/samples, leading to the final concentration of extract/sample as 1000, 500 and 100 μg/ml (DPPH solution). The Mixtures were shaken well and incubated in dark at 37 °C for 1 h. Absorbance was measured at 517 nm using spectrophotometer (Agilent, 8453). Pure DMSO was used as negative control and ascorbic acid (AsA) was used as positive control. Each test was performed in triplicates. Percentage scavenging of free radical by extracts/samples was measured using the following formula and IC50 values were calculated using table curve software. % scavenging effect = ( 1 - As / Ac ) × 100 where “Ac” is absorbance of negative control and “As” is absorbance of test sample.

3

3 Results

Three different transgenic lines of A. dubia used in this study were earlier obtained by transformation with A. tumefaciens containing rol ABC genes confirmed by PCR and Southern blotting as described in our previous report (Kiani et al., 2012).

3.1

3.1 Morphological characters

Considerable variation in the morphology of transgenic lines was observed as compared to controlled plants, like decrease in plant height with small and narrow leaves, terminal inflorescence and hard texture. The detailed morphological differences studied are listed in Table 1.

Table 1 Morphological differences observed among transgenic and control plants of Artemisia dubia.
Morphological characters Control plants Transgenic plants
T1 T2 T3
Plant height 87 cm + 0.5 70 cm + 0.3 62 cm + 0.3 67 cm + 0.3
Stem Straight, unbranched and soft in texture Branched and hard in texture Highly branched and hard in texture Branched and hard in texture
Leaves Large size and broad Small size and narrow Small size and narrow Small size and narrow
Inflorescence Axial, without hairs Terminal, excessive hairy Terminal and hairy Terminal, excessive hairy

3.2

3.2 Phytochemical analyses

3.2.1

3.2.1 Total flavonoid content

The total flavonoid content of crude extracts of transformed and untransformed plants of A. dubia was determined in terms of rutin equivalent as shown in Fig. 1. Highest flavonoid content (2.06%) was observed in shoots of transgenic line T2, followed by T1 (1.92%) and T3 (1.71%) as compared to control shoots (1.20%). Among roots of different transgenic lines highest amount of flavonoids i.e., 0.26% was observed in RT1 and RT2 compared to the 0.20% of control roots.

Comparative analysis of total flavonoid contents in terms of rutin equivalent (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 1 Comparative analysis of total flavonoid contents in terms of rutin equivalent (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

3.2.2

3.2.2 Total phenolic content

The total phenolic content of the crude extracts was determined in terms of gallic acid equivalent as shown in Fig. 2. In contrast to the previous results where T2 showed comparatively higher activity/quantity than T1 and T3, here shoots from the transgenic line 1 (T1) showed higher quantity (9.73%) of total phenolics followed by T2 (6.82%) and T3 (5.70%), which was higher than of untransformed control shoots (4.64%). Similarly among roots of the respective shoots, the highest phenolic content was found in RT1 (5.73%) followed by RT2 (3.99%) and RT3 (3.73%) compared to the 3.32% phenolics of RTC.

Comparative analyses of total phenolic contents in terms of gallic acid equivalent (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 2 Comparative analyses of total phenolic contents in terms of gallic acid equivalent (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

3.2.3

3.2.3 HPLC analysis of some important phytochemicals

HPLC analysis of the extracts of these plants showed the presence of catechin, caffeic acid and gallic acid (Fig. 3), while rutin, kaempferol, myricetin, and quercetin were not detected.

HPLC analyses of some important phytochemicals (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 3 HPLC analyses of some important phytochemicals (% w/w dry extracts) of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

An increase in the amount of caffeic acid was observed in transformed shoots of T2, T1 and T3 showing 0.08%, 0.07% and 0.05% content respectively compared to the 0.01% of control shoots. Catechin was only found in the roots of all the three transgenic lines (RT2, 0.09% and RT1 and RT3 0.07% each), while Gallic acid; was only found in control shoots.

3.2.4

3.2.4 Comparative TLC profiling

TLC analysis was done using four different mobile phases so as to obtain 4 different TLC finger prints for each extract/samples (transgenic and non-transgenic plant). By this way total 22 spots (where each spot is representing a compound or mixture of compounds) were identified on 4 different TLC plates and named as C1–C22 (just for better understanding). Intensity of a particular spot on TLC was distinguished by numerical numbers from 0 to 5 (greater the number higher is the intensity of that spot in a specific sample). TLC finger prints are shown in Fig. 4 and the presence/absence and intensity of a particular spot is elaborated in Table 2. Among shoots of different transgenic lines, T2 showed an increase in quantity of several compound/s like C2, C5, C11, 12, 13 and C14 while T3 showed a comparative increase only in C20. C11 appeared only in T2 and T1. Among roots of different transgenic lines, an increase in quantity of compounds was only observed in RT3 where C2, C11 and C16 were compounds that appeared only in RT3 but not in control.

Comparative analysis of TLC fingerprinting of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively.
Figure 4 Comparative analysis of TLC fingerprinting of transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively.
Table 2 Comparative TLC fingerprinting of transformed and untransformed root and shoots of Artemisia dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively.
Spots on TLC Comparative quantitative grading of the spots (compound/s) among transgenic lines and non-transgenic controlled plants
T1 T2 T3 C RT1 RT2 RT3 RC
C1 0 0 0 2 3 1 2 4
C2 3 4 0 3 0 0 2∗ 0
C3 1 1 0 1 0 0 0 0
C4 2 3 1 3 1 0 2 1
C5 2 4 0 2 0 0 2 1
C6 0 0 0 2 0 0 0 0
C7 2 2 1 2 1 0 2 1
C8 2 2 0 2 1 0 2 1
C9 1 2 1 2 1 1 2 1
C10 1 1 1 1 1 0 1 1
C11 1∗ 1∗ 0 0 0 0 1∗ 0
C12 1 2 1 1 0 0 0 1
C13 1 2 1 1 0 0 2 1
C14 2 3 1 2 1 1 2 1
C15 2 5 1 5 0 0 4 1
C16 1 2 0 2 0 0 2∗ 0
C17 1 1 0 3 0 0 2 1
C18 2 3 2 5 3 1 5 3
C19 0 1 1 3 0 0 0 0
C20 0 0 3 2 0 0 0 0
C21 0 0 4 4 1 1 1 1
C22 0 1 5 5 2 2 2 2

T1, T2 and T3 represent three transgenic lines of shoots, RT1, RT2 and RT3 represent transformed roots of transgenic lines T1, T2, T3 respectively. C and RC represent control shoots and roots respectively. Numbering in the table is representing the intensity of a particular compound/s (scale 0–5) in a specific plant (greater the number higher is the intensity). C1–C22 represent spots of compound or mixture of compounds.

Numbers with ∗ are representing the compound/s appeared only in the transgenic line/s.

Numbers in bold are representing a comparative increase in the compound/s quantity.

Numbers in italic are representing a comparative decrease in the compound/s quantity.

3.3

3.3 Biological activities

3.3.1

3.3.1 Antibacterial activity

All transformed shoots and roots showed moderate antibacterial activity against all bacterial strains tested, whereas untransformed control shoots and roots showed no antibacterial activity. Among shoots of different transgenic lines, the highest antibacterial activity was observed in T2 which showed an MIC of 50 μg/disk against 3 of the tested bacteria strains i.e., S. aureus, B. subtilis and E. coli. Among roots of different transgenic, the same 50 μg/disk MIC value (as that of shoot) was observed for RT1 against S. aureus and B. subtilis and RT2 against S. aureus and E. coli (Table 3).

Table 3 Antibacterial and antifungal activity of extracts of transformed and untransformed Artemisia dubia plant. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively.
Samples Antibacterial activity⁎ Antifungal activity⁎
MIC (μg/disk) MIC (μg/disk)
S. aureus B. subtilis M. luteus E. coli E. aerogenes Mucor A. niger A. flavis A. fumigatus F. solani
T1 50 50 100 100 100 50 100 100 50 50
T2 50 50 100 50 100 10 10 50 10 50
T3 50 100 100 50 100 50 50 100 50 100
TC – – – – – – – – – –
RT1 50 100 100 100 100 50 50 50 50 50
RT2 50 50 100 100 100 10 10 10 10 10
RT3 100 100 100 100 100 50 50 50 100 50
RTC – – – – – 500 500 500 100 500
Cef. 6.25 6.25 12.5 12.5 12.5 NA NA NA NA NA
Ter. NA NA NA NA NA 3.21 5.80 3.21 3.21 5.80

E. coli:- Escherichia coli; B. sub:- Bacillus subtillus; E. aerog:- Enterobacter aerogenes; S. aureus:- Staphylococcus aureus; M. luteus:- Micrococcus luteus; F. solani:- Fusarium solani; A. niger:- Aspergillus niger; A. flavis:- Aspergillus flavis; Ter:- Terbinafine; Cef:- Cefotaxime; A. fumigatus:- Aspergillus fumigatus; mm:- Millimeter.

– :- No zone of inhibition.

NA:- Not applicable.

⁎ The data represent the mean values of three replicates.

3.3.2

3.3.2 Antifungal activity

All the extracts/samples (except the control shoots) showed some antifungal activity (Fig. 5, Table 3). Among shoots of different transgenic lines, the highest antifungal activity was found in T2 which showed MIC values of 10 μg/disk against 3 (Mucor, A. niger, A. flavis and A. fumigatus) out of five tested fungal strains followed by T1 and T3 which showed MIC values of 50 μg/disk only. Among roots of the different transgenic lines highest antifungal activity was observed in RT2 which showed MIC value of 10 μg/disk against all the tested fungal strains followed by RT1 and RT3 which showed the MIC of 50 μg/disk only.

Pictorial representation of the comparative antifungal activity of transformed and untransformed extracts of Artemisia dubia against (a) Mucor species (b) Aspergillus flavis. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. T is terbinafine (positive control).
Figure 5 Pictorial representation of the comparative antifungal activity of transformed and untransformed extracts of Artemisia dubia against (a) Mucor species (b) Aspergillus flavis. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. T is terbinafine (positive control).

3.3.3

3.3.3 Brine shrimp cytotoxicity

Transformed shoots and roots showed higher percentage mortality compared to untransformed plants (Fig. 6). Among shoots of the different transgenic lines, the highest cytotoxic activity was found in T2 with IC50 value 24.8 μg/ml followed by T1 (31.6 μg/ml) and T3 (39.1 μg/ml) compared to control shoots (57.7 μg/ml). Whereas among roots the highest cytotoxic activity was observed in RT2 with IC50 value 49.7 μg/ml compared to control root (90.2 μg/ml).

Comparative analysis of percentage mortality caused by transformed and untransformed extracts of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 6 Comparative analysis of percentage mortality caused by transformed and untransformed extracts of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

3.3.4

3.3.4 Antitumor activity

Transgenic lines showed higher antitumor activity as compare to non-transgenic control plants. Among shoots of transgenic lines, the highest antitumor activity was observed in T1 which inhibited tumor formation up to 96% at 10,000 μg/ml with IC50 value 90.01 μg/ml followed by T2 (IC50 114.0 μg/ml) and T3 (IC50 138.4 μg/ml) compared to control shoots (IC50 234.6 μg/ml). Among roots, the highest antitumor activity was found in RT3 with IC50 value of 144.0 μg/ml followed by RT2 (159.6 μg/ml) and RT1 (181.7 μg/ml) compared to the 282.12 μg/ml of control roots (Fig. 7).

Comparative analysis of tumor inhibition in transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 7 Comparative analysis of tumor inhibition in transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

3.3.5

3.3.5 Free radical scavenging activity

The free radical scavenging activity of the crude extracts was determined spectrophotometrically by monitoring the disappearance of DPPH free radicals at 517 nm. Transformed shoots showed stronger antioxidant activity compared to its respective roots and control shoots. The highest antioxidant activity observed was in T1 with IC50 value 131.6 μg/ml followed by T2 (IC50 169 μg/ml) compared to 257.2 μg/ml of TC (Fig. 8). Moreover T1 has shown up to 87.9% of free radicals scavenging potentials at 1000 μg/ml.

Comparative analysis of antioxidant activity in transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.
Figure 8 Comparative analysis of antioxidant activity in transformed and untransformed plants of A. dubia. T1, T2 and T3 and RT1, RT2 and RT3 represent shoots and roots of transgenic lines while TC and RTC represent control shoots and roots respectively. The average of three replicates together with standard deviation is shown.

4

4 Discussion

The rol ABC genes have been shown to enhance the production of secondary metabolites (Putalun et al., 2007; Wang et al., 2006) in plants, possibly through stimulation of the defense pathway (Bulgakov, 2008). This report examines the potential of genetic transformation with rol ABC genes via A. tumefaciens to enhance pharmacological potentials in A. dubia through increased production of secondary metabolites. As secondary metabolites play important role in plant defense mechanism therefore, increased pharmacological activities can be correlated with increased secondary metabolites. The increase in secondary metabolites can be determined via phytochemical analysis as well. According to McLaughlin et al. (1998) crude botanical extracts can be comprised of very effective mixtures of bioactive compounds, and it is quite possible to sort out which activities are due to which components with the help of simple bioassay procedures and various separation techniques. In the present study, methanolic extract of transformed and untransformed shoots and roots of A. dubia were subjected to phytochemical analysis via spectrophotometer, HPLC and TLC and evaluation of biological activities like antimicrobial, cytotoxicity, antitumor and antioxidant activities.

Morphological variations were observed and compared between transgenic lines and controlled plants. Shoots of the transgenic lines showed a considerable variation in morphology of the stem, leaves and inflorescence. Transformation with rol genes caused decrease in plant height and leaf area. Transformation also caused axial inflorescence without hairs compared to the terminal inflorescence with excessive hairs of untransformed controlled plants. These transformed plants contain similar morphological variations as reported earlier (Bulgakov, 2008; Piispanen et al., 2003; Casanova et al., 2005).

Phytochemical analysis was carried out on dried plant material (shoots and roots) of transformed and non-transformed plants of A. dubia. Our phytochemical analysis revealed that transformed shoots and roots produced higher amount of flavonoids and phenolics as compared to untransformed shoots and roots. Bensaddek et al. (2008) reported that rol ABC genes are reliable source for increased production of secondary metabolites and hence flavonoids and phenolics in plants transformed with rol ABC genes. Similarly, Wang et al. (2006) and Putalun et al. (2007) also described that rol genes greatly enhance the production of secondary metabolites in transformed plants. In the current research, transformed shoots of T2 showed an increase of 71.1% in flavonoids followed by 60% increase in T1. Similarly in case of phenolics transformed shoot T1 showed an increase of 110.8% which was 70% higher than its respective root RT1 which showed an increase of 72.2% only. From the above discussion it is clear that increase in flavonoid phenolics contents was observed both in shoot and root however shoot showed higher increase in flavonoid and phenolics compared to their respective roots. The increase in flavonoids and phenolics from moderate to appreciable amounts in transformed plants of A. dubia can be unambiguously correlated with the introduction of tans rol ABC genes. Similarly the increase in caffeic acid and decrease in gallic acid in transformed shoots as determined by HPLC analysis is also due to the rol ABC genes and in accordance with the results of Inyushkina et al. (2009).

Similarly, an earlier report showed that high expression of the rol genes in transformed plant cells dramatically increased the biosynthesis of different secondary metabolites (Shkryl et al., 2008). Our HPLC analysis also revealed the production of catechin in roots of all the three transgenic line RT1, RT2 and RT3 which was not observed in shoots and roots of the untransformed controlled plant of A. dubia (Fig. 3).

TLC profiling is very important parameter for the proper isolation and identification of different compound in medicinal plants (Chothani et al., 2012). It is often used to provide the first characteristic finger prints of herbs (Liang et al., 2004). In current study TLC analysis was done with 4 different mobile phases in the order of increasing polarity. These combinations were used to get the maximum separation of the compounds that can best be compared on TLC plate (Fig. 4). The increase in quantity of compound/s and the appearance of compound/s for the first time in transformed plants/transgenic lines of A. dubia can be clearly correlated with the introduction rol ABC genes. This is the first report on the production of different compounds in rol genes transformed A. dubia plants, however early investigations used TLC for the determination of the artemisinin production in Artemisia annua (Rimada et al., 2009).

Biological analysis was carried out on dried shoots and roots of transformed and non-transformed plants of A. dubia. Transformation has caused transgenic A. dubia to show antibacterial activity which otherwise was not shown by untransformed plants. Shoots and roots showed almost similar antibacterial potentials. Same was the case with antifungal activity where T2 and its respective RT2 showed antifungal activity with MIC value of 10 μg/ml against all the tested fungal strains which was near to MIC values of positive control terbinafine i.e., 3.21 μg/ml and 5.8 μg/ml against Mucor and F. solani. More importantly transformation has caused shoots of transgenic lines to show antifungal activity which otherwise was not shown by untransformed controlled. The result suggests that transformation via rol ABC can be a good strategy to induce or increase antimicrobial activity in medicinally important plants. Since rol ABC genes induce genetic modification affecting hormone sensitivity and plant morphology, an effect on plant resistance to pathogens may be expected. Bettini et al. (2001) found higher resistance to toxins of Fusarium oxysporum in rol ABC transformed tomato plants, suggesting that modifications induced by the rol genes improved the plant defense response. These metabolites can be involved in defense mechanism of plant to protect them from antimicrobial attack. So transformation with rol ABC can serve to increase plant microbial defense through increased production of medicinally important secondary metabolites.

Brine shrimp cytotoxicity assay (Mayerhof et al., 1991) and potato disk antitumor assays are suggested to be convenient probe for the pharmacological activities in plant extracts. To the best of our knowledge, this is the first report on the effect of transformation (with rol ABC genes) on antitumor and cytotoxic potentials of A. dubia. Results showed that transformation with rol ABC genes has increased the cytotoxicity and antitumor potentials of A. dubia (Figs. 6 and 7).

DPPH free radical scavenging assay is a non-enzymatic method currently used to provide basic information about the ability of compounds to scavenge free radicals. Reduction of DPPH by an antioxidant results in the loss of absorbance at 517 nm (Fukumoto and Mazza, 2000). Transformed shoots showed more antioxidant activity compare to transformed roots and untransformed controls. High antioxidant activity of the shoot was as expected because shoots showed comparatively high flavonoids and phenolic contents to their roots (Figs. 1 and 2). The relationships of phenolic and flavonoids with the antioxidant activity of medicinal plants are well documented (Younes, 1981; Das and Pereira, 1990; Velioglu et al., 1998; Kahkonen et al., 1999) therefore increase in phenolics and flavonoids by transformation with rol ABC genes may have led to increased antioxidant potentials.

To know how the rol ABC genes change the biosynthetic pathways of different important secondary metabolites and hence the pharmacological activities, although, were not the scope of this study, however it can be assumed that rol ABC genes have to do something with the biosynthetic pathways to alter the phytochemistry and pharmacology of transgenic lines. Evidence indicates that the rol genes mediate uncommon signal transduction pathways in plants. They act on phytoalexin production independently of plant defense hormones and the calcium dependent NADPH oxidase pathway (Bulgakov, 2008). The extent of secondary metabolism activation varies between plant species, from 2- to 300-fold depending on the group of secondary metabolites and the plant species (Bulgakov, 2008). Transformation with the rol genes provokes a biphasic effect with an initial suppression and the subsequent activation of biosynthesis for particular groups of secondary metabolites (Bulgakov et al., 2005; Bulgakov, 2008; Inyushkina et al., 2009). The information about the effect of rol genes on secondary metabolism is still limiting and transformation in some cases causes unpredictable results. Therefore it is yet to understand that how rol ABC genes exert their effect on biochemical pathways to alter the phytochemistry and how these genes play their role in enhancing the pharmacological activities.

5

5 Conclusion

These data allow us to present a novel model for the effect of rol ABC genes on enhanced production of pharmacologically active compounds in transformed plants of A. dubia. Crude methanolic extract of transformed A. dubia showed significant increase in antifungal, antioxidant, cytotoxic and antitumor activities. The transformed plants also showed an increased amount of different phytochemicals like caffeic acid and catechin. This may explain that transformation with Agrobacterium rol genes has a significant positive effect on the different pharmacological activities and phytochemistry of transformed A. dubia plants.

Authors contribution

  • Dr. Bushra Hafeez Kiani; has worked on transformation and Biological activities of plant and helped in data arrangement and manuscript writing.

  • Dr. Ihsan-ul-Haq and Dr. Nazif Ullah; have worked on phytochemistry and pharmacology.

  • Dr. Bushra Mirza; supervisor of the Project and reviewer.

Acknowledgments

We thank Higher Education Commission of Pakistan for financial support.

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