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Phytochemical Analysis of Nigella sativa and its Antibacterial Activity against Clinical Isolates Identified by Ribotyping.

Byline: Shabina Ishtiaq, Muhammad Ashraf, Muhammad Qasim Hayat and Mudassir Asrar

Abstract

The study was carried out to find in vitro anti-bacterial activity of Nigella sativa extracts against seven clinical isolates identified by ribotyping. Crude Extracts of N. sativa in eight organic solvents (Aqueous, Methanol, Ethanol, Cloroform, Butanol, Diethyl ether, n-Hexane and Acetone) were evaluated at 5 different concentrations 100 mg/mL, 50 mg/mL, 25 mg/mL, 10 mg/mL and 5 mg/mL by using disk diffusion method against human pathogenic bacterial strains including Gram- positive bacteria (Enterococcus faecalis IARS1, Staphylococuss aureus IARS4) and Gram-Negative bacteria (Acinetobacter junii IARS2, Escherichia coli IARS3, Proteus mirabilis IARS5, Serratia marcescens IARS6, Enterobacter cloacae IARS7). All the organic N. sativa extracts with minimum concentration of 5 mg/mL showed effective growth inhibition against the tested pathogenic bacterial strains. Standard antibiotics ceftriaxone 100 mg/mL, Amoxycillin 100 mg/mL, Gentamycin 100 mg/mL were used as a positive control.

Out of eight extracts Methanolic, Ethanolic, Chloroform, n-Hexane and Acetonic extracts showed maximum antibacterial activity. N. sativa extracts were screened for qualitative detection of secondary metabolites and it showed that steroids, tannins, flavanoids, coumarins, cardiac glycosides, saponins and diterpenes were present in methanolic and ethanolic extract of N. sativa. The results of this study will provide an insight to characterize bioactive compounds from these extracts, which can act as strong bacterial growth inhibitor against wide range of infectious disease caused by pathogenic bacteria. (c) 2013 Friends Science Publishers

Keywords: Phytochemical analysis; Nigella sativa; Anti-bacterial activity; Disk diffusion; Ribotyping

Introduction

Medicinal plants are the richest bioresource of drugs for traditional systems of medicine, nutraceuticals, food supplements, modern medicines, pharmaceutical intermediates, folk medicines and chemical entities for synthetic drugs. According to World Health Organization (WHO), up to 80% of the people depends on traditional medicinal plants for their medicines (Arunkumar and Muthuselvam, 2009).

Plant products derived from barks, flowers, roots, leaves, seeds, fruits are the part of phytomedicines (Criagg et al., 2001). For synthesis of complex chemical compounds knowledge of the chemical constituents of plants is desirable (Mojab et al., 2003; Parekh and Chanda, 2007; 2008).

Phytochemical components such as tannins, carbohydrates, alkaloids, terpenoids, phenolic compounds, steroids and flavonoids are responsible for various pharmacological activities of the plants (Abbas et al., 2012a, b; Shah et al., 2011; Zaman et al., 2012). These phytochemical compounds are synthesized by primary or secondary metabolism of living organisms. Secondary metabolites are taxonomically and chemically diverse compounds with vague function. They are extensively used in agriculture, human therapy, veterinary and related scientific research etc. (Vasu et al., 2009; Mansoor et al.,2011).

N. sativa is an indigenous herbaceous plant belongs to the Ranunculaceae family that is more commonly known as the fennel flower plant. This plant has finely divided foliage and blue flowers, which produce black seeds and it grows to a maximum height of about 60 cm. The plant is known by many other names e.g. kalonji (Urdu), habba-tu sawda (Arabic), black cumin (English), shonaiz (Persian), kalajira (Bangali) (Khan, 1999). It is cultivated extensively in Pakistan and India, and also grows in the Mediterranean countries. In Islamic medicine, the use of the black seeds is recommended in daily use because it is regarded as one of the greatest forms of healing medicine available. Prophet Muhammad (PBUH) once stated that the black seed can heal every disease-except death-as narrated in the following hadith "Hold onto the use of the black seeds for in it is healing for all diseases except death"(Sahih Bukhari vol. 7 book 71 # 592).

N. sativa seeds, as nutritional and medicinal plant, have traditionally been used for thousands of years as folk medicine and some of its active compounds were reported against many ailments (Toncer and Kizil, 2004).

Different pharmacological effects such as gastric ulcer healing (Javed et al., 2010), anti-microbial effect (Mariam and Al-Basal,2009), anti-cancer activity (Shafi et al., 2009), cardiovascular disorders (Sultan et al., 2009), gastroprotective and antioxidant activity (El-Abhar et al.,2003), immunomodulatory, anti-inflammatory and anti- tumor effects (Majdalawieha et al., 2010), antitussive effect (Hosseinzadeh et al., 2008), anti-anxiety effect (Boskabady et al., 2010), anti-asthmatic effect (Navdeep et al., 2009), anti-inflammatory effects in pancreatic cancer cells (Salem,2010), anti-helicobacter activity (Tingfang et al., 2008), tumor growth suppression (Eugene et al., 2011), anti-viral activity against cytomegalovirus (Salem and Hossain,2000), hepatoprotective activity (Khan, 1999) have been reported for this medicinal plant.

This study was carried out to find the antibacterial activity of N. sativa aqueous and organic extracts against seven human pathogenic bacterial strains which were identified by ribotyping. Ribotyping is a molecular technique, which is used to identify bacteria on the basis of 16S ribosomal RNA gene. Materials and Methods Collection of Bacterial Strains Human pathogenic bacterial strains including Gram-positive bacteria Enterococcus faecalis IARS1, Staphylococuss aureus IARS4 and Gram-Negative bacteria Acinetobacter junii IARS2, Escherichia coli IARS3, Proteus mirabilis IARS5, Serratia marcescens IARS6, Enterobacter cloacae IARS7 were isolated from blood samples obtained from microbiology laboratory, Pakistan Institute of Medical Sciences Hospital, Islamabad, Pakistan.

Identification of Bacterial Strains by Ribotyping

Bacterial template DNA was extracted by overnight grown

5 mL bacterial culture. Microcentrifuged 1.5 mL at 14000 rpm for 2 min or until a compact pellet forms. The pellet was resuspended in 567 mL TE buffer. Moreover, 30 mL of 10% SDS and 3 mL of 20 mg/mL proteinase K, were added to it and mixed thoroughly and the mixture was incubated for 1 hour at 37degC and followed by addition of 100 mL of 5M NaCl and 80 mL of CTAB/NACL and incubation at 65degC. 1 vol (0.7 to 0.8 ml) of 24:1 chloroform/isoamyl alcohol was added into the mixture, mixed thoroughly and microcentrifuged at 14000 rpm for 4 to 5 min. Supernatant was transferred to fresh tube. 0.6 volume of isopropanol was added to supernatant and mixed gently to precipitate DNA. It was then microcentrifuged at 14000 rpm for 2 min at room temperature, supernatant was discarded and 70% ethanol was added to the pellet. Then the mixture was centrifuged for 5 min at room temperature to precipitate the pellet, which was dried in a lypholizer. Finally the pallet was suspended in 100 mL TE buffer.

Molecular identification of the bacterial strains was confirmed by sequencing 16S rRNA gene. Primers RS-1 (5- AAACTCAAATGAATTGACGG-3) and RS-3 (5- ACGGGCGGTGTGTAC-3) were used to ampilify 16S rRNA gene from bacteria by PCR (Zahoor and Rehman,2009).

Total of 50 mL Reaction mixture was prepared for PCR reaction using Finnzyme kit. Denaturation of DNA at 94degC for 1 min was followed by 30 cycles of amplifications (94degC for 30 sec, 60degC for 30 sec, 72degC for 1:30 min) and final extension at 94degC for 7 min. The PCR product was analyzed on 1% agarose gel. These eluted PCR product was sequenced and Basic Local Alignment Search tool BLAST was then performed for 16S rRNA sequences to identify the sequence by alignment.

Collection of N. sativa Seeds

N. sativa seeds were purchased from local herb shop in Jeddah, Saudi Arabia. The plant species was confirmed by taxonomist Dr. Muhammad Qasim Hayat (ASAB, NUST) and it was deposited at Medicinal Plant Laboratory of the Atta-ur-Rahman School of Applied Biological Sciences for future records.

Extract Preparation

N. sativa seeds were first dried at room temperature and then grinded into fine powder by using electric grinder. Eight different solvent extracts of N. sativa seeds were prepared to screen the anti-bacterial activity.

Finely grinded N. sativa seed powder was subjected to aqueous and seven organic solvents (Methanol, Ethanol, Chloroform, Diethyl ether (DEE), n-Hexane, Acetone, Butanol) separately with ratio 10:100 in a flask and then kept it on shaker at room temperature on continuous shaking for 24-48 h. The solution was then centrifuged for 15 min at 2000 rpm. The supernatant then collected and filtered through Whatmann filter paper 1. The filtrate then dried in a rotary evaporator at 50-60degC until all the solvents gets evaporated and only dry extract left behind. The dry extract then stored at 4degC for further research use.

In-vitro Antibacterial Activity

Antibacterial activities of all eight extracts of N. sativa were performed by disk diffusion method (Malika et al., 2004). Mueller Hinton Agar cultures of test microorganism were prepared with standardized inoculums maintaining bacterial culture count at 1 x 108 cells/mL. During antibacterial testing, 40 mL of extracts with different concentrations 100 mg/mL, 50 mg/mL, 25 mg/mL, 10 mg/mL, 5 mg/mL were placed separately on 6 mm filter paper disk with the help of Micropipette. Five filter paper disks with different concentrations of the same extract were placed in a single petri plate. The disk impregnated with standard antibiotics ceftriaxone 100 mg/mL, Amoxycillin 100 mg/mL, Gentamycin 100 mg/mL served as positive control. The disks soaked and evaporated in eight different solvents namely Aqueous, Methanol, Ethanol, Chloroform, DEE, Acetone, Butanol, Hexane and DMSO act as negative control. The plates were then incubated for 8-12 h at 37degC after which zone of inhibition was measured.

The experiments were performed in triplicate. The minimum concentrations of extract that inhibited bacterial growth were recorded as minimum inhibitory concentration.

Statistical analysis was carried out using the student's t-test, for the estimation of results as mean +- SD (standard deviation).

Qualitative Phytochemical Secreening for Secondary Metabolites

N. sativa seed extracts (Methanol, Ethanol, Chloroform, Hexane, DEE, Acetone, Butanol and Aqueous) were subjected to phytochemical screening for secondary metabolites. Qualitative phytochemical analysis for steroids, tannins, terpenoids, flavanoids, anthocyanins, leucoanthocyanins, coumarins, cardiac glycosides, saponins and diterpenes was carried out by standard protocols (Harborne, 1973; Trease and Evans, 1989; Sofowra, 1993).

Results

Genomic DNA of seven different human pathogenic bacterial strains were extracted (Fig. 1) and then these Genomic DNA were amplified to 470 bp amplicon, and sequenced (Fig. 2). The resulted sequences were submitted to NCBI genbank JQ863232 (E. faecalis IARS1), JQ863233 (A. junii IARS2), JQ863234 (E. coli IARS3), JQ863235 (S. aureus IARS4), JQ863236 (P. mirabilis IARS5), JQ863237 (S. marcescens IARS6), JQ863238 (E. cloacae IARS7) and aligned to search for similar sequences. These Bacterial sequences showed 99% similarity with database sequences.

All eight extracts with different concentrations showed effective results against seven pathogenic bacterial strains. Antibacterial activity of N. sativa extracts against seven pathogenic bacterial strains were summarized in Table 1. A. juni, E. coli and S. marcescens showed sensitivity against aqueous extract of N. sativa. Minimum inhibitory concentration of aqueous extract was found to be 5 mg/mL against these bacterial strains.

E. faecalis, Acinetobater Junii, E. coli, P. mirabilis, S. marcescens were sensitive to ethanolic extract with MIC value 5 mg/mL, while S. aureus isolates were resistant to ethanolic extract. MIC of methanolic extract is 5 mg/mL against all the tested bacterial strains, while it is 25 mg/mL against S. aureus. MIC value of chloroform extract against S. aureus, E. cloacae was 10 mg/mL and 25 mg/mL against E. coli. MIC value of Acetonic extract against E. faecalis was 100 mg/mL, while it was 50 mg/mL against S. aureus and E. cloacae. MIC value of n-hexane

Fig. 1: Lane 1: 1 kb ladder, Lane 2-8 shows genomic DNA of seven bacterial strains. Lane 2= Enterococcus faecalis, Lane 3= Acinetobacter junii, Lane 4= Escherichia coli, Lane 5= Staphylococuss aureus, Lane 6= Proteus mirabilis, Lane 7= Serratia marcescens Lane 8= Enterobacter cloacae

Fig. 2: PCR-based amplification of seven pathogenic bacterial 16S rRNA gene. M: Hindi III ladder, Lane 1-7 shows bands of amplified DNA fragments at approximately 500 bp. Lane 1= Enterococcus faecalis, Lane 2= Acinetobacter junii, Lane 3= Escherichia coli, Lane 4= Staphylococuss aureus, Lane 5= Proteus mirabilis,Lane 6= Serratia marcescens Lane 7= Enterobacter cloacae

extract against E. coli, S. aureus and P. mirabilis is 100 mg/mL, 50 mg/mL and 25 mg/mL respectively, while rest of the strains were sensitive to it with MIC value 5 mg/mL. S. aureus and P. mirabilis were resistant to the diehtyl ether

Table 1: Zone of inhibition of eight extracts against seven pathogenic bacterial strains

Zone of Inhibition (mm)

Identified by Ribotyping

###Extract###E. faecalis###A. junii###E. coli###S. aureus###P. mirabilis###S. marcescens###E. cloacae

###100 mg/ml###N.Z###15+1.0###14.0+1.0###N.Z###N.Z###12.0+1.0###N.Z

Aqueous###50 mg/ml###N.Z###13.3+0.57###12.33+1.0###N.Z###N.Z###12.3+0.5###N.Z

###25 mg/ml###N.Z###12.6+0.57###10.66+1.1###N.Z###N.Z###13.5+1.3###N.Z

###10 mg/ml###N.Z###11.5+0.8###9.1+1.2###N.Z###N.Z###13.6+1.5###N.Z

###5 mg/ml###N.Z###9.5+0.8###8.83+0.7###N.Z###N.Z###14.0+1.0###N.Z

###100 mg/ml###15.33+1.5###15.16+1.0###13.5+1.3###N.Z###12.0+0.0###17.33+1.1###14.4+0.5

Ethanolic###50 mg/ml###13.33+0.5###14.0+1.0###12.0+1.0###N.Z###11.0+1.0###14.7+1.5###13.4+1.1

###25 mg/ml###12.66+0.7###12.67+0.5###11.33+1.2###N.Z###9.6+1.0###14.0+1.0###12.3+0.5

###10 mg/ml###11.33+0.5###11.5+0.5###10.0+1.3###N.Z###10.0+0.0###12.33+0.5###11.0+1.0

###5 mg/ml###8.67+0.5###10.5+0.5###9.5+1.0###N.Z###8.6+0.5###10.33+0.5###N.Z

###100 mg/ml###16.33+0.5###15.33+0.5###15.0+1.0###14.6+1.5###13.33+0.5###15.33+0.5###15.0+1.0

Methanolic###50 mg/ml###14.0+0###14.33+1.5###12.6+0.5###12.0+1.0###11.66+0.5###13.66+.5###14.4+1.1

###25 mg/ml###13.33+1.1###14.33+1.5###12.33+0.51###9.3+1.5###10.16+0.7###12.0+1.0###12.7+0.5

###10 mg/ml###11.33+1.1###12.33+0.7###11.33+1.2###N.Z###9.3+0.7###11.33+0.5###10.7+1.1

###5 mg/ml###11.33+1.1###11.0+0.1###10.8+1.4###N.Z###8.83+1.0###10.0+1.0###9.0+0.0

###100 mg/ml###12.83+1.0###13.0+1.0###12.0+1.5###15.33+1.1###14.0+1.0###11.66+0.5###13.7+1.5

###50 mg/ml###13.83+0.2###13.0+1.0###11.66+1.5###14.0+1.0###13.33+0.5###13.33+1.5###13.$+1.2

Chloroform###25 mg/ml###14.83+0.2###14.67+0.7###11.0+1.3###12.0+2.0###10.5+0.5###13.6+1.5###11.0+1.7

###10 mg/ml###15.5+0.5###14.83+0.7###N.Z###9.1+0.7###10.0+0.0###15.0+1.7###11.0+1.4

###5 mg/ml###16.33+0.5###16.16+0.2###N.Z###N.Z###8.6+0.5###15.0+1.7###N.Z

###100 mg/ml###11.5+0.8###14.67+0.5###12.66+1.1###13.66+1.5###12.66+1.1###15.0+1.0###15.4+1.1

Acetonic###50 mg/ml###N.Z###14.0+0.0###11.83+1.4###10.33+2.5###11.33+2.3###13.6+0.5###14.0+1.1

###25 mg/ml###N.Z###12.83+0.7###10.1+0.2###N.Z###10.0+2.6###12.3+0.5###N.Z

###10 mg/ml###N.Z###12.1+1.0###9.5+1.0###N.Z###9.16+0.7###12.4+0.5###N.Z

###5 mg/ml###N.Z###11.3+1.5###N.Z###N.Z###8.83+0.76###12.0+0.0###N.Z

###100 mg/ml###14.86+0.8###16.0+1.0###7.75+0.3###15.8+1.0###15.0+1.0###14.0+1.0###17.4+1.1

Hexane###50 mg/ml###12.83+2.0###14.67+0.5###N.Z###13.0+1.0###12.6+0.5###12.5+0.5###17.0+1.0

###25 mg/ml###11.33+1.5###13.16+1.2###N.Z###N.Z###9.7+0.5###10.8+0.7###14.7+1.1

###10 mg/ml###11.0+1.7###10.8+0.28###N.Z###N.Z###N.Z###10.0+0.0###12.6+0.5

###5 mg/ml###10.33+2.5###10.0+0.0###N.Z###N.Z###N.Z###9.3+0.7###10.83+0.7

###100 mg/ml###N.Z###13.33+0.5###13.1+1.0###N.Z###N.Z###16.7+0.5###13.66+0.5

Diethyl Ether###50 mg/ml###N.Z###12.67+0.5###11.3+1.1###N.Z###N.Z###14.4+0.5###12.0+0.0

###25 mg/ml###N.Z###12.16+0.7###10.2+1.2###N.Z###N.Z###13.7+0.5###11.33+1.1

###10 mg/ml###15.83+1.0###10.8+0.28###N.Z###N.Z###N.Z###12.0+1.0###10.0+1.0

###5 mg/ml###11.67+1.5###10.0+0.0###N.Z###N.Z###N.Z###11.0+1.0###N.Z

###100 mg/ml###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z

Butanol###50 mg/ml###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z

###25 mg/ml###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z###13.7+1.5

###10 mg/ml###12.0+ 0###N.Z###N.Z###N.Z###N.Z###N.Z###11.7+1.5

###5 mg/ml###8.0+0###N.Z###N.Z###N.Z###N.Z###N.Z###10.33+1.5

Gentamycin###100 g/ml###22+0.5###25+0.1###24+0.2###20+0.2###28+0###20+0.1###24+.1

Amoxycillin###100 g/ml###20+0###N.Z###N.Z###18+0###18+0.2###N.Z###10+0

Ceftriaxone###100 g/ml###25+0###18+0.2###15+0.3###32+0.1###N.Z###17+0###N.Z

Negative control DMSO###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z###N.Z

E. faecalis=Enterococcus faecalis, A. junii=Acinetobacter junii, E. coli= Escherichia coli, S. aureus= Staphylococcus aureus, P. mirabilis=Proteus

mirabilis, S. marcescens=Serratia marcescens, E. cloacae=Enterobacter cloacae. Values are expressed as mean+- SD (n = 3). N.Z= No zone

Table 2: Phytochemical Analysis of Nigella Sativa linn. Seed Extracts

EXTRACTS###Methanol###Ethanol###Chloroform###DEE###Acetone###Aqueous###Butanol###Hexane

Steroids###+###+###++###++###+++###-###-###+

Tannins###++###++###-###-###-###++###+###-

Terpenoids###-###-###+++###-###-###-###+++###-

Flavanoids###++###++###+###-###-###+++###-###-

Anthocyanins###-###-###-###-###-###-###-###-

Leucoanthocyanins###-###-###-###-###-###-###-###-

Coumarins###+++###++###+###+###+###+++###-###+

Cardiac Glycosides###++###+###+###++###++###-###+###+

Saponins###++###++###-###+###-###-###+###-

Diterpens###++###++###-###-###-###+###+###-

+ = weakly present, ++ = Moderately present, +++= Strongly Present, - = Not present

extract, while it is sensitive to E. faecalis, A. junii, E. coli, S. marcescens E. cloacae. Butanolic extract is only sensitive to E. faecalis and E. cloacae, while it is resistant to rest of bacterial strains. Results on the phytochemical screening of aqueous and seven organic extracts of N. sativa seeds are summarized in Table 2.

Discussion

In the Current study, antibacterial activity of organic and aqueous extracts of N. sativa seeds in comparison with standard drugs Ceftriaxone, Amoxycillin, Gentamycin was determined and found to proceed in dose dependent manner against major clinical isolates. Methanolic extract of N. sativa seeds shows antibacterial activity against all bacterial strains under investigation. Thymol is present in the methanol extract of N. sativa (Enomoto et al., 2001) that has been reported to possess antibacterial activity (Karapinar and Aktug, 1987). It was also investigated by (Mason et al., 1987) that thymol is responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds, possibly through nonspecific interactions with the proteins.

In present study, phytochemical screening for secondary metabolites shows that steroids, tannins, flavanoids, coumarins, cardiac glycosides, saponins and diterpenes were present in methanolic extract. Previously it was reported that N. sativa seeds contain tannins, which is extracted in methanolic extract (Eloff et al., 1998). Tannins forms complexes with proteins through forces such as hydrophobic effects, hydrogen bonding and covalent bond formation, thus, tannins act as antibacterial agent by inactivating microbial adhesins, enzymes, cell envelope transport proteins (Hashem and El-Kiey, 1982).

The use of N. sativa oil in preserving food has shown the oil to be a potent inhibitor of food hazardous and spoilage bacteria (El-Sayed et al., 1996; Zuridah et al.,2008).

Current study also proves that oil extracted by different N. sativa seed extracts show antibacterial activity against food spoilage bacteria including E. coli and S. aureus with MIC value 5 mg/mL.

Present study shows that chloroform extract possess antibacterial activity against all human pathogenic bacterial strains under study. This antibacterial activity is due to the presence of steroids, terpenoids, flavonoids, coumarins and cardiac glycosides in the chloroform extract. Aqueous extract of N. sativa seed showed less antibacterial activity.

This may be due to lower accessibility of the aqueous extract to the micro-organism or low extraction of antibacterial agents into this extract (Morsi, 2000).

All the extracts except aqueous extract contain oily content due to which extract appears oily. It was investigated three decades ago that volatile oil of N. sativa had antimicrobial activity, which is responsible for growth inhibition of both gram positive and gram negative bacteria (Toppozada et al., 1965). This is proved by current study that oil extracted by methanolic, ethanolic, diethyl ether, acetonic and n-hexane shows effective antibacterial activity against both gram positive and gram negative bacteria.

In conclusion, bacterial resistance against antibiotics is a great challenge. The results of this study shows that N. sativa seeds extracts have antibacterial activity against most common bacterial strains involved in human pathogenesis. In addition, phytochemicals evaluation of N. sativa seed provides information about number of medicinally important secondary metabolites, which impart antibacterial characteristics. Further studies are recommended to find the antiviral activity of N. sativa against life threatening viral diseases e.g. HCV, HBV and Dengue.

Acknowledgements

Authors acknowledge the financial support of Research Directorate, National University of Sciences and Technology (NUST), H-12 Islamabad, Pakistan.

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Medicinal Plants Research Group, Department of Plant Biotechnology, Atta-Ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), H-12 Islamabad, Pakistan

Pakistan Council for Science and Technology (PCST), Ministry of Science and Technology (MoST) Islamabad, Pakistan

Corresponding author's e-mails: ashrafjahanian@yahoo.com; ashrafjahanian@asab.nust.edu.pk
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Author:Ishtiaq, Shabina; Ashraf, Muhammad; Hayat, Muhammad Qasim; Asrar, Mudassir
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2013
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