Authors Accepted Manuscript

Isolation, characterization and HPLC quantification

of compounds from Aquilegia fragrans Benth:
Their in vitro antibacterial activities against bovine
mastitis pathogens

Saleem Mushtaq, Mushtaq A Aga, Parvaiz H Qazi,

Md. Niamat Ali, Aabid Manzoor Shah, Sajad
Ahmad Lone, Aiyatullah Shah, Aehtesham www.elsevier.com/locate/jep

Hussain, Faheem Rasool, Hafizullah Dar, Zeeshan

Hamid Shah, Shabir H Lone

PII: S0378-8741(15)30241-5
DOI: http://dx.doi.org/10.1016/j.jep.2015.11.039
Reference: JEP9838
To appear in: Journal of Ethnopharmacology
Received date: 30 April 2015
Revised date: 23 October 2015
Accepted date: 23 November 2015
Cite this article as: Saleem Mushtaq, Mushtaq A Aga, Parvaiz H Qazi, Md.
Niamat Ali, Aabid Manzoor Shah, Sajad Ahmad Lone, Aiyatullah Shah,
Aehtesham Hussain, Faheem Rasool, Hafizullah Dar, Zeeshan Hamid Shah and
Shabir H Lone, Isolation, characterization and HPLC quantification of
compounds from Aquilegia fragrans Benth: Their in vitro antibacterial activities
against bovine mastitis pathogens, Journal of Ethnopharmacology,
http://dx.doi.org/10.1016/j.jep.2015.11.039
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 Isolation, characterization and HPLC quantification of compounds from Aquilegia
fragrans Benth: Their in vitro antibacterial activities against bovine mastitis pathogens

Saleem Mushtaqa,d, Mushtaq A Agab, Parvaiz H Qazi a,*, Md. Niamat Alid Aabid Manzoor
Shaha, Sajad Ahmad Lonea, Aiyatullah Shaha, Aehtesham Hussaina, Faheem Rasoolc,
Hafizullah Darc, Zeeshan Hamid Shaha and Shabir H Loneb

a) Microbial Biotechnology Division, CSIR - Indian Institute of Integrative Medicine,

Sanatnagar, Srinagar, Jammu & Kashmir - India, 190005.
b) Bioorganic Chemistry Division, CSIR - Indian Institute of Integrative Medicine,
Sanatnagar, Srinagar, Jammu & Kashmir - India, 190005.
c) Medicinal Chemistry Division, CSIR - Indian Institute of Integrative Medicine,
Sanatnagar, Srinagar, Jammu & Kashmir - India, 190005.
d) Centre of Research for Development (CORD), University of Kashmir - India, 190006.

*Corresponding author: Microbial Biotechnology Division, CSIR - Indian Institute of

Integrative Medicine, Sanatnagar, Srinagar- India, 190005
Email: pervaizqazi@yahoo.com /qphassan@iiim.ac.in , Cell No: +91-9596050105. Fax:
+91-01942441331.
Abstract:

Ethno-pharmacological relevance: The underground parts of Aquilegia fragrans are

traditionally used for the treatment of wounds and various inflammatory diseases like bovine
mastitis. However, there are no reports on the phytochemical characterization and
antibacterial studies of Aquilegia fragrans.

Aim of the study: To isolate compounds from the methanol extract of the underground parts of
Aquilegia fragrans and determine their antibacterial activity against the pathogens of bovine
mastitis. The study was undertaken in order to scientifically validate the traditional use of
Aquilegia fragrans.

Materials and methods: Five compounds were isolated from the methanol extract of the
underground parts of Aquilegia fragrans using silica gel column chromatography. Structural
elucidation of the isolated compounds was done using spectral data analysis and comparison
with literature. High performance liquid chromatography (HPLC) was used for the
qualitative and quantitative determination of isolated compounds in the crude methanol
extract. The methanol extract and isolated compounds were evaluated for antibacterial
activities against mastitis pathogens using broth micro-dilution technique.

Results: The five isolated compounds were identified as 1) 2, 4-dihydroxyphenylacetic acid

methyl ester 2) -sitosterol 3) Aquilegiolide 4) Glochidionolactone-A and 5)
Magnoflorine. A quick and sensitive HPLC method was developed for the first time for
qualitative and quantitative determination of four isolated marker compounds from Aquilegia
fragrans. The crude methanol extract and compound 5 exhibited weak antibacterial activities
that varied between the bacterial species (MIC = 5003000 g/ml).

Conclusions: The above results show that the crude methanol extract and isolated compounds
from Aquilegia fragrans exhibit weak antibacterial activities. Further phytochemical and
pharmacological studies are required for proper scientific validation of the folk use of this
plant species in the treatment of various inflammatory diseases like bovine mastitis.

Keywords: Bovine mastitis, Aquilegia fragrans, Magnoflorine, HPLC, antibacterial activity,

minimum inhibitory concentration (MIC).
Chemical compounds studied in the article: -sitosterol (PubChem CID: 222284);
Magnoflorine (PubChem CID: 73337).
1. Introduction:

Mastitis i.e.; mammary gland inflammation, is the most common and expensive disease
affecting the dairy industry worldwide. The annual economic losses due to mastitis in India
and worldwide have been estimated at $1.1 billion (Mir et al., 2014) and $35 billion
(Wellenberg et al., 2002), respectively. Mastitis is often caused by bacterial infection, mostly
by Staphylococci, Streptococci, and gram negative bacteria like Escherichia coli and
Klebsiella pneumoniae (Contreras and Rodriguez, 2011). Coagulase negative staphylococci
(CNS) such as Staphylococcus xylosus and Staphylococcus chromogenes are opportunistic
pathogens that have recently emerged as important mastitis pathogens (Pyorala and Taponen,
2009). The most common treatment method available against these pathogens is the
intramammary infusion of antibiotics. However, their indiscriminate use has led to the
emergence of antibiotic resistant strains of bacteria (Gindonis et al., 2013). Therefore, there
is a need to search for alternative approaches for the treatment of bovine mastitis.

Aquilegia fragrans Benth; Ranunculaceae (Columbine) is a perennial herb distributed in the

North-West Himalayan region, in Eastern Afghanistan, Pakistan and North-Western India. Its
popular name is Sweet-scented columbine and locally known as Zaoneel, Kalumb and Jangli
kuth. In India, the plant grows in the wild at high altitudes of Kashmir Valley (Mathew and
Sinnott, 2003). Here, the underground parts of the plant are used traditionally in the treatment
of wounds and various inflammatory diseases (Beigh et al., 2014). It is also used to treat
various veterinary diseases like bovine mastitis. The aim of the present study was to isolate
compounds from the methanol extract of the underground parts of Aquilegia fragrans and
determine their antibacterial activity against the pathogens of bovine mastitis. The study was
undertaken to scientifically validate the traditional use of Aquilegia fragrans.

2. Materials and methods.

2.1. Plant material. Proper permission was sought from the state forest department to collect the
underground parts of Aquilegia fragrans. Only minimum quantity of sample for sole research
purposes was collected in presence of personnel from the concerned forest department. The
plant material of Aquilegia fragrans was collected from Aharbal area of Kashmir Valley,
India in the month of September 2013. Aharbal is situated at an altitude of 2,266 m
(7,434 ft) having latitude 33 North and longitude 74 East. The plant
 material was identified with the help of taxonomists at Centre for Biodiversity and
Taxonomy (CBT), Department of Botany, University of Kashmir. A Voucher specimen No.
2039-KASH, was deposited in the Departments herbarium. The plant name has been
verified with www.theplantlist.org.

2.2. Extraction and isolation. The plant material was properly washed, shade dried and
pulverized into powder using a mechanical grinder. The powdered material was extracted
with methanol using the cold maceration technique. The extracts obtained were kept at 4C
for further use.

A portion (30g) of the methanol extract was dissolved in minimum amount of

dichloromethane (DCM) and adsorbed on silica gel, air dried and chromatographed over
silica gel (60120mesh). The column was eluted with petroleum ether: ethyl acetate in the
ratio of 1:1 to afford compound 1. The same column then eluted with pure DCM yielded
compound 2. Finally, the column was further eluted with a step gradient mixture of
DCM/methanol in the ratio of 99:1, 98:2, 95:5, 90:10 and 85:15 to afford compounds 3, 4,
and 5.

2.3. High-performance liquid chromatography (HPLC) analysis. Liquid chromatography

separation was performed using Nexera UHPLC composed of quaternary pump, prominence
degassing unit, Autosampler, Column oven and Diode Array Detector (DAD).
Chromatographic separation was carried out using Enable RP C18 Column
(250mm4.5mm, 5m) at 25C. Elution was performed at a flow rate of 0.6ml/min and the
injection volume was 5l. The injection volume was kept constant at 5l for the extract as
well as the standards. Proper dilutions were made to the extract to keep it in the range of
LOD and LOQ. Solvents used were 0.1% formic acid in water (A) and methanol (B). All
solvents were filtered through a 0.45m nylon filter after ultrasonic degassing. Isocratic
flow of 80:20 A: B was used for analysis. The chromatograms were recorded at 278nm and
data was acquired using Lab solutions software. For quantitative analysis, the extract was
analyzed under the conditions described above. Compounds were run at four different
concentrations and found to be linear in the range with a correlation coefficient (r2) of
0.999-0.997. The estimation of the compounds content in the extract was performed using
 linear regression analysis. The standard solution was prepared by dissolving 1 mg of the
compound in 1 ml of methanol.

2.4. Antibacterial activity.

2.4.1. Test organisms. The in vitro antibacterial activity of the methanol extract and isolated
compounds was tested against different pathogens viz.; Staphylococcus aureus (MTCC 96),
Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 109), Streptococcus
pyogenes (MTCC 442), and clinical isolates of bovine mastitis viz., Staphylococcus xylosus
(SM-1001), Staphylococcus equorum (SM-1002), Enterococcus faecaelis (SM-1004) and
Pantoae sp (SM-1006).

2.4.2. Preparation of stock and working solutions: The stock solutions were prepared at a
concentration of 10 mg/ml for compounds and 100mg/ml for extract, respectively, in 100%
dimethyl sulphoxide (DMSO, Sigma Aldrich). The working solutions were prepared by
diluting stock solutions in Mueller Hinton broth. The reference antibiotic, Ciprofloxacin was
obtained from Sigma Aldrich. The concentration of the stock solution was 1mg/ml.
Ciprofloxacin is a broad spectrum antibacterial drug active against both gram positive and
gram negative bacteria. It is also used in the treatment of bovine mastitis.

2.4.3. Determination of Minimum inhibitory concentration (MIC): The MIC was determined
by broth micro-dilution method and as per the guidelines of Clinical and Laboratory
Standards Institute (CLSI, 2006, M7-A7), with some modifications. Briefly, 200 l of sample
mixed with Mueller Hinton Broth (MHB) was added to the first wells of each row. All the
remaining wells initially received 100 l of MHB. Then, two-fold serial dilutions were
performed by transferring 100 l from column 1 to column 2 and continued through column
10. 100 l of excess medium was discarded from the wells in column 10. For the preparation
of bacterial inocula, 24h cultures were suspended in 5ml of sterile normal saline. The
turbidity of each bacterial suspension was adjusted to 0.5 McFarland standards (1.5 x 108
CFU/ml). The bacterial suspension was further diluted in MHB and 100l of the same was
added to each well of micro-titre plate to obtain a required inoculum of 5105 CFU/ml in the
well. The final concentration of samples ranged from 12.0 to 6000 g/ml for extract and from
2.0 to 1000 g/ml for compounds, respectively. Columns 11 and 12 served as positive and
negative controls. The plates were then incubated at 37C for 24 h and were visually read for
 the absence or presence of turbidity. The MIC was considered as the lowest concentration of
the sample showing no visible growth or turbidity.

3. Results:

3.1. Phytochemical composition. A total of five known phytochemical compounds [(1) 2, 4-

dihydroxyphenylacetic acid methyl ester, a phenylacetic acid derivative (2) -sitosterol, a
sterol (3) Aquilegioilide, a butenolide (4) Glochidionolactone-A, a butenolide glucoside
and (5) Magnoflorine, an aphorphine alkaloid] were isolated from the methanol extract of
underground parts of Aquilegia fragrans. The structures of the isolated compounds were
established using various spectroscopic techniques and direct comparison with literature
(Supplemental File 1). This study reports for the first time the isolation of compounds from
Aquilegia fragrans. Furthermore, the first two isolated compounds viz.; 2, 4-
dihydroxyphenylacetic acid methyl ester and -sitosterol are reported for the first time from
the Genus Aquilegia.

3.2. HPLC analysis: A simple and sensitive HPLC method was developed for identification of
the standard compounds isolated from the methanol extract of Aquilegia fragrans. The
chromatographic separation of these compounds was achieved in less than 40 minutes with
retention time 6.7, 10.2, 11.02 and 35.64 respectively. Figure 1 shows the chromatograms
obtained from methanol extract and standards isolated from the underground parts of
Aquilegia fragrans. The peaks corresponding to the individual compounds are symmetrical
and well resolved from other co-extracted material. The method ensured good column
performance and reproducible retention times and peak areas for the compounds investigated.
The external standard method was used for the quantification process. Quantification of the
compounds by HPLC-DAD showed their content to be 0.13 g (2, 4-dihydroxyphenylacetic
acid methyl ester), 2.4 g (Aquilegiolide), 3.1 g (Glochidionolactone-A) and 0.82 g
(Magnoflorine) per gram of the crude methanol extract. The limit of detection (LOD) and
limit of quantification (LOQ) were calculated using the equations: LOD= 3.3 /S and LOQ=
10 /s where and S represent the standard deviation of response and the slope of the
calibration curve respectively. Limit of detection represents the lowest concentration of the
analyte in a sample that can be detected by HPLC under the developed method while as limit
of quantification represents the lowest concentration that can be quantified under the
 operating conditions. The data of retention time, regression equation, correlation coefficient
(r 2), linear range as well as limits of detection (LOD) and quantification (LOQ) of each
compound are summarized in Table 1.

Table 1

Compound name Retention Regression equation r2 Linear range LOD LOQ

time (g/ml) (g/ml) (g/ml)
2,4-dihydroxy 6.7 58412.4*x-2309.82 0.999 1-8 0.123 4.09
phenyl acetic acid
methyl ester
Aquilegiolide 10.2 2189.02*x+1232.85 0.999 2-16 0.157 0.52
Glochidionolactone A 11.02 8008.64*x+2180.84 0.997 10-80 4.67 15.56
Magnoflorine 35.64 104328*x+1648.14 0.997 0.5-4 0.152 0.506

Figure 1

A) B)

3.3. Antibacterial activity and Minimum inhibitory concentration (MIC). The crude
methanol extract exhibited weak antibacterial activity with MIC values ranging from 1500-
3000 g/ml. Compounds 4 and 5 also showed weak antibacterial activities. Compound 4 i.e.;
Glochidionolactone A was weakly active against Staphylococcus aureus and Klebsiella
pneumonia with MIC value of 1000 g/ml. Compound 5 i.e.; Magnoflorine showed weak
antibacterial activity against different gram-positive and gram-negative bacteria with MIC
values in the range of 500-1000 g/ml (Table 2).
 Table 2

Sample MIC values of the tested samples in g/ml

S. a E. c K. p S. p S. x S. e E. f P. sp.

Methanol extract 1500 3000 1500 3000 1500 1500 3000 3000

Glochidionolactone A 1000 NA 1000 NA NT NT NT NT

Magnoflorine 500 1000 1000 1000 500 500 1000 1000

Ciprofloxacin 50 25 25 50 50 50 25 25

MIC, Minimum inhibitory concentration: S. a = Staphylococcus aureus; E. c = Escherichia coli; K. p =

Klebsiella pneumoniae; S. p = Streptococcus pyogenes; S. x = Staphylococcus xylosus; S. e = Staphylococcus equorum; E. f =
Enterococcus faecalis.; P. sp = Pantoae sp., NA = Not active at a concentration of 1000 g/ml; NT= Not tested.

4. Discussion: Bovine mastitis continues to be the most frequent and costly disease of dairy
cattle all over the world (Seegers et al., 2003). The use of conventional antibiotics against
bovine mastitis has led to the emergence of antibacterial resistant strains (Tenhagen et al.,
2006). Medicinal plants have been used for millennia in the treatment of infection and can
serve as an alternative source for combating multidrug resistant bacteria (Wright and
Sutherland, 2007). To the best of our knowledge, the present study reports for the first time
the phytochemistry and antibacterial studies of Aquilegia fragrans against pathogens of
bovine mastitis. The crude methanol extract and two isolated compounds i.e.;
Glochidionolactone A and Magnoflorine weakly inhibited the growth of various mastitis
pathogens such as Staphylococcus aureus and coagulase negative Staphylococci.

5. Conclusion: The results of this study show that the crude methanol extract and isolated
compounds from the underground parts of Aquilegia fragrans have weak antibacterial
activity. Further phytochemical and pharmacological studies are required for proper scientific
validation of the folk use of this unexplored plant species especially in the treatment of
various inflammatory diseases like bovine mastitis.

Acknowledgements: The first author is grateful to University Grants Commission (UGC), New
Delhi for providing fellowship. The authors are thankful to Mr. Akhtar H. Malik (Curator)
Centre for Biodiversity and Taxonomy (CBT), Department of Botany, University of Kashmir
for identification of the plant material. The role of Dr. Tabasum Shaheen, Laboratory Officer,
 Disease Investigation Laboratory, Animal Husbandry Department, Srinagar, is well
acknowledged. The authors are also thankful to Dr. Y.S. Bedi (Head) and Dr. Ram A.
Vishwakarma (Director) for providing the necessary facilities during this period.
5. References:
1. Beigh, B.A., Ramamoorthy, D., Wani, B.A., 2014. Population status and conservation
prioritization of some threatened Medicinal plants of Kashmir Himalayas.
International Journal of Applied Biology and Pharmaceutical Technology 5(4), 1-14.
2. CLSI, Clinical and Laboratory Standards Institute. 2006. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard.
M7-A7. 7th edition Wayne, PA.
3. Contreras, G.A., Rodrguez, J.M., 2011. Mastitis: Comparative Etiology and
Epidemiology. J Mammary Gland Biol Neoplasia 16, 339-356.
4. Gindonis, V., Taponen, S., Myllyniemi, A.L., Pyorala, S., Nykasenoja, S., Salmenlinna, S.,
Lindholm, L., Rantala, M., 2013. Occurrence and characterization of methicillin
resistant staphylococci from bovine mastitis milk samples in Finland. Acta Veterinaria
Scandinavica 55(61), 1-8.
5. Mathew, B., Sinnott, M., 2003. AQUILEGIA FRAGRANS-Ranunculaceae. Royal
Botanic Gardens, Kew. Blackwell Publishing Ltd. Page 151.
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dairy farms in Punjab: prevalence, distribution of bacteria and current antibiogram.
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8. Pyorala, S., Taponen, S., 2009. Coagulase-negative staphylococciemerging mastitis
pathogens. Vet. Microbiol 134, 3-8.
9. Seegers, H., Fourichon, C., Beaudeau, F., 2003. Production effects related to mastitis
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Pathogens and Their Resistance against Antimicrobial Agents in Dairy Cows in
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 Graphical abstract

Figure1. A) HPLC chromatogram of the methanol extract solution of the underground parts of
Aquilegia fragrans B) HPLC chromatogram of the standards isolated from the
underground parts of Aquilegia fragrans.

Table Captions:

Table1. Retention time, regression equation, correlation coefficient (r2), linear range and limits of
detection (LOD) and quantification (LOQ) for isolated standards from Aquilegia fragrans

Table2. Antibacterial activity and Minimum inhibitory concentrations (g/ml) of the methanol
extract and isolated compounds from the underground parts of Aquilegia fragrans