Journal of Pharmacognosy and Phytochemistry 2019; 8(3): 2668-2673

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(3): 2668-2673 Isolation, purification and characterization of
Received: 01-03-2019
Accepted: 05-04-2019 antibacterial bioactive compounds from
Madhusudhan KN
Bougainvillea spectabilis Leaf.
Silkworm Molecular Biology Lab,
Central Sericultural Research and
Training Institute, Central Silk Madhusudhan KN, Vinayarani G, Moorthy SM, Satish L, Thirupathaiah,
Board, Mysore, Karnataka, India
Y, Maheshwari C, Prakash HS, Teotia RS and Sivaprasad V
Vinayarani G
Department of Biotechnology,
University of Mysore, Abstract
Manasagangotri, Mysore, Karnataka, Bacterial diseases are the major threats for the mankind and silkworm. Hence, present study was initiated
India to evaluate the antibacterial potential of different solvent extracts of Bougainvillea spectabilis leaves. The
initial screening of different solvents against mulberry silkworm infecting bacteria, Bacillus
Moorthy SM
Silkworm Molecular Biology Lab, thuringenesis revealed that, chloroform extract showed more inhibition of test pathogen. Further, the
Central Sericultural Research and chloroform extract was evaluated for the antibacterial potential against Salmonella typhi and Shigella
Training Institute, Central Silk flexneri, human pathogens. Chloroform extract of B. spectabilis inhibited the both pathogens, which
Board, Mysore, Karnataka, India confirms the antibacterial character of extract on both gram negative and positive bacteria. TLC
Satish L fingerprinting of chloroform extract revealed presence of five major bands with different retardation
Silkworm Molecular Biology Lab, factor value (RF value). The screening of individual bands for antibacterial activity revealed that, the
Central Sericultural Research and band with Rf value of 0.84. The HPLC profiling of showed the reduction in number of compounds in
Training Institute, Central Silk partially purified extract comparison with crude extract. GC-MS analysis of antibacterial band revealed
Board, Mysore, Karnataka, India
the presence of Nineteen different compounds having different important biological activity viz.,
Thirupathaiah, Y antibacterial, anti-inflammatory, antidiabetic, antifungal, antioxidant and antiprotozoal characters. The
Silkworm Molecular Biology Lab, results of the present work confirms that, the chloroform extract of B. spectabilis leaf can be used for the
Central Sericultural Research and management of bacterial diseases of humans and silkworm.
Training Institute, Central Silk
Board, Mysore, Karnataka, India
Keywords: Bougainvillea spectabilis, Antibacterial activity, Silkworm, TLC, HPLC, GC-MS
Maheshwari C
Silkworm Molecular Biology Lab,
Central Sericultural Research and
Introduction
Training Institute, Central Silk Plant based medicines has become a part of traditional healthcare in majority parts of the
Board, Mysore, Karnataka, India world for thousands of years. Several medicinal plants are being used daily in Ayurvedic
Prakash HS
practices. In India more than 7,000 medicinal plant species are known, which contains many
Department of Biotechnology, biological active compounds having antimicrobial property and were used as an antimicrobial
University of Mysore, anasagangotri, drugs in traditional medicines. As per the report of World Health Organization, nearly 80% of
Mysore, Karnataka, India
world’s populations depends on traditional medicine for their primary health care needs.
Teotia RS Recently, medical practitioners are switching over to such plant based medicines, as there is a
Silkworm Molecular Biology Lab, high rate of developing of resistant pathogens due to excessive selection pressures created by
Central Sericultural Research and
Training Institute, Central Silk misuse and rampant use of classical antimicrobials like antibiotics (Bayane et al., 2016) [4].
Board, Mysore, Karnataka, India Bougainvillea is a genus of flowering plant belonging to the family of Nyctaginaceae native in
Sivaprasad V
South America. It has about 18 species and generally used in the arid landscapes for
Silkworm Molecular Biology Lab, beautification, horticulture, pharmaceutical industries, agriculture and environmental industries
Central Sericultural Research and on account of the large flexibility in different agro climatic regions of the world. Bougainvillea
Training Institute, Central Silk
Board, Mysore, Karnataka, India spectabilis is another widely used species from Brazil. It grows to 6 feet. Its leaves are darker
than the rest of bougainvilleas, with hairy leaves, covered with very prominent hairs on the
underside. It flowers during summer and its bracts are bright red or purple.
They found literally thousands of phytochemicals from plants as safe and broadly effective
alternatives with less adverse effect. Many beneficial biological activity such as antibacterial,
anti-inflammatory, antiulcer, anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic
and wound healing activity were reported.
In this study, Bougainvillea plant was investigated for the bioactive compounds present in its
leaf. Extraction of the plant material was done with n-butanol, methanol, chloroform and
distilled water and the bioactivity of each extract was tested against mulberry silkworm
Correspondence pathogen, Bacillus thuringiensis and human pathogens like Salmonella typhi, Shigella flexneri.
Madhusudhan KN The separation and characterization of bioactive compounds present in the leaf was carried out
Silkworm Molecular Biology Lab,
Central Sericultural Research and by using TLC, HPLC and GC-MS.
Training Institute, Central Silk
Board, Mysore, Karnataka, India

~ 2668 ~
Journal of Pharmacognosy and Phytochemistry

Materials and Methods Rf = distance travelled by centre of component /distance

Maintainance of pathogens travelled by solvent front.
Pre-maintained culture of bacterial pathogens viz., Bacillus The different bands from TLC plate were detached (scrapped)
thuringiensis was collected by culture collection center of separately. The individual bands were dissolved in chloroform
CSRTI, Mysore, where as Salmonella typhi and Shigella and the obtained supernatant was subjected to antibacterial
flexneri were collected from the Department of activity against B. thuringenesis. The antibacterial activity
Biotechnology, University of Mysuru, Mysuru. The sub- exhibiting bands was further used for purity and analyis of
culturing of the pathogens was carried out under a-septic compounds.
condition to evaluate the antibacterial activity aganist leaf
extract. The strains were grown in NB medium and incubated Analytical HPLC of crude and partially purified
at 37 ºC for overnight. After incubation the culture were chloroform extract of B. spectabilis
centrifuged and the pellets were collected to check the The partially purified chloroform extract was subjected to
antimicrobial sensitivity test. HPLC for purity analysis. Samples were filtered through an
ultra-membrane filter (pore size 0.45 µm) prior to injection in
Collection of plant material the sample loop. The HPLC system (Spinco laboratories) was
The fresh green leaves of Bougainvillea spectabilis visibly used for analysis. Chromatographic analysis was carried out
free from disease were collected from the garden area of using a C-18 column at ambient temperature under isocratic
Central Sericulture Research and Training Institute, Central condition with mobile phase of Acetonitrile: Methanol in the
Silk Board, Mysore. ration of 80:20 at flow rate 0.500 ml/min; and detection at
260nm. The total volume of the sample injected to HPLC was
Preparation of extract 100µl.
The leaves of B. spectabilis were washed thoroughly several
times with potable water and then allowed to dry naturally on GC-MS analysis
room temperature. Dried leaves were crushed by using liquid The GC-MS MS analysis was carried out using Varian 4000
nitrogen by using pestle and mortar. The fine powder was ion trap GC-MS with fused silica 15m×0.2mm 1D×1µm of
soaked separately in 50ml of n-butanol, 50ml of methanol, capillary column. The instrument was set to an initial
50ml of chloroform and 50ml of distilled water for 24hours. temperature of 70 ºC, and maintained at this temperature for
The extracts were filtered through Whatman No.1 filter paper 2min. At the end of this period the oven temperature was rose
and freshly prepared extracts were used for analysis. up to 260 ºC, at the rate of an increase of 6ºC/min, and
maintained for 9min. Injection port temperature was ensured
Determination of Antimicrobial activity as 250 ºC and Helium flow rate as 1ml/min. The ionization
Antimicrobial susceptibility test of the selected pathogens was voltage was 70CV. The samples were injected in spilt mode
done by Disc diffusion method. Whatman filter paper disc as 10:1. Mass spectral scan range was set at 45-450(m/z).
with diameter about 6mm was used for preparing discs. Each Using computer searches on a NIST ver 2.1ms data library
disc was immersed in the respective leaf extracts and placed and comparing the spectrum obtained through GC-MS-MS
on the surface of pathogenic bacteria inoculated plates. These compounds present in the plants sample were identified.
freshly prepared discs were used for the determination of
antibacterial activity. All the tests were performed on nutrient Results
agar plates under aseptic condition in Laminar Air Flow Initial evaluation of different solvent extracts of B.
(LAF). Each pathogenic bacterial suspension was prepared in spectabilis leaf on B. thuringiensis
sterile distilled water. Suspension of microbial cultures (50µl) The antibacterial activity of different solvent extracts of B.
was inoculated on the surface of the nutrient agar media in a spectabilis was evaluated against Bacillus thuringiensis.
petriplate by spread plate method using L shaped glass rod. Based on the presence of clear zone of inhibition, among the
The sterile discs of diameter 6mm were immersed in the four organic solvents used in the study, chloroform extract
respective leaf extract and positive control (ampicillin) markedly inhibited the growth of organism. The zone of
solutions placed onto inoculated Nutrient agar plates. The inhibition was measured. Standard antibiotic, Ampicillin
experimental Nutrient agar plates were incubated at 37 ºC for showed the more inhibition zone in comparison with other
24-48hours. After incubation the plates was observed for the solvent extracts. (Table 1 & Fig 2).
presence of inhibition zone around each disc. The diameter of
zone of inhibition of bacteria was recorded in milli metres. Evaulation of antibacterial activity against the bacterial
pathogens of silkworm and humans.
Thin layer chromatography The antibacterial activity of chloroform extract was evaluated
The concentrated fractions collected from the column by under in vitro condition against Salmonella typhi, Shigella
chromatography were subjected to thin layer chromatography flexneri (gram negative bacteria) and Bacillus thuringiensis
(TLC). Briefly, a spot of each fraction was carefully applied (gram positive bacteria). Clear zone of inhibition was noticed
onto a thin layer chromatographic plate (coated with silica) against the three pathogen used in the study. Among the three
and left to dry. After about five minutes, the plates were organism tested, S. typhi showed more sensitivity towards
dipped in a suitable solvent (methanol: chloroform: acetic chloroform extract of B. spectabilis followed by Shigella
acid in the ratio 9:1:1drop) which allowed the compounds in flexneri and Bacillus thuringiensis (Table 2 & Fig 3).
the spot to move upwards by capillary attraction. The plate
was then removed from the solvent and left to dry. The Characterization of compounds by using TLC
position of different compounds was observed by The seperation of compounds on TLC revealed the presence
fluorescence under UV-light and Rf value was calculated by of different compounds in the chloroform extract of B.
using the following formula: spectabilis. The Retardation factor value (Rf value) of

~ 2669 ~
Journal of Pharmacognosy and Phytochemistry

different seperated compounds was calculated by using the tested with varied effectiveness. The ethanol and chloroform
formula. extracts have shown relatively greater activity than that of any
other extracts at 40μl concentration (Swamy et al., 2012). The
results of our study revealed that, among different solvent
extracts utilized the chloroform extract showed more
inhibition of B. thuringenesis (Fig. 2). Subsequent evaluation
The different compounds seperated in TLC were collected by of chloroform extract on the human pathogens Salmonella
scrapping the band from TLC and evaluation of their typhi, Shigella flexneri (gram negative bacteria) under in vitro
antibacterial potential was carried out against B. condition showed clear zone of inhibition (Table 2 & Fig 3).
thuringenesis. The promising compound present in Rf value These results confirms that, chloroform extract of B.
0.84 was further characterization (Table 3 & Fig 5). spectabilis can be used as potent antibacterial extract for
inhibiting bacterial infections in silkworm as well as human
Evaluation of purity of partially purified compounds by beings. Similarly, Dhankhar et al. (2013) [5] evaluated the
using HPLC antibacterial activity of various solvent extracts (water,
The isocratic sepeartion of crude and partially purified methanol, acetone, chloroform, petroleum ether) of the leaf of
chloroform extract revealed the presence of different B. spectabilis.
compounds. The HPLC chromatogram of crude extract Natural products, such as plants extract, either as pure
revealed the presence of 10 compounds (peaks) (Fig.5), where compounds or as standarized extracts, provide unlimited
as partially purified extracts showed the presence of 3 opportunities for new drug discoveries because of the
compounds (peaks) (Fig.6). unmatched availability of chemical diversity. Earlier study
carried out by Maheshwari et al. (2008) [24] have analyzed
Characterization of compounds present in the partially qualitatively by phytochemical screening for the presence of
purified extract by using GC-MS amino acids, proteins, anthroquinones, saponins, triterpenoids,
The characterization of compounds present in the partially flavonoids, carbohydrates, alkaloids, phytosterols, glycosidal
purified extract has revealed the presence of nineteen sugars, tannins, phenols and furanoids in different solvent
antimicrobial compounds (Table 4 & Fig 7). The majority of extracts of B. spectabilis. The authors opinied that,
compounds identified in the present study have antimicrobial phytochemicals present in exatracts may be responsible for
activity. Some of the compounds identified possess anti- the antibacterial acitvity of the plant leaf extract. Hajare et al.
inflammatory, antidiabetic, antifungal, antioxidant and (2015) [7] evaluated the antibacterial activity of various
antiprotozoal characters. solvent extracts of the leaf of B.spectabilis. Similarly, in the
present study, the characterization of compounds present in
Discussion the partially purified extract has revealed the presence of
The medicinal plants are of greatest interest pertaining to nineteen antimicrobial compounds which are having
human health. Medicines derived from plants have been a antimicrobial activity (Table 4 & Fig 7). The a number of
component of traditional healthcare in most parts of the world compounds identified from GC-MS possess anti-
for centuries or thousands of years. In Ayurveda practices, inflammatory, antidiabetic, antifungal, antioxidant and
exploitation of many medicinal plants is being carried out. antiprotozoal characters.
More than 7,000 medicinal plant species are reported from
India. Due to application in pharmaceutical, cosmetic, Table 1: Initial evaluation of different solvent extracts of B.
agricultural and food industry medicinal plants gained major spectabilis leaf on B. thurengenisis.
attention from different scientific corners of the world. Due to
Sl. No. Sample Inhibition zone (in cm)
presence of numerous biologically active compounds in 1. Positive control (Ampicillin) 0.5±0.57
plants, many of these have been shown to exhibit 2. Chloroform 0.5±0.27
antimicrobial properties and hence, they have been exploited 3. Methanol 0.2±0.43
as antimicrobial drugs in traditional medicines. As per the 4. n- Butanol 0.1±0.33
report of World Health organization, still 80% of world’s The values are the mean of three independent experiments ± Standar
populations for their primary health needs depends on error.
traditional medicine. The screening of plant extracts or plant
derived substances systematically still remains an attention- Table 2: Antibacterial activity of leaf extract against human
grabbing strategy to find new lead compounds (Maheshwari bacterial pathogens
et al., 2006) [14]. Sl No. Organism Positive control(cm) Chloroform extract(cm)
Bougainvillea spectabilis belonging to family Nyctaginaceae 1. B. thuringiensis 0.8±0.17 0.2±0.07
is an important horticultural plant. Leaf extracts of B. 2. Shigella flexneri 0.5±0.13 0.2±0.17
specatibilis showed strong antiviral activity against plant 3. Salmonella typhi 1.0±0.23 0.5±0.21
viruses (Madhusudhan et al., 2011) [13]. The antiviral protein The values are the mean of three independent experiments ± Standar
error.
present in B. spectabilis was characterized by Balasaraswathi
et al. (1998) [3] and anti-inflammatory activities were also
Table 3: The Retardation factor value (Rf) of different seperated
observed by Mandal et al. (2015) [15]. compounds
Several reports says that, B. spectabilis have factors
responsible for controlling and preventing diabetes (Jwala et Sl No. Rf value
al., 2012) [9]. In vitro antibacterial activity of Bougainvillea 1. 0.59
spectabilis leaves extracts is been reported by Umamaheswari 2. 0.68
et al. (2008) [24]. The considerable antimicrobial activity was 3. 0.84
4. 0.89
observed in B. spectabilis. All flower extracts of B. spectabilis
5. 0.93
inhibited the growth of few of the bacterial and fungal strains
~ 2670 ~
Journal of Pharmacognosy and Phytochemistry

Table 4: GC-MS Analysis of chloroform extract of Bougainvillea spectabilis

Sl.
Compound name Remarks References
No
1. Cyclotetradecane Antibacterial, antifungal Afrouzan et al., (2018) [1]
2. 5-Isopropyl-4-(Trifluoro methyl)-1H-pyrimidin-2-one Antibacterial Sengottuvel et al., (2015) [20]
3. 2-tert-butyl-4-trifluromethyl-1-methylimidazole Antibacterial Sangeetha et al.,(2015) [19]
4. 2-tert-butyl-4-isopropyl-5-methylphenol Antibacterial Sangeetha et al., (2015) [19]
5. 2-Allyl-5-t-butylhydroquinone Antibacterial Pradheesh et al., (2017) [17]
6. 15-Methyltricyclo pentadeca-1,3,5,7 Antibacterial Mohana Priya and Senthilkumar (2014) [16]
Antibacterial, antifungal
7. 9,11,13-Heptene Veena et al., (2016) [25]
and anti-inflammatory
8. Isochiapin- B Antibacterial Senthilkumar et al., (2012) [21]
9. 2(1H)Naphthalenone Antibacterial Senthilkumar et al.,(2012) [21]
10. Octahydro-1-methyl-1-(2-propenyl) Antibacterial Kusuma and Mahfed (2017) [12]
Antiproliferation and
11. Olean-18-ene Hussain et al.,(2016) [18]
anti-inflammatory
12. D-Galactopyranoside, methyl Antibacterial Kawsar et al., (2016) [11]
13. 1,4-Naphthoquinone-2-acetyl-5,8-dihydroxy-3-methoxy Antibacterial Chahra et al., (2016) [6]
14. 1,2-Benzenedicarboxylic acid Antibacterial Shoge et al.,(2016) [22]
15. Celidoniol, deoxy Anti-inflammatory and Anti-cancer Kanimozhi et al., (2012) [10]
16. Pthalic acid Antibacterial Akter et al., (2017) [2]
17. 2,3-bis-o-(trimethylsilyl)-, cyclic butylboronate (CAS) Antibacterial Rzhepishevska et al., (2011) [18]
18. Germanicol Antiplasmodial Zahid et al., (2013) [26]
19. 7,9-di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione Antibacterial Pradheesh et al., (2017) [17]

Fig 1: Initial screening of different solvent extracts of B. spectabilis

leaf on B. thuringiensis

Fig 3: TLC fingerprinting of chloroform extract of B. spectabilis leaf

Fig 4: HPLC-Chromatogram of crude chloroform extract of B.

Fig 2: Inhibition of different pathogens by chloroform extract of B. spectabilis leaf.
spectabilis leaf.

~ 2671 ~
Journal of Pharmacognosy and Phytochemistry

active principles responsible for these activities of B.

spectabilis. Since, B. spectabilis possess significant
antimicrobial activity that, following additional studies, the
potential antimicrobial compound could replace commercially
known antibiotics.

References
1. Afrouzan H, Tahghighi A, Zakeri S, Es-haghi A.
Chemical Composition and Antimicrobial Activities of
Iranian Propolis. Iranian biomedical journal. 2018; 22:50.
2. Akter M, Graham H,. Iji PA. Interactions between
phytase and different dietary minerals in in vitro systems.
South African journal of animal science. 2017; 47:5.
Fig 5: HPLC-Chromatogram of partially purified chloroform extract 3. Balasaraswathi R, Sadasivam S, Ward M, Walker JM. An
of B. spectabilis leaf. antiviral protein from Bougainvillea spectabilis roots;
purification and characterisation. Phytochemistry 1998;
47:1561-1565.
4. Beyene B, Beyene B, Deribe H. Review on application
and management of medicinal plants for the livelihood of
the local community. Journal of Resources Development
and Management. 2016; 22:33-39.
5. Dhankhar S, Sharma M, Ruhil S, Balhara M, Kumar M,
Chhillar AK. Evaluation of antimicrobial and antioxidant
activities of Bougainvillea spectabilis. International
Journal of Pharmceutical Sciences. 2013; 34:178-182.
6. Chahra D, Ramdani M, Lograda T, Chalard P, Figueredo
G. Chemical composition and antimicrobial activity of
Evernia prunastri and Ramalina farinacea from Algeria.
Issues in Biological Sciences and Pharmaceutical
Research. 2016; 4:35-42.
7. Hajare CN, Inamdar FR, Patil RV, Shete CS, Wadkar SS,
Patil KS et al. Antibacterial activity of the leaves of
Bougainvillea spectabilis against E. coli NCIM 2832 and
M. aureus NCIM 5021. Int J Pharm Sci Rev Res 2015;
34:194-196.
8. Hussein HM, Hameed IH, Ibraheem OA. Antimicrobial
activity and spectral chemical analysis of methanolic
leaves extract of Adiantum Capillus- Veneris using GC-
MS and FT-IR spectroscopy. International Journal of
Pharmacognosy and Phytochemical Research 2016;
8:369-385.
9. Jawla S, Kumar Y, Khan MSY. Hypoglycemia activity of
Bougainvillea spectabilis stem bark in normal and
alloxan induced diabetic rats. Asian Pac J Trop Biomed
2012; 2:19-23.
10. Kanimozhi D, Ratha bai V. Evaluation of Phytochemical
antioxidant antimicrobial activity determination of
bioactive components of ethanolic extract of aerial and
underground parts of Cynodon dactylon L. International
Journal of Scientific Research and Reviews. 2012; 1:33-
Fig 6: GC-MS spectra and fragmentation pattern of partially purified
chloroform extract of B. spectabilis leaf. 48.
11. Kawsar SM, Nishat SS, Manchur MA, Ozeki Y.
Conclusion Benzenesulfonylation of Methyl [alpha]-D-
The results of the present study suggested that, the different Glucopyranoside: Synthesis, Characterization and
solvents antibacterial action of chloroform extract of Antibacterial Screening. International Letters of
Bougainvillea spectabilis leaves may signify their prospective Chemistry, Physics and Astronomy. 2016; 64:95.
as antibacterial herbal remedies. The compounds extracted 12. Kusuma HS, Mahfud M. GC-MS analysis of essential oil
from Bougainvillea spectabilis showed the antibacterial of Pogostemon cablin growing in Indonesia extracted by
activity against bacterial pathogens viz., Salmonella typhi, microwave-assisted hydrodistillation. International Food
Shigella flexneri and Bacillus thuringiensis. The study also Research Journal. 2017; 24:1525-1528.
shows that, bioactive compounds isolated from Bougainvillea 13. Madhusudhan KN, Vinayarani G, Deepak SA, Niranjana
spectabilis showed inhibition against both gram positive and SR, Prakash HS, Singh GP et al. Antiviral Activity of
gram negative bacteria. Hence, further studies are suggested Plant Extracts and other Inducers against Tobamoviruses
to be undertaken the mechanism of such actions scientifically. Infection in Bell Pepper and Tomato Plants. International
This will emphasize on the isolation and characterization of Journal of Plant Pathology. 2011; 2:35-42.
~ 2672 ~
Journal of Pharmacognosy and Phytochemistry

14. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC.

Multiple biological activities of curcumin: a short
review. Life sciences 2006; 78:2081-2087.
15. Mandal G, Chatterjee C, Chatterjee M. Evaluation of
anti-inflammatory activity of methanolic extract of leaves
of Bougainvillea spectabilis in experimental animal
models. Pharmacogonsy Research. 2015; 7:18-22.
16. Mohana Priya R, Senthilkumar P. Pharmacological Study
of Trichoderma indicum. International Journal of Biotech
Trends and Technology. 2014; 4:1-5.
17. Pradheesh G, Suresh J, Suresh S, Alex Ramani V, Hong
SI. Antimicrobial activity and identification of potential
compounds from the chloroform extract of the medicinal
plant Cyathea Nilgirensis Holttum. World Journal of
Pharmacy and Pharmaceutical Sciences. 2017; 6:1167-
1184.
18. Rzhepishevska O, Ekstrand-Hammarström B, Popp M,
Björn E, Bucht A, Sjöstedt A et al. The antibacterial
activity of Ga3+ is influenced by ligand complexation as
well as the bacterial carbon source. Antimicrobial agents
and chemotherapy. 2011; 55:5568-5580.
19. Sangeetha C, Krishnamoorthy AS, Nakkeeran S,
Ramakrishnan S, Amirtham D. Evaluation of bioactive
compounds of Ophiocordyceps sinensis sacc. Against
Fusarium spp. Biochem Cell Arch. 2015; 2:431-435.
20. Sengottuvel T, Kannappan N, Sivakumar T,
Pytochemical and GC-MS analysis of bioactive
compounds present in the Adiantum incisum forsk. Der
Pharma Chemica. 2015; 7:137-142.
21. Senthilkumar N, Murugesan S, Vijayalakshmi KB. GC-
MS-MS analysis of Trichilia connaroides (Wight and
Arn.) Bentv (Meliaceae): A tree of ethnobotanical
records. Asian Journal of Plant Science and Research.
2012; 2:193-197.
22. Shoge M, Garba S, Labaran S. Antimicrobial activity of
1,2-Benzenedicarboxylic acid, butyldecal ester isolated
from the seeds and pods of Acacia nilotica Linn. Basic
research Journal of Microbiology. 2016; 3:8-11.
23. Swamy MK, Sudipta KM, Lokesh P, Neeki MA, Rashmi
W, Bhaumik SH et al. Pythochemical screening and in
vitro antimicrobial activity of Bougainvillea spectabilis
flower extracts. International Journal of Phythomed 2012;
4:375-379.
24. Umamaheshwari R, Shreevidya A. Nuni, In vitro
antimicrobial activity of Bougainvillea spectabilis leaves
extracts. Advances in Biological Research. 2008; 2:01-
05.
25. Veena ME, Niranjana P, Rajeshwara Gas NA.
chromatography-Mass spectrometry analysis of bioactive
components from the rhizome extract of Nardostachys
Jatamansi. Asian Journal of Pharmaceutical and Clinical
Research. 2016; 9:115-118.
26. Zahid IA, Habib-ur-Rehman, Fiaz AM, Ashfaq AA.
Antiplasmodial activity of compounds isolated from
Viburnum nervosum. International Journal of
Pharmaceutical Science Invention. 2013; 2:19-24.

~ 2673 ~