ABSTRACT

The research was done to determine the antibacterial activity of flowers of Rosa
Alba, Rosa Hybrida and leaves of Melia Azedarach against selected Gram negative
bacterial strains. Hot water, Cold water, and Methanol extracts of these plants were
applied using Agar well(Miller et al, 1939) and disk diffusion method (Bauer, et al,
1966). The results obtained shows that Methanolic extract of Rosa Alba and Rosa
Hybrida is very effective against E. coli, Salmonella typhimurium, Shigella sonnei,
Klebsiella pneumoniae and Pseudomonas aeruginosa while Methaolic extract of Melia
Azedarach shows high level of inhibition zone against Salmonella typhimurium, Shigella
sonnei and Klebsiella pneumoniae. Smilarly Hot water extract of Rosa Alba is only
effective against Salmonella typhimurium. Effect of all other extracts tested against
bacterial strains show random number of conclusions. From outcome it is concluded that
Methanolic extract of Rosa Hybrida, Rosa Alba and Melia Azedarach have capability to
enhance the antibacterial effect of different antibiotics used in the market or may also
used in the formation of new antibiotics.

INTRODUCTION
Since ancient times man has explored secrets of nature. One of the useful secrets
are related to medicinal plants and their healing properties as they ameliorate diseases or
sufferings (Chevallier, 1996). Medicinal plants are defined by Hasan et al., as plants with
healing properties. These plants are used from centuries as traditional or folk medicine
and are cheaper, easily approchable and effective with minimum number of
disadvantages.

Rose and Melia Azedarach from long time are being used for traditional
medicinal purposes. Rose water was first produced by Muslim chemists in the medieval
Islamic world through the distillation of roses (AlHassan, 1998). Rose water was
utililized for drinking and perfumery industries (AlHassan, 1998). It was also used in
traditional Iranian medicine to treat rheumatic heart disease, to strengthen gastric nerves,
and to treat some forms of headache and sickness (World Health Organization, 2002).
Rose hips are important in lowering blood pressure and improves the digestive efficiency
(South, 2005). Honey together with roses has always been used as an effective remedy
for children’s gums irritations, especially when first teeth appear (Wilcox, 2008).

Similarly Melia Azedarach is also being utilized because of its insecticidal

property. The seed oil of Melia Azedarach is used for rheumatism and skin diseases such
as ringworm and scabies (reference?). Internally, the oil is useful in malaria fever and
leprosy (Ramya et al., 2009).

Most of the people belonging to Asian and African countries relies on traditional
medicine (reference ?). Up to 80% of the population in Africa depends on traditional
medicine for its health care needs (World Health Organization, 2002). However,
according to World Health Organization (WHO) "inappropriate use of traditional
medicines or practices can have negative or dangerous effects" and that "further research
is needed to ascertain the efficacy and safety" of ssuch practices and medicinal plants
(World Health Organization, 2008). In recent years modren procedures are utilized for the
use of medicinal plants i-e extraction of active chemicals from plants and their utilization
in the formation of drugs.

Medicinal plants and their extracts are not only being used in treatment of body
imbalances but are also used to treat diseases due to other other organisms like becteria,
viruses, fungi etc. Scientists and chemist are making efforts to enhance the drugs by
utilizing these plants as well as other chmicals. “Efforts of scientists in establishing plants
with promising antimicrobial activity are yielding fruitful results” (Ndukwe et al., 2005
p.?). For example Hirulkar and Agrawal (2010) conducted experiments on the use of
different extracts of rose against diseases caused due to bacteria. They concluded that
alcoholic extract showed higher antimicrobial activity as compare to high dilution. Their
results further showed that Petroleum ether extract showed maximum 29 mm zone of
inhibition for Pseudomonas aeruginosa as compared to other bacterial strains which they
tested. Furthermore, alcoholic extract showed higher inhibitory effect on Streptococcus
pneumoniae (30mm), Enterobacter aerogens (28 mm), Staphylococcus epidermidis (25
mm), Bacillus subtilis (30 mm), Pseudomonas Aeruginosa (32 mm) and aqueous extract
showed higher inhibition against E.coli (21 mm), Enterobacter aerogens (25mm) and
Bacillus subtilis (28 mm) as compare to other bacterial strains (Hirulkar and Agrawal,
2010). Sharafi et al., in 2010 determined the Cytobiochemical potentials of Rosa
Damascena Mill extract and draw conclusion that rose extract are applicant as a natural
antioxidant and health promoting agent (Sharafi et al., 2010).

Efforts are also done in field of exploring the hidden secrets from Melia
Azedarach such as Khan et al., (2001). Their results showed broad spectrum of
antibacterial activity with methanol extracts of leaves, root and stem barks of Melia
Azedarach. An increase in activity was seen when fractionation (petrol, dichloromethane,
ethyl acetate), particularly in the dichloromethane fraction of the stem bark of Melia
Azedarach was used (Khan et al., 2001). Similarly significant inhibition against gram
positive and gram negative bacteria’s was seen with Methanol, Ethyl acetate and
Aqueous extracts of Melia Azedarach (Ramya et al., 2009). The seed oil of Melia
Azedarach is the most active medicinal product of the plant (reference ?). It is used as
antiseptic for sores and ulcers that show no tendency to heal (Ramya et al., 2009). One
another conclusion about Melia Azedarach is that alcholic extract (50% EtOH) of stem
bark of Melia Azedarach is anticancerous, antispasmodic, antiviral (Rastogi and
Mehrotra, 1991; 1993; Rastogi, 1998).

Khan et al., (2002) describes that fresh leaf extract is applied externally against
burns and used as mouth wash against Gingivitis disease.(Khan et al., 2002). Khan et al.,
further elaborated that bark infusion 30-50 ml is administered orally twice a day is
effective against Gonorrhea. They further describes that for the treatment of Piles disease
leaf extract 5 ml is administered orally thrice a day and Leaf extract 5-10 ml is
administered orally twice a day for 7 days against Pyrexia disease.

Various genera of Rosa Damesena (Reference), Rosa china (reference?) and have
been tested on becteria as literature revirew above suggests. However, there is no
research in our knowledge that tested the effects of Rosa alba and Rosa hybrida as
antibecterial agents. This present studies therefore was conducted to determine the
antibacterial activity by use of different genera of rosa alba and hybrida. .

METHOLOGY
About 2 kg flowers of each Rosa Alba (white Rose) and Rosa Hybrida (yellow
Rose) and 1 kg leaves of Melia Azedarach (Bakain) were dried under shade in open air.
Petals of white and yellow roses and leaves of bakain were dried for an average 12 days.
Dired petals and leaves were then crushed to get saw which were then filtered to get
powdered form.
Three different extracts of each sample were prepared in lab. These extracts are
given in the following table
Table 1. Extracts of plants and their solvent concentrations
Extracts Plant Sample(powder) Solvent
amount concentration
Cold water Extract 10 gm 200 ml water
Rosa Alba

Hot water Extract 10 gm 200 ml water

Methanolic Extract 5 gm 100 ml methanol
Cold water Extract 10 gm 200 ml water
Hybrida
Rosa

Hot water Extract 10 gm 200 ml water

Methanolic Extract 5 gm 100 ml methanol
Cold water Extract 10 gm 100 ml water
Azedarach
Melia

Hot water Extract 10 gm 100 ml water

Methanolic Extract 10 gm 100 ml methanol
Table 3. Extracts for Melia Azedarach (Bakain)

 These extracts were prepared by taking sample amounts along with solvent
concentration given in Table above and poured in 500ml flasks. These flasks were
then covered with aluminum foil.
 Three cold water extracts and three methanol extracts of every sample were placed on
a shaker at 150 rpm for 24 hours. Similarly three flasks of hot water extracts were
placed in the incubator for 24 hours at 40 ˚C.
 After 24 hours all extracts were centrifuge for 7 minutes on 4400 rpm and the
supernatant layer was filtered. This procedure was repeated thrice. After last filtration
we get the 100% pure supernatant extract. These pure extracts were then used directly
for sensitivity test.
 ANTIBECTERIAL ASSAY
Agar well method (Miller et al, 1939) and Disc diffusion method (Bauer, et al,
1966) were used in this experiment to determine the effect of antibacterial agents. In
these methods zone of inhibition or effectiveness of antibacterial agent against bacterium
were measured. These methods were carried on in the following steps.
 First of all Laminar flow cabinet was disinfected with spirit.
 Sterilized autoclaved media was poured into sterilized Petri plates in Laminar flow by
using spirit lamp flame to avoid any contamination.
 To solidify the media electric fan was then turned on for some time.
 The bacterium’s dilutions were swabbed uniformly across different culture plates
according to number of extracts.
 In case of Agar well diffusion method 5mm well was made in each plate using borer
then this well was filled with different extracts.
 While in case of Disk diffusion method sterlized filter paper disks of 10 mm diameter
were dipped in different extracts using forcep and then placed in center of the media
plate.
 Plates were then placed in an incubator at 37 ˚C for 24 hours. At this temperature
bacteria show their maximum activity but effective extracts produces a clear
inhibition zone.
 This zone was then measured using vernier caliper after 24 hours.
RESULTS
Table 4. Zone of inhibition of different extracts against different bacterial strains
Inhibitory Mean Inhibitory
Extracts Bacterial zones(mm) (mm) zone(mm) by
Strains by Agar well Disk
method diffusion
method
E. coli 18 16 17 -
Hot water Salmonella typhimurium 20 21 20.5 24
extract of Shigella sonnei - - - 14
Rosa Alba Klebsiella pneumoniae 15 17 16 19
Pseudomonas aeruginosa - - - 16
E. coli - - - 16
Cold water Salmonella typhimurium 14 19 16.5 18
extract of Shigella sonnei - - - 11
Rosa Alba Klebsiella pneumoniae 12 16 14 -
 Pseudomonas aeruginosa - - - -
E. coli 13 9 11 17
Methanolic Salmonella typhimurium 22 28 25 28
extract of Shigella sonnei 15 24 19.5 23
Rosa Alba Klebsiella pneumoniae 20 14 17 28
Pseudomonas aeruginosa 22 26 24 27
E. coli 12 13 12.5 14
Hot water Salmonella typhimurium 17 20 18.5 21
extract of Shigella sonnei - - - -
Rosa Hybrida Klebsiella pneumoniae 16 21 18.5 22
Pseudomonas aeruginosa - 16 - -
E. coli - - - -
Cold water Salmonella typhimurium 23 29 26 23
extract of Shigella sonnei - - - -
Rosa Hybrida Klebsiella pneumoniae 13 18 15.5 18
Pseudomonas aeruginosa - - - -
E. coli 21 15 18 26
Methanolic Salmonella typhimurium 22 15 18.5 20
extract of Shigella sonnei 13 17 15 21
Rosa Hybrida Klebsiella pneumoniae 22 20 21 30
Pseudomonas aeruginosa 24 17 20.5 21
E. coli 15 12 13.5 -
Hot water Salmonella typhimurium 15 13 14 -
extract of Shigella sonnei - - - -
Melia Azedarach Klebsiella pneumoniae - - - -
Pseudomonas aeruginosa 11 19 15 18
E. coli 14 10 12 -
Cold water Salmonella typhimurium - - - -
extract of Shigella sonnei - - - 15
Melia Azedarach Klebsiella pneumoniae - - - 13
Pseudomonas aeruginosa - - - -
E. coli - - - -
Methanolic Salmonella typhimurium 14 29 21.5 35
extract of Shigella sonnei 17 17 17 20
Melia Azedarach Klebsiella pneumoniae 18 20 19 23
Pseudomonas aeruginosa - - - 14

RESULTS DISCUSSION
Due to lesser side effects many scientists are concentrating toward natural sources
of antibiotics especially plants. “Efforts of scientists in establishing plants with promising
antimicrobial activity are yielding fruitful results” (Ndukwe et al., 2005). Hirulkar and
Agrawal, (2010) results for example showed that alcoholic extract showed higher
antimicrobial activity against selected bacterial strains (Hirulkar and Agrawal, 2010).
Efforts are also done in field of exploring the hidden secrets from Melia
Azedarach such as Khan et al., (2001). Their results showed that “The methanol extracts
of leaves, root and stem barks of Melia Azedarach showed a broad spectrum of
antibacterial activity”.Similarly Ramya et al., (2009) concluded that “Methanol, Ethyl
acetate and Aqueous extracts of Melia Azedarach showed significant inhibition against
gram positive and gram negative bacteria’s”.
These studies mentioned above are algined with the study in this research and
determine the antibacterial activity of flowers of Rosa Alba, Rosa Hybrida against
selected Gram negative bacterial strains. Hot water, Cold water, and Methanol extracts of
these plants were prepared and then applied using Agar well and disk diffusion method.
The results obtained shows that Methanolic extract of Rosa Alba and Rosa Hybrida is
very effective against E. coli, Salmonella typhimurium, Shigella sonnei, Klebsiella
pneumoniae and Pseudomonas aeruginosa while Methaolic extract of Melia Azedarach
shows high level of inhibition zone against Salmonella typhimurium, Shigella sonnei and
Klebsiella pneumoniae. Smilarly Hot water extract of Rosa Alba is only effective against
Salmonella typhimurium. Effect of all other extracts tested against bacterial strains show
random number of conclusions. Some results varies to that of other researches like [give
references of the papers with whom your results vary and also list the variation points].
This may be due to enviromental differencesbetween this research and the above
mentioned researches. [Please also try to come up with a list of the key environmental
differences between this research and other mentioned researches]

CONCLUSIONS
From outcome it is concluded that Methanolic extract of Rosa Hybrida and Rosa
Alba have capability to enhance the antibacterial effect of different antibiotics used in the
market or may also used in the formation of new antibiotics.

REFERENCES