In recent old ages, the antimicrobic and antioxidant actions of substances have received much attending. This is because of the increasing involvement in human wellness and have been studied in vitro and in vivo by many research workers. The antioxidant agents may be utile in retarding oxidative harm to cellular components which lead to cell hurt and decease. This has been associated with pathogenesis of assorted chronic diseases, e.g. carcinomas, coronary bosom disease, and many other wellness jobs related to progress age1-3. The antimicrobic agents protect populating beings from amendss ensuing in the bar of assorted infective diseases4.
There is a turning involvement in substances exhibiting antimicrobic and antioxidant belongingss that are supplied to human and animate beings as nutrient constituents or as specific pharmaceutics5. Plants are the primary beginnings of of course happening antioxidants for worlds. It has been good known that indispensable oils and works infusions have antimicrobic and antioxidant effects6.
N. jatamansi DC, is a perennial herb, belongs to the household Valerianceae and normally found in Himalayas. The works has a rich history of medicative usage and has been valued for centuries in Ayurvedic and Unani systems of medical specialty. It is used as a stimulation, antiseptic, insect repellant and for the intervention of epilepsy, craze, spasmodic fondnesss, stomach ache, irregularity and cholera. The indispensable oil of N. jatamansi besides has medicative belongingss. In combination with cold H2O, the oil is considered to be effectual against sickness, stomach ache, flatulency, liver jobs, icterus, kidney ailments, insomnia and concern. Externally, the oil is added to a steaming bath to handle redness of the womb. The oils are besides used in oculus compounds and as toxicant counterpoisons. Oil is reported to be utile in the intervention of atrial flutter7, 8.
The purpose of the present survey was to find the chemical composing of N. jatamansi indispensable oil and antioxidant and antibacterial activities was aimed to happen natural antioxidant and bacteriocides that are safe to worlds and environment.
Materials and Methods
Extraction of oil
The N. jatamansi roots were collected from the local market. They were cleaned from immaterial affair. The indispensable oil was extracted through hydro-distillation by rearward Dean Stark apparatus9. The steam distillation was removed, dried over anhydrous Na sulfate and stored at low temperature.
The analysis of the indispensable oil was carried out on GC-MS of Agilent Technologies, Model 6890N, runing in EI manner at 70 electron volts equipped with a split-splitless injector. Helium used as a bearer gas at the flow rate of 1mL/min, while HP-5MS ( 30 m – 0.25 millimeter, 0.25 µm ) capillary column was used. The initial temperature was programmed at 50-140 & A ; deg ; C at the rate of 5 & A ; deg ; C/min and so 100-250 & A ; deg ; C at the rate of 3 & A ; deg ; C/min followed by a changeless temperature at 260 & A ; deg ; C for period of 20 proceedingss. Sample ( 2µL ) was injected to column programmed at 200 & A ; deg ; C and declarations of constituents were attained. The constituents were identified by their keeping clip and peak sweetening with standard samples in gas chromatographic manner and NIST library hunt from the derived atomization form of the assorted constituents of the oil.
In vitro antimicrobic surveies were carried out on six bacterial strains including Bacillus subtilis ATCC6633, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacter aerogenes. Among the tried micro-organism Bacillus subtilis ATCC6633 was obtained from microbiology research lab of PCSIR labs composite Lahore and other were collected from pathological research lab of a local infirmary. All clinical isolates were characterized to specie degree harmonizing to standard microbiological techniques described by Monica Cheesbrough10. The civilizations of bacteriums were maintained in the research lab on alimentary agar angles at 4 & A ; deg ; C throughout the survey.
Paper disc diffusion method as reported by Bauer et al,11 was applied with little alteration to prove the antimicrobic activity of N. jatamansi indispensable oil. Normal strength food agar medium ( OXOID, England ) was prepared and autoclaved at 121±1 & A ; deg ; C for 15 proceedingss. For antibacterial check 24 hours old bacterial civilizations grown at 37 & A ; deg ; C were used. Cultures were diluted 10-1 in unfertile toller solution 12 to put inoculums denseness of about 106CFU/mL which was used farther for the trial. Thirty micro-liters of these bacterial suspensions were inoculated to home bases incorporating unfertile alimentary agar medium utilizing a unfertile cotton swab.
Each filter paper phonograph record ( 6mm in diameter ) impregnated with different concentrations of
N. jatamansi indispensable oil individually ( 10?L, 15?L and 20?L ) were placed on pre-inoculated civilization media under sterile conditions individually and incubated at 37 & A ; deg ; C for 24hours. The zone of suppression was measured as the diameter ( in millimetres ) of the clear zone around the phonograph record. All experiments were performed in extra. PenicillinG and streptomycin were used as positive controls. Control antibiotics solutions were prepared in appropriate sum ( 0.01g/10mL ) so 25?L of each antibiotic solution was dropped on paper phonograph record used during present survey.
Antioxidant activity of N. jatamansi oil:
Antiradical activity was evaluated by mensurating the scavenging activity of the examined N. jatamansi oil on the 2,2-diphenyl-1-picrylhydrazil ( DPPH ) group. The DPPH check was performed as described by Epsin et al,13. The samples ( 100 µL each ) were assorted with 3 milliliters of DPPH solution. The optical density of the resulting solutions and the space ( with merely DPPH and no sample ) were recorded after an incubation clip of 30 proceedingss at room temperature against ascorbic acid as a positive control. For each sample, 3 replicates were recorded. The disappearing of DPPH was measured spectrophotometrically at 517 nanometer. The per centum of extremist scavenging activity was calculated utilizing the undermentioned equation ;
DPPH scavenging consequence ( % ) = ( A0-A1 ) /A0 – 100
Where A0 is the optical density of the control at 30 proceedingss and A1 is the optical density of the sample at 30 proceedingss.
RESULTS AND DISCUSSION
The indispensable oil was extracted by hydrodistillation from the N. jatamansi roots, collected from local market. The output of oil was 0.245 % . The gas chromatography coupled with mass spectrometric analysis revealed the presence of 26 compounds, out of which 10 chief constituents were, identified ( Table 1 ) . It was recorded that ledene oxide [ II ] ( 13.021 % ) , and sesquiterpine intoxicant patchouly intoxicant ( ( 9.582 % ) were major constituent of the oil. [ – ] -spathulenol ( 2.672 % ) , globulol ( 1.876 % ) , 4- [ 3,3-dimethyl-but-1-ynyl ] -4-hydroxy-2,6,6-trimethylcyclohex-2-enone ( 1.849 % ) , magastigma-4,6 [ E ] , 8 [ Z ] -triene ( 1.015 % ) , aristolene ( 0.997 % ) , ? -vatirenene ( 0.932 % ) , were present in considerable measure. These constituents were identified first clip from indispensable oil of Nardostachys jatamansi DC. roots.
The informations related to the in vitro antimicrobic potency of N. jatamansi root indispensable oil against Gram positive and Gram negative bacteriums along with control antibiotics are presented in Table 2. The consequences indicated that among Gram positive bacteriums Bacillus subtilis exhibited maximal antimicrobic activity at 20?L concentration of N. jatamansi indispensable oil with an suppression zone diameter ( IZD ) of 23.00±0.88mm followed by Staphylococcus aureus with an IZD of 18.75±0.35mm at same concentration. These consequences are supportive to the findings reported by Sohail et Al, 14 who stated that ethanolic infusion of N. jatamansi roots was effectual against both Bacillus subtilis and Staphylococcus aureus. During present survey, the biological activity of N. jatamansi indispensable oil was besides evaluated against four G-ve bacteriums including Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. The consequences indicated that among tried G-ve bacterium merely K. pneumoniae and E. aerogenes were sensitive to N. jatamansi indispensable oil as shown in Table 2. The IZD of E. aerogenes was 15.00±0.81mm which is greater than K. pneumoniae ( 11.00±0.35mm ) at 20µL concenteration of indispensable oil. Sohail et al,14 reportd that ethanolic infusion of N. jatamansi root showed suppression zone diameter of 34-24mm at different concentrations ( 20mg/mL to 5mg/mL severally ) against K. pneumoniae. This is far higher than IZD of merely 11.00±0.35mm observed for N. jatamansi indispensable oil used against K. pneumoniae during current survey. It may hence be concluded that indispensable oil of N. jatamansi does non incorporate the active ingredient which may be present in the ethanolic infusion of the same. This is in documentation with Nimri et al,15 who has reported the consequence of assorted factors i.e. consequence of dissolvers and method of extraction, adulthood of beginning works and/or sensitiveness of the trial strains on antimicrobic potency of the stuff tested. Further among trial beings both S. typhimurium and P. aeruginosa were opposition to N. jatamansi indispensable oil. Present probe sing P. aeruginosa is in conformity to Girgune et al,16 who reported that N. jatamansi oil showed no zone of suppression against P. aeruginosa. In instance of positive controls ( antibiotics ) Streptomycin was found to be efficacious against all tried micro-organisms as shown in Table-2. Maximum zone of suppression observed in instance of B. subtilis i.e 31.00±0.35mm whereas the minimal IZD was 17.00±0.35mm exhibited by K. pneumoniae. Penicillin G was found to be effectual against merely B. subtilis and S. aureus as depicted in table-1.
Several natural compounds are known to slake free radicals17. In the current survey ( Figure 1 ) indispensable Peel oil was able to cut down the stable extremist DPPH to yellow-colored DPPH-H making 99.04 % of DPPH scavenging consequence. The comparing of DPPH scavenging activity of N. jatamansi roots oil with good known antioxidant BHT showed that root oil has stronger antioxidant potency at all concentrations.
Table 1: GS/MS analysis of indispensable oil of Nardostachys jatamansi.
M+ ( 204,34 ) ( 189,43 ) ( 161,45 ) ( 147,43 ) ( 133,37 ) ( 105,100 ) ( 91,46 ) ( 79,14 ) ( 55,8 )
M+ ( 202,100 ) ( 187,27 ) ( 159,55 ) ( 145,63 ) ( 131,88 ) ( 117,38 ) ( 105,38 ) ( 95,26 ) ( 91,51 ) ( 77,24 ) ( 69,13 ) ( 55,9 )
Magastigma-4,6 [ E ] , 8 [ Z ] -triene
M+ ( 161,100 ) ( 176,91 ) ( 147,20 ) ( 133,50 ) ( 119,82 ) ( 105,39 ) ( 91,77 ) ( 77,30 ) ( 65,15 ) ( 51,10 )
[ – ] -Spathulenol
M+ ( 220,6 ) ( 205,100 ) ( 187,33 ) ( 177,16 ) ( 159,50 ) ( 147,38 ) ( 119,58 ) ( 105,47 ) ( 79,38 ) ( 67,22 ) ( 55,17 )
M+ ( 222,55 ) ( 189,62 ) ( 175,21 ) ( 161,100 ) ( 147,42 ) ( 135,42 ) ( 121,62 ) ( 109,89 ) ( 93,68 ) ( 69,57 )
Ledene oxide [ II ]
M+ ( 222,24 ) ( 202,26 ) ( 187,46 ) ( 177,39 ) ( 159,100 ) ( 151,38 ) ( 123,34 ) ( 109,60 ) ( 91,51 ) ( 81,39 ) ( 67,34 ) ( 55,24 ) ( 51,4 )
M+ ( 222,100 ) ( 207,34 ) ( 189,14 ) ( 179,17 ) ( 161,35 ) ( 138,35 ) ( 121,18 ) ( 109,28 ) ( 98,42 ) ( 67,24 ) ( 55,24 ) ( 51,2 )
4- [ 3,3-dimethyl-but-1-ynyl ] -4-hydroxy-2,6,6-trimethylcyclohex-2-enone
M+ ( 234,40 ) ( 219,66 ) ( 191,48 ) ( 177,44 ) ( 163,100 ) ( 145,32 ) ( 105,40 ) ( 91,46 ) ( 77,31 ) ( 67,20 ) ( 55,23 )
Table 2: Appraisal of antimicrobic activity of N. jatamansi indispensable oil against six micro-organisms.
Gram staining/ Colony morphology
Antimicrobial activity of Nardostachys jatamansi indispensable oil and some standard Antibiotics.
Zone of suppression ( millimeter )
Bacillus subtilis ATCC 6333
G +ve rods/
White, dry surface
Staphylococcus aureus HIb
G +ve rods/
yellow, unit of ammunition, shiny
G -ve rods/
R degree Celsius
Pseudomonas aeruginosa HI
G -ve rods/ viridity, flat- spreaded
Klebsiella pneumoniae HI
G -ve rods/ gray-white, mucoid
G -ve rods/
big, white non-mucoid
aPaper phonograph record ( 6mm diameter )
bHospital isolated pathogen
cResistant to antibiotic
± Standard divergence, ( – ) No suppression zone
Figure 1: Percentage antioxidant activity of indispensable oil of N. jatamansi DC. roots in comparing with BHT as standard mention by DPHH check.
In decision, this the first survey measuring the antibacterial and antioxidant activities of indispensable oil from N. jatamansi DC roots. The consequences has shown that oil has strong in vitro antibacterial avtivity against B. subtilis ATCC 6633, K. pneumoniae, S. typhimurium, P. aeruginosa, P. fluorescens, S. aureus, E. coli and Enterobacter and antioxidant activity. So, the indispensable oil can be used in intervention of diseases caused by these bugs straight or by incorporation into medical specialties used for the intervention of these complaints. Further surveies are required to find the mechanism of action of the indispensable oil for bactericide and antioxidant activity.
The writers are thankful to Dr. Zia ur Rehman, Senior Scientific Officer, PCSIR Laboratories Complex, Ferozpur Road Lahore-54600, Pakistan for GC-MS analysis.