Anacyclus Pyrethrum's Antimicrobial Abilities

    

1. OBJECTIVE

The multi drug resistance to several type of antibiotic agent that causing major problems in the clinic condition. One of the main causes of multi drug resistance is broad spectrum antibiotics overuse, abuse, in some cases misuse due to incorrect diagnosis. Antibiotics use in animal husbandry is also creating some drug resistance bacteria which can be transmitted to humans. The origin of resistance are said to be start with some bacteria that said to be have innate resistance against antibiotics and this typically reflects variation in the structure of  their cell envelope. Resistance may also br phenotypic resulting from adaption to growth within a specific environment. The origin of antibiotics resistance genes are unclear however resistance can be achieved by horizontal acquisition of resistance genes, mobilized via insertion sequences, transposons and conjugative plasmids by recombination of foreign DNA into the chromosome or by mutations in different chromosomal loci. There are a number of resistant organisms causing concern at present. The Gram-positive organisms include methicillin resistance staphylococcus aureus and coagulase-negative staphylococci, glycopeptides-intermediate sensitivity s.aureus, vancomycin-resistant enterococcus species and penicillin-resistant streptococcus pneumonia. Concerns among the Gram-negative organisms include multidrug-resistant pseudomonas aeruginosa, stenotrophomonas maltophilia and acinetobacter baumanii and members of the enterobacteriaceae with extended spectrum β-lactamases. Mycobacterium tuberculosis and M. avium complex pose major health threat worldwide. ^{[31, 32]}

In view to overcome the situation of drug resistance world is looking towards natural resources. Here the present study aims to review the potential ability of plant Anacyclus pyrethrum as antimicrobial. Anacyclus pyrethrum has been used for many years for its different properties especially for its insecticidal ability. The present work carried out to prepare different extract of different part of Anacyclus pyrethrum. This extract has been used for in vitro analysis of the antibacterial efficacy against various gram +ve and gram –ve microorganisms. Obtain results evaluated and extract showed maximum antibacterial efficacy against microorganisms has been taken for MIC and MBC study. Extract showing maximum efficacy were used to carried out GC/MS for phytochemical evaluation of Anacyclus pyrethrum.    

2. INTRODUCTION

2.1 PLANT PROFILE

Pyrethrum belongs to the genus Anacyclus in the family of Asteraceae. The flowers of the species Cinerariefolium have long been exploited commercially for their insecticidal properties. These properties were probably discovered accidentally in 1840, by a German woman in Dalmatia, who received a bouquet of flowers on her birthday. After a night long partying, she threw the flowers into a corner. In the morning the flowers were surrounded by dead insect’s. The deaths of the insects were associated with the insecticidal properties of the insects. Since then the flower has undergone extensive research establishing its complete, effective and safe commercial exploitation as a source of the natural insecticides collectively known as pyrethrins. ^{[1, 2]}

2.2 CLASSIFICATION

Botanical name: -       Anacyclus pyrethrum

Botanical sources: -    Flower, Root, Arial part

Synonym: -                 Pellitory, akalakari, akarakara, dalmation

Kingdome: -                Plantae

Division: -                   Spermatophyta

Sub-division:-            Angiosperms

Class: -                       Dicotyledons

Sub class: -                Metachlamydae

Order: -                      Companulate

Family: -                    Asteraceae

Genus: -                   Anacyclus

Species: -                  Pyrethrum

2.3 GEOGRAPHICAL SOURCES

 Anacyclus pyrethrum, is a widely-distributed plant known in different countries under different names Pellitory, akarakara, dalmation etc. Kenya is the leading producer of pyrethrum extract producing approximately 70% of the world consumption. Other large producers of pyrethrum are Rwanda, Tanzania and Tasmania in Australia. In India it is found along the Himalaya, Jammu and Kashmir, and Bengal.^[3,4,5]

2.4 CULTIVATION AND COLLECTION

Pyrethrum is cultivated in tropical zone at an attitude of 1500 to 3500 meters, depending upon the distance from equator. The soil requirements depend upon rain and other climatic conditions in a particular area. A rain fall between 800 to 1300 mm consider suitable for pyrethrum cultivation. Since the plant is very sensitive to frost, sunny periods interrupting rainfall are desired condition for cultivation. It needs a temperature between 15°C to 25°C^. The seeds are soaked in water and are then wrapped in sacking and buried in dampens and for four or five days. They are then mixed with dry sand and sown in well drained, sunny seed bed having carefully plawed, soft sandy soil which has been freed from stones and clouds. Fertilizer consisting of manure and superphosphate is worked into the bed before sowing; excessive use of fertilizer causes too rapid growth and is avoided. One pint of seed is used to 150 square yards of bed; this will yield seedlings enough for an area ten times as great. After sowing the seeds are covered with earth or ashes, the beds are shaded with screens with and in periods of drought they are carefully watered. The seedlings appears in about twelve days, when they are two to three inches high, fertilizer is added. After 4-5 months the seedlings reach a height of about four inches and are ready for transplanting. This must be done early enough to permit the roots to establish themselves firmly before cold weather, otherwise they will winter kill. The field is carefully plowed weeded, manure and leveled. The seedlings are planted in rows at intervals of 7 to 12 inches between rows. The rows are raised or ridged to prevent water collecting around the roots. If the plants are set too deeply, few flowers are produced. The harvesting periods extends for 14 to 18 days in given locality and the flower are picked up when they are about 70% open. The flower picking was done by hands. Drying generally requires about 5 to 7 days and is accomplished by spreading the flower heads and root straw mats, in day and placing them indoors at night. After the flower and root are thoroughly dried so that they can easily be crumbled between the fingers, they are packed in straw bag and store^. Average yield of the drug per hectare is 300 to 400 kg. ^{[5, 6]}                 

2.5 MACROSCOPIC CHARACTER

It is a perennial, procumbent herb, resembling chamomile. Stems lie on the ground for part of their length, before rising erect. Each bears one large terminal flower, the disk being yellow and the ray’s white, tinged with purple beneath. The leaves are smooth, alternate, and pinnate, pale green, with deeply cut segments. Fruit obovate achene. The root is almost cylindrical, very slightly twisted and tapering and often crowned with a tuft of grey hairs. Externally it is brown and wrinkled, with bright black spots. The fracture is short, bark with 1-2 circles of resin ducts, closely adhering to yellowish radiate porous wood in which occur 1-3 rows of resin ducts; odor distinct; taste sweetish, pungent, very acrid, tingling, sialagogue effect.

                                                     Figure 1: Anacyclus Pyrethrum Plant 

2.6 MICROSCOPIC CHARACTER

2.6.1 Root - Mature root shows cork consisting of tabular cells, many of which developed as sclerenchyma; a few inner cork cells contain rosette crystals of calcium oxalate; secondary cortex consisting of isodiametric or tangentially, elongated, thin-walled, parenchymatous cells, a few sclerenchymatous cells also found scattered in secondary cortex; secondary phloem consisting of usual elements, cambium 2-5 layered, secondary xylem very wide consisting of xylem vessels, tracheids and xylem parenchyma; vessels pitted, more or less in groups distributed throughout xylem, more and wider vessels found towards peripery, xylem fibers thick-walled, 1.37-28.8 μ in width, 53.2 - 231 μ in length having narrow lumen, medullary rays numerous, running straight, bi to tri and multiseriate, uniseriate rays very rare, starting from primary xylem and reaching up to secondary cortex; ray cells thick-walled, radially elongated, inulin.

Figure 2: Photomicrograph of cross section of root:  A- cork, B-Ring of stone cells, C-Parenchyma of primary cortex,  D-Cambian, E-Medullary ray, F-Xylem

present in cells of secondary cortex, secondary phloem and medullary rays; oleo-resinous schizogenous glands found scattered in secondary cortex, secondary phloem and medullary rays; calcium oxalate crystals in rosette form present in secondary cortex, secondary phloem, secondary xylem and medullary ray cells.^[7],[8]

2.6.2 Powder - Ash colored; shows vessels having scalariform thickening, rosette crystals of calcium oxalate and fragments of sclerenchyma; also gives positive tests for inulin.                                                                                                                                    

2.7 PHYTOCHEMICAL CONSTITUENTS OF ANACYCLUS PYRETHRUM

2.7.1 Flower: - Anacyclus pyrethrum flower’s principles are located in the oleoresin secretion of floral parts of partially open or closed flowers. Although pyrethrin I and pyrethrin II are the main active constituents, it also contains other active compounds called cinerin I, cinerin II, jasmoline I and Jasmoline II. All these constituents are chemically esters.^[5]

2.7.2 Root: - Root contains alkyl amides, which active constituent’s pyrethrin. Alkyl amide fraction of roots of Anacyclus pyrethrum is made up of the following isobutylamides and tyramine amides. The root contain Anacyclin, Pellitorine enetriyne alcohol (pyrethrins ), hydrocarolin, inulin (50%), traces of volatile oil and (+)-sesamin. They also contain N-(2-P-hydroxy phenylethyl) deca,dodeca, and tetradeca- trans-2,a new series of tyra mine amides corresponding to the isobutylamides.^[9]

2.7.3 Arial part: - Anacyclus pyrethum arial parts contain active constituent is Anacyclin, N-methylanacyclin, Nmethyl- N-(2-methyl propyl) 2, 8-decadiene 4, 6-diynamide along with very low quantity of pyrethrin I and pyrethrin II.^[9] Amongs others Anacylcus herb contains various chemical constituents such as pellitorine, anacyclin, enetriyne alcohol, inulin, hydrocarolin, sesamin and traces of volatile oils. Pyrethrin, an alkaloid is considered to be an active constituent in this herb. The roots of the herb is rich in rubefacient and stimulant properties. Various medicinal benefits of the herb are discussed in this article.

2.7.4 Medicinal uses

Pyrethrum plant is used for various medicinal the main used are enlisted as:

1.   The powdered root forms a good snuff to cure chronic catarrh of the head and nostrils and to clear the brain, by exciting a free flow of nasal mucous and tears.

2.   Pyrethrum kills insect by disrupting their nervous systems. Pyrethrins are toxic to the “sodium channel” the cellular structure that allows sodium ions to enter a cell as part of the process of transmitting a nerve impulse.

3.  Pyrethrum root used almost exclusively as a sialagogue in headache, neuralgic and rheumatic affections of the face, toothache, etc., or as a local stimulant in epalsy of the tongue or throat, or relaxation of the uvula.

4.   Pyrethrum contains anacycline, isobutylamide, inulin and a trace of essential oil. Use of  the drug in patients with insulin dependent diabetes mellitus reduces the dose of insulin. It decreased the plasma glucose and serum cholesterol levels after oral administration for 3–6 weeks.

5.   Ues to kill lice and their eggs and in mosquito coil.^{[10], [11], [12}

2.7.5 Ayurvedic Properties

1 Guna (Properties) – Ruksha (Dry), Tikshan (Sharp)

2. Virya (Potency) – Ushan (Hot)
3. Rasa (Taste) – Katu (Pungent)
4. Prabhav (Impact) – Shukrashodhan
5. Karma : Vatahara, Pittahara, Kaphahara, saukrala, Vajikara, Svedakara, Dipana, Buddhivardhaka, Balakrka.^[7]

3.  Material and Method

1        Test plant: leaves / root /whole plant  powder

2        Solvents  : Ethanol & Methanol  (500 ml each)         

3        Glassware: Conical flasks  - 50ml /8nos,  Beakers  (8nos.),Petri dish/cover ,Glass rod

4        Measuring cylinders

5        Cotton                   

6        Whattman filter paper no.1

7        Muller Hinton agar/Nutrient agar/Nutrient Broth  medium   

8        Test organisms: Staphyllococcus aureus ,E.coli, Pseudomonas aeruginosa , Salmonella abony , Proteus vulgeris.

3.1 LIST OF CHEMICALS AND INSTRUMENTS

3.1.1 List of Chemicals

              Table No. 1: List of Chemicals

Sr. No.	Chemicals	Manufacture
1.	Readymade Agar Media	Hi-Media
2.	Beef extract	Hi-Media
3.	Peptones	Hi-Media
4.	Ethanol	Rankem
5.	Methanol	Rankem
6.	Sodium Chloride	Merk

3.1.2. List of instruments

              Table No.2: List of Instrument

Sr. No.	Instruments	Manufacturer
1.	Laminar Air Flow	Veekay Blower Industries, India.
2.	Autoclave	D4 Surgicals India
3.	GC/MS	SHIMADZU Japan
4.	BOD Incubator	Vertical Industries, India
5.	pH meter	Orion 4 star
6.	Analytical balance	Sartorius BP 210
7.	Microbial Spreader	-
8.	Cork Borer	-

3.2. LIST OF TEST MICROORGANISM

             Table No.3: Test Microorganism       

Sr.No.	Microorganisms	ATCC NO.
1.	Staphyllococcus aureus	6538
2.	Escherichia coli	10346
3.	Pseudomonas aeruginosa	9027
4.	Salmonella abony	6014
5.	Proteus vulgeris	6380

          

 (All cultures were 18hrs old.)

All the bacterial culture used for antimicrobial analysis was old. The bacteria were maintained on nutrient broth (NB) at 37°C.

3.5 PREPARATION OF INOCULUM

The gram positive S.aureus and gram negative E.coli, Proteus and Psedomonas were subculture in nutrient broth and kept overnight in oven at temperature of 37 °C. For subculture 7-10 days old culture were used. The subculture microorganisms are used in antimicrobial study.

3.6 Preparation of the Plant Material & Extraction

The whole plant of the Anacyclus pyrethrum was collected from Lord’s Herbal and Medicinal plant centre, Maharashtra and authticated. The completely dried parts washed with distilled water & cut into pieces.

Dried roots were crushed into molten and pestle 10 gms of powder were added worth  100 ml of methanol & ethanol each. The flask was closed with cotton plugs & incubated at room temperature (30 deg. Celsius) for 24 hours. Extract was filtered using Whatman filter paper no.1. Label the extract as TS1, TS2 respectively. The dried powder of aerial part of the plants was also mix with 100ml of methanol & ethanol each. The mixture was incubated for 24 hours at room temperature (30 deg. Celsius).Now the extract was filtered using Whatman filter paper no.1 and label the extract as TS3, TS4 respectively.

4.7 Antimicrobial assay    

Agar well diffusion assay method was used for determining the zone of inhibition. 25ml of nutrient agar was added in the Petri dish, the nutrient agar was allowed to settle for 10-15 minutes. Now punch the agar with the cork borer (10mm). Add 0.1ml of test microorganism on the plate and spread with a sterile spreader. Now load the extracts in the wells (0.1ml).Allow the plates for incubation for 24hrs at 37˚C.

Four test samples were used for antibacterial assay. Antibacterial activity determined by the agar well diffusion method. Agar cup bioassay was employed for testing antimicrobial activity of plant extract. The ready-made agar medium (Hi-media, 39g) was suspended in distilled water and autoclaved at pressure of 15 lb/inc2 for 20 minutes. Test organisms are inoculated in nutrient broth and incubated at 37ᵒc for 18-24 hours are used. 10µl test organism was used to inoculate agar plate with help of sterile spreader uniformly and wait for 5-6 minutes after inoculation allow the liquid culture to soak into the agar. Then four wells were made on the surface of the agar plate by the use of sterile cork borer and named as set-1 i.e TS1, TS3, -ve(methanol) and antibiotic or set-2 i.e TS2, TS4, negative control (ethanol) and antibiotic. Now with help of sterile micropipette 10 µl of methanolic or ethanolic extract was added in wells. Remaining wells were filled with10 µl of negative control (Methanol or Ethanol) and 10 µl of standard antibiotic. Wait for 5-6 minutes after inoculation to allow the liquid extract to soak into the agar. After that plates are incubated at 37ᵒC for 24-48 hours and zone of inhibition were measured. Five replicates were maintained for each treatment and the result of average ranges is documented in the result section.

                           Figure 3: ZonE of inhibition of E.coli, 1-ts2, 2-ts-4, 3 -ethanol 4 - antibiotic

      Figure 4: Zone of inhibition of PSEUDOMONAS, 1- ts2, 2- ts-4, 3- ethanol 4- antibiotic.

             Figure 5:zone of inhibition of s.aureus,1- ts2, 2- ts-4,   3- ethanol 4- antibiotic

3.8 Minimum Inhibitory Concentration (MIC):-

Minimum inhibitory concentration is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of microorganism after overnight incubation. Minimum inhibitory concentration is used by diagnostic laboratories mainly to confirm resistance but most often as research tool to determine the minimum inhibitory concentration breakpoints. There are two method of MIC testing   i) Broth dilution method and ii) Agar dilution method.

Here broth dilution method was used to determine the MIC of TS1, TS2, TS3, TS4. For MIC sterile graduated pipettes of 5ml, 2ml and 0.5ml, small capped test tubes and overnight broth cultures are required. Six sterile caped test tube taken and labeled as A,B,C,D,E,F  to each tube 4ml of freshly prepared broth added follow by addition of  0.5 ml of overnight grown broth culture of E.coli having turbidity equivalent 0.5  McFarland. Different concentration of anacyclus pyrethrum was prepared by serial dilution method with given strength of 10,000 µl/ml. Five sterile test tube taken and labeled as 1,2,3,4,5 to each tube add 4.5ml of freshly prepare broth. Now transfer 0.5ml of stock solution of Anacyclus pyrethrum to test tube no.1 this will give the concentration of 500µl/ml. Similarly different concentration are prepared 50 µl/ml, 5 µl/ml, 0.5 µl/ml, 0.05 µl/ml by following-

Add 0.5 ml of test tube no.1 to test tube no.2 - (50µl/ml).

Add 0.5ml of test tube no.2 to test tube no.3 - (5µl/ml).

Add 0.5 ml of test tube no. 3 to test tube no.4 - (0.5µl/ml).

0.5ml of different concentration of Anacyclus pyrethrum from test tube1, 2, 3, 4 added to A, B, C, D tube respectively and to tube F 0.5ml of freshly prepared broth added as a control. Incubate the test tube for 24 hours and results are documented.

3.9 Minimum Bactericidal Concentration

Minimum bactericidal concentration is the lowest concentration of antimicrobial that will prevent growth of an organism after subculture on to antibiotic free media.0.5 ml of test tube 1,2,3,4 are poured into freshly prepared agar plate separately and incubated overnight. After incubation the plate showing no growth of organisms are recorded as MBC .

3.10 GAS CHROMATOGRAPHY-MASS SPECTROSCOPY (GC-MS) ANALYSIS:-

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions as well as the phase properties. The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute (come off of) from the column at different times (called the retention time), and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio. These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample.

GC-MS analysis of the samples was carried out using Mass Sensitive Detector, equipped with a cross linked methyl silicone gum phase capillary column (25m x 0.32mm). Hydrogen was used as the carrier gas and the temperature programming was set with initial oven temperature at 40°C and held for 2 min and the final temperature of the oven was 280°C with rate at 10°C/ min. A 2 μL sample was injected with splitless mode. Mass spectra were recorded over 35-650 amu range with electron impact ionization energy 70 eV. The total running time for a sample is 45 min.

4. Result and DISCUSSION

4.1 Preparation of the Plant MATERIAL & EXTRACTION

Four samples were subject for extraction and the resulted extract labeled as the TS 1 TS2 TS3 TS4 respectively. TS1 and TS3 are methanolic extract and TS2 and TS4 are ethanolic extract of AP. TS1 and TS2 are root part of AP where as TS3 and TS4 are aerial. These extracts were analyzed for their antibacterial effects immediately after their preparation.

4.2 Antimicrobial assay

Effects of different extract of Anacyclus pyrethrum were tested against five different microorganisms. All extract inhibited the microorganism with varying degree of sensitivity. Result obtained in the present study relieved that the tested medicinal plant extract posses potential antibacterial activity against S. aureus, E.coli, Pseudomonas aeruginosa, Proteus vulgeris. When tested by well diffusion method by using methanolic and ethanolic extract of Anacyclus pyrethrum.

Table 4: Antimicrobial activity of Methanol extract of Anacyclus Pyrethrum

Micro-organism	Test Solution 1	Test Solution 3	Methanol ( -ve )	Antibiotic
S. Aureus	12 mm	10 mm	8 mm	28 mm
E. Coli	18 mm	10 mm	8 mm	30 mm
Pseudomonas	16 mm	14 mm	10 mm	28 mm
S.Abony	14mm	12mm	8mm	28mm
Proteus	18 mm	12 mm	8 mm	30 mm

The methanolic extract of root (TS1) showed significant activity against E.coli and Proteus, around 18mm, the highest antibacterial and least activity recorded in S. aureus measured 12 mm. Methanolic extract of aerial part of Anacyclus pyrethrum (TS3) exhibit low activity as compare to TS1.the highest activity of TS3 is for Pseudomonas 14 mm and lowest in E.coli and s. aures 10mm. The antibacterial activity was more for TS1 as compared to TS2. The diameter of inhibition zones ranged from 10 mm to 18 mm among different microorganism species and increased with the increase in concentration of test solution. The maximum zone of inhibition was found for E.coli, Proteus with TS1 and minimum zone of inhibition found to for 10mm for TS3.

Figure 7: Effect of Methanol Extract of AP on Microbes

Table 5: Antimicrobial activity of Ethanol extract of Anacyclus Pyrethrum

Micro-organism	Test Solution 2	Test Solution 4	Ethanol ( -ve )	Antibiotic
S. Aureus	16  mm	15 mm	8  mm	28 mm
E. Coli	26  mm	18 mm	8  mm	29 mm
Pseudomonas	24 mm	20 mm	8  mm	28 mm
S.Abony	14 mm	12 mm	8 mm	28 mm
Proteus	23 mm	22 mm	8 mm	30 mm

Ethanolic extract of root (TS2) and aerial part (TS4) showed significant activity against S. aureus, E.coli Pseudomonas aeruginoisa, S. abony and Proteus. Ethanolic extract of root TS2 show around 26mm, the highest antibacterial with 24mm and 23mm respectively for Pseudomonas and Proteus and least activity recorded in S. abony measured 14 mm. Ethanolic extract of aerial part of Anacyclus pyrethrum (TS4) exhibit low activity as compare to TS2.The highest activity of TS4 is for Proteus 22 mm and lowest in S. abony 12 mm . The antibacterial activity was more for TS2 as compared to TS4. The diameter of inhibition zones ranged from 12 mm to 26 mm among different microorganism species. The maximum zone of inhibition was found for E.coli, with TS2 and minimum zone of inhibition found to for 12 mm for TS4.

Figure 8: Effect of Ethanol extract of AP on Microbes

Overall Ethanolic extract of root and aerial part posses more antibacterial potency as compare to methanolic extract of root and aerial part of AP. Among methanolic and ehthanolic extract, root extracts shows more antibacterial potency as compare to aerial part extract. Ethanol extract of root and aerial part posses more potency as compare to methnolic extract of root and aerial part. Hence among alls ethanolic extract of root (TS3) shows good antimicrobial activity with maximum potency against E.coli. Minimum activity was observed for Methanolic extract of aerial part of anacyclus pyrethrum against S. aureus and E.coli. From these data it is clear that ethanolic extracts are more potent antimicrobials as compare to methanolic extracts. It also indicate that root part of AP is posses more antimicrobial potency as compare to aerial part of AP. As the maximum antimicrobial activity is posses by TS3 against E.coli it is consider for further investigation.

4.3 Minimum Inhibitory Concentration (MIC)

0.5ml of different concentration of Anacyclus pyrethrum from test tube1, 2, 3, 4 added to A, B, C, D tube respectively and to tube F 0.5ml of freshly prepared broth added as a control. Incubate the test tube for 24 hours and results and documented the minimum concentration showing clear solution are observed and recorded as MIC that is 500µl/ml overnight grown culture of test tube are used for minimum bactericidal concentration.

 Table 6: Minimum Inhibitory concentration of TS2 against E. coli

Microorganism	Minimum inhibitory Concentration of TS2
E.coli	500µl/ml

4.4 Minimum Bactericidal Concentration  

Minimum bactericidal concentration is the lowest concentration of antimicrobial that will prevent growth of an organism after subculture on to antibiotic free media.0.5 ml of test tube 1,2,3,4 are poured into freshly prepared agar plate separately and incubated overnight. After incubation the minimum concentration containing plate showing no growth of organisms are recorded as MBC. 

 Table 7: Minimum Bactericidal concentration of TS2 against E.coli

Microorganism	Minimum Bactericidal concentration of TS2
E.coli	50µl/ml

4.5 THIN LAYER CHROMATOGRAPHY (TLC)

Chromatography technique used to separate the mixtures of constituents. Thin layer chromatography was performed on a sheet of glass aluminum foil, which was coated with a thin layer of adsorbent material silica gel. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate with the help of capillary tube, a solvent mixture of Toluene: ethyl acetate ( 85:15 ) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. This separated analytes are cut and dissolve in ethanol and use for GC-MS analysis.

Figure 9: TLC of TS2

4.6 GC-MS ANALYSIS

Gas chromatography-mass spectrometry (GC-MS) analysis of the PEE of A. pyrethrum were carried out in order to characterize the extract .GC-MS of the PEE indicated the presence of a compound with the fragmentation of which matched with compounds enlisted below

Analytical conditions maintained were as follow

temperatures:	250°C (column) 250°C (injector) 300°C (detector)
column gas flow:	1.2 mL/min (hydrogen)
make-up gas flow:	60 mL/min (nitrogen)
injection size:	1.0 µL
injection split:	55 to 1
column:	SPB-l, 1.0-µm film thickness, 30 m × 0.32-mm i.d. fused silica (Supelco Inc.)
retention times: (min)	2.38 (p-chlorobiphenyl) 16 (cinerin I) 7.31 (jasmolin I) 12.83 (cinerin II)

Figure 10: GC/MS Analysis

Compound	CAS no.	R1	R
pyrethrin I	[121-21-1]	CH3	CH2CH=CHCH=CH2
pyrethrin II	[121-29-9]	COOCH3	CH2CH=CHCH=CH2
cinerin I	[25402-06-6]	CH3	CH2CH=CHCH3
cinerin II	[121-20-0]	COOCH3	CH2CH=CHCH3
jasmolin I	[4466-14-2]	CH3	CH2CH=CHCH2CH3
jasmolin II	[1172-63-0]	COOCH3	CH2CH=CHCH2CH3

The pyrethrins are a naturally-occurring group of six chemically-related esters, each of which is insecticidally active. Three ( pyrethrins I) are esters of chrysanthemic acid, and three (pyrethrins II) of pyrethric acid. The alcohol moieties are pyrethrelone in pyrethrin I and II, cinerolone in cinerin 1 and 2, and jasmolone in jasmolin 1 and 2. Table 1 gives the structures of the acid and alcohol moieties.

               Table 8: Components moieties of pyrethrin esters

ACID	ALCOHOL

The six pyrethrin esters are designated collectively by the ISO common name “pyrethrins”. Pyrethrin 1 predominates. Information on the individual esters is provided below, where the IUPAC chemical names are according to Rothamsted nomenclature

4.6.1. Pyrethrin I

Chemical names:-

IUPAC: (Z)-(S)-2-methyl-4-oxo-3-(penta-2,4-dienyl)cyclopent-2-enyl (1R)- trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

CAS: [1R-[1α[S*(Z)],3β]]-2-methyl-4-oxo-3-(2,4-pentadienyl)  cyclopenten-1-yl 2,2-dimethyl-3-(2-methyl-1-  propenyl)cyclopropanecarboxylate

 CAS No.: 121-21-1

Molecular formula: C21H28O3 Molecular weight: 328.4

Pyrethrin I

Figure 11: Structure of Pyrethrin I

4.6.2. Cinerin 1

Chemical names:-

IUPAC:-  (Z)-(S)-3-(but-2-enyl)-2-methyl-4-oxocyclopent-2-enyl (1R)-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

CAS:-[1R-[1α[S*(Z)],3β]]-3-(2-butenyl)-2-methyl-4-oxo-2-cyclopenten-1-yl2,2 dimethyl-3-(2-methyl-1-propenyl) cyclopropanecarboxylate

CAS No.:- 25402-06-6

Molecular formula: C20H28O3 Molecular weight: 316.4

Cinerin I

   

 Figure 12: Structure of Cinerin I

4.6.3 Jasmolin 1

Chemical names:-

IUPAC: (Z)-(S)-2-methyl-4-oxo-3-(pent-2-enyl)cyclopent-2-enyl (1R)-trans-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropanecarboxylate

CAS: [1R-[1α[S*(Z)],3β]]-2-methyl-4-oxo-3-(2-pentenyl)-2-cyclopenten-1-yl 2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropanecarboxylate

 CAS No.:- 4466-14-2

Molecular formula: C21H30O3 Molecular weight: 330.4

   

  

Jasmolin I

Figure 13: Structure of Jasmolin I

5.6.4. Pyrethrin II

Chemical names:-

IUPC: - (Z)-(S)-2-methyl-4-oxo-3-(penta-2, 4-dienyl)cyclopent-2-enyl (E)-(1R)-trans-3-(2-methoxycarbonylprop-1-enyl)-2, 2-dimethylcyclopropanecarboxylate

CAS:-[1R-[1α[S*(Z)], 3β (E)]]-2-methyl-4-oxo-3-(2,4-pentadienyl)-2-cyclopenten-1-yl 3-(3-methoxy-2-methyl-3-oxo-1-propenyl)-2, 2-dimethylcyclopropanecarboxylate

CAS No.: 121-29-9

Molecular formula: C22H2805 Molecular weight: 372.4

Pyrethrin II

Figure 14: Structure of Pyrethrin II

5.6.5 Cinerin II

Chemical names:-

IUPAC: (Z)-(S)-3-(but-2-enyl)-2-methyl-4-oxocyclopent-2-enyl (E)-(1R)-trans -3-(2 methoxycarbonylprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate

 CAS: [1R-[1α[S*(Z)], 3β (E)]]-3-(2-butenyl)-2-methyl-4-oxo-2-cyclopenten-1-yl 3-(3-methoxy-2-methyl-3-oxo-1-propenyl)-2,2-dimethyl cyclopropanecarboxylate

CAS No.: 1172-63-0

Molecular formula: C21H28O5 Molecular weight: 360.4

Cinerin II

Figure 15: Structure of Cinerin II

5.6.6 Jasmolin II

Chemical names:

IUPAC: (Z)-(S)-2-methyl-4-oxo-3-(pent-2-enyl)cyclopent-2-enyl (E)-(1R)-trans-3-(2 methoxycarbonylprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate

CAS: [1R-[1α[S*(Z)],3β(E)]]-2-methyl-4-oxo-3-(2-pentenyl)-2-cyclopenten-1-yl 3-(3-methoxy-2-methyl-3-oxo-1-propenyl)-2,2-dimethylcyclopropanecarboxylate

CAS No.: 121-20-0

Molecular formula: C22H30O5 Molecular weight: 374.4

Jasmoline II

Figure 16: Structure of Jasmoline II

5. conclusions

Emergence of multi-drug resistance in human and animal pathogenic bacteria as well as undesirable side effects of certain antibiotics has triggered immense interest in the search for new antimicrobial drugs of plant origin. Some strains of bacteria were found to be sensitive to the plant extracts but showed resistant against antibiotics. Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay. Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants. Some of these observations have helped in identifying the active principles responsible for such activities and in the developing drugs for the therapeutic use in human beings. However, not many reports are available on the exploitation of antimicrobial property of plants for developing commercial formulations for applications in crop protection. In the present study, the methanol and ethanol extract of  root and aerial part of  plant Anacyclus pyrethrum showed the activity against S. aureus, E coli, Pseudomonas aeruginosa, Salmonella abony  and Proteus vulgeris and Plant based products have been effectively proven for their utilization as source for antimicrobial compounds. For instance, both extracts of Anacyclus pyrethrum exhibited inhibitory extracts of root and aerial part of Anacyclus pyrethrum were evaluated by the well diffusion method against five S. aureus, E coli, Pseudomonas aeruginosa, Salmonella abony and Proteus vulgeris. Methanol extract of aerial and root of Anacyclus pyrethrum exhibits microbial growth on agar media but ethanol extract of aerial and root extract showed more potent antimicrobial activity. 

The traditional use of Anacyclus pyrethrum against enteric pathogens proves the best in particular to the E.coli. The co relation and the exact mode of these active components as a potential novel entities need to be explore in future research. The results of present investigation clearly indicate that the antibacterial and antifungal activity vary with the species of the plants and plant material used. Thus, the study ascertains the value of plants used in Ayurveda, which could be of considerable interest to the development of new drugs.

7. References

1.      Casida JE, Quisdad GB, Pyrethrum Flowers, Production, Chemistry, Toxicology, and Uses, Oxford University Press, New York, 1995; 46-53.

2.      Casida JE, Pyrethrum, The Natural Insecticide, Academic Press, New York 1973; 3: 43-45.

3.      Lloyd UJ, History of the vegetable drug’s U.S.P 1911.

4.      Kenya’s Pyrethrum Industry published by Ministry of Agriculture and Rural development of Keneya, 2005:1-10.

5.      Kokate CK, Porohit AP, Gokhale SB, Pharmacognosy, Nirali Prakashan, New Delhi Edittion 33, 2007; 578-581.

6.      Gnadinger CB, Pyrethrum flower, Biotech Publisher, Spain, 2001; 1: 1-34.

7.      The Ayurvedic pharmacopeia of India, part- I, Volume-II, 1999; 1:1-2.

8.      Lamnuer D and Kamal B., A guide to medicinal plant In North Africa, Anacyclus Pyrethrum, IUCN Mangla, 2005:35-37

9.      The wealth of India “A dictionary of Indian raw material and Industrial product, 1948; 9:250-254.

10.  Ursulla B., Federal biology researches center for agriculture and forestry, Germany, 2002; 1: 685-749.

11.  Odinga WA and Angedu CA, The relationship between Pyrethrin and the yellow pigmentation in pyrethrum, African journal of science and technology science and engineering series, 2003;4(2):116 –123.

12.  Insecticidal Factsheet, Journal of pesticide reform, Spring, 2002; 22(1): 14-15.

13.   Tattesfield F, Pyrethrum Post, Official publication of Pyrethrum board of keneya 1948; 1: 1-2.

14.  Casida JE; Pyrethrum flowers and Pyrethroid insecticides, Enviromental health perspective, 1980; 34: 189-202.

15.  Burgess IF, Brown CM and Burgess NA; Synergized pyrethrin mouses a new approach to head lice eradication; Pyrethrum post, Official publication of Pyrethrum board of keneya 1994; 19; (2): 41-46.

16.  Mbaria JM, Maitho TE, Milema ES and Muchiri DJ; Comparative efficacy of pyrethrum marc with Albendazole against sheep gastrointestinal nematodes; Tropical Animal Health and Production, 1998; 30: 17-22.

17.  Panchal GM, Venkatakrishna Bhat, Devasankaraiah G, Gopalakrishna G, Patel VK; Anaesthetic activity of Anacyclus Pyrethrum in laboratory animal; Indian Journal of Pharmacology 2001;33:296.

18.  Bendjeddou D, Lalaoui K, Satta D, Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis , J Ethnopharmacol. 2003; 88 ;(2):155-60.

19.  Sharma V, Thakur M, Singh N, Kumar V ;Evaluation of the Anabolic, Aphrodisiac and Reproductive Activity of Anacyclus Pyrethrum DC in Male Rats Scientia Pharmaceutica, 2009; 77; 97–110.

20.  Badhe SR, Badhe RV, Ghaisas MM, Chopade VV, Deshpande AD.;Evaluations of antidepressant activity of Anacyclus pyrethrum root extract; International Journal of Green Pharm 2010;4:79-82.

21.  Gautam OP, Savita Verma, Jain SK, Anticonvulsant and Myorelaxation activity of Anacyclus Pyrethrum DC. Root extract, Pharmacologyonline 2011; 1: 121-125.

22.   Sujith K, Suba V and Ronald DC; Neuropharmacological profile of ethanolic extract of Anacyclus pyrethrum in Albino wistar rats, IJPSR, 2011; 2; (8): 2109-2114.

23.   Chaouki S, Benali O, Boufeldja T, Lahcen L, Yahia H, Green corrosion inhibitor: inhibitive action of aqueous extract of Anacyclus pyrethrum L. for the corrosion of mild steel in 0.5 M H2SO4, J. Mater. Environ. Sci. 2012; 3(1): 206-219.

24.  Sukumaran K, Kuttan R Inhibition of tobacco-induced mutagenesis by eugenol and plant extracts available at http://www.ncbi.nlm.nih.gov/pubmed/7753104 accessed on 5 march 2012.

25.  Antnthanarayan and Paniker, Text book of microbiology, Orient longman, Hyderabad, 2009; 8: 195-199.

26.  Harris L, Foster SJ and Richards RG, An introduction to staphylococcus aureus and techniques for identifying and quantifying s.aureus adhesins in relation to adhesion to biomaterial, 2002; 4: 39-60.

27.  Antnthanarayan and Paniker, Text book of microbiology, Orient longman, Hyderabad, 2009; 8: 270-281.

28.  Antnthanarayan and Paniker, Text book of microbiology, Orient longman, Hyderabad 2009; 8: 315-317.

29.  David pollack, Student presentation on Salmonella entrica typhi, University of connecticut, Department of molecular and cell biology; 2003.

30.  Proteus microbilis, Technical information, Manson chemical Company 2010; 1-9.

31.  Stephen PD, Norman AH, Sean PG, Hugo and Russell’s, Pharmaceutical Microbiology, Blackwell Science Malden, 2004; 7: 119-131.

32.  Allen DA, Austin B, and Colwell RR; Antibiotic resistance patterns of metal-tolerant bacteria isolated from an estuary, Antimicrob Agents Chemother, 1977; 12:545-547.