Description:
FIELD OF INVENTION:
The present invention relates to the field of assessing the natural degradation products of promethazine compounds. The invention specifically relates to the use of natural degradation products of promethazine compound as an anti-microbial .

BACKGROUND OF THE INVENTION:
Promethazine is a neuroleptic medication and first-generation antihistamine. It is an important drug and used in many cough syrups and anti-allergic formulations. Promethazine is used to treat allergy symptoms such as itching, runny nose, sneezing, itchy or watery eyes, hives, and itchy skin rashes. It also prevents motion sickness, and treats nausea, vomiting or pain after surgery. It is also used as a sedative or sleep aid.
It has been reported in the art that promethazine is photosensitive. In aqueous solution, promethazine hydrochloride is degraded by heat and light more rapidly in air or oxygen. Thus it is very important to study the degradation analysis of promethazine. Degradation analysis is performed on a secondary product that is obtained by degradation of the original product of interest by means of exogenous factors such as heat, humidity, oxidation, change of pH, photosensitivity, hydrolysis etc. This is of particular relevance to pharmaceutical industry since bulk or raw drugs are often stored for years in an atmosphere that exerts stress factors over the drugs as mentioned above. This in turn, potentiates chances of degradation of that product which may lead to formation of either less potent and/or toxic product which may contaminate the active pharmaceutical ingredient (API) itself.
Degradation analysis is done to analyze the difference of the original product and the degraded product throughout several different processes. By this practice amount of drug is degraded or changed can be determined. Labels are encrypted over the bulk drug about the limit up to which the percentage of the API is acceptable to make the dosage form. Some manufacturers also suggest re-assay of the API should be carried out periodically after a few years in order to find out if the API is within the acceptance limit or not. However, cares are overlooked about the degradation product that might be toxic and create side effects in the human subjects. Hence it is extremely important to evaluate the toxicity and side effects of the degradation product which necessitates obtaining the degradation product in pure and isolated form. Degradation can be performed by (a) a forced degradation study or (b) a real time or natural degradation study.
Forced degradation is a degradation of new drug substance and drug product at conditions more severe than accelerated conditions (severe temperature, humidity, oxidation and hydrolysis). It is required to demonstrate specificity of stability indicating methods and also provides an insight into degradation pathways and degradation products of the drug substance and helps in elucidation of the structure of the degradation products. Forced degradation studies show the chemical behavior of the molecule which in turn helps in the development of formulation and package. In addition, the regulatory guidance is very general and does not explain about the performance of forced degradation studies. Forced degradation is accepted as per ICH Guidelines and hence is followed because it requires comparatively lesser time to carry out the study than real time degradation analysis. Research paper entitled “Oxidative degradation of pharmaceutically important phenothiazines I: Isolation and identification of oxidation products of promethazine” and “Oxidative degradation pharmaceutically important phenothiazines II: Quantitative determination of promethazine and some degradation products” by Underberg WJ; describes the degradation products of promethazine compound by forced degradation methods. Though forced degradation is a standard procedure as per ICH but it is not optimum because the parameters cannot be same as real time or natural degradation. Moreover, forced degradation study cannot simulate the actual scenario since it is controlled exploitation of one or two specific stress parameters in a short time regimen. However, real time degradation usually combines all the stress parameters for the slow uncontrolled degradation including the seasonal climatic change all over the year. Hence it would be worthy to perform a natural or real time degradation study on promethazine compound. Till date, real time degradation study of active pharmaceutical ingredient (also known as “API”) is less studied worldwide and thus the actual bioactivity potential (either positive or negative) of bulk drug degradation products (over years) is not investigated at all.
There exists a need in the art to come up with a way of assessing loss incurred due to natural degradation of promethazine compound. This is currently an excruciating problem in pharmaceutical industry. The estimation of promethazine purity is often guided by assay methodology as described in official monographs; however, this often fails to distinguish promethazine entirely from contaminating degradation products of the same phenothiazine group. Hence, there is a compelling need of promethazine batch calibration in order to find out degree of degradation which in turn, helps in investigating loss of potency and/or enhancement of toxicity of the drug compound.
In case the sample turns out to be toxic or have a higher degree of degradation it may be considered as waste and may undergo mass elimination. This could lead to economic loss to an organization. Finding out ways to re-use or recycle the waste sample can provide economic advantages to organization. Moreover, promethazine being bioactive; the degradation product may also have bioactivity potential against some other targets which may be a promising for drug discovery. One such use of the degraded product can be as an anti-microbial .
Patent no. US 2495270 A, US3196074 A , research articles “Antimicrobial activity of phenothiazines” by Amaral L et al, and “Phenothiazine: the parent molecule “by Mitchell SC; teaches the use of phenothiazine as an antifungal, antibacterial, anthelmintic and insecticidal . Research article “Synthesis and antimicrobial activities of novel biologically active heterocycles: 10H-phenothiazines, their ribofuranosides, and sulfone derivatives” by Dixit Y et.al teaches the synthesis and anti-microbial activity of a series of novel substituted 10H-phenothiazines and its derivatives.
However, there exists a need in the art to explore the anti-microbial activity of the naturally (real time) degraded product/s of promethazine compound against a set of plant and human pathogens.
Hence, the current invention also aims to address the need of the art by assessing the anti-microbial activity of natural degradation product/s of promethazine compound against plant and animal pathogens.

OBJECTS OF THE INVENTION:
The principal object of the instant invention is to study the natural (real time) degradation product of promethazine compound.
Yet another object of the present invention is to isolate the natural degradation product of promethazine compound.
Still another object of the current invention is to determine the percentage of degradation of the drug sample.
Still another object of the current invention is to find out ways to use the natural degraded product of promethazine compound.
Another object of the instant invention is to assess the anti-microbial activity of natural degradation product of promethazine compound against a set of plant and animal pathogens.

SUMMARY OF INVENTION:
The above-mentioned shortcomings, disadvantages and problems are addressed herein which will be understood by reading and understanding the following specification.

In one embodiment of the present invention, describes an anti-microbial comprising; a natural degradation product of promethazine compound, which comprises a phenothiazine compound having a formula (I)

(I)
Wherein R is selected from the group consisting of hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine.
In another embodiment of the invention R is selected from group consisting of N,N dimethyl amino propyl (Promazine or Chlorpromazine), 2-(N,N dimethyl)-propyl (Phenothiazine), 3-(4-methyl-1-piperazinyl) propyl (Prochlorperazine), 10-propyl piperazin-1-yl ethanol (Fluphenazine or Perphenazine), 2-[(RS)-1-Methylpiperidin-2-yl]ethyl (Thioridazine or Mesoridazine), 3-(4-methylpiperazin-1-yl) propyl (Trifluperazine) and so others. Degradation product of promethazine compound can be one or more compounds.

In another embodiment of the present invention, R may be an alkyl group. In yet another embodiment of the present invention, the degradation product comprises a phenothiazine compound having a formula (II) and formula (III)

N-propyl phenothiazine
(II)

10H phenothiazine

(III)

The antimicrobial is used against plant and animal microbes. Bacillus pumilus, Pseudomonas syringaepathovars, Ralstonia solanacearum, Arabidopsis thaliana, Agrobacterium tumefaciens, Xanthomonas oryzae, pv. Oryzae, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Erwinia amylovora,Xylella fastidiosa, Dickeya (dadantii and solani), Pectobacterium carotovorum and Pectobacterium atrosepticum. Animal microbe is selected from the group consisting of Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholera, Bacillus pumilus and Escherichia coli. In one embodiment, Bacillus pumilus is selected as a model plant microbe from a genre of reported plant pathogens.

Yet another embodiment of the invention describes a process for natural degradation analysis of a promethazine compound, wherein the degradation product comprises a phenothiazine compound having a formula (I)
(I)
wherein R is selected from the group consisting of hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine.
In yet another embodiment of the invention R is selected from 2-(N,N dimethyl) propyl, methyl or unsaturated nitrogen (sp2 hybridized)). In another embodiment of the present invention, R is hydrogen, n-propyl or mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: 3D Chromatogram of standard and sample applications. Lane 1-5: Standard degraded product 1, 2, 5, 8, 10 µg and Lane 6-7: Sample (degraded batch sample) each containing 10 µg.
Figure 2: TLC profile of the sample as per example 1.
Figure 3: IR spectroscopy of compound no 3 & 6 (Table 1).
Figure 4: Mass spectroscopy of compound no 3 & 6 (Table 1).
Figure 5: NMR spectroscopy of compound no 3 & 6 (Table 1).
Figure 6: Zone of inhibition for Drug 1/D1, Drug 2/D2, Promethazine and Standard (Ciprofloxacin).
Figure 7: Zone of inhibition for Drug 1/D1, Drug 2/D2, Promethazine and Standard (Ciprofloxacin) for Bacillus pumilus 8241 and Salmonella typhi 743.

DETAILED DESCRIPTION OF THE INVENTION:
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
The term “anti-microbial ” is an that kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. ‘Plant microbes” is the set of microbes which affects the plants and “Animal microbes” are the set of microbes which causes disease in the animals.
The term “natural degradation” or “real time degradation” as used herein, generally refers to analysis which is normally performed for longer duration of the test period in order to allow significant product degradation under recommended natural storage conditions. Real time or natural degradation is the best possible degradation study as here the sample that is analysed is allowed to degrade naturally for a long time period and under controlled or uncontrolled environmental conditions. No accelerated condition is forced upon the sample and so the degradation is more realistic. The period of the test depends upon the stability of the product which should be long enough to indicate clearly that no measurable degradation occurs and must permit one to distinguish degradation from inter-assay variation. During the testing, data is collected at an appropriate frequency such that a trend analysis is able to distinguish instability from day-to-day ambiguity. The reliability of data interpretation can be increased by including a single batch of reference material for which stability characteristics have already been established. Stability of the reference material also includes the stability of reagents as well as consistency of the performance of the instrument to be used throughout the period of stability testing. However, system performance and control for drift and discontinuity resulting from changes in both reagents and instrumentation must be monitored.

Process for conducting a natural/ real time degradation testing involves:
1. Storage: a product is stored at recommended storage conditions and monitored for a period of time (ttest).
2. Natural degradation: Product will degrade below its specification, at some time, denoted ts, and we must also assure that ts is less than or equal to ttest. The estimated value of ts can be obtained by modeling the degradation pattern. Good experimental design and practices are needed to minimize the risk of biases and reduce the amount of random error during data collection.
3. Testing: Testing should be performed at time intervals that encompass the target shelf life and must be continued for a period after the product degrades below specification. It is also required that at least three lots of material be used in stability testing to capture lot-to-lot variation, an important source of product variability.
“Promethazine compound” refers to all salts, polymorphs, isomers, enantiomers, derivatives and mixture of Promethazine either as an API. or finished dosage form. Finished dosage form includes tablets, capsules, solution, suspensions, emulsions, etc. Degradation product of promethazine compound can be one or more compounds.
Embodiments of the invention as disclosed herein provide a natural degradation product of promethazine compound, wherein the degradation product comprises a phenothiazine compound having a formula (I)

(I)

Wherein R is selected from the group consisting hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine.
In another embodiment of the invention R is selected from group consisting of N,N dimethyl amino propyl (Promazine or Chlorpromazine), 2-(N,N dimethyl)-propyl (Phenothiazine), 3-(4-methyl-1-piperazinyl) propyl (Prochlorperazine), 10-propyl piperazin-1-yl ethanol (Fluphenazine or Perphenazine), 2-[(RS)-1-Methylpiperidin-2-yl]ethyl (Thioridazine or Mesoridazine), 3-(4-methylpiperazin-1-yl) propyl (Trifluperazine) and so others. Degradation product of promethazine compound can be one or more compounds.
In another embodiment R is selected from gropup ofhydrogen, 2-(N,N dimethyl) propyl, methyl or unsaturated nitrogen (sp2 hybridized). Most preferably R is hydrogen, n-propyl or mixtures thereof.

Another embodiment of the present invention describes an anti-microbial agent comprising; a natural degradation product of promethazine compound, wherein the degradation product comprises a phenothiazine compound having a formula (I)

(I)

Wherein R is selected from the group consisting of hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine. Most preferably degradation product comprises a phenothiazine compound having a formula (II) and formula (III)

N-propyl phenothiazine
(II)

10H phenothiazine

(III)

The antimicrobial agent is used against plant and animal microbes. Plant microbe is selected from Bacillus pumilus, Pseudomonas syringaepathovars, Ralstonia solanacearum, Arabidopsis thaliana, Agrobacterium tumefaciens, Xanthomonas oryzae, pv. Oryzae, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Erwinia amylovora,Xylella fastidiosa, Dickeya (dadantii and solani), Pectobacterium carotovorum and Pectobacterium atrosepticum. Animal microbe is selected from the group consisting of Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholera, Bacillus pumilus and Escherichia coli.
Yet another embodiment of the instant invention describes process for natural degradation analysis of a promethazine compound, comprising:

a. Procuring a sample of a drug containing the promethazine compound.
The samples are procured from storehouse of any pharmaceutical organizations or any other location wherein the drug is stored under natural environmental conditions. During procuring the same the “age of the samples” comes into picture. The age of the sample is the period of which it has been stored in the warehouse under natural climatic condition of the store. Natural climatic condition also takes into account the seasonal climatic changes that happen during the year. Preferably the drug samples collected are at least 5- 10 years old, more preferably they are 7-8 years old.

b. Isolating a degradation product of the drug
The whole procedure of isolation is carried out in a photoprotective semi-dark room at controlled temperature. Isolation of the degraded products is performed using thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC) and column chromatography (CC). Solvent system selection is a critical factor while performing the isolation and purification of the degraded products.
The analysis of the degraded product is performed using NMR, IR and MASS Spectroscopy to elucidate the structures of the compound. The structure of the degradation product helps to decipher possible pathway of degradation. This in turn, may reveal the possible factors responsible for degradation.

c. Evaluating the amount of degradation product in the drug.
The amount of the degradation product in the drug is measured in terms of “percentage of degradation” and is calculated using the formula below:

Weight of the degraded product in sample
Percentage of degradation = ---------------------------------------------------------- X 100
Weight of the actual drug sample

In case the “percentage of degradation” is found out to be more than at least 5%, more preferably more than at lease 7-10% the sample is considered for rejection.

d. Using the degradation product as an anti-microbial .
The degradation product, if biologically significant, can lead to finding a new therapeutic entity which may serve as a potential lead for new drug discovery. This may come out as a future dimension of drug discovery together with a cost effective technology for the industry where they may recycle the “waste” as a more significant entity. Promethazine has one thiazine ring. This has similarity with the thiazole ring of beta-lactam antibiotic. Thus its degradation product has a potential chance for possessing antimicrobial activity.
An example embodiment of the present invention is described below:

Example 1: Isolation of degraded products using CC.
The samples were procured from storehouse of a pharmaceutical industry in Kolkata, WB, India. Age of the samples was: 7-8 years. The samples were stored at seasonal climatic condition on the southern part of west Bengal.

The whole procedure was carried out in a photoprotective semi-dark room at controlled temperature (22-25 °C) with relative humidity at 50± 5 %. The isolated compounds were stored in aluminium foiled containers (for light protection) at 4°C. The real time degradation products of promethazine were isolated by column chromatography guided isolation technique.

Step 1: The degradation mixture from industry was charged on to silica gel (column grade, mesh #200) into a column with mixture: silica gel (1:8 w/w).

Step 2: The column was run with gradient elution technique where the mobile phase was used Chloroform (CHCl3): Ethyl Acetate (C2H5COOC2H5): Methanol (CH3OH) (100:0:0 to 67:25:8 v/v). The solvent elution profile has been chosen as described in Table 2.

Step 3: Five-six fractions were collected for each system of mobile phase. After fraction collections, each fraction was monitored for its compound elution profile on to a silica gel GF254 plate by Thin Layer Chromatography (TLC) using a mobile phase Benzene (C6H6): Chloroform : Ethyl Acetate: Methanol (4:3:2:1).

Step 4: After pooling the fractions with similar compound elution, each pooled fraction was evaporated in vacumn distillation unit under reduced pressure.

Step 5: Eight compounds were isolated (as shown in Figure 2) which showed close similarity in polarity on to the silica gel plate. Amongst them, based on substantial difference on polarity, two compounds were selected and characterized. TLC profile is provided in Table 1.

Step 6: Compound no 3 & 6 (Table 1) seemed to be different and pure. Those compounds were selected for spectroscopic evaluation like IR (Figure 3), Mass spectroscopy(Figure 4) and NMR (Figure 5) analysis.

Observation: Compounds identified were N-propyl phenothiazine (Drug1 /D1), 10H phenothiazine (Drug 2/D2).

Table 1: TLC profile

Compound number Identity (As per fraction collection) Rf Value
1 Degraded product (3) 0.72
2 Degraded product (2) 0.61
3 Degraded product (9.1) 0.5464
4 Degraded product (1) 0.56
5 Degraded product (3.1) 0.579
6 Degraded product (3.3) 0.74
7 Degraded product (23.1) 0.78
8 Mother Promethazine 0.83

Table 2: The solvent elution profile for column chromatography

Mobile phase Chloroform (CHCl3 ) Ethyl Acetate (C2H5COOC2H5) Methanol (CH3OH)
1 100 0 0
2 95 5 0
3 90 10 0
4 85 15 0
5 80 20 0
6 75 25 0
7 70 30 0
8 67 30 3
9 67 28 5
10 67 25 8

Example 2: Analysis of percent of degradation.
The samples were procured from storehouse of a pharmaceutical industry in Kolkata, WB, India. Age of the samples was: 7-8 years. The samples were stored at seasonal climatic condition on the southern part of West Bengal.
The whole procedure was carried out in a photoprotective semi-dark room at controlled temperature (22-25 °C) with relative humidity at 50± 5 %. The isolated compounds were stored in aluminium foiled containers (for light protection) at 4°C. The real time degradation products of promethazine were isolated from the degraded product mixture by column chromatography using gradient elution method.

Step 1: The solvent system used is Benzene: Choloform: Ethyl acetate: Methanol (4:3:2:1) for separation of compound and degradation products on a HPTLC viable pre-coated silica gel plate.

Step 2: HPTLC was performed on aluminium-backed Silica Gel 60 GF254 TLC plate (10 cm x 10 cm) from Merck, India. Sample solution was applied as 6 band and 4 spacing at a constant application rate of 10 µl by means of Linomat V sample applicator fitted with a 100-µl syringe.

Step 3: The compounds were separated by upward elution on the TLC plate in a CAMAG twin trough chamber with mobile phase consisting of as described in TLC section. The chamber saturation with mobile phase vapor was performed for 30 min at ambient temperature (22 ± 2 ºC) and relative humidity 65 ± 5%.

Step 4: The plates were allowed to run at a distance of 7.0 cm. The plates were subsequently dried using hair dryer. Plates were evaluated by spectrodensitometric measurement at 269 nm by means of a CAMAG TLC scanner III with Wincat software version 4.0.1. The corresponding chromatogram has been shown in Table 3 and Table 4.

Result: From calibration, the degradation of the degraded batch sample has been found as 92.786% (w/w). Figure 1 describes the 3D HPTLC Chromatogram of standard and sample applications. Lane 1-5: Standard degraded product 1, 2, 5, 8, 10 µg and Lane 6-7: Sample (degraded batch sample) each containing 10 µg.

Table 3: Assay Validation sheet
Accuracy 95.320 ± 7.06
Slope 1.761
Intercept 0.0176
Linearity range 0.1 - 1.0 µg/ml
Correlation coefficient 0.9896
SE of intercept 0.0648
SD of intercept 0.144897205
LOD 0.271527982
LOQ 0.822812067

Table 4: Recovery study

No. of observation Added concentration (µg/ µl) Added amount (µg) Area (*10^4) Recovered concentration (µg/ µl) Recovered amount (µg) % Recovery Average percentage recovery Standard Deviation
1 1 10 1.7309 0.992902 9.929018 99.29018 101.6525 10.25163
2 1 10 1.6164 0.927882 9.278819 92.78819
3 1 10 1.9702 1.12879 11.2879 112.879

Example 3: Anti-microbial activity of the degradation products.
Method employed: Agar disc diffusion (Disc=Standard filter paper, Whatman No.1 ); Medium: Nutrient Agar (10 g/L peptone. 10 g/L beef extract, 5 g/L NaCl, 1.8 % agar); Microbial cell population: Overnight culture (106 ~108 CFU/ml), just required to cover the surface of the agar; Filter paper: 4 , absorbing 0.01 ml of the sample solution; Incubation: 37 0C, overnight (16 ~18 hrs.
Sample: Promethazine pure, D1/Drug 1 (N-propyl-phenothiazine), D2/Drug 2 (10H phenothiazine) and Reference standard (Ciprofloxacin & Ceftriaxone)
Details of the microorganism strains used and standard taken is provided in table 5 and the:
Table 5: Strains and standards used for the study
Strains used Standard taken Zone of inhibition reference table
Salmonella typhi 743 Ciprofloxacin Table 6 ; Figure 7
Staphylococcus aureus 6538 Ceftriaxone Table 7
Pseudomonas aeruginosa APC Ciprofloxacin Table 8
Vibrio cholerae 569B Ciprofloxacin Table 9
Bacillus pumilus 8241 Ciprofloxacin Table 10; Figure 7
Escherichia coli K12 ROW Ciprofloxacin Table 11

Table 6: Zone of inhibition for Salmonella typhi
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CIPROFLOXACIN 5 5.2 5 5.06
PROMETHAZINE
(PURE) 0.9 0.6 0.8 0.76
D1 1 0.6 0.9 0.83
D2 0.7 0.7 0.7 0.7

Table 7: Zone of inhibition for Staphylococcus aureus 6538
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CEFTRIAXONE 1.2 1.3 1.2 1.23
PROMETHAZINE
(PURE) 1 1.1 0.9 1
D1 0.6 0.7 0.6 0.63
D2 0.7 0.7 0.7 0.7

Table 8: Zone of inhibition for Pseudomonas aeruginosa APC
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CIPROFLOXACIN 3.8 4.2 4.2 4.06
PROMETHAZINE
(PURE) 1 0.9 1 0.96
D1 0.7 0.8 0.8 0.76
D2 0.7 0.8 0.7 0.73

Table 9: Zone of inhibition for Vibrio cholerae 569B
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CIPROFLOXACIN 2.2 1.9 2.1 2.06
PROMETHAZINE
(PURE) 2 2.5 1.8 2.1
D1 0.7 0.6 0.8 0.7
D2 1 0.8 1 0.93

Table 10: Zone of inhibition for Bacillus pumilus 8241
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CIPROFLOXACIN 2.2 2.5 2.5 2.4
PROMETHAZINE
(PURE) 2.6 2.9 2.8 2.76
D1 2.3 1.9 1.8 2
D2 2.6 3 2.9 2.83

Table 11: Zone of inhibition for Escherichia coli K12 ROW
DRUG (cm) AVERAGE
OBS 1 OBS 2 OBS 3
CIPROFLOXACIN 4.3 4.4 3.9 4.2
PROMETHAZINE
(PURE) 1.4 1.5 2 1.63
D1 1.1 1.1 1.6 1.26
D2 1.2 0.8 1.1 1.03

Observation: Both the degradation products showed antimicrobial activity. However, D2 (10H phenothiazine) (D2) showed more promising results than D1 (N-propyl-phenothiazine). 10H phenothiazine shows activity against all strains of microorganism used in the study. 10H phenothiazine produced remarkable activity against Bacillus pumilus which is predominantly a plant & human pathogen and moderate activity against Staphylococcus aureus which causes respiratory tract infection in humans. The anti-microbial activity of D2 was found to be more effective than promethazine.
It has been reported in the art that administration of phenothiazine daily in human subjects could relieve urinary tract infections (UTI) effectively without toxic effects. Hence, Promethazine natural degraded compound 10H phenothiazine alone, or its mixture with N-propyl phenothiazine could be used against Bascillus pumilus and other human pathogens.. In addition, Bascillus pumillus is a plant pathogen especially destroying potato, mango and ginger. Since plant infection in this case is a superficial infection, the compound/s can be simply sprayed on to the plants as suitable solution that is easily washable by later treatments. This can be of immense help for the agricultural field.
Since this product is recycled from waste, the natural degraded products of promethazine compound that is usually thrown away by industries, can be easily used back by instant methodology to generate antimicrobial products. It reduces the cost of production of the antimicrobial compound significantly, becomes beneficial to the industries, agricultural field as well as to the patients suffering from food poisoning or UTI infection. It also reduces the environmental hazard that might be caused due to phenothiazine exposure by waste disposal of promethazine.
Other aspects and advantages of the invention will be further appreciated and understood when considered in conjunction with the following description and accompanying drawings. While the following description may contain specific details describing particular embodiments of the invention, this should not be construed as limitations to the scope of the invention but rather as an exemplification of preferable embodiments. For each aspect of the invention, many variations are possible as suggested herein that are known to those of ordinary skill in the art. A variety of changes and modifications can be made within the scope of the invention without departing from the spirit thereof.

Claims:1. An anti-microbial comprising;
a natural degradation product of promethazine compound, wherein the degradation product comprises a phenothiazine compound having a formula (I)

(I)

Wherein R is selected from the group consisting of hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine.

2. The anti-microbial of claim 1, wherein R is hydrogen, or n-propyl.

4. The anti-microbial of claim 1, wherein the is employed against plant and animal microbes.

5. The anti-microbial of claim 3, wherein the plant microbe is selected from Bacillus pumilus, Pseudomonas syringaepathovars, Ralstonia solanacearum, Arabidopsis thaliana, Agrobacterium tumefaciens, Xanthomonas oryzae, pv. Oryzae,Xanthomonas campestris pathovars,Xanthomonas axonopodis pathovars,Erwinia amylovora,Xylella fastidiosa,Dickeya (dadantii and solani), Pectobacterium carotovorum and Pectobacterium atrosepticum.

5. The anti-microbial of claim 3, wherein the animal microbe is selected from the group consisting of Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholera, Bacillus pumilus and Escherichia coli.

6. A process for natural degradation analysis of a promethazine compound, comprising:
a. procuring a sample of a drug containing the promethazine compound.
b. isolating a degradation product of the drug
c. evaluating the amount of degradation product in the drug.
d. using the degradation product as an anti-microbial .

7. The process for natural degradation analysis of a promethazine compound of claim 6, wherein the isolation is performed using TLC, HPTLC and column chromatography.

8. The process for natural degradation analysis of a promethazine compound of claim 6, wherein using the degradation product as an anti-microbial is done in case the percentage of degradation is more than at least 10%.

9. The process for natural degradation analysis of a promethazine compound of claim 6, wherein the degradation product comprises a phenothiazine compound having a formula (I)
(I)

Wherein R is selected from the group consisting of hydrogen, alkyl, aryl, hydroxyl, carboxyl, nitro, amide, amine.

10. The process for natural degradation analysis of a promethazine compound of claim 6, wherein the degradation product comprises a phenothiazine compound having a formula (I)

(I)
wherein R is hydrogen, or n-propyl.