WO2013021258A1

WO2013021258A1 - Anti-tubercular compounds

Info

Publication number: WO2013021258A1
Application number: PCT/IB2012/001503
Authority: WO
Inventors: Swati Pramod Joshi; Roshan Rajan Kulkarni; Ketaki Dilip SHURPALI; Sampa Sarkar; Dhiman Sarkar
Original assignee: Council Of Scientific & Industrial Research
Priority date: 2011-08-05
Filing date: 2012-08-06
Publication date: 2013-02-14

Abstract

The present invention relates to pharmaceutical composition comprising at least one compound of formula 1 and formula 2 for the treatment of Mycobacterial tubercular infections. More particularly, present invention discloses use of compound of formula 1 and formula 2 for the treatment of Mycobacterial tubercular infections.

Description

ANTI-TUBERCULAR COMPOUNDS

The following specification particularly describes the nature of the invention and the manner in which it is to be performed:

TECHNICAL FIELD OF THE INVENTION

The present invention relates to pharmaceutical composition comprising at least one compound of formula 1 and formula 2 for the treatment of Mycobacterial tubercular infections. More particularly, present invention discloses use of compound of formula 1 and formula 2 for the treatment of Mycobacterial tubercular infections. Also, present invention provides a process for the isolation and characterization of triterpene such as corosolic acid of formula land flavonoid such as 5-Hydroxy-6,7,3',4'-tetramethoxyflavone of formula 2 from Plectranthus mollis.

BACKGROUND AND PRIOR ART OF THE INVENTION

Plectranthus mollis (also known as Plectranthus incanus, Plectranthus cordifolia) (Family: Lamiaceae), native to Asia, is a small, erect, fleshy, annual herb, growing up to 30-50 cm tall. Leaves are ovate-cordate, acute or acuminate, crenate. Flowers are pale lilac to light blue in colour. Fruits are round, brown, dotted and purple coloured. The oil of the plant possesses anti-bacterial activities. It the plant is reported to also act as cardiac depressant, respiratory stimulant, vaso-contraictor and relaxant on smooth and skeletal muscles.

Plectranthus mollis infusion of the leaves has been used in the folk medicine to treat inflammations, respiratory infections, vasoconstriction and mental retardations. In India, several studies have shown that Plectranthus mollis is used as a febrifuge (medicine used to reduce fever). Plectranthus mollis contains diterpenoids fenchone, limonene, piperitenone, β-hisabolene, β-cubebene and a-humulene. Terpenoids, steroids, flavones and phenolics are the most common secondary metabolites in Plectranthus and/which are known to be antioxidants. Mycobacterium tuberculosis (MTB) is a pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases of tuberculosis (TB). Tuberculosis is a major and still neglected cause of death and disability with around 2 million deaths worldwide in 2009 and 9 million infections. There is more tuberculosis today than at any other time in History.

One third of the world's population is thought to have been infected with M. tuberculosis, with new infections occurring at a rate of about one per second. In 2007, there were an estimated 13.7 million chronic active cases globally, while in 2010, there were an estimated 8.8 million new cases and 1.5 million associated deaths, mostly occurring in developing countries. The absolute number of tuberculosis cases has been decreasing since 2006, and new cases have decreased since 2002. The distribution of tuberculosis is not uniform across the globe; about 80% of the population in many Asian and African countries test positive in tuberculin tests, while only 5-10% of the United States population tests positive. More people in the developing world contract tuberculosis because of compromised immunity, largely due to high rates of HIV infection and the corresponding development of AIDS.

Tuberculosis typically attacks the lungs, but can also affect other parts of the body. It is spread through the air when people who have an active TB infection cough, sneeze, or otherwise transmit their saliva through the air. Most infections are asymptomatic and latent, but about one in 10 latent infections eventually progresses to active disease which, if left untreated, kills more than 50% of those so infected.

The physiology of M. tuberculosis is highly aerobic and requires high levels of oxygen. Primarily a pathogen of the mammalian respiratory system, MTB infects the lungs. The most frequently used diagnostic methods for TB are the tuberculin skin test, acid-fast stain, and chest radiographs.

The classic symptoms of active TB infection are a chronic cough with blood-tinged sputum, fever, night sweats, and weight loss. Infection of other organs causes a wide range of symptoms. Diagnosis of active TB relies on radiology, as well as microscopic examination and microbiological culture of body fluids. Diagnosis of latent TB relies on the tuberculin skin test (TST) and/or blood tests. Treatment is difficult and requires administration of multiple antibiotics over a long period of time. Social contacts are also screened and treated if necessary. Antibiotic resistance is a growing problem in multiple drug-resistant tuberculosis (MD -TB) infections. Prevention relies on screening programs and vaccination with the bacillus Calmette-Guerin vaccine.

An article titled "The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strains" by Castellar Aline et al, published in Rev. bras. Farmacogn, May 06, 2011, discloses the isolation and identification of the active antimycobacterial compounds from Lippia lacunose. Compounds isolated were evaluated for their in vitro activity against Mycobacterium tuberculosis (susceptible and rifampicin resistant strain) using a redox bioassay. From the dichloromethane extract of L. lacunosa leaves, seven methoxy-flavones named cirsimaritin, eupatilin, eupatorin, salvigenin, 3'-0-methyl-eupatorin, 3',7-dimethoxy-5,6,4'- trihydroxyflavone, and 7'-0- methylapigenin, and one triterpene, named oleanolic acid, were isolated. All compounds were found to display antimycobacterial activity against susceptible strain, with MIC ranging from 25 to 200μg/mL.

Korean Patent Publication No. 20080005724, titled "Method for separation and purification of corosolic acid from corosolic acid-containing materials" discloses a method of extracting and purifying corosolic acid from a material containing corosolic acid to obtain high-purity corosolic acid efficiently by a successive process containing an extraction, chromatography, precipitation and high performance liquid chromatography. The material containing corosolic acid contains a plant body, callus, cell or cell suspension cultures of a plant selected from the group consisting of Lagerstroemia speciosa, Eriobotrya japonica, Ternstroemia gymnanthera, Crataegus pinnatifida and Tiarella polyphylla.

Chinese Patent Publication No. 101434636 titled "Method for extracting corosolic acid from plant" discloses a method for extracting corosolic acid from plants, which extracts and separates out high-purity corosolic acid by taking the plants containing the corosolic acid as raw materials, such as crape myrtle or loquat.

Review article titled "Natural product growth inhibitors of Mycobacterium tuberculosis" by Brent R Copp and Norrie Pearce, published in Nat. Prod. Rep., 2007, 24, 278-297, discloses natural products (secondary metabolites) with reported growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. The natural (or related derivative) antimycobacterial agents exhibit wide-ranging in- vitro potency towards M. tuberculosis, with minimum inhibitory concentrations (MIC) of O^g/ml or less. The review discloses triterpenes isolated from plants Ruprechtia triflora and Calceolaria pinnifolia, urosolic acid, 24-hydroxyursolic acid and oleanic acid isolated from Valeriana laxiflora; oleanic acid analogues isolated from Sapium haematospermum, 3-acetlyoleanolic acid derivative isolated from roots and stems of Prismatomeris fragrans, isoiguesterin and 20-epi- isoiguesterinol isolated from root extracts of Salacia madagascariensis.

Review article titled "Structure-activity relationships of oleanane- and ursane- type triterpenoids" by Chun HU et. al., published in Botanical Studies (2006) 47: 339-368, discloses the isolation and structure modifications of oleanane- and ursane- type triterpenoids with an emphasis on their structure-activity relationships. Six triterpenes namely urosolic acid, oleanolic acid and 4-7 were isolated from stem bark of Physocarpus intermedius, and their ED50 values against five different tumor cells were evaluated. Isolated compound 6, corosolic acid displayed both cytotoxicity and PKC inhibition. Besides, it incompletely inhibited rat brain PKC activity in vitro at concentrations higher than 20 μg/mL.

An article titled "New Prenylated Flavonoids of Orthosiphon stamineus Grown in Malaysia", by M. Amzad Hossain and Zhari Ismail, published in Asian Journal of Biotechnology, 3: 200- 205, discloses two prenylated flavonoid derivatives; 5,7,3',5'-tetramethoxy-8-C-prenyl- flavone, 5,7,3', 5'-tetramethoxy-6-C-prenylflavone, together with four known flavonoids 5,6,7,3',4'-pentamethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, ladanein and 6- hydroxy-5,7,4'-trimethoxyflavone isolated from the leaves extracts of Orthosiphon stamineus. All the isolated compounds were tested for their cytotoxicity towards highly liver metastatic murine colon 26-L5 carcinoma cells.

An article titled "Byrsonima fagifolia Niedenzu Apolar Compounds with Antitubercular Activity", by C. T. Higuchi et al., published in Evidence-Based Complementary and Alternative Medicine Volume 2011, discloses a study to evaluate by Microplate Alamar Blue Assay (MABA), the potential antitubercular activity of B. fagifolia leaves extracts, enriched fractions and pure compounds, identified by phytochemical analysis. The triterpene basic acid showed strong antitubercular activity with MIC values of 2.5 μgmL-l.

Review article "A Review on Anti-Tubercular Plants" by Vikrant Arya, published in International Journal of PharmTech Research, Vol. 3, No.2, pp. 872-880, discloses about 48 plants which have the potential of anti-tubercular activity. Wide ranges of phytoconstituents which are responsible for anti-tubercular activity include alkaloids, glycosides, tannins, phenolics, xanthones, quinones, sterols, triterpenoids etc. These phytoconstituents present in plants exert desired pharmacological effect on body and thus act as natural anti-tubercular agents. The paper also discloses various plant drugs along with their chemical constituents responsible for anti-tubercular activity.

The emergence of drug resistant strains and confluence with HIV epidemic has turned TB into a global public health crisis. Although, available drug regimens can cure most patients, emergence MDR, SDR and XDR-TB coupled with insufficient global drug pipeline, justifies continued efforts towards development of new drugs with new mode of action and novel structures. It necessary to identify new leads molecules for the development of new tuberculostatic drugs. In the search for new molecules for such activity, the inventors investigated a hitherto unknown source for compounds that have not been known to exhibit activity against Mycobacteria.

Though terpenes and flavonoids isolated from Plectranthus mollis are known compounds having anti-bacterial activity, used as anti-inflammatory agents, cardiac depressant, respiratory stimulant, vaso-contrictor, however, the same have not been studied for potential anti mycobacterial tubercular property.

OBJECTS OF THE INVENTION

Main objective of the present invention is to provide a pharmaceutical composition comprising at least one compound of formula 1 and 2 along with pharmaceutical acceptable excipients.

Another object of the present invention is to provide use of compound of formula 1 and formula 2 for the treatment of Mycobacterial tubercular infections. Another object of the present invention is to provide isolation and characterization of triterpene such as corosolic acid of formula 1 and flavonoid such as 5-Hydroxy-6,7,3',4'- tetramethoxyflavone of formula 2 from Plectranthus mollis for the treatment of Mycobacterial tubercular infections.

SUMMARY OF THE INVENTION

Accordingly, present invention provides a pharmaceutical composition comprising at least one compound of formula 1 or formula 2 together with atleast one pharmaceutical acceptable excipient for the treatment of M. Tuberculosis.

Formula 1 Formula 2

In an embodiment of the present invention, use of compound of formula 1 and formula 2 is for the treatment of Mycobacterial tubercular infections

In an embodiment of the present invention, pharmaceutical acceptable excipients are selected from the group consisting of diluents, binders, lubricants, wetting agents and disintegrant.

In another embodiment of the present invention, a process for the isolation of compound of formula 1 and compound of formula 2 from Plectranthus mollis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition comprising at least one compound of formula 1 and 2 along with pharmaceutical acceptable excipients.

In one embodiment, the compound of formula 1 or formula 2 are present in range of 0.1 to 99 % w/w of total composition. In another aspects, present invention provides a process for isolation and characterization of triterpene such as corosolic acid of formula 1 and flavonoid such as 5-Hydroxy-6,7,3',4'- tetramethoxyflavone of formula 2 from Plectranthus mollis, useful for the treatment of mycobacterial infections.

The present invention describes the the process for isolation and characterization of compounds from Plectranthus mollis and use of their anti-mycobacterial properties.

Plectranthus mollis, whole plants, in flowering, are collected along roadside from Kas area, District Satara. A herbarium is deposited in Botanical Survey of India, Western Circle, Pune (No. SPJll). Roots are separated and aerial parts are cleaned off adhering dust and unwanted plant material. Aerial parts are dried in shade, cut and pulverized.

Corosolic acid is a pentacyclic triterpene acid, also found in Lagerstroemia speciosa, is used in dietary supplements as a glucose-lowering agent and insulin mimetic. It is similar in structure to ursolic acid, differing only in the fact that it has a 2-a-hydroxy attachment.

Corosolic acid and flavonoid 5-hydroxy-6,7,3',4'-tetramethoxyflavone according to the present invention are isolated from acetone extract of aerial parts of Plectranthus mollis.

Corosolic acid 5-Hydroxy-6,7,3',4'-tetramethoxyflavone

Compound 1 Compound 2

In an embodiment of the present invention, use of compound of formula 1 and formula 2 is for the treatment of Mycobacterial tubercular infections In an embodiment of the present invention, pharmaceutical acceptable excipients are selected from the group consisting of diluents, binders, lubricants, wetting agents and disintegrant.

In another embodiment of the present invention, a process for the isolation of compound of formula 1 and compound of formula 2 from Plectranthus mollis comprising the steps of; a) extracting dried, pulverized aerial parts of P. mollis with acetone at temperature in the range of 25 to 30°C;

b) filtering and concentrating the acetone soluble as obtained in step (a) under reduced pressure in the range of 50 to 100 mm of Hg to provide a greenish acetone extract;

c) separating the acetone extract as obtained in step (b) by column chromatography (CC) using acetone: petroleum ether gradient to obtain 20 fractions (PM1-PM20);

d) Subjecting Fraction PM16 as obtained in step (c) to CC in 5% methanol: chloroform to collect six fractions (PM16a-PM16f);

e) Subjecting Fraction PM17 as obtained in step (c) to CC in 5% methanol: chloroform to collect seven fractions (P 17a-PM17g);

f) combining Fractions PM16a as obtained in step (d) and PM17a as obtained in step (e) followed by washing successively with chloroform and acetone to obtain compound of formula 1;

g) subjecting Fraction PM 18 as obtained in step (c) to CC in gradient of methanol in chloroform from 1 to 5% to collect seven fractions (PM 18a-PM18g); combining Fractions PM18a and PM18b as obtained in step (g) followed by washing with acetone to obtain compound of formula 2.

In yet another embodiment, present invention provides a method of treating a subject suffering from mycobacterial tubercular infections comprising administering at least one compound of formula 1 or formula 2 with at least one pharmaceutical acceptable excipient.

In yet another embodiment, present invention provides Use of compound of formula 1 and 2 for the treatment of M. Tuberculosis. In yet another embodiment of the present invention, said compound exhibit IC₅₀ value in the range of 4.37 to 4.93 μ§/ΐΎΐΙ.

In one embodiment, the present invention provides use of compound of formula 1 and 2 for the treatment of infection caused by M. Tuberculosis.

In one embodiment, the compound of formula 1 is corosolic acid and compound of formula 2 is 5-Hydroxy-6,7,3',4'-tetramethoxyflavone.

In one embodiment, the compounds of present invention are from Plectranthus mollis and having anti mycobacterial tubercular property.

The present invention provides the process for isolation of Corosolic acid and 5-hydroxy- 6,7,3', 4'-tetramethoxyflavone from Plectranthus mollis includes the following steps:

a) extracting dried, pulverized aerial parts of P. mollis with acetone (3x3 Lx 14 h) at room temperature in the range of 25 to 30°C;

b) filtering and concentrating the acetone solubles under reduced pressure in the range of 50 to 100 mm of Hg to provide a greenish acetone extract;

c) separating the acetone extract of step (b) by column chromatography (CC) using acetone: petroleum ether gradient to give 20 fractions (PM 1-PM20); d) Subjecting Fraction PM16 to CC in 5% methanol: chloroform to collect six fractions (PM16a-PM16f).

e) Subjecting Fraction PM17 to CC in 5% methanol: chloroform to collect seven fractions (PM17a-PM17g).

f) from combined Fractions PM16a and PM17a, corosolic acid (35mg) was obtained as white precipitate. It was purified by washing the precipitate successively with chloroform and acetone.

g) Subjecting Fraction PM18 to CC in gradient of methanol in chloroform from 1 to 5% to collect seven fractions (PM18a-PM18g).

h) from combined Fractions PM18a and PM18b, 5-Hydroxy-6,7,3',4'- tetramethoxyflavone was obtained as yellow precipitate (40mg) which was purified further by washing with acetone. According to the process described, roots are separated and aerial parts are cleaned off adhering dust and unwanted plant material. Aerial parts are dried in shade, cut and pulverized. Pulverized aerial parts of P. mollis (2.0 kg) are extracted with acetone (3 ^χ 3 L ^χ 14h) at room temperature. The acetone solubles are filtered and concentrated under reduced pressure to provide a greenish acetone extract (50. Og). Acetone extract, 48g, is separated by column chromatography (CC) with acetone: petroleum ether gradient to give 20 fractions (PM 1-PM20). Fractions PM 16 and PM 17 have compound 1. Fraction PM 16 was subjected to CC in 5% methanol: chloroform to collect six fractions (PM 16a-PM 16f). Fraction PM 17 was subjected to CC in 5% methanol: chloroform to collect seven fractions (PM 17a- PM 17g). From combined fractions PM 16a and PM 17a, compound 1 (35mg) was obtained as white precipitate. It was purified by washing the precipitate successively with chloroform and acetone. Fraction PM 18 was subjected to CC in chloroform with gradient of methanol from 1-5% to collect seven fractions (PM 18a-PM 18g). From fractions PM 18a and PM 18b, compound 2 was obtained as yellow precipitate (40mg) which was purified further by washing with acetone

Characterization of Compound 1 - Corosolic acid

N MR(Pyridine-d₅) δ= 0.97 (3H, d, J = 5.8 Hz, H-29), 0.99(3H, s, H- 26), 1.01 (3H, d, 6.6 Hz, H- 30), 1.06 (3H, s, H-25), 1.09 (3H, s, H-24), 1.23 (3H, s, H-27), 1.29 (3H, s, H-23), 2.64 (1H, d, 11.1 Hz, H-18), 3.42 (1H, d, 9.16 Hz, H-3), 4.12 (1H, ddd, 10.98, 9.3,4.27 Hz, H-2),5.48 (1H, t, 3.36Hz, H-12); for ¹³C NMR (Pyridine-cf₅) δ= 17.1 (C-24), 17.6(C-25), 17.6(C-29), 17.8(C-26), 18.9(C-6), 21.5(C-30), 23.8(C-16), 24.0 (C-27), 25.0(C-11), 28.7(C-15), 29.5(C-23), 31.2(C-21), 33.6(C-7), 37.5 (C-22), 38.5 (C-10), 39.5(C-19), 39.6 (C-20), 40.0(C-4), 40.1 (C-8), 42.6 (C-14), 48.1 (C-l), 48.1 (C-17), 48.2 (C-9), 53.6 (C-18), 56.0 (C-5), 68.7 (C-2), 83.9 (C-3), 125.7 (C-12), 139.4 (C-13), 180.0 (C-28).

Characterization of Compound 2:- 5-Hydroxy-6,7,3',4' -tetramethoxyflavone

Compound 2 was identified as 5-Hydroxy-6,7,3',4'-tetramethoxyflavone by detailed study of ^XH and ¹³ C NMR spectroscopy. Table 1: ^XH and ¹³C NMR data for Compound 2 (in CDCI₃)

The present invention provides a pharmaceutical composition comprising an extract of Plectranthus mollis consisting of atleast one compound selected from corosolic acid and 5- hydroxy-6_;7,3',4'-tetramethoxyflavone together with at least one pharmaceutical acceptable excipient, useful in the treatment of mycobacterial tubercular infections.

The pharmaceutical acceptable excipients are selected from diluents, binders, lubricants, wetting agents and disintegrant etc. These pharmaceutical excipients are conventional and known to the person skilled in the art. The active ingredients and excipients can be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical composition of the present invention may be any pharmaceutical form such as solids in the form of tablets, capsules, a liquid suspension or an injectable composition.

The composition may be administered as sustained, controlled, modified or in immediate dosage form.

The quantity of the compound used in pharmaceutical composition of the present invention will vary depending upon the body weight of the patient and the mode of administration and can be of any effective amount to achieve the desired therapeutic effect.

The present invention provides a method of treating a subject suffering from mycobacterial tubercular infections, comprising administering a pharmaceutical composition comprising an extract of Plectranthus mollis consisting of atleast one compound selected from corosolic acid and 5-hydroxy-6,7,3',4'-tetramethoxyflavone together with atleast one pharmaceutical acceptable excipient.

The present invention provides the use of a pharmaceutical composition comprising an extract of Plectranthus mollis consisting of atleast one compound selected from corosolic acid and 5-hydroxy-6,7,3',4'-tetramethoxyflavone together with atleast one pharmaceutical acceptable excipient, for the treatment of mycobacterial tubercular infections.

EXAMPLES

The following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1

Roots are separated and aerial parts are cleaned off adhering dust and unwanted plant material. Aerial parts are dried in shade, cut and pulverized. Pulverized aerial parts of P. mollis (2.0 kg) are extracted with acetone (3 ^χ 3 L ^χ 14h) at room temperature. The acetone solubles are filtered and concentrated under reduced pressure to provide a greenish acetone extract (50. Og). Acetone extract, 48g, is separated by column chromatography (CC) with acetone: petroleum ether gradient to give 20 fractions (PM 1-PM20). Fractions PM16 and PM 17 have compound 1. Fraction PM16 was subjected to CC in 5% methanol: chloroform to collect six fractions (PM 16a-PM 16f). Fraction PM 17 was subjected to CC in 5% methanol: chloroform to collect seven fractions (PM 17a-PM 17g). From combined fractions PM 16a and PM 17a, compound 1 (35mg; 0.00175% based on dry plant) was obtained as white precipitate. It was purified by washing the precipitate successively with chloroform and acetone. Fraction PM18 was subjected to CC in chloroform with gradient of methanol from 1-5% to collect seven fractions (PM 18a-PM 18g). From fractions PM 18a and PM 18b, compound 2 was obtained as yellow precipitate (40mg; 0.002% based on dry plant) which was purified further by washing with acetone.

Example 2

The compounds obtained after extraction in accordance with Figure 3 were assayed for their anti-mycobacterial property and the results of some compounds are shown in Table 2 be'ow:

Table 2: Anti-tubercular activities of compounds against M. Tuberculosis. These activities has been carried out by known and conventional procedure in the art.

Example 3

Composition

Corosolic acid 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and fill in pouch or bottle.

Mode of administration

Disperse the powder in water/juice.

Example 4

Composition

Corosolic acid 20.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and fill in pouch or bottle.

Mode of administration

Disperse the powder in water/juice.

Example 5

Composition

Corosolic acid 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and may be filled in a capsule of suitable size.

Mode of administration: The capsule may be had with water of juice Example 6

Composition

Corosolic acid 20.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration: The capsule may be had with water of juice

Example 7

Composition

Corosolic acid 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavour 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100 %w/w

Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and may be compressed as tablet.

Mode of administration

Mode of administration: The tablet may be had with water of juice

Example 8

Composition

Corosolic acid 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w Mannitol q.s. to 100.0%w/w

Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adso color and flavor on mannitol. Mix active ingredient and Magnesium stearate and may be compressed as tablet.

Mode of administration: The tablet may be had with water of juice

Example 9

Composition

Corosolic acid 5.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration

Disperse the powder in water/juice.

Example 10

Composition

5-Hydroxy-6,7,3',4'-tetramethoxyflavone 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration

Disperse the powder in water/juice. Example 11

Composition

5-Hydroxy-6,7,3',4'-tetramethoxyflavone 20.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration

Disperse the powder in water/juice.

Example 12

Composition

5-Hydroxy-6,7_;3',4'-tetramethoxyflavone 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration: The capsule may be had with water of juice

Example 13

Composition

5-Hydroxy-6,7,3',4'-tetramethoxyflavone 20.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and may be filled in a capsule of suitable size.

Mode of administration: The capsule may be had with water of juice

Example 14

Composition

5-Hydroxy-6,7,3',4'-tetramethoxyflavone 10.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavour 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration

Mode of administration: The tablet may be had with water of juice

Example 15

Composition

5-Hydroxy-6,7,3',4'-tetramethoxyflavone 210.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration: The tablet may be had with water of juice Example 16

Composition

5-Hydroxy-6,7,3', 4' -tetramethoxyflavone 5.0% w/w

FDA approved colour 0.3% w/w

FDA approved flavor 0.7% w/w

Magnesium stearate 2.0% w/w

Mannitol q.s. to 100.0%w/w

Mode of administration

Disperse the powder in water/juice.

ADVANTAGES OF THE INVENTION

After assaying the compounds viz. corosolic acid and 5-hydroxy-6,7,3',4'- tetramethoxyflavone of the present invention, these compounds are found to be effective against Mycobacterium tuberculosis due to their anti-tubercular property.

Claims

im

A pharmaceutical composition comprising at least one compound of formula 1 o formula 2 together with atleast one pharmaceutical acceptable excipient for th« treatment of M. Tuberculosis.

Formula 1 Formula 2

2. The composition as claimed in claim 1, wherein the compound of formula 1 or formula 2 are present in range of 0.1 to 99 % w/w of total composition.

3. The composition as claimed in claim 1, wherein pharmaceutical acceptable excipients are selected from the group consisting of diluents, binders, lubricants, wetting agents and disintegrant.

4. A process for the isolation of compound of formula 1 and compound of formula 2 from Plectranthus mollis comprising the steps of; a) extracting dried, pulverized aerial parts of P. mollis with acetone at temperature in the range of 25 to 30°C;

e) Subjecting Fraction PM17 as obtained in step (c) to CC in 5% methanol: chloroform to collect seven fractions (PM17a-PM17g); f) combining Fractions PM16a as obtained in step (d) and PM17a as obtained in step (e) followed by washing successively with chloroform and acetone to obtain compound of formula 1;

g) subjecting Fraction PM18 as obtained in step (c) to CC in gradient of methanol in chloroform from 1 to 5% to collect seven fractions (PM18a-PM18g);

combining Fractions PM18a and PM18b as obtained in step (g) followed by washing with acetone to obtain compound of formula 2.

A method of treating a subject suffering from mycobacterial tubercular infections comprising administering at least one compound of formula 1 or formula 2 with atleast one pharmaceutical acceptable excipient.

Use of compound of formula 1 and 2 for the treatment of M. Tuberculosis.

Formula 1 Formula 2

7. Use of compound as claimed in claim 6, wherein said compound exhibit IC₅₀ value in the range of 4.37 to 4.93 μg/ml.

8. Use of compound as claimed in Claim 6, wherein compound of formula 1 is corosolic acid.

9. Use of Compound as claimed in Claim 6, wherein compound of formula 2 is 5- Hydroxy-6,7,3',4'-tetramethoxyflavone.

10. Use of compounds as claimed in any one of Claims 6-9, wherein the compounds are from Plectranthus mollis and having anti mycobacterial tubercular property.