WO2011083397A1

WO2011083397A1 - Herbal composition for skin disorders

Info

Publication number: WO2011083397A1
Application number: PCT/IB2011/000007
Authority: WO
Inventors: Rangesh Paramesh; Uddagiri Venkanna Babu; Ekta Saxena
Original assignee: Himalaya Global Holdings Ltd
Current assignee: Himalaya Global Holdings Ltd
Priority date: 2010-01-05
Filing date: 2011-01-05
Publication date: 2011-07-14
Anticipated expiration: 2012-07-05

Abstract

Disclosed herein is blend of extract of Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata and pharmaceutically acceptable carrier. Further, the invention provides methods of obtaining the extracts, composition thereof and methods of using said composition.

Description

HERBAL COMPOSITION FOR SKIN DISORDERS

Field of the Invention

This invention, in general relates to a herbal composition for skin disorders. In particular, the present invention provides a herbal composition comprising blend of extract of Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata and pharmaceutically acceptable carrier, methods of obtaining the same, pharmaceutical formulations thereof and methods of using said composition.

Background of the Invention

Dermatitis is a broad term used for any inflammation of the skin, which results in scaling, thickening, flakes, itching, blistering, and change of skin colour. Various skin diseases such as chronic lichen planus, psoriasis, chronic lichen simplex, chronic eczema and various allergies lead to dermatitis. Generally, a lot of toxins are accumulated in the body through various foods, pollution and drugs that are consumed by human. Due to this accumulation of toxins, the liver gets damaged which results in various dermatological diseases. A large number of medications are available in the market for the treatment of skin diseases. Use of complementary therapies in the management of healthy skin have gained acceptance due to their effectiveness and safety and much work is being carried out in this direction.

It is therefore a very important and challenging task to develop a safe and effective herbal medication that can be useful in the treatment of dermatitis due to different skin diseases and also as detoxifier and safe for prolonged use in humans.

Related Art

Patent Publication No. US20060263449A1 by Hsu et. al. discloses a herbal composition comprises the extract of Andrographis paniculata Nees. The patent further provides a method for treating arthritic disorders, skin inflammatory disorders and pain, comprising administering to a subject an extract of Andrographis paniculata Nees. Further, it discloses that the skin inflammatory disorders includes psoriasis, eczema and dermatitis. The formulation is in the form of powder, capsule, tablet, liquid or syrup.

Patent Publication No. WO2006122160A2 by Jia and Hong discloses a composition of bakuchiol for prevention and treatment of diseases and conditions of the skin, mouth, teeth or gums. The patent also provides the methods of making the same. The bakuchiol is isolated from a plant selected from Psoralea corylfolia L. or Psoralea glandulosa L.

US Patent No. 7615239B2 by Santo et. al. discloses a drinkable tea composition for therapy of dermatitis by improvement of physical constitution itself of a patient. The tea composition contains extracts drawn from one, two or more medicinal herbs selected from the group consisting of Lightyellow Sophora Root, Isatis Leaf, and Terminalia Fruit. The tea composition is in the form of liquid, powder or granule.

US Patent No. by Nguyen discloses the treatment of medical disorders and ailments comprising the administration of an effective amount of turmeric. The turmeric is especially useful for the treatment of skin disorders, such as acne, when administered orally. It can also be used to treat liver and stomach disorders, skin discoloration, constipation, and hemorrhoids.

Summary of the Invention

In accordance with one preferred embodiment of the present invention, there is provided an herbal composition for skin disorders comprising blend of extract of Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata and pharmaceutically acceptable carrier.

In accordance with one other preferred embodiment of the present invention, there is provided a herbal composition for skin disorders comprising blend of extract of blend of extract of Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata and pharmaceutically acceptable carrier, wherein said herbs are in a ratio of 25:11 :23:14:27.

In accordance with one other preferred embodiment of the present invention, there is provided a herbal for skin disorders comprising blend of extract of seeds of Psoralea corylifolia, rhizomes of Curcuma longa, whole parts of Sphaeranthus indicus, leaves of Azadirachta indica and aerial parts of Andrographis paniculata.

Brief Description of the Drawings

Further objects of the present invention together with additional features contributing thereto and advantages accruing there from will be apparent from the following description of preferred embodiments of the invention which are shown in the accompanying drawing figures, wherein: Figure 1 illustrates the effect of various ER PPU extracts on ear inflammation in rats-induced by topical application of croton oil

Detailed Description of the Invention

Further objects of the present invention together with additional features contributing thereto and advantages accruing there from will be apparent from the following description of preferred embodiments of the invention which are shown in the accompanying drawing figures.

The present invention involves the selection and identification of the herbs and obtaining the extract by subjecting the same to different extraction techniques including conventional solvent extraction and super critical fluid extraction. The bioassay guided fractionation of the extract or combination thereof to identify the active markers or active fraction and to develop effective and safe composition as detoxifier and helps in improvement of liver functions and removal of toxic metabolic products in various systemic and skin infections and useful in renormalization of disturbed physiological metabolic processes.

According to the present invention a herbal composition comprising extracts of Psoralea corylifolia seeds and/or Curcuma longa rhizomes and/or Sphaeranthus indicus whole parts and/or Azadirachta indica leaves and/or Andrographis paniculata aerial parts for treatment of skin allergies, dermatitis and other skin disorders. The composition acts as detoxifier and helps in improvement of liver functions and removal of toxic metabolic products in various systemic and skin infections and useful in renormalization of disturbed physiological metabolic processes in humans using the said composition and pharmaceutical formulations thereof.

According to the present invention the extract of the herb is obtained by employing percolation, hot soxhlation, enzymatic extraction or super critical fluid extraction, wherein the percolation or hot soxhlation is performed in presence of a solvent selected from n-hexane, ethyl alcohol, acetone, methanol, water or mixture thereof and wherein methanol and water are preferably used in a ratio of 1 : 1.

The enzymatic extraction is performed using an enzyme cellulase or pectinase or mixture thereof, wherein the enzymes are used in a range of 0.01% to 10 %.

The composition according to the present invention is in a suitable dosage form, preferably tablet, capsule and syrup. The following non-limiting examples illustrate specific embodiments of the present invention. They are, not intended to be limiting the scope of present invention in any way.

Example 1

Preparation of extract from Psoralea corylifolia by percolation method:

The shade dried material of Psoralea corylifolia seeds was pulverized to coarse powder and about 10 Kg each of powdered material placed in different percolators and extracted with n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1:1), methanol and water (1:1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 2

Preparation of extract from Psoralea corylifolia by hot-soxhlation method:

The coarse powdered material of Psoralea corylifolia seeds was subjected to hot-soxhlation by placing 10 Kg of material in each soxhlator using solvents n- hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (PC-1), acetone extract (PC-2), ethyl alcohol extract (PC-3), methanol extract (PC-4), ethyl alcohol and water (1 :1) extract (PC-5), methanol and water (1 : 1) extract (PC-6) and water extract (PC-7) prepared from the seeds of Psoralea corylifolia by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

Example 3

Preparation of extract from Psoralea corylifolia by Enzyme extraction

The shade dried material of Psoralea corylifolia seeds was pulverized to powder and about 10 Kg each of powdered material placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 4

Preparation of extract from Psoralea corylifolia by Super Critical Fluid Extraction

The shade dried material of Psoralea corylifolia seeds was pulverized to coarse powder and about 100 Kg of powdered material placed in a SCF extractor at the temperature of 40-50°C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract thus obtained was free from any solvent residues and in highest pure form.

Example 5

Preparation of extract from Curcuma longa by percolation method:

The shade dried material of Curcuma longa rhizomes was pulverized to coarse powder and about 10 Kg each of powdered material placed in different percolators and extracted with n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 6

Preparation of extract from Curcuma longa by hot-soxhlation method:

The coarse powdered material of Curcuma longa rhizomes was subjected to hot-soxhlation by placing 10 Kg of material in each soxhlator using solvents n- hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1:1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (CL-1), acetone extract (CL-2), ethyl alcohol extract (CL-3), methanol extract (CL-4), ethyl alcohol and water (1 :1) extract (CL-5), methanol and water (1 :1) extract (CL-6) and water extract (CL-7) prepared from the rhizomes of Curcuma longa by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

Example 7

Preparation of extract from Curcuma longa by Enzyme extraction

The shade dried material of Curcuma longa rhizomes was pulverized to powder and about 10 Kg each of powdered material placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 8

Preparation of extract from Curcuma longa by Super Critical Fluid Extraction

The shade dried material of Curcuma longa rhizomes was pulverized to coarse powder and about 100 Kg of powdered material placed in a SCF extractor at the temperature of 40-50°C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract thus obtained was free from any solvent residues and in highest pure form.

Example 9

Preparation of extract from whole plant of Sphaeranthus indicus by percolation method:

The shade dried material of Sphaeranthus indicus whole plants was pulverized to coarse powder and about 10 Kg each of powdered material placed in different percolators and extracted with n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 : 1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 10 Preparation of extract from whole plant of Sphaeranthus indicus by hot-soxhlation method:

The coarse powdered material of whole plant of Sphaeranthus indicus was subjected to hot-soxhlation by placing 10 Kg of material in each soxhlator using solvents n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (SI-1), acetone extract (SI-2), ethyl alcohol extract (SI-3), methanol extract (SI-4), ethyl alcohol and water (1 :1) extract (SI-5), methanol and water (1 :1) extract (SI-6) and water extract (SI-7) prepared from the whole plant of Sphaeranthus indicus by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

Example 11

Preparation of extract from Sphaeranthus indicus by Enzyme extraction

The dried material of whole plant of Sphaeranthus indicus of was pulverized to powder and about 10 Kg each of powdered material placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

ExampleU

Preparation of extract from Sphaeranthus indicus by Super Critical Fluid Extraction The dried material of Sphaeranthus indicus whole plant was pulverized to coarse powder and about 100 Kg of powdered material placed in a SCF extractor at the temperature of 40-50°C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract thus obtained was free from any solvent residues and in highest pure form.

Example 13

Preparation of extract from Azadirachta indica by percolation method:

The dried material of leaves of Azadirachta indica was pulverized to coarse powder and about 10 Kg each of powdered material placed in different percolators and extracted with n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure .

Example 14

Preparation of extract from leaves of Azadirachta indica by hot-soxhlation method:

The shade dried leaves of Azadirachta indica was subjected to hot-soxhlation by placing 10 Kg of material in each soxhlator using solvents n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1:1), methanol and water (1 :1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (AI-1), acetone extract (AI-2), ethyl alcohol extract (AI-3), methanol extract (AI-4), ethyl alcohol and water (1 :1) extract (AI-5), methanol and water (1 :1) extract (AI-6) and water extract (AI-7) prepared from the leaves of Azadirachta indica by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

Example 15

Preparation of extract from of Azadirachta indica by Enzyme extraction

The dried material of Azadirachta indica leaves was pulverized to powder and about 10 Kg each of powdered material placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 16

Preparation of extract from Azadirachta indica by Super Critical Fluid Extraction The dried material of leaves of Azadirachta indica was pulverized to coarse powder and about 100 Kg of powdered material placed in a SCF extractor at the temperature of 40-50C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract thus obtained was free from any solvent residues and in highest pure form.

Example 17

Preparation of extract from Androeraphis paniculata by percolation method:

The shade dried material of Andrographis paniculata aerial parts was pulverized to coarse powder and about 10 Kg each of powdered material placed in different percolators and extracted with n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1:1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 18

Preparation of extract from Androeraphis paniculata by hot-soxhlation method:

The coarse powdered material of Andrographis paniculata aerial parts was subjected to hot-soxhlation by placing 10 Kg of material in each soxhlator using solvents n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (AP-1), acetone extract (AP-2), ethyl alcohol extract (AP-3), methanol extract (AP-4), ethyl alcohol and water (1 :1) extract (AP-5), methanol and water (1 :1) extract (AP-6) and water extract (AP-7) prepared from Andrographis paniculata aerial parts by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

Example 19

Preparation of extract from Androsraphis paniculata by Enzyme extraction

The shade dried material of Andrographis paniculata aerial parts was pulverized to powder and about 10 Kg each of powdered material placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 20

Preparation of extract from Androsraphis paniculata by Super Critical Fluid Extraction

The shade dried material of Andrographis paniculata aerial parts was pulverized to coarse powder and about 100 Kg of powdered material placed in a SCF extractor at the temperature of 40-50C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract thus obtained was free from any solvent residues and in highest pure form.

Example 21

Preparation of extract from Herbal Blend by percolation method:

The shade dried material of herbal blend of Psoralea corylifolia seeds and/or rhizomes of Curcuma longa and/or whole plants of Sphaeranthus indicus and/or leaves of Azadirachta indica and/or aerial parts of Andrographis paniculata in the ratio of 25: 11 :23:14:27 respectively mixed and pulverized to coarse powder and about 10 Kg each of herbal blend placed in different percolators and extracted with n- hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure.

Example 22

Preparation of extract from Herbal Blend by hot-soxhlation method:

The shade dried material of herbal blend of Psoralea corylifolia seeds and/or rhizomes of Curcuma longa and/or whole plants of Sphaeranthus indicus and/or leaves of Azadirachta indica and/or aerial parts of Andrographis paniculata in the ratio of 25:11 :23:14:27 respectively was subjected to hot-soxhlation by placing 10 Kg of herbal blend in each soxhlator using solvents n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water at refluxing temperature of each solvent and recycled the process until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature.

All extracts such as n-hexane extract (NP34-1, also coded as ERNPPU-1 ), acetone extract (NP34-2, also coded as ERNPPU-3 ), ethyl alcohol extract (NP34-3, also coded as ERNPPU-2 ), methanol extract (NP34-4), ethyl alcohol and water (1 :1) extract (NP34-5), methanol and water (1 :1) extract (NP34-6, also coded as ERNPPU- 4) and water extract (NP34-7, also coded as ERNPPU-5 ) prepared from the herbal blend by percolation method or hot-soxhlation method were subjected to HPTLC (High Performance Thin Layer Chromatography) and HPLC (High performance Liquid chromatography) in various mobile phases on precoated TLC plates (Merck) and ODS column for qualitative and quantitative estimation of marker compounds and active principles.

The shade dried material of herbal blend of Psoralea corylifolia seeds and/or rhizomes of Curcuma longa and/or whole plants of Sphaeranthus indicus and/or leaves of Azadirachta indica and/or aerial parts of Andrographis paniculata in the ratio of 25:11 :23:14:27 respectively was subjected to pulverization to a coarse powder. About 250 Kg of herbal blend powder was subjected to water extraction by hot soxhalation method. The subsequent extractions were combined and concentrated to soft extract of 30-35% total solids. The soft extract thus obtained was subjected to spray drying to get spray dried powder.

Example 23

Preparation of extract from herbal blend by Enzyme extraction The shade dried material of herbal blend of Psoralea corylifolia seeds and/or rhizomes of Curcuma longa and/or whole plants of Sphaeranthus indicus and/or leaves of Azadirachta indica and/or aerial parts of Andrographis paniculata in the ratio of 25:11 :23:14:27 respectively was pulverized to powder and about 10 Kg each of powdered herbal blend was placed in Stainless Steel container and 0.5% to 5% of cellulase and pectinase enzyme combination was added in 4 volumes of distilled water. The enzyme extraction was processed at the temperature of 55°C to 60°C under occasional stirring upto 4-6 hours, and then plant extracts were filtered and concentrated to dryness (coded as ERNPPU-6) on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. The extractive yields of enzyme extraction from each herbal material and their combined extract are given in table- 1.

Table- 1

Example 24

Preparation of extract from herbal blend by Super Critical Fluid Extraction

The shade dried material of herbal blend of Psoralea corylifolia seeds and/or rhizomes of Curcuma longa and/or whole plants of Sphaeranthus indicus and/or leaves of Azadirachta indica and/or Andrographis paniculata aerial parts in the ratio of 25:11:23:14:27 respectively was pulverized to coarse powder and about 100 Kg of powdered herbal blend was placed in a SCF extractor at the temperature of 40-50C at high pressure of 300-350 bar using carbon dioxide as super critical fluid for extraction upto 4 to 6 hours and then the extract was collected in the collection vessel and evaporated at room temperature to remove any further residues of carbon dioxide. The extract (coded as ERNPPU-7) thus obtained was free from any solvent residues and in highest pure form.

Example 25

Yields of various ERNPPU extracts are mentioned in Table -2

Table -2

The various extracts were standarised by in house methods. The chemical constituents are mentioned in Table-3

Table-3

Example 26

The various extracts were also standarised by LCMSMS method: LC-MS/MS conditions for NP-034 series extract

LC-MS/MS optimization conditions:

The LC-MS/MS analysis performed with Shimadzu LC-20 AD Prominence series HPLC system. LC separation was performed on a Reverse phase CI 8 (250 X4.6mm, 5um) Thermo ODS HYPERSIL column. The mobile phase consisted of 0.1% formic acid in water and acetonitrile in a ratio of 1:1 (v/v), delivered at a flow rate of 0.750 ml/min. The autosampler SIL-HTC (Shimadzu maker) injection volume was set at 20μ1. UV detector chromatograms were monitor at 420nm and 371 nm.

The API-2000 (Applied Biosystems, Canada) tandem triple quadrupole mass spectrometer equipped with an APCI source coupled with LC separation system in positive mode was used to ionize the molecules. Analyst 1.5 version was used for the control of equipment, acquisition and data analysis. APCI source conditions as follows: declustering potential (DP) 20 V, source gas (GS1) 70 psi, curtain gas (CUR) 25psi, Nebulizer current (NC) 2 V, focusing potential (FP) 400 V and source temperature (TEM) 440 C were optimized with respect to ionization intensity response of the peak.

Sample solution preparation: Individual sample solutions were prepared in the concentration of lmg/ml by weighing accurately 10 mg of individual samples in a separate 10 ml clean volumetric flask. The aqueous extracts were dissolved in water and remaining solvent extracts were dissolved in methanol by sonication for 10 min in an ultrasonic bath and finally made up to the volume respectively. The final sample solutions were filtered through 0.2μπι syringe filter before loading to the autosampler.

The chemical constituents were detected and identified by LCMSMS were curcuminoids, turmerone, turmerin, arturmirone, zingiberene, beta- sesquiphellandrene, psoralen, andrographolide, andrographin, , azadiractin, azadirone, nimbolidine,azadirol, nimbidin , nimbisidine , para-methoxy cinnamaldehyde , phenylurethan , corylifolin, bakuchicin , bavacoumestans etc.

List of chemical constituents of combined extract by LC-MS/MS

Curcuma longa Curcumin 368.37 cineole 154.24 borneol 154.25 zingiberene 204.35 turmerone 218.33 sabinone 150.21 p-cymene 134.21

Trans- β-farnesone 204.35 β-bisabolene 222.36 β-sesquiphellandrene 204.35 tarmerone 218.33

Psoralea corylifolia Psoralen (Ficusin) 186.16

Isopsoralen (Angellicin) 186.16

Bakuchiol 256.38

Bavachinin 338.39

Bavachalcone 324.37

Bavachin 324.37 ·

Isobavachin 324.37 isobavachalcone 324.37

Andrographis 14-deoxy-l 1,12- 332.0 paniculata dihydroandrographolide

Neoandrographolide 480.0

14-deoxyandrographolide 496.0

Panicolin 314.0

Homo-androgdrapholide 344.0

14-deoxy-l 2- 350.0 hydroxyandrographolide

14-deoxy-l l-oxoandrographolide 348.0

14-deoxyandrographolide 334.0

3, 14-dihydroxyandrographolide 318.0 Andrographiside 512.0

Andrographin 328.0

Andrographane 562.0

4 Azadirachta indica Azadirachtin 720.71

Salannin 596.70

Nimbin 540.6

Azadirone 436.58

Azadiradione 450.56

Nimbolin A 642.77

Nimbolin B 674.77

Nimbolide 466.52

Meliantriol 490.71

Nimbalidin B 698.79

Meldenin 440.57

Nimbolidin C 686.78

Nimbolidin D 738.86

5. Sphaeranthus indicus a-ionone 192.29 d-codinene 204.35 p-methoxy cinnamaldehyde 162.18 β-sitosterol 414.7 n-triacontanol 438.81

Phenylurethan 165.18 n-pentacosane 352.68

Example 27

Screening of various ERNPPU extracts for hepatoprotective activity in vitro

Methods

Cell culture: HepG-2 cells were maintained in DMEM High glucose with 10%

FBS, 100μg/ml of streptomycin and 100 IU/ml pencillin at 370 degree C in a humidified atmosphere of 5% C02. Drug Preparation: The drugs/toxicants were dissolved in DMEM-HG with 2% FBS to attain 2mg /ml. This was further diluted by two-fold dilution method with same medium to attain different concentrations and all the dilutions were prepared freshly.

Hepatoprotective studies

HepG-2 cultures of 24 h with 70-80% confluency in 96 well microtitre plate were used for the study. Cultures, except cell control group were treated with 0.1 ml of 30 mM of CC14 solution for 60 min. Supematants were discarded and washed with fresh medium. Different non toxic doses of each test and standard drugs (0.1 ml to each well) were added to cultures. Cell controls and toxicant controls were

maintained. The cultures were incubated at 370 C with 5% C02 for 24 h and the cultures were observed microscopically to record visible changes in their morphology. Cell viability was determined by performing MTT assay and protective effect of test drugs was evaluated against CC14 treatment. The results were expressed as percentage protection offered by the drugs over CC14 control.

Results: The Hepatoprotective activity results are mentioned in Table-4.

Table- 4

Cytoprotective effect of test drugs in CC14 intoxicated HepG-2 cell cultures

The drugs tested exhibited medium to moderate activity by offering cytoprotection to CC14 treated HepG2 cells with percentage protection ranging from 30.09 to 34.71. Standard drug, Silymarin protected the cells with 54.47% protection over toxicant control.

Example 28

Anti bacterial activity of ERNPPU samples against test organisms Propionibacterium acne:

To evaluate the inhibitory effect of test drugs against Propionibacterium acne by Agar well diffusion technique.

Microorganisms and media:

Propionibacterium acne (MTCC 11827) was procured from MTCC, IMTECH, Chandigarh and subcultured regularly as per the suppliers instructions. Media and reagents were procured form Himedia labs, Mumbai.

Procedure:

1) Drug stock preparation:

Drugs were weighed and solubilized initially in ΙΟΟμΙ DMSO and thereafter with broth to make a stock concentration of 5mg/ml. further dilutions were prepared using broth as diluents.

2) Inoculum preparation:

To prepare the stock inoculum Propionibacterium acne was inoculated into 5 ml of broth and incubated at 37°C for 5-7 days under anaerobic conditions to obtain cell population approximately 7x10⁹Cfu/ml

3) Preparation of standard antibiotic:

Stock solution was prepared by dissolving 5mg of Tetracyclene in ΙΟΟμΙ 0.02N HCL and thereafter with broth to make a stock concentration of lmg/ml. further dilutions were prepared using broth as diluents.

Procedure:

1. Reinforced Clostridial agar plates/Sheep blood agar plates were taken and test inoculum of approximately 7xl0⁹ Cfu/ml cell population was spreaded on the agar surface using sterile swabs.

2. Wells were bored on to the agar plates using sterile well borer of diameter 10mm.

3. 100 μΐ of drugs from the stock of 5mg/ml were loaded to each well.

4. Negative control, Solvent control and positive control plates were maintained.

5. Incubated the plates at 37°C for 5-7 days at anaerobic conditions. 6. After incubation the zone of inhibition was measured in mm and results were recorded.

Assay was carried out by agar well diffusion method. The test organisms were Propionibacterium acne (MTCC -11827). The media used was sheep blood agar plates (SBA) and Reinforced clostridial agar (RCA). The incubation was carried out in anaerobic conditions at 37 degree C observed the visual growth and MIC was calculated. Results are mentioned in Table-5

Table 5

Agar well diffusion method using Sheep blood agar plates (SBA)

Table-6

Agar well diffusion method using Reinforced clostridial agar plates (RCA)

Note: As haemolysis was observed on Sheep blood agar plates, Reinforced clostridial agar plate was taken for the assay. s Name of the Drug Concentrations MIC value

No tested ( Zone of inhibition in mm)

1 ERNPPU-1 5mg/ml 36mm

2 ERNPPU-2 5mg/ml 26mm

3 ERNPPU-3 5mg/ml 26mm

4 ERNPPU-4 5mg/ml No zone observed

5 ERNPPU-5 5mg/ml 15mm

6 ERNPPU-6 5mg/ml 10mm

7 ERNPPU-7 5mg/ml 31mm

8 Standard drug -Tetracycline lOug/ml 23mm

9 Vehicle control DMSO No zone observed

10 Media blank

Inference:

In step 1: Agar well diffusion method using Sheep blood agar plates (SBA) ERNPPU-1, ERNPPU-2, ERNPPU-3, and ERNPPU-7 showed inhibition against the test organism in comparison with the standard drug tetracycline (15ug/ml).

In step 2: Agar well diffusion method using Reinforced clostridial agar plates (RCA) ERNPPU-1, ERNPPU-2, ERNPPU-3, and ERNPPU-7 showed inhibition against the test organism in comparison with the standard drug tetracycline (lOug/ml).

Example 29

Evaluation of ERNPPU variants for anti-inflammatory activity using experimental model of croton oil -induced ear edema in rats.

Objective: The objective of the present study was to evaluate the antiinflammatory activity of ERNPPU variants against cutaneous inflammation induced by croton oil in rats.

Procedure: Laboratory bred Wistar rats of either sex weighing between 120- 150 gms, were used for the experiment. Eighty males were randomized into 8 groups, each comprising of 5 animals of each sex. The group classification were as follows; Group I animals served as control and were administered with water lOml/kg, group II to group VIII animals were treated with ERNPPU-1 to ERNNPU-7 respectively at a dose of 500mg/kg b.wt per day for 3 consecutive days. After 2 days of assigned treatment, animals were kept for fasting and deprived of water overnight before starting of the test. On Day-3, initial ear thickness was recorded for all the animals with digital Vernier Calliper (0.01 mm accuracy). After initial readings, all the animals were administered with respective extract 60 min's prior to induction of cutaneous inflammation using croton oil. The topical inflammation /ear oedema was induced by applying 20 μΐ of croton oil preparation on both sides (inner and outer surface) of left ear. The animals were observed for erythema and edema of the ear and thickness of same were measured every hour for four hour.

Statistical analysis:

The oedema / ear thickness value observed was expressed as Mean ± SEM . The results were analyzed statistically using one-way ANOVA followed by post Dunnet's multiple comparison tests using Prism software package to find out the level of significance. The minimum level of significance was fixed at p<0.05

Results: The results are presented in figure- 1

Conclusion: Topical application of croton oil promoted a significant increase in the thickness of the ear, indicating edema and inflammation. Pre-tretament with ERNNPU extracts orally, at a dose of 500mg/kg body weight, reduced the ear edema in rats but to a varying extent. It was found that ERNNPU- 1 , ERNNPU-2, ERNNPU- 3, ERNNPU-6 and ERNNPU-7 extarcts reduced the ear inflammation to a statistically significant extent at 60 mins post croton oil challenge. However, the antiinflammatory effect of ERNNPU- 1, ERNNPU-2 and ERNNPU-7 was found to be persistent even at 120 mins post croton oil challenge.

Example 30

Manufacturing of NP-34 tablets

Direct compression with excipients

PROCEDURE: Active and Excipients were sieved through a 36 mesh sieve, resulting blend was then compressed into tablets using suitable capsule shaped tooling to give capsule shaped tablets. The composition of the tablets was as follows Table-7

Table-7 Formula Formula Formula Formula

S.No Ingredients

l(in mg) 2(in mg) 3 (in mg) 4 (in mg)

1 NP-34 Extract 450 450 450 450

Microcrystalline cellulose

2 250 375 170 220

102

3 Maize starch 170 45 - -

4 Croscarmellose sodium 35 - 25 -

5 Magnesium stearate 5 - 5 -

6 Sodium starch glycolate - 25 - -

7 Lactose DC - - 250 -

8 Colloidal Silicon Dioxide - 5 - 5

Dicalcium phosphate

9 - - - 200 Dihydrate

10 PVP K 30 - - - 25

Total 900 900 900 900

Result: Direct compression tablets report Table-8

Table-8

Example 31

Manufacturing of NP34 tablets/capsules by wet granulation by using Purified Water as a granulating fluid

PROCEDURE: Active and Excipients were sieved through a 36 mesh sieve into a suitable mixer and granulated with a suitable quantity of boiled cooled purified water to form a medium /heavy granule. The granules were dried in a suitable oven at 60°C, until the LOD was <2%. BLENDING: The resulting dried granule was then passed through a 16 mesh sieve to give a granule and then blended with excipients. The resulting blend was then compressed into tablets using suitable capsule shaped tooling to give capsule shaped tablet-9, the composition of the tablets was as follows Table-9

Table-9

RESULT: Wet Granulation by using excipients and Purified water as a granulating fluid was used to prepare tablets and their evaluation is mentioned Table- 10 Table-10

Example 32

Capsule filling

By using Purified Water as a granulating fluid.

PROCEDURE: Active and Excipients were sieved through a 36 mesh sieve into a suitable mixer and granulated with a suitable quantity of boiled cooled purified water to form a medium /heavy granule. The granules were dried in a suitable oven at 60°C, until the LOD was <2%.

BLENDING: The resulting dried granule was then passed through a 16 mesh sieve to give a granule and then blended with excipients. The resulting blend was then filled in suitable size capsules; the composition of the capsule was as follows Table - 11 Table-ll

RESULT: Granules were filled in size '00' capsules Table-12 mentions the properties of the final capsules.

Table-12

Manufacturing formula for Syrup (Table- 13)

Table- 13

Procedure for manufacturing Syrup

Step-1: Heated water to 85 degree C, added parabens, dissolved completely, followed by active herbal extracts, cooled to 40 degree C.

Step-2: Dissolved separately sodium citrate, citric acid, disodium EDTA ans sodium saccharine in water separately and added to step-1

Step-3: Dispersed xanthan gum in propylene glycol and added to step -1 , mixed. Step-4; added sorbitol and flavours, make up the volume. Many modifications and variations of this invention can be made without departing from scope of this invention, as will be apparent to those skilled in the art. Invention is to be limited only by the terms of the appended claims, along with the full scope of the equivalents to which claims are entitled.

References

1. Su SL, Wu QN, Ouyang Z, Wu DK, Chen J. Zhongguo Zhong Yao Za Zhi. Study on the SFE condition for curcumin in Curcuma longa. 29(9):857-60, 2004.

2. A.C. Kumoro and Masitah Hasan. Supercritical Carbon Dioxide

Extraction of Andrographolide from Andrographis paniculata: Effect of the Solvent Flow Rate, Pressure, and Temperature. Chin. J. Chem. Eng., 15(6) 877— 883, 2007.

3. R Ajaya Kumar, K Sridevi, N Vijaya Kumar, S Nanduri and S Rajagopal. Anticancer and immunostimulatory compounds from Andrographis paniculata. Journal of Ethnopharmacology, 92(2-3), 291-295, 2004.

4. Andrographolide and kalmegh {Andrographis paniculata) extract: effect on rat liver and serum transaminases. IRCS Med. Sci. 1984; 12(6): 466; Chem. Abstr. 1984, 101 :104063t.

5. Poonam Kulyal, UK Tiwari, A Shukla and AK Gaur. Chemical constituents isolated from Andrographis paniculata. Indian Journal of Chemistry

2010; Vol. 49B, PP. 356-359.

6. Handa SS, Sharma A and Chakarborti KK. Natural Products and plants as liver protecting drugs. Fitoterapia 1986; 57: 307.

7. Kapil A, Koul I B, Banerjee SK and Gupta BD. Biochemical Pharmacology 1993 ; 46: 182.

8. Neha Trivedi and UM Rawal. Hepatoprotective and toxicological evaluation of Andrographis paniculata on severe liver damage. Indian J. of Pharmacology 2000, 32, 288-293. Indian J. Exp. Biol. 1978; 6: 830.

9. Olalde Rangel; Jose Angel. Hepato Phyto-Nutraceutical Synergistic Composition United States Patent Application, 20080160042, Kind Code Al July 3,

2008.

10. C. Balasundaram and R. Harikrishnan. P26: Immunomodulatory effect of Andrographis paniculata on Asian Catfish, Clarias batrachus against Aeromonas hydrophila Experimental and Toxicologic Pathology, 61(3), 293-294, 2009.

11. Naik, Suresh R. and Amolkumar Hule. Evaluation of immunomodulatory activity of an extract of andrographolides from Andrographis paniculata. Planta Medica 75(8), 785—791, 2009. 12. Olalde Rangel; Jose Angel. Synergistic Inmune Phyto-Nutraceutical Composition United States Patent Application 20080118582, Kind Code Al May 22, 2008

13. Holt; Stephen. Herbal compositions, methods of stimulating immunomodulation and enhancement of immunomodulating agents using the herbal compositions. United States Patent Application, 20070196381, Kind Code Al August 23, 2007.

14. Li-Hsun Chang, Ting-Ting Jong, Ho-Shin Huang, Yung-Feng Nien, Chieh-Ming J. Chang. Supercritical carbon dioxide extraction of turmeric oil from Curcuma longa Linn and purification of turmerones. Separation and Purification Technology, 47(3), 119-125, 2006.

15. Ge F, Shi Q, Tan X, Li Z, Jing X. Technological study on the supercritical-C02 fluid extraction of Curcuma longa oils. Zhong Yao Cai. 20(7), 345- 50, 1997.

16. Gopalan B, Goto M, Kodama A, Hirose T. Supercritical carbon dioxide extraction of turmeric {Curcuma longa). J Agric Food Chem. 48(6), 2189-92, 2000.

17. Nilesh N. Kurmudle, Sandip B. Bankar, Ishwar B. Bajaj, Mahesh V. Bule, Rekha S. Singhal. Enzyme-assisted three phase partitioning: A novel approach for extraction of turmeric oleoresin. Volume 46, Issue 1, Pages 423-426, 2011.

18. Yue GG, Chan BC, Hon PM, Lee MY, Fung KP, Leung PC, Lau CB. Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa. Food Chem Toxicol. 48(8-9):2011-20, 2010.

19. Managoli; Nandkishor Bapurao. Herbal composition for treatment and maintenance of hormone dependent conditions, osteoporosis, circulatory conditions, and for use as an immunostimulant. United States Patent Application, 20070122497, Kind Code, Al, May 31, 2007.

20. Managoli; Nandkishor Bapurao. Herbal composition for treatment of immunocompromised conditions. United States Patent Application, 20070122496,

Kind Code, Al, May 31, 2007.

21. B. Manohar and K. Udaya Sankar. Enrichment of bakuchiol in supercritical carbon dioxide extracts of chiba seed {Psoralea corylifolia L.) using molecular distillation-Response surface methodology. Biotechnology and Bioprocess Engineering, 14(1), 112-117,

22. Huang F, Huang X, Liang W, Ge F, Wu H. Studies on the supercritical C02 fluid extraction and separation of psoralen, isopsoralen and fatty oils from Psoralea corylifolia. Zhong Yao Cai. 23(5), 266-7, 2000.

23. Wang Yi, Chengtao Hong, Chenguang Zhou, Dongmei Xu, Hai-bin Qu and Yiyu Cheng. Screening antitumor compounds psoralen and isopsoralen from Psoralea coryifolia L. seeds. eCAM 2009.

24. Latha P.G., Evans D.A., Panikkar K.R., Jayavardhanan K.K. Immunomodulatory and antitumour properties of Psoralea corylifolia seeds.

Fitoterapia, 71(3), 223-231, 2000.

25. Patrizia Ambrosino, Rosa Fresa, Vincenzo Fogliano, Simona M. Monti, Alberto Ritieni. Extraction of Azadirachtin A from Neem Seed Kernels by Supercritical Fluid and Its Evaluation by HPLC and LC/MS. J. Agric. Food Chem. 47(12), 5252-5256, 1999.

26. Shaun Johnson, E. David Morgan. Supercritical fluid extraction of oil and triterpenoids from Neem seeds. Phytochemical Analysis, Special Issue: Supercritical Fluid Extraction, 8(5), 228-232, September/October 1997.

27. Upadhyay SN, Dhawan S, Garg S, Talwar GP. Immunomodulatory effects of neem {Azadirachta indica) oil. Int J Immunopharmacol. 14(7): 1187-93,

1992.

28. Van der Nat JM, Klerx JP, van Dijk H, de Silva KT, Labadie RP. Immunomodulatory activity of an aqueous extract of Azadirachta indica stem bark. J Ethnopharmacol. 19(2):125-31, 1987.

29. Bafna AR, Mishra SH. Immunomodulatory activity of petroleum ether extract of flower heads of Sphaeranthus indicus Linn. J Herb Pharmacother. 7(1):25- 37, 2007.

30. S.Sangeetha, P. Marimuthu, P.Doraisamy, D.V.L. Sarada. Evaluation of antioxidant activity of the antimicrobial fraction from Sphaeranthus indicus. International Journal of Applied Biology and Pharmaceutical Technology, 1(2), 431- 436, 2010. 31. Shekhani M.S., Shah P.M., Yasmin A., Siddiqui R., Perveen S., Khan K.M. Kazmi S.U., Atta Ur R. An immunostimulant sesquiterpene glycoside from Sphaeranthus indicus. Phytochemistry 29, 2573-2576, 1990.

32. Shirwaikar A., Prabhu K.S., Punitha I.S.R. In vitro antioxidant studies of Sphaeranthus indicus (Linn). Indian J. Experimental Biology, 44, 993-996, 2006.

33. Atta Ur R., Shekhani M.S., Perveen S., Habib Ur R. Afshanyasmin, Zia-Ul-Haque A., Shaikh D. 7Hydroxyfrullanolide an antimicrobial sesquiterpene lactone from Sphaeranthus indicus Linn. J. Chemical Res. 68, 1989.

Claims

We Claim:

1. A herbal composition for skin disorders comprising blend of extract of Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata and pharmaceutically acceptable carrier.

2. The herbal composition according to claim 1, wherein the blend comprises Psoralea corylifolia, Curcuma longa, Sphaeranthus indicus, Azadirachta indica and Andrographis paniculata in a ratio of 25:11:23:14:27.

3. The herbal composition according to claim 1, wherein the extract of Psoralea corylifolia is obtained using seeds.

4. The herbal composition according to claim 1, wherein the extract of Curcuma longa is obtained using rhizomes.

5. The herbal composition according to claim 1, wherein the extract of Sphaeranthus indicus is obtained using whole plant.

6. The herbal composition according to claim 1, wherein the extract of

Azadirachta indica is obtained using leaves.

7. The herbal composition according to claim 1, wherein the extract of Andrographis paniculata is obtained using aerial parts.

8. The herbal composition according to claim 1, wherein the extract of the herb is obtained by employing percolation, hot soxhlation, enzymatic extraction or super critical fluid extraction.

9. The herbal composition according to claim 8, wherein the percolation or hot soxhlation is performed in presence of a solvent selected from wherein the herbal blend was extracted with n-hexane, ethyl alcohol, acetone, methanol : water (1 :1), water or mixture thereof.

10. The herbal composition according to claim 8, wherein the enzymatic extraction is performed using an enzyme cellulase and pectinase or mixture thereof.

11. The herbal composition according to claim 10, wherein the enzymes are used in a range of 0.01% to 10% in water medium.

12. The herbal composition according to claim 1, wherein the composition is in a suitable dosage form.

13. The herbal composition according to claim 12, wherein the dosage form is tablet, capsule and syrup.

14. The herbal composition according to claim 1, wherein the skin disorders comprises anti-inflammatory, antibacterial and hepatoprotective activity.