WO2011083398A2

WO2011083398A2 - Herbal composition for diarrhoea

Info

Publication number: WO2011083398A2
Application number: PCT/IB2011/000009
Authority: WO
Inventors: Rangesh Paramesh; Uddagiri Venkanna Babu; Ekta Saxena
Original assignee: Himalaya Global Holdings Limited
Priority date: 2010-01-05
Filing date: 2011-01-05
Publication date: 2011-07-14
Also published as: WO2011083398A3

Abstract

Disclosed herein is a herbal composition for diarrhoea comprising extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale and a pharmaceutically acceptable carrier. Further, the invention provides methods of obtaining the extracts, composition thereof and methods of using said composition.

Description

HERBAL COMPOSITION FOR DIARRHOEA

Field of the Invention

This invention relates to a herbal composition for diarrhoea. In particular, the present invention provides a herbal composition comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica, Zingiber officinale and a pharmaceutically acceptable carrier, methods of obtaining the same, pharmaceutical formulations thereof and methods of using the composition.

Background of the Invention

Diarrhoea is defined as an increase in the stool frequency, a change to fluid consistency or mean stool weight in excess. Diarrhoeas often result from an infection of the intestinal tract. The infectious agents, viruses, bacteria, or parasites, enter the intestinal tract and act directly on the luminal side of the enterocytes. The loss of water and electrolytes produces dehydration, the severity of which depends upon the frequency of diarrhoea and the amount of water and electrolyte loss. The disturbances of the entero- systemic cycle of water explain the signs and symptoms of diarrhoea.

Diarrhoea can be caused by motility disorders includes Irritable bowel syndrome and rapid transit with incomplete absorption, osmotic disorders eg. lactose intolerance, permeability defects due to distorted mucosal architecture results in impaired permeability, active transport disorders eg impaired membrane transporters and secretory diarrhoeal disease

The associated symptoms in patients with diarrhoea includes abdominal discomfort, gas formation, pain during defecation, mucus in stools etc.The symptoms may be due to chronic and/or acute amebiasis etc.

Anti-motility agents, loperamide, diphenoxylate are being used for treatment in chronic diarrhoea. However their use is limited or even contraindicated in patients with acute diarrhoea, especially in those with bloody bowel movements. They should not be taken without the advice of physician. Other treatments that are available, depending on the cause of diarrhoea, include the bulk agent psyllium and the binder cholestyramine.

A herbal medication from seeds of Plantago psyllium is being used for treatment of diarrhoea and bouts of irritable bowel syndrome, a condition in which periods of diarrhoea alternates with periods of constipation. Both United States health authorities and the German Commission E have approved the use of psyllium to treat diarrhoea. The use of psyllium is reported to cause increased abdominal gas, stomach rumbling, and a feeling of bloating. It is therefore a very important and challenging task to develop a safe and effective herbal medication that can address all symptoms of diarrhoea and treatment for irritable bowel syndrome and also safe for prolonged use in humans.

Related Art

Patent Publication No. DE202006010905U1 by Ghosh discloses a preparation of supplementary food and/or a medicament, useful to treat or prevent diarrhoea. The preparation comprises plant/vegetable a component e.g. leaves and fruits, and flavoring materials optionally containing dextrose. The preparation further discloses vegetable component as a single plant or mixture from two or more substances in powder form of the intact plants or plant components such as leaves, fruits and/or root and/or aqueous or alcoholic content of e.g. Zingiber officinalis, Magnifera indica, Trachyspermum amni, Foeniculum vulgare, Piper nigrum, Pimpinella anisum, Curcuma longa, Allium sativum, Cuminum cyminum and Acacia catechu.

US Patent No. 7172772 by Pushpangadan et. al. discloses a novel herbal antidiarrhoeal dosage form for the treatment of functional gastrointestinal disorders such as irritable bowel syndrome and diarrhea. The herbal formulation comprises the decoction of Cissampelos pareria, Mangifera indica, Cinnamomum sp. and Buchanania lanzan with the conventional additives to form the oral dosage forms, which include syrup, tablets, capsules and powders ready for suspension.

Patent Publication No. US20090226550A1 by Smith discloses an organic composition comprising ginger, turmeric, cumin, fennel, cinnamon, and red pepper, preferably in a ratio of about 1:2:1 :1 :1:1. The composition is useful for relieving pain in the joints, relieving stomach distension caused by an accumulation of gas in the stomach, suppressing the appetite, increasing physical endurance, and/or reducing the intensity or occurrence of hot flashes, when taken in therapeutically effective amounts.

Patent Publication No. US20010046523A1 by Newmark and Schulick discloses a herbal composition contains therapeutically effective amounts of a supercritical extract of ginger (preferably of certified organic ginger) and rosemary. The composition is used to promote stomach, liver and intestinal health; reduce inflammation; support blood platelet health and cardiovascular function; and provide antioxidant benefits. The herbal composition is preferably administered orally or parenterally in the form of capsules, tablets, elixirs, powders, granules, suspensions, sachets, food additives, beverage additives or tea.

Summary of the Invention

In accordance with one preferred embodiment of the present invention, there is provided an herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale and a pharmaceutically acceptable carrier.

In accordance with one other preferred embodiment of the present invention, there is provided a herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale and a pharmaceutically acceptable carrier, wherein said herbs are in a ratio of 07:24:53:08:08.

In accordance with one other preferred embodiment of the present invention, there is provided a herbal for diarrhoea comprising blend of extract of fruits of Aegle marmelos, rhizomes of Cyperus rotundus, bark of Holarrhena antidysenterica, seeds of Mangifera indica and rhizomes of Zingiber officinale.

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 NP-40 variants on time of onset of diarrhoea in rats challenged with castor oil.

Figure 2 illustrates the effect of NP-40 variants on percentage delay in time of onset of diarrhoea in rats challenged with castor oil.

Figure 3 illustrates the effect of NP-40 variants on dry faecal weight of rats challenged with castor oil.

Detailed Description of the Invention

While this specification concludes with claims particularly pointing out and distinctly claiming that, which is regarded as the invention, it is anticipated that the invention can be more readily understood through reading the following detailed description of the invention and study of the included examples. 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 antidiarrhoeal and useful in symptomatic control and relief in painful flatulence, abdominal pain, and other dysentery related diseases etc in humans.

According to the invention, there is provided an herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale and a pharmaceutically acceptable carrier.

The extract of Aegle marmelos is obtained using fruits of the herbs.

The extracts of Cyperus rotundus and Zingiber officinale are obtained using rhizomes of the herbs.

The extract of Holarrhena antidysenterica is obtained using bark of the herbs.

The extract of Mangifera indica is obtained using seeds of the herbs.

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, dichloromethane, acetone, ethyl acetate, ethyl alcohol, methanol or water or mixture thereof.

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%.

According to the present invention, there is provided a herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale wherein the blend extract is preferably methanol: water extract.

According to the present invention, there is provided a herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale, wherein the blend extract is preferably water extract. According to the present invention, there is provided a herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale, wherein the blend extract is preferably enzyme extract.

According to the invention, the composition exhibits potent anti-diarrhoeal activity.

In accordance with the invention, a unit dose of the extract is between 50 mg to 1000 mg.

The composition according to the present invention is in a suitable dosage form, preferably tablet, capsule, powder, tincture and so forth.

The herbal composition according to the invention is used as an anti-diarrhoeal and in symptomatic control and relief in painful flatulence, abdominal pain, and other dysentery related diseases in humans.

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 Aesle marmelos by percolation method: The shade dried material of Aegle marmelos fruits 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 Aesle marmelos by hot-soxhlsoxhlation method:

The coarse powdered material of Aegle marmelos fruits 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 (AM-1), acetone extract (AM-2), ethyl alcohol extract (AM-3), methanol extract (AM-4), ethyl alcohol and water (1:1) extract (AM-5), methanol and water (1 :1) extract (AM-6) and water extract (AM-7) prepared from the fruits of Aegle marmelos 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 Aesle marmelos by Enzyme extraction

The shade dried material of Aegle marmelos fruits 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 Aeele marmelos by Super Critical Fluid Extraction

The shade dried material of Aegle marmelos fruits 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 Cyperus rotundus by percolation method: The shade dried material of Cyperus rotundus 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 Cyperus rotundus by hot-soxhlation method:

The coarse powdered material of Cyperus rotundus 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 (CR-1), acetone extract (CR-2), ethyl alcohol extract (CR-3), methanol extract. (CR-4), ethyl alcohol and water (1 :1) extract (CR-5), methanol and water (1:1) extract (CR-6) and water extract (CC-7) prepared from the rhizomes of Cyperus rotundus 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 Cyperus rotundus by Enzyme extraction

The shade dried material of Cyperus rotundus 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 Cyperus rotundus by Super Critical Fluid Extraction

The shade dried material of Cyperus rotundus 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 Holarrhena antidysenterica by percolation method:

The shade dried material of Holarrhena antidysenterica bark 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.

ExamplelO

Preparation of extract from bark of Holarrhena antidysenterica by hot-soxhlation method:

The coarse powdered material of Holarrhena antidysenterica bark 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 (HA-1), acetone extract (HA-2), ethyl alcohol extract (HA-3), methanol extract (HA-4), ethyl alcohol and water (1 : 1) extract (HA-5), methanol and water (1 : 1) extract (HA-6) and water extract (HA-7) prepared from the bark of Holarrhena antidysenterica 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 Holarrhena antidysenterica by Enzyme extraction

The dried material of rhizomes of Holarrhena antidysenterica 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.

Examplel2

Preparation of extract from Holarrhena antidysenterica by Super Critical Fluid Extraction The dried material of Holarrhena antidysenterica bark 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 Mangifera indica by percolation method: The dried material of Mangifera indica seeds of 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 Mansifera indica seeds by hot-soxhlation method:

The seeds of Mangifera 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 (MI-1), acetone extract (MI-2), ethyl alcohol extract (MI-3), methanol extract (MI-4), ethyl alcohol and water (1 :1) extract (MI-5), methanol and water (1 :1) extract (MI-6) and water extract (MI-7) prepared from the seeds of Mangifera 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 Mansifera indica by Enzyme extraction The dried material of Mangifera indica 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 16

Preparation of extract from Mangifera indica by Super Critical Fluid Extraction

The dried material of Mangifera indica 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 17

Preparation of extract from Zingiber officinale by percolation method:

The shade dried material of Zingiber officinale 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 18

Preparation of extract from Zingiber officinale by hot-soxhlation method:

The coarse powdered material of Zingiber officinale 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 (ZO-1), acetone extract (ZO-2), ethyl alcohol extract (ZO-3), methanol extract (ZO-4), ethyl alcohol and water (1 :1) extract (ZO-5), methanol and water (1 :1) extract (ZO-6) and water extract (ZO-7) prepared from Zingiber officinale rhizomes 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 Zingiber officinale rhizomes by Enzyme extraction

The shade dried material of Zingiber officinale 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 20

Preparation of extract from Zingiber officinale by Super Critical Fluid Extraction The shade dried material of Zingiber officinale 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 21

Preparation of extract from Herbal Blend by percolation method: The shade dried material of herbal blend of Aegle marmelos fruits and/or rhizomes of Cyperus rotundus and/or bark of Holarrhena antidysenterica and/or Mangifera indica seeds and/or Zingiber officinale rhizomes in the ratio of 07:24:53:08:08 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 Aegle marmelos fruits and/or rhizomes of Cyperus rotundus and/or bark of Holarrhena antidysenterica and/or Mangifera indica seeds and/or Zingiber officinale rhizomes in the ratio of 07:24:53:08:08 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 (NP40-1 also coded as ERNP-102), acetone extract (NP40-2 also coded as ERNP-104), ethyl alcohol extract (NP40-3 also coded as ERNP-103), methanol extract (NP40-4 ), ethyl alcohol and water (1 :1) extract (NP40-5), methanol and water (1 :1) extract ( P40-6 also coded as ERNP-105) and water extract (NP40-7 also coded as ERNP-106 ) 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. All these extracts were subjected to pharmacological evaluation. Example-23

The herbal blend of Aegle marmelos fruits and/or rhizomes of Cyperus rotundus and/or bark of Holarrhena antidysenterica and/or Mangifera indica seeds and/or Zingiber officinale rhizomes in the ratio of 07:24:53:08:08 and 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. This extracts was coded as NP-040 and submitted for pharmacological evaluation.

Example 24

Preparation of extract from herbal blend by Enzyme extraction The shade dried material of herbal blend of Aegle marmelos fruits and/or rhizomes of Cyperus rotundus and/or bark of Holarrhena antidysenterica and/or Mangifera indica seeds and/or Zingiber officinale rhizomes in the ratio of 07:24:53:08:08 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 on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. This extract was coded as NP040/ERNP-107 and subjected to pharmacological evaluation. The extractive yields of enzyme extraction from each herbal material and their combined extract are given in table- 1.

Table- 1

Example 25

Preparation of extract from herbal blend by Super Critical Fluid Extraction

The shade dried material of herbal blend of Aegle marmelos fruits and/or rhizomes of Cyperus rotundus and/or bark of Holarrhena antidysenterica and/or Mangifera indica seeds and/or Zingiber officinale rhizomes in the ratio of 07:24:53:08:08 respectively was pulverized to coarse powder and about 100 Kg of powdered herbal blend was placed in a SCF extractor at the temperature of 40-50°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. This extract was coded as NP040/ERNP-184 and subjected to pharmacological evaluation.

Example 26

Identification of chemical constituents of combined extract from the herbal blend by LC-

MS MS

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) Phenomenex 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 0.600 ml/min. The autosampler SIL-HTC (Shimadzu maker) injection volume was set at 20μ1. UV detector chromatograms were monitor at 21 Onm and 254nm.

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. ESI source conditions as follows: declustering potential (DP) 20 V, nebulizing gas (GS1) 60 psi, curtain gas (CUR) 25psi, nebulizer current (NC) 2 V, focusing potential (FP) 400 psi and source temperature (TEM) 430 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 list of chemical constituents identified in the combined extract is summarized in the below table-2

Table-2

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

Alpha-cyperone 234.33

Copadiene 202.33

Cyperenol 220.35

Cyperolone 236.34

Eugenol 164.20

Isocyperol 220.35

Beta-rotunol 234.33 obusone 222.32

Isokobusone 222.32

Holarrhena and Conessine 356.58 dysenterica

Conkurchine 312.49

Conessidine 326.51

Conimine 328.53

Kurchine dihydrochoride 415.48

Holarrhimine 332.52

Conamine 328.53

Kurchiline 359.54

Holarosine A 517.69

Holantosine A 493.67

Holantosine B 475.66

Holantosine D 475.66

Holadysine 415.49

Holanamine 325.44

Mangifera indica Friedelin 426.71

Mangiferin 422.33

Protocatechuic acid 154.12

Catechin 290.26

Gallic acid 172.13

Ellagic acid 302.19 m-Digallic acid 322.22

Gallotanin 626.46

Fisetin 286.23 Leucocyanidin 306.26

Mangiferolic acid 456.70

Hydroxymangiferolic acid 458.67

Mangoferonic acid 454.68

Ambonic acid 468.71

Homomangiferin 452.36

Cycloartanol 428.73

Amyrin 426.71

Lupeol 426.71

Butin 272.25

5. Zingiber officinalis Zingiberene 204.35

Gingirone 194.22

Shogaol 276.37

Dihydrogingirol 192.21

(6) Dehydrogingerdione 290.75

(10) Dehydrogingerdione 346.46

(6) Gingerdione 292.37

(10) Gingerdione 348.47

(6) Gingerol 294.38

Beta-Bisabolene 204.35

Zerumbone 218.33

Zerumbodienone 220.35

Geranial 152.23

Camphene 136.23

Terpinolene 136.23

Curcumene 368.37

Alpha- Farnesene 204.35

Linalool 154.24

Alpha- Sesquiphellan 204.35 drene Example-27

The objective of the present study was to compare the anti-diarrhoeal activity of various solvent extracts in an experimental model of castor oil-induced diarrhoea model in rats.

Effect of NP-40 variants on castor oil induced diarrhoea in rats

40 male Wistar rats of body weight 250-300 gms were divided in to five groups eight each. The animals were fasted for 18 hours before the experiment, Food but not water was withheld and it was provided ad libitum. The Group I rats received 10 ml/kg of dimineralised water and serves as control and group II to IV received NP-040, NP- 040/ERNP-105 and NP-040/ERNP-107 at the dose of 250mg/kg b.wt. by oral route. The rats in group V was treated with standard drug loperamide (1 mg/kg, p.o.). One hour after the treatment, all the animals were challenged with 5ml/ kg body wt. of castor oil orally, by gavage. The time of onset of diahrrea was recorded. After 4 hours, consistency of fecal material was scored. In addition to this, the evacuated faeces were dried at constant temperature and weighed.

Statistical analysis

The Individual values were expressed as Mean ± SEM. The results were analyzed statistically using One- Way ANOVA to find out the level of significance followed by Dunnet's post comparison test. The minimum level of significance was fixed at 0.05. The analysis was carried out using GraphPad Prism version 4.01. The "n ' ranges from 7 to 8 per group. Results are depicted in Fig 1 to Fig. 3

Results & Conclusion

It was found that administration of castor oil to rats caused significant secretory diarrhoea in rats, as indicated by increase in faecal consistency score, weight of dry faeces etc. The time of onset of diarrhoea was considered as an important parameter to evaluate the anti-diarrhoeal activity. Loperamide, a standard drug was taken as reference standard in the experiment.

NP-040 extracts were screened for anti-diarrhoeal activity at a dose of 250 mg/kg body weight orally. It was found that pre-treatment with extract NP-40 ERNP-107 significantly delayed the onset of diarrhoea in rats and reduced the mean weight of dry faces compared to untreated control. The other two extracts (NP-040 and NP-040/ERNP- 105), also showed mild delay in onset of diahrrea. It can be concluded that NP-40 ERNP- 107 extract was found to be more potent among the different variants tested in delaying the castor oil-induced diarrhoea in rats.

Example-28

Manufacturing of tablets using combined water extract NP-040 by direct compression method.

Extract 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 final composition of the tablets is given in the below table-3

Table-3

Pharmaceutical properties of compressed tablets are given in the below table-4

Table-4

Formula 2 900 10 0.7 23

Formula 3 950 11 0.9 27

Formula 4 1000 13 0.6 32

Example-29

Manufacturing of tablets using combined extract NP-040 by wet granulation method.

Active extract and excipients were sieved through a 36 mesh sieve into a suitable mixer and granulated with a suitable quantity of Isopropyl Alcohol to form a medium /heavy granule. The granules were air dried first and then dried in a suitable oven at 60°C, until the LOD was <2%. 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, The final composition of the tablets is given in the below table -5.

Table-5

12 Croscarmellose sodium 20 - - - 30

Microcrystalline

13 - - - - 67

cellulose

14 Magnesium stearate 5 5 - - 3

Sodium starch

15 - 15 - 20 - glycolate

Colloidal Silicon

16 - - 5 5 - Dioxide

17 Crospovidone - - 15 - -

Total 800 850 900 950 1000

Phamaceutical properties of tablets compressed are given in table-6

Table-6

Example-30

Manufacturing of Capsules from the combined extract NP-040 by wet granulation method.

Active extract 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%. 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 final composition of the capsule is given in table-5 and results of capsules given in table-7

Table-7 Formula 1 Formula 2

Ingredients

S.No. (in mg) (in mg)

1 NP-040 SPD Extract 460 460

2 Calcium Carbonate 100 100

3 Microcrystalline cellulose 150 250

4 Croscarmellose sodium 25 -

5 Maize starch 90 -

6 Sodium starch glycolate - 20

7 Purified Water Qs Qs

Blending

8 Croscarmellose sodium 20 15

9 Magnesium stearate 5 .5

Total 850 850

Table-8

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 & Bibiliography

1. Xingang Shi; Xiao Wang; Daijie Wang; Yanling Geng; Jianhua Liu. Separation and Purification of a-Cyperone from Cyperus rotundus with Supercritical Fluid Extraction and High-Speed Counter-Current Chromatography. Separation Science and Technology, 1520-5754, Volume 44, Issue 3, 2009, Pages 712 - 721.

2. Uddin SJ, Mondal K, Shilpi JA, Rahman MT. Antidiarrhoeal activity of Cyperus rotundus. Fitoterapia. 2006, 77(2): 134-6.

3. S. Balachandran, S.E. Kentish, R. Mawson. The effects of both preparation method and season on the supercritical extraction of ginger. Separation and Purification in the Food Industry, Volume 48, Issue 2, Pages 94-105, 2006.

4. Chu-Chin Chen, Robert T. Rosen, Chi-Tang Ho. Chromatographic analyses of gingerol compounds in ginger (Zingiber officinale roscoe) extracted by liquid carbon dioxide. Journal of Chromatography A, Volume 360, pages 163-173, 1986.

5. Ute Schweiggert, Silke Hofmann, Mareike Reichel, Andreas Schieber, Reinhold Carle. Enzyme-assisted liquefaction of ginger rhizomes (Zingiber officinale

Rose.) for the production of spray-dried and paste-like ginger condiments Journal of Food Engineering volume 84, Issue 1, Pages 28-38, 2008.

6. Poonam G, Daswani, S. Brijesh, Pundarikakshudu Tetali, Noshir H. Antia and Tannaz J. Birdi. Antidiarrhoeal activity of Zingiber officinale (Rose.) CURRENT SCIENCE, 2010, 98(2), pp. 222-229.

7. Poonam G. Daswani, Tannaz J. Birdi, D.S. Antarkar, Noshir H. Antia. Investigation of the Antidiarrhoeal activity of Holarrhena antidysenterica. Indian J. Exp. Pharmaceut. Biol. 2002, March- April, 164-167.

8. Kavitha D, Shilpa PN, Devaraj SN. Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL. Indian J Exp Biol. 2004 42(6):589-94.

9. K. Sairam, S. Hemalatha, Ashok Kumar, T. Srinivasan, Jai Ganesh, M. Shankar and S. Venkataraman. Evaluation of anti-diarrhoeal activity in seed extracts of Mangifera indica. Journal of Ethnopharmacology, 2003, 84(1), pp. 11-15.

10. Pushpangadan, et al. Herbal composition for gastrointestinal disorders. United States Patent, 7, 172,772, February 6, 2007. 11. S Brijesh, Poonam Daswani, Pundarikakshudu Tetali, Noshir Antia and Tannaz Birdi. Studies on the antidiarrhoeal activity of Aegle marmelos unripe fruit: Validating its traditional usage. BMC Complementary and Alternative Medicine 2009, 9(47),

12. Mazumder R, Bhattacharya S, Mazumder A, Pattnaik A , Tiwary PM,

Chaudhary S. Antidiarrhoeal evaluation of Aegle Marmelos (Correa) Linn, root extract. Phytother Res. 2006; 20(1): 82-4.

13. Surve V.S., Ghangale G.R., Tamhankar S.P., Gante M.M. Studies on antidiarrhoeal activity of Aegle marmelos (Bael) in rats. The J. Bombay Veterinary College. 2008, 16 (1).

Claims

We Claim:

I . A herbal composition for diarrhoea comprising blend of extract of Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale and a pharmaceutically acceptable carrier.

2. The herbal composition according to claim 1, wherein the blend comprises

Aegle marmelos, Cyperus rotundus, Holarrhena antidysenterica, Mangifera indica and Zingiber officinale in a ratio of 07:24:53:08:08.

3. The herbal composition according to claim 1, wherein the extract of Aegle marmelos is obtained using fruits.

4. The composition according to claim 1, wherein the extracts of Cyperus rotundus and Zingiber officinale are obtained using rhizomes.

5. The herbal composition according to claim 1, wherein the extracts of Holarrhena antidysenterica is obtained using bark.

6. The herbal composition according to claim 1, wherein the extracts of Mangifera indica is obtained using seeds.

7. 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.

8. The herbal composition according to claim 7, wherein the percolation or hot soxhlation is performed in presence of a solvent selected from n-hexane, acetone, ethyl alcohol, methanol, ethyl alcohol and water (1 :1), methanol and water (1 :1) and water or mixture thereof.

9. The herbal composition according to claim 7, wherein the enzymatic extraction is performed using an enzyme cellulase or pectinase or mixture thereof.

10. The herbal composition according to claim 9, wherein the enzymes are used in a range of 0.01% to 10%.

I I . The herbal composition according to claim 1 , wherein the blend extract is methanol: water extract.

12. The herbal composition according to claim 1, wherein the blend extract is water extract.

13. The herbal composition according to claim 1, wherein the blend extract is enzyme extract.

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

15. The herbal composition according to claim 14, wherein the dosage form is tablet, capsule, powder and tincture.

16. The herbal composition according to claim 1, wherein the extract is used in a dose from 50 mg to 1000 mg.

17. The herbal composition according to claim 1, wherein the diarrhoea includes painful flatulence, abdominal pain, and other dysentery related diseases in humans.