Title of Invention	A PROCESS OF PREPARING HERBOSOMES WITH ANDROGRAPHOLIDE HAVING BETTER HEPATOPROTECTIVE ACTIVITY AND PRODUCT THEREOF.
Abstract	This invention relates to Herbosomes with Andrographolide, a novel compound comprising lipophilic complexes of Andrographolide with phospholipids and the preparation of these complexes by non- conventional methods. Herbosomes are the concept of value addition to phytoconstituents to develop a specific dosage form with more therapeutic potency than using it as such, which increase the availability of the constituents for a longer time in blood and help in curing the diseases to a higher extant. This Herbosomes is a value added herbal formulation with isolated phytoconstituents having potential hepatoprotective activity. The novel compounds persisted for a longer period of time in plasma and subsequently produced prolonged significant hepatoprotective activity than the pure molecule at the same dose levels. The resulting improvement in the phramacokinetics and pharmacological parameters is such that the compound can be used advantageously in the treatment of acute or chronic liver diseases.

Title of Invention

A PROCESS OF PREPARING HERBOSOMES WITH ANDROGRAPHOLIDE HAVING BETTER HEPATOPROTECTIVE ACTIVITY AND PRODUCT THEREOF.

Abstract

This invention relates to Herbosomes with Andrographolide, a novel compound comprising lipophilic complexes of Andrographolide with phospholipids and the preparation of these complexes by non- conventional methods. Herbosomes are the concept of value addition to phytoconstituents to develop a specific dosage form with more therapeutic potency than using it as such, which increase the availability of the constituents for a longer time in blood and help in curing the diseases to a higher extant. This Herbosomes is a value added herbal formulation with isolated phytoconstituents having potential hepatoprotective activity. The novel compounds persisted for a longer period of time in plasma and subsequently produced prolonged significant hepatoprotective activity than the pure molecule at the same dose levels. The resulting improvement in the phramacokinetics and pharmacological parameters is such that the compound can be used advantageously in the treatment of acute or chronic liver diseases.

Full Text	The present invention relates to the improved process of preparing of Herbosomes with Andrographolide, a novel compound obtained by chemicai interaction Detween one or more phospholipids and pure andrographolide or andrographolide in standardized methano! extract of Andrographis paniculata, which increased hepatoprotective activity in liver disorders than Andrographolide alone. Herbosomes are defined as value added herbal formulation with standardized Botanicals. DISCUSSION OF THE RELATED ART: Herbosomes with Andrographolide having better hepatoprotective activity along with other biological activities are known from prior art: U.S. Patent No. 4,764,508 describes novel complexes of phospholipids with the main constituents of silymarin, a known standardized extract obtained from seeds of Silybum marianum and used for the treatment of liver disease of varying origin. According to Chinese Patent application CN1552320 an Andrographolide compound (which can be used as medicine with improved solubility) can be prepared by mixing the Andrographolide with solubilizer like cyclodextrin, amino acid, phosphatide, water soluble high polymer and vegetable oil, water or organic cosolvent, mechanical grinding, ultrasonic processing and mixing with medicinal auxiliary. Another Chinese Patent application (CN1528290) relates to an andrographolide soft capsule and its production method. Every soft capsule contains 10-300 mg of andrographolide, and contains bees wax and medicinal auxiliary material. Said medicinal auxiliary material includes diluting agent, suspension adjuvant and defoaming agent, and the interior of said capsule shell, contains (by weight ratio) 10 portions of gelatin and 4.0-5.5 portions of glycerine. Its production process includes the following steps: adding water to dissolve gelatin, adding glycerine and proper quantity of preservative, vacuum deaerating, adding suspension adjuvant into vegetable oil, mixing andrographolide, diluting agent and defoaming agent uniformly, die-pressing to obtain said invented soft capsule. Its quality is stable, administration is convenient and its effective components can be quickly resolved out. The Process of preparation of one kind of soft andrographolide capsule is described in a Chinese Patent application CN1481792 where this capsule medicine liquid contains enough andrographolide as its effective component, and proper amount of medicinal supplementary material is added, including diluent, suspension assistant, antioxidant, preservative, etc. The capsule shell consists of mainly gelatin, glycerine and water. The capsule of the present invention has stable quality, convenient administration, fast leaching out of the effective component and timely acting. An International Patent application WO9617605 relates to the discovery a group of compounds (which are Andrographolide compounds) isolated from the extract of Andrographis paniculata that causes the reduction of expression of p34kinase. Process of preparation of novel derivatives of Andrographolide, their stereo isomers, their polymorphs, their pharmaceutically acceptable salts and their pharmaceutically acceptable solvates and their uses as antiviral, anti-malarial, antibacterial, hepatoprotective and immunomodulating agents and for the treatment of other metabolic disorders are described in U.S Patent applications US 6,486,196 and US 6,410,590. Background of the invention: Now -a- days development of a new molecule with therapeutic potential is very expensive as well as time consuming, irrespective of the source, it may be natural or synthetic. Sometimes the newly developed molecule fails clinical trial due to severe toxicity and hardly can come into market as a therapeutically potent drug molecule. Due to these shortcomings of new drug development, the researchers worldwide have engaged themselves in the development of novel drug delivery system for the exiting molecules so as to bring some new therapeutic measures in the treatment of different types of human ailments. Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years. The plants and phytoconstituents in the traditional systems of medicine of many countries continue to play an essential role in healthcare. Thorough research in the field of Herbal Drug Delivery System as a means of improving the therapeutic indices of drugs is of prime importance. Toxicity, short duration of action and limited absorption of different drug molecules obtained from herbal sources are particularly difficult problems in patients undergoing treatment of different diseases, which leads to sub-optimal therapy. From this view point development of novel herbal dosage form can be of paramount importance. Liver diseases of different origin remain one of the serious health problems and a major cause of mortality. In the absence of reliable liver protective drugs in allopathic medical practices, herbs and plants play an important role in the management of various liver disorders (Trivedi and Rawal, 2000). Numerous literature related to the hepatoprotective activity of phytomolecules from herbal sources have shown that there is a vast array of phytoconstituents having potent hepatoprotective efficacy. Andrographolide is one such hepatoprotective herbal molecule. Jt is a wel-known phytoconstituents obtained from Andrographis paniculata Nees, having very bitter taste. It is a bicyclic diterpenoid lactone with multiple pharmacological activities. The traditional uses include treatment of fever, inflammation, common cold, upper respiratory infection, tonsillitis, pharyngitis, laryngitis, pneumonia, tuberculosis, pyelonephritis, hepatic impairment etc. (Barilla, 1999; Caceres etal., 1997, 1999; Hancke et al., 1995; Handa and Sharma, 1990; Kapiletal., 1993; Melchior et al., 1997; Visen et al., 1993). As per reported literature, andrographolide possesses potent antihyperglycemic (Yu et al 2003), antipyretic (Deng, 1978; Madav et al., 1995; Vedavathy and Rao, 1991), Antiinflammatory (Wang et al 2004, Shen et al 2002), anticancer (Rajagopal et al 2003), antileishmanial (Sinha et al 2000), antifertility (Akbarsha and Murugaian 2000), anti HIV (Calabrese et al 2000), cardiovascular (Zhang and Tan 1997), immunomodulatory (Puri et al 1993), Choleretic (Shukla et al 1992) activities. It also inhibits PAF-induced platelet aggregation (Amroyan et al 1999). It has got preventive activities against common cold (Caceres et al., 1997, 1999; Hancke et al., 1995; Melchior et al., 1997; Puri et al., 1993) and has potent antimicrobial (Misra et al., 1992; Singha et al., 2003; Dutta and Sukul NC, 1982; Raj, 1975; Chander et al., 1995) as well as antidiarrhoeal activities (Deng, 1978; Gupta et al., 1990). The molecule is also useful in the management of respiratory tract infection (Coon and Ernst, 2004; Poolsup et al., 2004). But the most important use of andrographolide is seems to be in the management of various liver disorders. The liver is subject to acute and potentially lethal injury by several substances including carbon tetrachloride, phalloidine, galactosamine, ethanol and other compounds (Middleton et al 2000). In Indian System of Medicine (Ayurveda), about 26 different polyherbal formulations containing Andrographis paniculata are mentioned as a popular remedy for the treatment of various liver disorders and an adequate number of pharmacological investigations have substantiated the therapeutic potential of andrographolide, principal chemical constituent of A. paniculata as a hepatoprotective agent. Andrographolide showed a significant dose dependent protective activity against paracetamol-induced toxicity on ex vivo preparation of isolated rat hepatocytes (Visen et al., 1993). It exhibits protective effects on hepatoxicity induced in mice by carbon tetrachloride or tert- butyl hydroperoxide (t-BHP) intoxication (Kapil et al., 1993; Handa and Sharma, 1990) Andrographolide has also been shown to produce ameliorative effect in galactosamine induced acute hepatitis in rats (Handa and Sharma, 1990) and prevents BHC induced increase in the activities of enzymes gamma-glutamyl transpeptidase, glutathione-S-transferase and lipid peroxidation (Trivedi and Rawal, 2001). It is useful in the treatment of infective hepatitis, an acute inflammatory condition of the liver. All the experimental models used to evaluate the hepatoprotective activity of andrographolide dealt with the chemicals responsible for damage of liver by causing lipid peroxidation, a process related to production of free radicals by the chemical attack lead to destruction of the cellular membrane that surrounded liver cells. So the hepatoprotective effect provided by andrographolide was attributed to the anti-oxidant ability of the compound, which scavenges the free radicals generated during hepatotoxin challenge (Kapil et al., 1993). Apart from antioxidant activity, inhibition of cytochrome P-450, stimulation of hepatic regeneration (Handa and Sharma, 1990a; Choudhury et al., 1987) and inhibition of microsomal enzymes or lipid peroxidation (Handa and Sharma, 1990b) could explain the hepatoprotective effect of andrographolide. According to the reported data in various literature the best protection against the liver intoxicating agents is obtained in the animal when the compounds are administered intraperitoneally or intravenously so as rapidly to obtain high concentrations in the bloodstream and the target organ. But in most of the cases it fails due to poor absorption of the compounds. In some cases, though it absorbed rapidly but due to faster elimination of the compound the needed therapeutic activity fails to be achieved. Despite this vast array of pharmacological activities and rapid absorption into the blood after oral administration, rapid clearance, lower elimination half life, as well as higher plasma protein binding of andrographolide necessitates the need of some novel compound with same dose of the conventional dosage form, which can maintain the concentration of andrographolide in blood for a longer period of time. Statement of the invention: The present invention deals with the production of Herbosomes with Andrographolide, a novel compound obtained by chemical interaction between one or more phospholipids and pure andrographolide or andrographolide in standardized methanol extract of Andrographis paniculate by some non-conventional methods. Herbosomes with Andrographolide are advanced form of herbal products containing andrographolide or standardized Andmgraphis paniculata extract and the phospholipids, having better utilization profiles in our body and subsequently produce better therapeutic efficacy than the conventional herbal extracts or the individual phytomotecule. This delivery system, having longer duration of action inside the body, can markedly alter the biodtstribution of their associated drugs, by increasing the drug concentration at the site of infection and inflammation. The invention deals with i) Preparation of Herbosomes with Andrographolide ii) Physicochemical evaluation of Herbosomes with Andrographolide iii) Hepatoprotective activity of Herbosomes with Andrographolide in carbon tetrachloride intoxicated rats in comparison to pure Andrographolide and iv) In-vitro release study of Andrographolide from pure Andrographolide suspension and Herbosomes with Andrographolide Brief description of accompanying drawings: Figure 1. HPTLC fingerprint of pure andrographolide; Figure 2. HPTLC fingerprint of andrographolide from Herbosomes with Andrographolide; Figure 3. In-vitro release of Andrographolide vs. time from Herbosomes with Andrographolide and pure Andrographolide suspension; and Figure 4. In-vitro release of Andrographolide vs. time from Herbosomes with standardized Andrographis paniculata extract and standardized Andrographis paniculata extract suspension Detailed description of the invention: This invention is explained in more detail as set forth hereunder 1. Collection of plant materials and chemicals: Aerial parts of Andrographis paniculate Nees were collected locally and authenticated from Botanical Survey of India, Shibpur, West Bengal, India. Pure andrographofide was obtained from Natural Remedies Pvt. Ltd., Bangalore, India as gift sample. The aerial parts of Andrographis paniculata Nees were extracted as per reported method (Handa and Sharma, 1990b) to get standardized Andrographis paniculata Nees extract (Standardized to contain 10% w/w andrographolide). Phospholipids like 1,2-Distearoyl-sn-Glycero-3-Phosphocholfne (DSPC), 1,2- Dimyristoyl-sn-Glycero-3-Phosphochoine (DMPC), 1,2-Oipaimitoy(-sn-Glycero-3-Phosphochotine (DPPC), 1,2-Distearoyl-sn-Glycero-3-hosphoethanolarnine (DSPE) were obtained from Lipoid GMbH, Germany. Dichloromethane, n-hexane and other solvents were obtained from Qualigens, Mumbai. 2. Preparation of Herbosomes with Andrographolide: Herbosomes with Andrographolide were prepared by a novel method of complexation using i) Pure Andrographolide ii) Andrographolide in standardized Andrographis paniculata extract (Standardized to contain 10% w/w andrographolide) and phospholipids. The phospholipids used in the preparations can be natural or synthetic. Preferably used is made of phospholipids from vegetable origin (soy phospholipids) such as 1,2-Distearoyl-sn-Glycero-3- Phosphocholine (DSPC) (Lipoid KG - Ludwigshafen, Germany). According to the invention, in one embodiment the novel complex was prepared by reacting 0.5 mole to 10 moles, but preferably 1 mole of a natural or synthetic phospholipids, wherein the phospholipid is a member selected from the group consisting of soybean lecithins, egg lecithin phospholipids from bovine or swine brain or dermis, phosphatidyl choline, phosphatidyl serine, phophatidyl ethanolamine in which the acyl groups are same or different, derived from palmitic, stearic, oleic, linoleic, linolenic acid with one mole of andrographolide, either alone or in the standardized A. paniculata extract, in aprotic organic solvents such as dioxane, dichloromethane or acetone, from which the complexes can be isolated by precipitation with non-polar solvents such as aliphatic hydrocarbons or by lyophilization or by spray drying. In the preferred embodiment the novel complex was prepared by the reaction of one mole Andrographolide isolated from the plant Andrographis paniculata or Andrographolide in standardized Andrographis paniculata extract (10% w/w) with weighed amount of one or more phospholipids. In the reaction process weighed amount of andrographolide and weighed amount of dipalmitoyl phosphatidyl choline (DPPC) or dimyristoyl phosphatidyl choline( DMPC) or distearoyl phosphatidyl choline (DSPC) was taken in a round bottom flask and dissolved in dichloromethane. The resulting suspension mixture was then refluxed over a water bath for 2-3hrs at a temperature not exceeding 80°C preferably at 55-60°C. All andrographolide thus goes to the solution and the resultant solution was then concentrated one third to its volume in a beaker and a non-polar solvent preferably n-hexane was added with continuous stirring and a complex was precipitated in the reaction mixture. The complex was then filtered and dried under vaccum to remove the traces of hexane. Andrgrapholide herbosomes thus obtained was kept in air-tight container for further evaluation. The method of preparing Herbosomes with Andrographolide can be represented schematically. Schematic diagram of Andrographolide Herbosomes formation 3. Physicochemical evaluation of Herbosomes with Andrographolide: The complex of andrographolide with phospholipids i.e. Herbosomes with Andrographolide were evaluated in terms of physicochemical properties and compared with that of pure andrographolide. The invention discloses that the starting material andrographolide, which is insoluble or sparingly soluble in chloroform, dichloromethane, ethyl ether or benzene, becomes extremely soluble in these solvents after forming the Herbosomes. This change in chemical and physical properties is due to the formation of a complex, as is clear from the modification in different parameters like texture, melting point, specific rotation, solubility etc (Table 1). Table 1. Comparative evaluation of macroscopic characteristics of andrographolide and Herbosomes with Andrographolide The microscopic view of the pure andrographolide shows prismatic crystals but the Herbosomes when suspended in water and observed under microscope it shows vesicular structures. The vesicles consist of phospholipids and the andrographolide is present in the lipid bi-layer as intercalated form. The chemical characteristics of the complex and pure andrographolide were evaluated in terms of High performance Thin Layer Chromatography (HPTLC) using Camag TLC scanner 3. Development of chromatogram Herbosomes with Andrographolide and free andrographolide solutions were spotted on the Silica Gel 6OF254 pre-coated TLC plates and chromatogram was developed in chromatographic chambers using specific solvent system at a room temperature of 30° C and at an angle of 70°. In this experiment, the mobile phase (solvent system) was allowed to run up to a distance of 10 cm from the origin. The time required for the development of chromatogram differed from 20 to 30 minutes. The plates were removed from the chamber after completion of the run and were allowed to dry in air. The plates were observed under UV light for the appearance of spots. The plates were scanned with the help of the scanner and the hRf values of the spots were recorded. The result has been furnished hereunder (Table 2, Fig.1 & Fig.2). Reference Solutions: Standard compound is prepared as 0.05% solutions in methanol and about 10ul is used for chromatography. The average detection limit is 5-10μg. Solvent system used: Chloroform: Methanol 7:1 Table 2. Comparative High performance thin layer chromatographic study of free andrographolide and Herbosomes with Andrographolide. Spectroscopic evaluation: UV Spectroscopic study of Pure Andrographoide and Herbosomes with Andrographolide: UV-visible spectroscopic analysis of pure andrographoide and Herbosomes with Andrographolide has been performed and it was observed that pure andrographolide has λmax at 223.7 nm in methanol. On contrary, Herbosomes with Andrographolide showed λmax at 229.7 nm in methanol. This change in λmax is due to formation of the complex. IR Spectroscopic study of Pure Andrographoide and Herbosomes with Andrographolide: IR spectrum of Andrographolide (Figure 4) shows occurrence of different bands at various wave numbers due to the presence of numbers of functional groups. But in case of the IR spectrum of Herbosomes with Andrographolide (Figure 5), broadening or shifting of these bands and in some cases disappearance of bands due to formation of the complex was observed. The bands appeared at λ = 3827.41 cm1 and λ = 3398.68 cm1 in the IR spectrum of Andrographolide were due to O-H stretching of furan ring and O-H stretching of naphthalene ring, respectively, but those bands disappeared in the spectrum of Herbosomes because of the interaction with the polar head group of phospholipids, which indicates formation of a complex. 400MHz 1H-NMR study of Pure Androoraoriolide and Herbosomes with Androorapholide: In the 1H-NMR spectrum of Andrographolide it was observed that there are number of peaks appeared at specific 5-values due to presence of various functional groups. Peaks, those appeared from 5-3.7 to 5 - 5.2 in the spectrum of Herbosomes with Andrographolide became suppressed due to formation of H-bonds between the phenolic hydroxyl group of andrographolide and the phosphate ion of the phospholipids. This can be deduced from the comparison of the NMR of the Herbosomes with that of pure Andrographolide. The signals of the fatty chain are almost unchanged. Such evidences inferred that the two long aliphatic chains of the phospholipid were wrapped around the active principle, producing a lipophilic envelop, which shielded the polar head of the phospholipid and andrographolide. Differential Scanning Calorimetric study of Pure Andrographolide and Herbosomes with Andrographolide Differential Scanning Calorimetric (DSC) is a fast and reliable method to screen drug - excipient compatibility and provides maximum information about the possible interactions. In DSC, an interaction is concluded by elimination of endothermic peak (s), appearance of new peak (s), change in peak shape and its onset, peak temperature / melting point and relative peak area or enthalpy. A sharp peak with a maximum occurrence at 235.23°C was appeared in the DSC thermogram of Pure Andrographolide. No additional endothermic or exothermic peaks were observed. In the DSC thermogram of Herbosomes with Andrographolide it was observed that the peak at 235.23°C was absent and two new peaks with maximum occurrence at 69.05°C and 85.57°C were appeared. The absence of original peak of Andrographolide and the appearance of two new peaks in the thermogram of Herbosomes indicated the origination of a new chemical entity. Thus the spectral data obtained from UV, IR and 1H-NMR study of Herbosomes with Andrographolide and pure Andrographolide showed a proof of complexation between the molecule and phospholipid. The thermograms obtained from the Differential Scanning Calorimetric (DSC) analysis of the complex and free molecule also strengthen the proof of complexation. From the above physicochemical studies of both free andrographolide and Herbosomes with Andrographolide, it was observed that physical as well as chemical properties of free andrographolide are different from Herbosomes with Andrographolide. Physical properties like melting point, solubility, optical rotation, chemical property like Rf value obtained from HPTLC study clearly indicating that andrographolide gone into complexation with phospholipids and resulted in a new chemical entity with different physicochemical properties. The microscopic view of the complex and the spectral as well as thermal analyses also support the claim of complexation strongly. 4. Evaluation of hepatoprotective activity of Herbosomes with Andrographolide: To prove the therapeutic superiority of Herbosomes with Andrographolide over pure Andrographolide, the hepatoprotective activity of both pure Andrographolide and Herbosomes with Andrographolide was evaluated in carbon tetrachloride -intoxicated albino rats. Test samples: Pure andrographolide (25 mg/kg body wt., p.o.) Pure andrographolide (50 mg/kg body wt., p.o.) Herbosomes with Andrographolide (Equivalent to pure andrographolide 25 mg/kg body wt., p.o.) Herbosomes with Andrographolide (Equivalent to pure andrographolide 50 mg/kg body wt., p.o.). Standardized Andrographis paniculata extract 200 mg/kg body wt., p.o. (Standardized to contain 10% w/w andrographolide, i.e. 20mg/kg pure andrographolide) Standardized Andrographis paniculata extract Herbosomes (Equivalent to Standardized Andrographis paniculata extract 200 mg/kg body wt., p.o.) Tested biochemical parameters: Serum glutamic -oxaloacetic transaminase (SGOT), Serum glutamic -pyruvic transaminase (SGPT), Serum alkaline phosphatase (SALP), Direct and total bilirubin, Total Protein (TP) Lactate dehydrogenase (LDH). Animals: In bred male albino rats (Wistar strain) weighing 180-200 g were used for this study. Animals were housed in groups of 7 - 8 in colony cages at an ambient temperature of 20-25° C and 45 - 55 % relative humidity with 12 hrs light / dark cycles. They had free access to pellet chow (Brook Bond, Lipton India) and water ad libitum. The experimentation on animals was performed based on the observations of animal ethical committee. Dosing: The adult male Wistar rats were divided into eight groups of ten animals each. The animals in different groups have received different substances as per the following protocol. Group I Received only distilled water with 0.3% CMC p.o. for seven days and served as normal. Group II (CCI4 - intoxicated) Received distilled water with 0.3% CMC p.o. for seven days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group III (Andrographolide 25 mg/kg body wt + CCI4) Animals were treated with andrographolide suspension in distilled water with 0.3% CMC at a dose level of 25 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group IV [Herbosomes with Andrographolide (equivalent to andrographolide 25 mg/kg body wt.) + CCI4] Animals were treated with Herbosomes with Andrographolide at a dose level of 25 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group V (Andrographolide 50 mg/kg body wt. + CCI4) Animals were treated with andrographolide suspension in distilled water with 0.3% CMC at a dose level of 50 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group VI [Herbosomes with Andrographolide (equivalent to andrographolide 50 mg/kg body wt.) + CCI4] Animals were treated with Herbosomes with Andrographolide at a dose level of 50 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group VII [Standardized Andrographis paniculata extract 200 mg/kg body wt. (Standardized to contain 10% w/w andrographolide, i.e. 20mg/kg pure andrographolide) + CCI4] Animals were treated with Standardized Andrographis paniculata extract at a dose level of 200 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. Group VIII [Standardized Andrographis paniculata extract Herbosomes (Equivalent to Standardized Andrographis paniculata extract 200 mg/kg body wt.) + CCI4] Animals were treated with Standardized Andrographis paniculata extract Herbosomes at a dose level equivalent to 200 mg/kg of Standardized Andrographis paniculata extract, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day. On the eighth, ninth and tenth day, blood samples were collected from the retro orbital plexus and centrifuged at 2000g for 10 minutes at 4°C to separate the serum and kept at 4°C to assay the activities of the serum enzymes. Assessment of liver function: The liver function test of the animals were performed by analyzing the level of serum enzymes namely Serum glutamic -oxaloacetic transaminase (SGOT), Serum glutamic -pyruvic transaminase (SGPT), Serum alkaline phosphatase (SALP), Lactate dehydrogenase (LDH) and the amount of total and direct bilirubin as well as Total Protein (TP). Levels of SGOT and SGPT in the blood serum of the experimental animals were estimated on day 8, 9 and 10 as per the method of Reitman and Frankel (1957). Serum alkaline phosphatase (SALP) was estimated according to the method of Kind and King (1954). Lactate dehydrogenase (LDH) was measured by the method of Wroblewski and LaDue as described by Varley (1967). Serum bilirubin and protein level were estimated by the method of Malloy and Evelyn (1937) and Lowry et al., 1951, respectively. Results: Hepatic damage induced by carbon tetrachloride caused significant rise in the serum level of marker enzymes like SGOT, SGPT, SALP and LDH and also increased the level of serum bilirubin (both direct and total). It also lowered the level of total protein. Oral administration of andrographolide, either pure or in the standardized Andrographis paniculata extract, reduced the elevated level of the enzymes and bilirubin and increased the reduced protein level. Herbosomes with Andrographolide also exhibited protective activity as evident from the experimental results. It was observed that the formulation produced better hepatoprotection for a longer period of time as compared to respective free or pure andrographolide (Table 3-9). It is also evident from the result that in case of all the marker enzymes, the Herbosomes, even at the lowest dose (group VIII, Standardized Andrographis paniculata extract Herbosomes (Equivalent to Andrographis paniculata extract 200 mg/kg body wt., standardized to contain 20mg/kg of pure andrographolide) produced better protective activity than the highest dose of pure andrographolide (50mg/kg body weight), at the end of the study (10th day). It proved that the Herbosomes acted for a longer period of time inside the body, which was further established with the help of in-vitro release study of andrographolide. 5. In-vitro release study of Andrographolide from pure Andrographolide suspension and Herbosomes with Andrographolide: In-vitro drug release from the Herbosomes was determined using dialysis sacks (retains proteins with molecular weight > 12,000), purchased from Sigma, 250 -7U, USA. The sacks were washed as per the instruction given by the manufacturer. After proper pretreatment, one end of the sack was tied and 1 ml of pure Andrographolide suspension / Herbosomes with Andrographolide / suspension of standardized A. paniculata extract / Herbosomes with standardized A. paniculate extract was placed inside the sack. The other end of the sack was tied and then suspended vertically into a beaker containing 200 ml of phosphate buffer saline (PBS) pH 7.4. The content of the beaker were stirred using a magnetic stirrer at 3711° C, At different time points the sacks were opened and 0.5 ml of sample was withdrawn and amount of drug release was determined spectrophotometrically at 223 nm. Results: It was evident from the result of the in-vitro release study of Andrographolide from Herbosomes with Andrographolide and pure Andrographofde suspension (free as well as in the standardized Andmgraphis paniculata extract) (Figure 10 and 11) that the release of Andrographolide from Herbosomes was sustained and persisted for a longer period of time as compared to free andrographolide and thus proved that the invention resulted in a value added formulation of andrographolide which helped in overcoming the problem of short duration of action of this phytomolecule. Table 3. Level of Serum glutamic -pyruvic transaminase (SGPT) Values are Mean ± SEM (n=10). * P treated group)]. Discussion: Carbon tetrachloride mediated hepatotoxicity was taken here as the experimental model for liver injury as the changes associated with CCI4-induced liver damage are similar to that of acute viral hepatitis (Rubinstein, 1962). CCI4 is accumulated in hepatic parenchymal cells activated metabolically by cytochrome P-450 dependant monooxygenase to form a trichloromethyl free radical (CCI3), which in turn disrupts the structure and function of lipid and protein macromolecules in the cellular membrane of different organs (majumdar et al., 1998) and markedly elevated the level of serum transaminases. In the present study it has been observed that CCI4 intoxication led to a significant increase in the activities of SGOT, SGPT, SALP, LDH and bilirubin. Level of total protein was also lowered. Pretreatment with pure andrographolide or standardized Andrographis paniculata extract or Herbosomes with Andrographolide attenuated these elevated enzyme activitiesproduced by CCI4. Stabilization of serum bilirubin and protein levels through administration of the formulations also clearly indicated the improved functional status of the liver cells. As suggested earlier, the hepatoprotective activity of andrographolide is due to its membrane stabilizing, antioxidant, microsomal enzyme and lipid peroxidation inhibitory activities (Handa and Sharma, 1990a; Choudhury et al., 1987; Handa and Sharma, 1990b). Since andrographolide has high plasma protein binding property and having lower elimination half life as well as mean residence time (Panossian et al., 2000), an effort has been given to formulate a novel dosage form of this molecule to overcome these shortcomings. Thus, the present invention, which deals with the production of Herbosomes with Andrographolide, a novel phospholipid complex of Andrographolide, resulted in better hepatoprotection of rat liver injury caused by CCI4 intoxication. The Herbosomes produced hepatoprotective activity for a longer period of time and normalized the adverse conditions of rat liver more efficiently than the free drug or the extract itself. Even the results showed that the complex at a lower dose (standardized Andrographis paniculata extract Herbosomes equivalent to 20mg/kg of free andrographolide) could exert similar effect like highest dose of free drug (50 mg/kg of andrographolide) on long run. Thus the invention can be helpful in reducing the amount and frequency of administered dose by virtue of its sustained release property. The results obtained from the in-vitro release study also pointed towards the sustained release property of Herbosomes and indicated the possibility of its longer retention inside the body, followed by prolonged duration of action. Based on the above observations it can be concluded that the effects of Herbosomes with Andrographolide at different dose levels persisted for a longer period of time in comparison to the molecule itself, which proves the sustained release property of the novel complex and thus solve the problem of rapid clearance, lower elimination half life associated with andrographolide. The product, according to the invention can be suitable for the treatment of acute or chronic liver diseases of toxic, metabolic and/or infective origin as evident for the preliminary animal experiments. The complex can be used in conventional pharmaceutical dosage forms like pills, capsules or in liquid forms in suspension. Different embodiments of the invention are possible to achieve the best process of performance and to obtain the good quality of product for better effect as stated earlier. It will be understood that skilled persons with many modifications, variations and adaptations may carry out the invention into practice without departing from its sprit or exceeding the scope of claims in describing the invention for the purpose of illustration. Claims: 1. A process of preparing of Herbosomes with Andrographolide (as a prototype molecule), a complex of Andrographolide having belter hepatoprotectrve activity than Andrographolide comprising the reaction of Andrographolide isolated from Andrographis paniculata or Andrographolide in standardized Andrographis paniculata extract with one or more phospholipids, wherein the phospholipid is a member selected from the group consisting of soybean lecithin, egg lecithin phospholipids from bovine or swine brain or dermis, phosphatidyl choline, phosphatidyl serine, phophatidyl ethanolamine in which the acyl groups are same or different, derived from palmitic, stearic, oteic, linoteic, linolenic acid. 2. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein there is equimolar ratio between Andrographolide and phospholipids. 3. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide having better hepatoprotectrve activity than AndrographoNde as claimed in claim 1 wherein phoshatidyl choline is dipalmitovl phosphatidyl choline or dimyristoyl phosphatidyl choline or distearoyl phosphatidyl choline and phosphatidyl ethanolamine is distearoyl phosphatidyl ethanolamine. 4. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein the mixture of Andrographolide and phospholipids in an organic solvent dichloromethane is refluxed over a water bath for 1-5hrs, preferably 2-3hrs at a temperature 40-80°C preferably 55-65°C. 5. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein Andrographolide complex in dichlofomethane solution was then concentrated one-third to its volume in a beaker and a non-polar solvent preferably n-hexane was added with continuous stirring and a complex was precipitated in the reaction mixture. The complex was then filtered and dried under vacuum to remove the traces of hexane. Andrgrapholide herbosomes thus obtained was kept in air-tight container for further evaluation. 6. Herbosomes with Andrographoide, a complex of Andrographofide with phospholipids at equimolar ratio, having better hepatoprotectrve activity than Andrographolide comprising the reaction of Andrographolide isolated from the plant Andrographis paniculata or Andrographolide in standardized Andrographis paniculate extract with one or more phospholipids, wherein the phosphoipid is a member selected from the group consisting of soybean lecithins, egg lecithin phospholipids from bovine or swine brain or denmis, phosphatidyl choline, phosphatidyl serine, phophatidyl ethanolamine in which the acyl groups are same or different, derived from palmitic, stearic, oteic, linoleic, linolenic acid. 7. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in claim 6, wherein phospholipids are dipalmitoyl phosphatidyl choline or dimyristoyl phosphatidyl choline or distearoyi phosphatidyl choline or distearoyl phosphatidyl ethanolamine. 8. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in claim 6, wherein the mixture of Andrographolide and phospholipids in an organic solvent dichloromethane is refluxed over a water bath for 1-5hrs, preferably 2-3hrs at a temperature 40-80°C preferably 55-65°C. 9. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in claim 6, wherein andrographolide complex in dichloromethane solution was then concentrated one third to its volume in a beaker and a non-polar solvent preferably n-hexane was added with continuous stirring and a complex was precipitated in the reaction mixture. The complex was then filtered and dried under vacuum to remove the traces of hexane. Herbosomes with Andrographolide thus obtained was kept in air-tight container for further evaluation. 10. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in claim 6, wherein the herbosomes can produce better therapeutic efficacy than the conventional herbal extracts or the individual phytomolecute. The delivery system of the herbosomes having longer duration of action inside the body can markedly alter the biodistribution of their associated drugs, by increasing the drug concentration at the site of infection and inflammation. 11. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in claim 6, wherein the Herbosomes is suitable for the treatment of acute or chronic liver diseases of toxic, metabolic and /or infective origin as evident from preliminary animal experiments and Herbosomes can be used in pharmaceutical dosage forms like pills, capsules or in liquid forms in suspension. 12. A process of preparing of Herbosomes with Andrographolide (as a prototype molecule), a complex of Andrographolide having better hepatoprotective activity than Andrographolide comprising the reaction of Andrographolide isolated from Andrographis paniculate or Andrographolide in standardized Andrographis paniculate extract with one or more phospholipids, as claimed in the preceding claims substantially herein described. 13. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at equimolar ratio, having increased hepatoprotective activity than Andrographolide comprising the reaction of Andrographolide isolated from the plant Andrographis paniculata or Andrographolide in standardized Andrographis paniculata extract with one or more phospholipids, as claimed in the preceding claims substantially herein described. This invention relates to Herbosomes with Andrographolide, a novel compound comprising lipophilic complexes of Andrographolide with phospholipids and the preparation of these complexes by non- conventional methods. Herbosomes are the concept of value addition to phytoconstituents to develop a specific dosage form with more therapeutic potency than using it as such, which increase the availability of the constituents for a longer time in blood and help in curing the diseases to a higher extant. This Herbosomes is a value added herbal formulation with isolated phytoconstituents having potential hepatoprotective activity. The novel compounds persisted for a longer period of time in plasma and subsequently produced prolonged significant hepatoprotective activity than the pure molecule at the same dose levels. The resulting improvement in the phramacokinetics and pharmacological parameters is such that the compound can be used advantageously in the treatment of acute or chronic liver diseases.

Full Text

The present invention relates to the improved process of preparing of Herbosomes with
Andrographolide, a novel compound obtained by chemicai interaction Detween one or more
phospholipids and pure andrographolide or andrographolide in standardized methano! extract of
Andrographis paniculata, which increased hepatoprotective activity in liver disorders than
Andrographolide alone. Herbosomes are defined as value added herbal formulation with
standardized Botanicals.
DISCUSSION OF THE RELATED ART:
Herbosomes with Andrographolide having better hepatoprotective activity along with other biological
activities are known from prior art:
U.S. Patent No. 4,764,508 describes novel complexes of phospholipids with the main constituents
of silymarin, a known standardized extract obtained from seeds of Silybum marianum and used for
the treatment of liver disease of varying origin.
According to Chinese Patent application CN1552320 an Andrographolide compound (which can be
used as medicine with improved solubility) can be prepared by mixing the Andrographolide with
solubilizer like cyclodextrin, amino acid, phosphatide, water soluble high polymer and vegetable oil,
water or organic cosolvent, mechanical grinding, ultrasonic processing and mixing with medicinal
auxiliary.
Another Chinese Patent application (CN1528290) relates to an andrographolide soft capsule and its
production method. Every soft capsule contains 10-300 mg of andrographolide, and contains bees
wax and medicinal auxiliary material. Said medicinal auxiliary material includes diluting agent,
suspension adjuvant and defoaming agent, and the interior of said capsule shell, contains (by
weight ratio) 10 portions of gelatin and 4.0-5.5 portions of glycerine. Its production process includes
the following steps: adding water to dissolve gelatin, adding glycerine and proper quantity of
preservative, vacuum deaerating, adding suspension adjuvant into vegetable oil, mixing
andrographolide, diluting agent and defoaming agent uniformly, die-pressing to obtain said invented
soft capsule. Its quality is stable, administration is convenient and its effective components can be
quickly resolved out.
The Process of preparation of one kind of soft andrographolide capsule is described in a Chinese
Patent application CN1481792 where this capsule medicine liquid contains enough andrographolide
as its effective component, and proper amount of medicinal supplementary material is added,
including diluent, suspension assistant, antioxidant, preservative, etc. The capsule shell consists of
mainly gelatin, glycerine and water. The capsule of the present invention has stable quality,
convenient administration, fast leaching out of the effective component and timely acting.
An International Patent application WO9617605 relates to the discovery a group of compounds
(which are Andrographolide compounds) isolated from the extract of Andrographis paniculata that
causes the reduction of expression of p34kinase.

Process of preparation of novel derivatives of Andrographolide, their stereo isomers, their
polymorphs, their pharmaceutically acceptable salts and their pharmaceutically acceptable solvates
and their uses as antiviral, anti-malarial, antibacterial, hepatoprotective and immunomodulating
agents and for the treatment of other metabolic disorders are described in U.S Patent applications
US 6,486,196 and US 6,410,590.
Background of the invention:
Now -a- days development of a new molecule with therapeutic potential is very expensive as well
as time consuming, irrespective of the source, it may be natural or synthetic. Sometimes the newly
developed molecule fails clinical trial due to severe toxicity and hardly can come into market as a
therapeutically potent drug molecule. Due to these shortcomings of new drug development, the
researchers worldwide have engaged themselves in the development of novel drug delivery system
for the exiting molecules so as to bring some new therapeutic measures in the treatment of different
types of human ailments. Plants have formed the basis of sophisticated traditional medicine
systems that have been in existence for thousands of years. The plants and phytoconstituents in the
traditional systems of medicine of many countries continue to play an essential role in healthcare.
Thorough research in the field of Herbal Drug Delivery System as a means of improving the
therapeutic indices of drugs is of prime importance. Toxicity, short duration of action and limited
absorption of different drug molecules obtained from herbal sources are particularly difficult
problems in patients undergoing treatment of different diseases, which leads to sub-optimal therapy.
From this view point development of novel herbal dosage form can be of paramount importance.
Liver diseases of different origin remain one of the serious health problems and a major cause of
mortality. In the absence of reliable liver protective drugs in allopathic medical practices, herbs and
plants play an important role in the management of various liver disorders (Trivedi and Rawal,
2000). Numerous literature related to the hepatoprotective activity of phytomolecules from herbal
sources have shown that there is a vast array of phytoconstituents having potent hepatoprotective
efficacy. Andrographolide is one such hepatoprotective herbal molecule. Jt is a wel-known
phytoconstituents obtained from Andrographis paniculata Nees, having very bitter taste. It is a
bicyclic diterpenoid lactone with multiple pharmacological activities. The traditional uses include
treatment of fever, inflammation, common cold, upper respiratory infection, tonsillitis, pharyngitis,
laryngitis, pneumonia, tuberculosis, pyelonephritis, hepatic impairment etc. (Barilla, 1999; Caceres
etal., 1997, 1999; Hancke et al., 1995; Handa and Sharma, 1990; Kapiletal., 1993; Melchior et al.,
1997; Visen et al., 1993). As per reported literature, andrographolide possesses potent
antihyperglycemic (Yu et al 2003), antipyretic (Deng, 1978; Madav et al., 1995; Vedavathy and Rao,
1991), Antiinflammatory (Wang et al 2004, Shen et al 2002), anticancer (Rajagopal et al 2003),
antileishmanial (Sinha et al 2000), antifertility (Akbarsha and Murugaian 2000), anti HIV (Calabrese
et al 2000), cardiovascular (Zhang and Tan 1997), immunomodulatory (Puri et al 1993), Choleretic
(Shukla et al 1992) activities. It also inhibits PAF-induced platelet aggregation (Amroyan et al 1999).

It has got preventive activities against common cold (Caceres et al., 1997, 1999; Hancke et al.,
1995; Melchior et al., 1997; Puri et al., 1993) and has potent antimicrobial (Misra et al., 1992;
Singha et al., 2003; Dutta and Sukul NC, 1982; Raj, 1975; Chander et al., 1995) as well as
antidiarrhoeal activities (Deng, 1978; Gupta et al., 1990). The molecule is also useful in the
management of respiratory tract infection (Coon and Ernst, 2004; Poolsup et al., 2004).
But the most important use of andrographolide is seems to be in the management of various liver
disorders. The liver is subject to acute and potentially lethal injury by several substances including
carbon tetrachloride, phalloidine, galactosamine, ethanol and other compounds (Middleton et al
2000).
In Indian System of Medicine (Ayurveda), about 26 different polyherbal formulations containing
Andrographis paniculata are mentioned as a popular remedy for the treatment of various liver
disorders and an adequate number of pharmacological investigations have substantiated the
therapeutic potential of andrographolide, principal chemical constituent of A. paniculata as a
hepatoprotective agent. Andrographolide showed a significant dose dependent protective activity
against paracetamol-induced toxicity on ex vivo preparation of isolated rat hepatocytes (Visen et al.,
1993). It exhibits protective effects on hepatoxicity induced in mice by carbon tetrachloride or tert-
butyl hydroperoxide (t-BHP) intoxication (Kapil et al., 1993; Handa and Sharma, 1990)
Andrographolide has also been shown to produce ameliorative effect in galactosamine induced
acute hepatitis in rats (Handa and Sharma, 1990) and prevents BHC induced increase in the
activities of enzymes gamma-glutamyl transpeptidase, glutathione-S-transferase and lipid
peroxidation (Trivedi and Rawal, 2001). It is useful in the treatment of infective hepatitis, an acute
inflammatory condition of the liver. All the experimental models used to evaluate the
hepatoprotective activity of andrographolide dealt with the chemicals responsible for damage of liver
by causing lipid peroxidation, a process related to production of free radicals by the chemical attack
lead to destruction of the cellular membrane that surrounded liver cells. So the hepatoprotective
effect provided by andrographolide was attributed to the anti-oxidant ability of the compound, which
scavenges the free radicals generated during hepatotoxin challenge (Kapil et al., 1993). Apart from
antioxidant activity, inhibition of cytochrome P-450, stimulation of hepatic regeneration (Handa and
Sharma, 1990a; Choudhury et al., 1987) and inhibition of microsomal enzymes or lipid peroxidation
(Handa and Sharma, 1990b) could explain the hepatoprotective effect of andrographolide.
According to the reported data in various literature the best protection against the liver intoxicating
agents is obtained in the animal when the compounds are administered intraperitoneally or
intravenously so as rapidly to obtain high concentrations in the bloodstream and the target organ.
But in most of the cases it fails due to poor absorption of the compounds. In some cases, though it
absorbed rapidly but due to faster elimination of the compound the needed therapeutic activity fails
to be achieved. Despite this vast array of pharmacological activities and rapid absorption into the
blood after oral administration, rapid clearance, lower elimination half life, as well as higher plasma

protein binding of andrographolide necessitates the need of some novel compound with same dose
of the conventional dosage form, which can maintain the concentration of andrographolide in blood
for a longer period of time.
Statement of the invention:
The present invention deals with the production of Herbosomes with Andrographolide, a novel
compound obtained by chemical interaction between one or more phospholipids and pure
andrographolide or andrographolide in standardized methanol extract of Andrographis paniculate by
some non-conventional methods.
Herbosomes with Andrographolide are advanced form of herbal products containing
andrographolide or standardized Andmgraphis paniculata extract and the phospholipids, having
better utilization profiles in our body and subsequently produce better therapeutic efficacy than the
conventional herbal extracts or the individual phytomotecule. This delivery system, having longer
duration of action inside the body, can markedly alter the biodtstribution of their associated drugs, by
increasing the drug concentration at the site of infection and inflammation.
The invention deals with
i) Preparation of Herbosomes with Andrographolide
ii) Physicochemical evaluation of Herbosomes with Andrographolide
iii) Hepatoprotective activity of Herbosomes with Andrographolide in carbon tetrachloride
intoxicated rats in comparison to pure Andrographolide and
iv) In-vitro release study of Andrographolide from pure Andrographolide suspension and
Herbosomes with Andrographolide
Brief description of accompanying drawings:
Figure 1. HPTLC fingerprint of pure andrographolide;
Figure 2. HPTLC fingerprint of andrographolide from Herbosomes with Andrographolide;
Figure 3. In-vitro release of Andrographolide vs. time from Herbosomes with Andrographolide and
pure Andrographolide suspension; and
Figure 4. In-vitro release of Andrographolide vs. time from Herbosomes with standardized
Andrographis paniculata extract and standardized Andrographis paniculata extract suspension

Detailed description of the invention:
This invention is explained in more detail as set forth hereunder
1. Collection of plant materials and chemicals:
Aerial parts of Andrographis paniculate Nees were collected locally and authenticated from
Botanical Survey of India, Shibpur, West Bengal, India. Pure andrographofide was obtained from
Natural Remedies Pvt. Ltd., Bangalore, India as gift sample. The aerial parts of Andrographis
paniculata Nees were extracted as per reported method (Handa and Sharma, 1990b) to get
standardized Andrographis paniculata Nees extract (Standardized to contain 10% w/w
andrographolide). Phospholipids like 1,2-Distearoyl-sn-Glycero-3-Phosphocholfne (DSPC), 1,2-
Dimyristoyl-sn-Glycero-3-Phosphochoine (DMPC), 1,2-Oipaimitoy(-sn-Glycero-3-Phosphochotine
(DPPC), 1,2-Distearoyl-sn-Glycero-3-hosphoethanolarnine (DSPE) were obtained from Lipoid
GMbH, Germany. Dichloromethane, n-hexane and other solvents were obtained from Qualigens,
Mumbai.

2. Preparation of Herbosomes with Andrographolide:
Herbosomes with Andrographolide were prepared by a novel method of complexation using
i) Pure Andrographolide
ii) Andrographolide in standardized Andrographis paniculata extract (Standardized to
contain 10% w/w andrographolide) and phospholipids.
The phospholipids used in the preparations can be natural or synthetic. Preferably used is made of
phospholipids from vegetable origin (soy phospholipids) such as 1,2-Distearoyl-sn-Glycero-3-
Phosphocholine (DSPC) (Lipoid KG - Ludwigshafen, Germany).
According to the invention, in one embodiment the novel complex was prepared by reacting 0.5
mole to 10 moles, but preferably 1 mole of a natural or synthetic phospholipids, wherein the
phospholipid is a member selected from the group consisting of soybean lecithins, egg lecithin
phospholipids from bovine or swine brain or dermis, phosphatidyl choline, phosphatidyl serine,
phophatidyl ethanolamine in which the acyl groups are same or different, derived from palmitic,
stearic, oleic, linoleic, linolenic acid with one mole of andrographolide, either alone or in the
standardized A. paniculata extract, in aprotic organic solvents such as dioxane, dichloromethane or
acetone, from which the complexes can be isolated by precipitation with non-polar solvents such as
aliphatic hydrocarbons or by lyophilization or by spray drying.
In the preferred embodiment the novel complex was prepared by the reaction of one mole
Andrographolide isolated from the plant Andrographis paniculata or Andrographolide in
standardized Andrographis paniculata extract (10% w/w) with weighed amount of one or more
phospholipids. In the reaction process weighed amount of andrographolide and weighed amount of
dipalmitoyl phosphatidyl choline (DPPC) or dimyristoyl phosphatidyl choline( DMPC) or distearoyl
phosphatidyl choline (DSPC) was taken in a round bottom flask and dissolved in dichloromethane.
The resulting suspension mixture was then refluxed over a water bath for 2-3hrs at a temperature
not exceeding 80°C preferably at 55-60°C. All andrographolide thus goes to the solution and the
resultant solution was then concentrated one third to its volume in a beaker and a non-polar solvent
preferably n-hexane was added with continuous stirring and a complex was precipitated in the
reaction mixture. The complex was then filtered and dried under vaccum to remove the traces of
hexane. Andrgrapholide herbosomes thus obtained was kept in air-tight container for further
evaluation.
The method of preparing Herbosomes with Andrographolide can be represented schematically.
Schematic diagram of Andrographolide Herbosomes formation

3. Physicochemical evaluation of Herbosomes with Andrographolide:
The complex of andrographolide with phospholipids i.e. Herbosomes with Andrographolide were
evaluated in terms of physicochemical properties and compared with that of pure andrographolide.
The invention discloses that the starting material andrographolide, which is insoluble or sparingly
soluble in chloroform, dichloromethane, ethyl ether or benzene, becomes extremely soluble in these
solvents after forming the Herbosomes. This change in chemical and physical properties is due to
the formation of a complex, as is clear from the modification in different parameters like texture,
melting point, specific rotation, solubility etc (Table 1).

Table 1. Comparative evaluation of macroscopic characteristics of andrographolide and
Herbosomes with Andrographolide

The microscopic view of the pure andrographolide shows prismatic crystals but the Herbosomes
when suspended in water and observed under microscope it shows vesicular structures. The
vesicles consist of phospholipids and the andrographolide is present in the lipid bi-layer as
intercalated form.
The chemical characteristics of the complex and pure andrographolide were evaluated in terms of
High performance Thin Layer Chromatography (HPTLC) using Camag TLC scanner 3.
Development of chromatogram
Herbosomes with Andrographolide and free andrographolide solutions were spotted on the Silica
Gel 6OF254 pre-coated TLC plates and chromatogram was developed in chromatographic chambers
using specific solvent system at a room temperature of 30° C and at an angle of 70°.
In this experiment, the mobile phase (solvent system) was allowed to run up to a distance of 10 cm
from the origin. The time required for the development of chromatogram differed from 20 to 30
minutes. The plates were removed from the chamber after completion of the run and were allowed
to dry in air. The plates were observed under UV light for the appearance of spots. The plates were
scanned with the help of the scanner and the hRf values of the spots were recorded. The result has
been furnished hereunder (Table 2, Fig.1 & Fig.2).
Reference Solutions: Standard compound is prepared as 0.05% solutions in methanol and about
10ul is used for chromatography. The average detection limit is 5-10μg.

Solvent system used: Chloroform: Methanol
7:1
Table 2. Comparative High performance thin layer chromatographic study of free
andrographolide and Herbosomes with Andrographolide.

Spectroscopic evaluation:
UV Spectroscopic study of Pure Andrographoide and Herbosomes with Andrographolide:
UV-visible spectroscopic analysis of pure andrographoide and Herbosomes with Andrographolide
has been performed and it was observed that pure andrographolide has λmax at 223.7 nm in
methanol. On contrary, Herbosomes with Andrographolide showed λmax at 229.7 nm in methanol.
This change in λmax is due to formation of the complex.
IR Spectroscopic study of Pure Andrographoide and Herbosomes with Andrographolide:
IR spectrum of Andrographolide (Figure 4) shows occurrence of different bands at various wave
numbers due to the presence of numbers of functional groups. But in case of the IR spectrum of
Herbosomes with Andrographolide (Figure 5), broadening or shifting of these bands and in some
cases disappearance of bands due to formation of the complex was observed. The bands appeared
at λ = 3827.41 cm1 and λ = 3398.68 cm1 in the IR spectrum of Andrographolide were due to O-H
stretching of furan ring and O-H stretching of naphthalene ring, respectively, but those bands
disappeared in the spectrum of Herbosomes because of the interaction with the polar head group of
phospholipids, which indicates formation of a complex.
400MHz 1H-NMR study of Pure Androoraoriolide and Herbosomes with Androorapholide:
In the 1H-NMR spectrum of Andrographolide it was observed that there are number of peaks
appeared at specific 5-values due to presence of various functional groups. Peaks, those appeared
from 5-3.7 to 5 - 5.2 in the spectrum of Herbosomes with Andrographolide became suppressed due
to formation of H-bonds between the phenolic hydroxyl group of andrographolide

and the phosphate ion of the phospholipids. This can be deduced from the comparison of the NMR
of the Herbosomes with that of pure Andrographolide. The signals of the fatty chain are almost
unchanged. Such evidences inferred that the two long aliphatic chains of the phospholipid were
wrapped around the active principle, producing a lipophilic envelop, which shielded the polar head
of the phospholipid and andrographolide.
Differential Scanning Calorimetric study of Pure Andrographolide and Herbosomes with
Andrographolide
Differential Scanning Calorimetric (DSC) is a fast and reliable method to screen drug - excipient
compatibility and provides maximum information about the possible interactions. In DSC, an
interaction is concluded by elimination of endothermic peak (s), appearance of new peak (s),
change in peak shape and its onset, peak temperature / melting point and relative peak area or
enthalpy. A sharp peak with a maximum occurrence at 235.23°C was appeared in the DSC
thermogram of Pure Andrographolide. No additional endothermic or exothermic peaks were
observed. In the DSC thermogram of Herbosomes with Andrographolide it was observed that the
peak at 235.23°C was absent and two new peaks with maximum occurrence at 69.05°C and
85.57°C were appeared. The absence of original peak of Andrographolide and the appearance of
two new peaks in the thermogram of Herbosomes indicated the origination of a new chemical entity.
Thus the spectral data obtained from UV, IR and 1H-NMR study of Herbosomes with
Andrographolide and pure Andrographolide showed a proof of complexation between the molecule
and phospholipid. The thermograms obtained from the Differential Scanning Calorimetric (DSC)
analysis of the complex and free molecule also strengthen the proof of complexation.
From the above physicochemical studies of both free andrographolide and Herbosomes with
Andrographolide, it was observed that physical as well as chemical properties of free
andrographolide are different from Herbosomes with Andrographolide. Physical properties like
melting point, solubility, optical rotation, chemical property like Rf value obtained from HPTLC study
clearly indicating that andrographolide gone into complexation with phospholipids and resulted in a
new chemical entity with different physicochemical properties. The microscopic view of the complex
and the spectral as well as thermal analyses also support the claim of complexation strongly.
4. Evaluation of hepatoprotective activity of Herbosomes with Andrographolide:
To prove the therapeutic superiority of Herbosomes with Andrographolide over pure
Andrographolide, the hepatoprotective activity of both pure Andrographolide and Herbosomes with
Andrographolide was evaluated in carbon tetrachloride -intoxicated albino rats.

Test samples:
Pure andrographolide (25 mg/kg body wt., p.o.)
Pure andrographolide (50 mg/kg body wt., p.o.)
Herbosomes with Andrographolide (Equivalent to pure andrographolide 25 mg/kg body wt., p.o.)
Herbosomes with Andrographolide (Equivalent to pure andrographolide 50 mg/kg body wt., p.o.).
Standardized Andrographis paniculata extract 200 mg/kg body wt., p.o. (Standardized to contain
10% w/w andrographolide, i.e. 20mg/kg pure andrographolide)
Standardized Andrographis paniculata extract Herbosomes (Equivalent to Standardized
Andrographis paniculata extract 200 mg/kg body wt., p.o.)
Tested biochemical parameters:
Serum glutamic -oxaloacetic transaminase (SGOT),
Serum glutamic -pyruvic transaminase (SGPT),
Serum alkaline phosphatase (SALP),
Direct and total bilirubin,
Total Protein (TP)
Lactate dehydrogenase (LDH).
Animals:
In bred male albino rats (Wistar strain) weighing 180-200 g were used for this study. Animals were
housed in groups of 7 - 8 in colony cages at an ambient temperature of 20-25° C and 45 - 55 %
relative humidity with 12 hrs light / dark cycles. They had free access to pellet chow (Brook Bond,
Lipton India) and water ad libitum. The experimentation on animals was performed based on the
observations of animal ethical committee.
Dosing:
The adult male Wistar rats were divided into eight groups of ten animals each. The animals in
different groups have received different substances as per the following protocol.

Group I
Received only distilled water with 0.3% CMC p.o. for seven days and served as normal.
Group II (CCI4 - intoxicated)
Received distilled water with 0.3% CMC p.o. for seven days and equal mixture of carbon
tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day.
Group III (Andrographolide 25 mg/kg body wt + CCI4)
Animals were treated with andrographolide suspension in distilled water with 0.3% CMC at a dose
level of 25 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon
tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day.
Group IV [Herbosomes with Andrographolide (equivalent to andrographolide 25 mg/kg body
wt.) + CCI4]
Animals were treated with Herbosomes with Andrographolide at a dose level of 25 mg/kg, p.o., from
1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil
(50% v/v, 1 ml/kg i.p.) on 7th day.
Group V (Andrographolide 50 mg/kg body wt. + CCI4)
Animals were treated with andrographolide suspension in distilled water with 0.3% CMC at a dose
level of 50 mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon
tetrachloride and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day.
Group VI [Herbosomes with Andrographolide (equivalent to andrographolide 50 mg/kg body
wt.) + CCI4]
Animals were treated with Herbosomes with Andrographolide at a dose level of 50 mg/kg, p.o., from
1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil
(50% v/v, 1 ml/kg i.p.) on 7th day.
Group VII [Standardized Andrographis paniculata extract 200 mg/kg body wt. (Standardized
to contain 10% w/w andrographolide, i.e. 20mg/kg pure andrographolide) + CCI4]
Animals were treated with Standardized Andrographis paniculata extract at a dose level of 200
mg/kg, p.o., from 1st to 7th day for seven consecutive days and equal mixture of carbon tetrachloride
and olive oil (50% v/v, 1 ml/kg i.p.) on 7th day.

Group VIII [Standardized Andrographis paniculata extract Herbosomes (Equivalent to
Standardized Andrographis paniculata extract 200 mg/kg body wt.) + CCI4]
Animals were treated with Standardized Andrographis paniculata extract Herbosomes at a dose
level equivalent to 200 mg/kg of Standardized Andrographis paniculata extract, p.o., from 1st to 7th
day for seven consecutive days and equal mixture of carbon tetrachloride and olive oil (50% v/v, 1
ml/kg i.p.) on 7th day.
On the eighth, ninth and tenth day, blood samples were collected from the retro orbital plexus and
centrifuged at 2000g for 10 minutes at 4°C to separate the serum and kept at 4°C to assay the
activities of the serum enzymes.
Assessment of liver function:
The liver function test of the animals were performed by analyzing the level of serum enzymes
namely Serum glutamic -oxaloacetic transaminase (SGOT), Serum glutamic -pyruvic transaminase
(SGPT), Serum alkaline phosphatase (SALP), Lactate dehydrogenase (LDH) and the amount of
total and direct bilirubin as well as Total Protein (TP).
Levels of SGOT and SGPT in the blood serum of the experimental animals were estimated on day
8, 9 and 10 as per the method of Reitman and Frankel (1957). Serum alkaline phosphatase (SALP)
was estimated according to the method of Kind and King (1954). Lactate dehydrogenase (LDH) was
measured by the method of Wroblewski and LaDue as described by Varley (1967). Serum bilirubin
and protein level were estimated by the method of Malloy and Evelyn (1937) and Lowry et al., 1951,
respectively.
Results:
Hepatic damage induced by carbon tetrachloride caused significant rise in the serum level of marker
enzymes like SGOT, SGPT, SALP and LDH and also increased the level of serum bilirubin (both
direct and total). It also lowered the level of total protein. Oral administration of andrographolide,
either pure or in the standardized Andrographis paniculata extract, reduced the elevated level of the
enzymes and bilirubin and increased the reduced protein level. Herbosomes with Andrographolide
also exhibited protective activity as evident from the experimental results. It was observed that the
formulation produced better hepatoprotection for a longer period of time as compared to respective
free or pure andrographolide (Table 3-9). It is also evident from the result that in case of all the
marker enzymes, the Herbosomes, even at the lowest dose (group VIII, Standardized Andrographis
paniculata extract Herbosomes (Equivalent to Andrographis paniculata extract 200 mg/kg body wt.,
standardized to contain 20mg/kg of pure andrographolide) produced better protective activity than
the highest dose of pure andrographolide (50mg/kg body weight), at the end of the study (10th day).
It proved that the Herbosomes acted for a longer period of time inside the body, which was further
established with the help of in-vitro release study of andrographolide.

5. In-vitro release study of Andrographolide from pure Andrographolide suspension and
Herbosomes with Andrographolide:
In-vitro drug release from the Herbosomes was determined using dialysis sacks (retains proteins
with molecular weight > 12,000), purchased from Sigma, 250 -7U, USA. The sacks were washed as
per the instruction given by the manufacturer. After proper pretreatment, one end of the sack was
tied and 1 ml of pure Andrographolide suspension / Herbosomes with Andrographolide / suspension
of standardized A. paniculata extract / Herbosomes with standardized A. paniculate extract was
placed inside the sack. The other end of the sack was tied and then suspended vertically into a
beaker containing 200 ml of phosphate buffer saline (PBS) pH 7.4. The content of the beaker were
stirred using a magnetic stirrer at 3711° C, At different time points the sacks were opened and 0.5
ml of sample was withdrawn and amount of drug release was determined spectrophotometrically at
223 nm.
Results:
It was evident from the result of the in-vitro release study of Andrographolide from Herbosomes with
Andrographolide and pure Andrographofde suspension (free as well as in the standardized
Andmgraphis paniculata extract) (Figure 10 and 11) that the release of Andrographolide from
Herbosomes was sustained and persisted for a longer period of time as compared to free
andrographolide and thus proved that the invention resulted in a value added formulation of
andrographolide which helped in overcoming the problem of short duration of action of this
phytomolecule.
Table 3. Level of Serum glutamic -pyruvic transaminase (SGPT)

Values are Mean ± SEM (n=10). * P treated group)].

Discussion:
Carbon tetrachloride mediated hepatotoxicity was taken here as the experimental model for liver
injury as the changes associated with CCI4-induced liver damage are similar to that of acute viral
hepatitis (Rubinstein, 1962). CCI4 is accumulated in hepatic parenchymal cells activated
metabolically by cytochrome P-450 dependant monooxygenase to form a trichloromethyl free
radical (CCI3), which in turn disrupts the structure and function of lipid and protein macromolecules
in the cellular membrane of different organs (majumdar et al., 1998) and markedly elevated the level
of serum transaminases.
In the present study it has been observed that CCI4 intoxication led to a significant increase in the
activities of SGOT, SGPT, SALP, LDH and bilirubin. Level of total protein was also lowered.
Pretreatment with pure andrographolide or standardized Andrographis paniculata extract or
Herbosomes with Andrographolide attenuated these elevated enzyme activitiesproduced by CCI4.
Stabilization of serum bilirubin and protein levels through administration of the formulations also
clearly indicated the improved functional status of the liver cells. As suggested earlier, the
hepatoprotective activity of andrographolide is due to its membrane stabilizing, antioxidant,
microsomal enzyme and lipid peroxidation inhibitory activities (Handa and Sharma, 1990a;
Choudhury et al., 1987; Handa and Sharma, 1990b).
Since andrographolide has high plasma protein binding property and having lower elimination half
life as well as mean residence time (Panossian et al., 2000), an effort has been given to formulate a
novel dosage form of this molecule to overcome these shortcomings. Thus, the present invention,
which deals with the production of Herbosomes with Andrographolide, a novel phospholipid complex
of Andrographolide, resulted in better hepatoprotection of rat liver injury caused by CCI4 intoxication.
The Herbosomes produced hepatoprotective activity for a longer period of time and normalized the
adverse conditions of rat liver more efficiently than the free drug or the extract itself. Even the
results showed that the complex at a lower dose (standardized Andrographis paniculata extract
Herbosomes equivalent to 20mg/kg of free andrographolide) could exert similar effect like highest
dose of free drug (50 mg/kg of andrographolide) on long run.
Thus the invention can be helpful in reducing the amount and frequency of administered dose by
virtue of its sustained release property. The results obtained from the in-vitro release study also
pointed towards the sustained release property of Herbosomes and indicated the possibility of its
longer retention inside the body, followed by prolonged duration of action. Based on the above
observations it can be concluded that the effects of Herbosomes with Andrographolide at different
dose levels persisted for a longer period of time in comparison to the molecule itself, which proves

the sustained release property of the novel complex and thus solve the problem of rapid clearance,
lower elimination half life associated with andrographolide.
The product, according to the invention can be suitable for the treatment of acute or chronic liver
diseases of toxic, metabolic and/or infective origin as evident for the preliminary animal
experiments. The complex can be used in conventional pharmaceutical dosage forms like pills,
capsules or in liquid forms in suspension.
Different embodiments of the invention are possible to achieve the best process of performance and
to obtain the good quality of product for better effect as stated earlier. It will be understood that
skilled persons with many modifications, variations and adaptations may carry out the invention into
practice without departing from its sprit or exceeding the scope of claims in describing the invention
for the purpose of illustration.

Claims:
1. A process of preparing of Herbosomes with Andrographolide (as a prototype molecule), a
complex of Andrographolide having belter hepatoprotectrve activity than Andrographolide
comprising the reaction of Andrographolide isolated from Andrographis paniculata or
Andrographolide in standardized Andrographis paniculata extract with one or more
phospholipids, wherein the phospholipid is a member selected from the group consisting of
soybean lecithin, egg lecithin phospholipids from bovine or swine brain or dermis,
phosphatidyl choline, phosphatidyl serine, phophatidyl ethanolamine in which the acyl
groups are same or different, derived from palmitic, stearic, oteic, linoteic, linolenic acid.
2. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide
having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein
there is equimolar ratio between Andrographolide and phospholipids.
3. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide
having better hepatoprotectrve activity than AndrographoNde as claimed in claim 1 wherein
phoshatidyl choline is dipalmitovl phosphatidyl choline or dimyristoyl phosphatidyl choline or
distearoyl phosphatidyl choline and phosphatidyl ethanolamine is distearoyl phosphatidyl
ethanolamine.
4. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide
having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein
the mixture of Andrographolide and phospholipids in an organic solvent dichloromethane is
refluxed over a water bath for 1-5hrs, preferably 2-3hrs at a temperature 40-80°C preferably
55-65°C.
5. A process of preparing of Herbosomes with Andrographolide, a complex of Andrographolide
having better hepatoprotectrve activity than Andrographolide as claimed in claim 1 wherein
Andrographolide complex in dichlofomethane solution was then concentrated one-third to its
volume in a beaker and a non-polar solvent preferably n-hexane was added with continuous
stirring and a complex was precipitated in the reaction mixture. The complex was then
filtered and dried under vacuum to remove the traces of hexane. Andrgrapholide
herbosomes thus obtained was kept in air-tight container for further evaluation.
6. Herbosomes with Andrographoide, a complex of Andrographofide with phospholipids at
equimolar ratio, having better hepatoprotectrve activity than Andrographolide comprising the
reaction of Andrographolide isolated from the plant Andrographis paniculata or

Andrographolide in standardized Andrographis paniculate extract with one or more
phospholipids, wherein the phosphoipid is a member selected from the group consisting of
soybean lecithins, egg lecithin phospholipids from bovine or swine brain or denmis,
phosphatidyl choline, phosphatidyl serine, phophatidyl ethanolamine in which the acyl
groups are same or different, derived from palmitic, stearic, oteic, linoleic, linolenic acid.
7. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in
claim 6, wherein phospholipids are dipalmitoyl phosphatidyl choline or dimyristoyl
phosphatidyl choline or distearoyi phosphatidyl choline or distearoyl phosphatidyl
ethanolamine.
8. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in
claim 6, wherein the mixture of Andrographolide and phospholipids in an organic solvent
dichloromethane is refluxed over a water bath for 1-5hrs, preferably 2-3hrs at a temperature
40-80°C preferably 55-65°C.
9. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in
claim 6, wherein andrographolide complex in dichloromethane solution was then
concentrated one third to its volume in a beaker and a non-polar solvent preferably n-hexane
was added with continuous stirring and a complex was precipitated in the reaction mixture.
The complex was then filtered and dried under vacuum to remove the traces of hexane.
Herbosomes with Andrographolide thus obtained was kept in air-tight container for further
evaluation.
10. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in
claim 6, wherein the herbosomes can produce better therapeutic efficacy than the
conventional herbal extracts or the individual phytomolecute. The delivery system of the
herbosomes having longer duration of action inside the body can markedly alter the
biodistribution of their associated drugs, by increasing the drug concentration at the site of
infection and inflammation.
11. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having better hepatoprotective activity than Andrographolide as claimed in
claim 6, wherein the Herbosomes is suitable for the treatment of acute or chronic liver
diseases of toxic, metabolic and /or infective origin as evident from preliminary animal

experiments and Herbosomes can be used in pharmaceutical dosage forms like pills,
capsules or in liquid forms in suspension.
12. A process of preparing of Herbosomes with Andrographolide (as a prototype molecule), a
complex of Andrographolide having better hepatoprotective activity than Andrographolide
comprising the reaction of Andrographolide isolated from Andrographis paniculate or
Andrographolide in standardized Andrographis paniculate extract with one or more
phospholipids, as claimed in the preceding claims substantially herein described.
13. Herbosomes with Andrographolide, a complex of Andrographolide with phospholipids at
equimolar ratio, having increased hepatoprotective activity than Andrographolide comprising
the reaction of Andrographolide isolated from the plant Andrographis paniculata or
Andrographolide in standardized Andrographis paniculata extract with one or more
phospholipids, as claimed in the preceding claims substantially herein described.

This invention relates to Herbosomes with Andrographolide, a novel compound comprising lipophilic
complexes of Andrographolide with phospholipids and the preparation of these complexes by non-
conventional methods. Herbosomes are the concept of value addition to phytoconstituents to
develop a specific dosage form with more therapeutic potency than using it as such, which increase
the availability of the constituents for a longer time in blood and help in curing the diseases to a
higher extant. This Herbosomes is a value added herbal formulation with isolated phytoconstituents
having potential hepatoprotective activity. The novel compounds persisted for a longer period of
time in plasma and subsequently produced prolonged significant hepatoprotective activity than the
pure molecule at the same dose levels. The resulting improvement in the phramacokinetics and
pharmacological parameters is such that the compound can be used advantageously in the
treatment of acute or chronic liver diseases.

Documents:

56-KOL-2006-FORM 27 1.1.pdf

56-KOL-2006-FORM 27.pdf

56-kol-2006-granted-abstract.pdf

56-kol-2006-granted-claims.pdf

56-kol-2006-granted-correspondence.pdf

56-kol-2006-granted-description (complete).pdf

56-kol-2006-granted-drawings.pdf

56-kol-2006-granted-examination report.pdf

56-kol-2006-granted-form 1.pdf

56-kol-2006-granted-form 18.pdf

56-kol-2006-granted-form 2.pdf

56-kol-2006-granted-form 26.pdf

56-kol-2006-granted-form 3.pdf

56-kol-2006-granted-others.pdf

56-kol-2006-granted-reply to examination report.pdf

56-kol-2006-granted-specification.pdf

« Previous Patent

Next Patent »

Patent Number

234595

Indian Patent Application Number

56/KOL/2006

PG Journal Number

24/2009

Publication Date

12-Jun-2009

Grant Date

10-Jun-2009

Date of Filing

19-Jan-2006

Name of Patentee

KAKALI MUKHERJEE

Applicant Address

SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA

Inventors:

#	Inventor's Name	Inventor's Address
1	PULOK K MUKHERJEE	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
2	BISHNU PADA SAHA	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
3	KAKALI MUKHERJEE	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
4	KUNTAL MAITI	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
5	KUNTAL MAITI	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
6	KAKALI MUKHERJEE	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
7	PULOK K MUKHERJEE	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032
8	BISHNU PADA SAHA	SCHOOL OF NATURAL PRODUCT STUDIES, DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, JADAVPUR UNIVERSITY, KOLKATA 700 032

PCT International Classification Number

A61K 31/704

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA