WO2007099560A2

WO2007099560A2 - Method for identification and quantification of polyherbal formulation ingredients

Info

Publication number: WO2007099560A2
Application number: PCT/IN2007/000080
Authority: WO
Inventors: Sateesh Kumar Chauhan; Deepak Bahri
Original assignee: Promed Exports Pvt. Ltd.
Priority date: 2006-03-02
Filing date: 2007-03-01
Publication date: 2007-09-07
Also published as: WO2007099560A3

Abstract

The present invention provides a novel method useful for identifying and then quantifying individual active ingredients derived from natural source from poly herbal formulation, said method comprising of converting the individual active ingredients into pellets/spansules, optionally with pharmaceutically active excipients, coating the pellets of individual ingredients with acceptable food colour distinct from one another, mixing these different color pellets as per the formulation which can be filled either in hard gelatin capsule or can be suspended in syrup or may be dispensed in the form of granules only. The different colour pellets from the formulation can be visually separated with naked eye or with the help of magnifying lens from one another; thus provides physical separation of individual ingredients from poly herbal formulation, which can be weighted and divided by the factor equivalent to the excipients. Further confirmation (qualitative or quantitative) may be established by different analytical means as already known in the art such as solvent extractive values, spectrophotometer, chromatography, titrimetry, gravimetric, etc.

Description

METHOD FOR IDENTIFICATION AND QUANTIFICATION OF POLYHERBAL FORMULATION INGREDIENTS Field of the invention

The present invention relates to a method for the identification of individual ingredients in formulations based on animal, marine, semi-synthetic or biotechnological ingredients. More particularly, the present invention relates to a method for the identification of individual ingredients present in polyherbal formulations. In particular, the present invention relates to a novel method by which all input herbal ingredients can be identified and analysed qualitatively and quantitatively from a poly herbal formulation used from different diseased profile in capsule or liquid dosage forms which are to be used for preventive or curative purpose, prepared as a drug or dietary supplements for human, veterinary or agriculture use. Background of the invention

Modern medical and pharmaceutical sciences typically attempt to treat disease by use of a single, highly purified and well-characterized pharmaceutical compound whose activity has been carefully measured so that a precise dosage may be administered. The use of such highly purified drugs facilitates the manufacture of uniform dosage forms because drug concentration can simply and accurately be used to predict the appropriate pharmacological dosage. However, it is also recognized that in several instances, the use of a single highly purified drug is not always appropriate or desirable. In some instances, nutrients and/or pharmacologically active compounds may act together, in synergy. For example, research indicates that several constituents in saw palmetto extracts operate in synchrony to inhibit proliferation of cells in benign prostatic hyperplasia (BHP).

The most frequent reason men consult a urologist is because of an impairment in urinary flow. In men over 45 years of age, the cause of impaired urinary flow is often benign prostatic hyperplasia (BHP). The cause of BHP is an abnormal, but nonmalignant, proliferation of cells and tissues within the prostate gland. Eventually, urethral obstruction leads to urinary retention, kidney damage, and infection. In advanced stages, surgical resection is the treatment of choice. To understand how medications may affect BPH, medical researchers seek the mechanism(s) that are believed to cause the condition. In the prostate, testosterone from the blood is converted by an enzyme to the more potent androgen, dihydrotestosterone (DHT). DHT increases the expression of proteins with resultant changes in cell metabolism and proliferation. In the process of normal growth, sex accessory organs are relatively insensitive to testosterone and DHT after puberty. However, in hyperplastic prostatic tissues the concentrations of DHT may be four to six times those of normal prostatic tissue. Thus, researchers infer that these high concentrations of DHT result in increased growth of the gland in mature males. Drugs have been developed to reduce the effects of androgens, for example, estrogens. However, while estrogens do reduce the effect of androgens they cause feminization, impotence, and cardiovascular toxicity in men—side effects which are highly undesirable.

In addition to androgenic stimulation, infiltration of prostate by inflammatory cells is an etiologic factor in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils, produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, derivatives of arachidonic acid have been extensively studied. Thus, medical research indicates that the best therapeutic regimen for BHP would address both androgenic and inflammatory mechanisms. Several plants contain compounds with antiandrogenic and anti-inflammatory properties, for example, saw palmetto which consists of the partially dried, ripe fruit of Serenoa repens. Saw palmetto was recognized as a "drug" in the United States from 1906 to 1950 and was once widely used for a variety of ailments, particularly those of the urogenital tract, until losing popularity in the United States after World War II. European scientists continued to study saw palmetto and recognized that, in patients suffering form BPH, an extract of the fruit produced increased urinary flow, reduced residual urine, increased ease in commencing micturition, and decreased frequency of urination.

While extensive clinical and laboratory studies have been undertaken and reported, the mechanism of action of saw palmetto is poorly understood. Studies have shown that a liposterolic extract of the berries reduced cellular uptake of both testosterone and DHT by more than 40 percent. This mechanism is confirmed by the observation that saw palmetto extract does not induce changes in the level of testosterone, or other hormones, in the plasma. Other studies indicate that an extract of saw palmetto reduces the conversion of less active testosterone to the more active DHT by inhibiting the enzyme 5 α reductase. In addition to their antiandrogenic properties, saw palmetto berries may also have anti-inflammatory activity. This could be due to inhibition of the cyclooxygenase and 5- lipoxygenase pathways, thereby preventing the biosynthesis of inflammation-producing prostaglandins and leukotrienes. Together, the antiandrogenic and anti-inflammatory effects seem to account for the beneficial role of the herb in treating BPH. Placebo-controlled, double-blind clinical studies carried out on more than 2,000 BPH patients in Germany have confirmed the effectiveness of a saw palmetto extract in such conditions.

A large number of possibly active ingredients have been isolated from saw palmetto including large amounts of beta-sitosterol-3-D-glucoside. Anthranilic acid, caffeic acid, chlorogenic acid, tannin, sugars, and polysaccharides, are also present. Unfortunately, the active antiandrogenic principles remain unidentified, although they are known to reside in the acidic lipophilic fraction of the berries. The inability to identify a single, active compound indicates that a combination of ingredients may be responsible for the beneficial activities of saw palmetto. Alteration of the combination through purification of single ingredients results in a concomitant loss of the original biological activity.

However, the pharmaceutical industry generally relies upon purifying and quantifying an active ingredient in order to standardize preparation of medications.

Some methods for detecting or identifying certain types of molecules have been developed which utilize phosphorylated proteins. For example, U.S. Pat. No. 5,496,703 to Babish et al., issued Mar. 5, 1996, discloses methods for detecting dioxins by exposing mammalian cells to a test sample which may contain such dioxins, examining a cell lysate of the mammalian cells for the presence of phosphorylated proteins and correlating the phosphorylation level of those phosphorylated cells with a standard set of phosphorylated proteins derived from mammalian cells known to be exposed to dioxin or dioxin-like molecules. U.S. Pat. No. 5,695,944 to Croce et al., issued Dec. 9, 1997, is directed to a method of identifying compounds that modulate bcl-2 mediated cell death by contacting a cell with a test compound and detecting whether the bcl-2 in the cell is phosphorylated at a higher or lower level than a control cell which is not contacted with the test compound. U.S. Pat. No. 5,672,470 to Hengstenberg et al., issued Sep. 30, 1997, is drawn to a method for detecting toxic substances, by incubating a sample with a suspension of bacteria which have a phosphotransferase system so that a substrate analog is phosphorylated and then hydrolyzed by an enzyme in the phosphotransferase system. U.S. Pat. No. 5,618,677 to Ni et al., issued Apr. 8, 1997, is directed to a method of testing whether a compound modulates a phosphate cotransporter protein by exposing a cell with the human inorganic phosphate cotransporter protein to a test compound and measuring a change in inorganic phosphate uptake relative to a control cell which is not exposed to the test compound. U.S. Pat. No. 4,871,661 to Webb et al., issued Oct. 3, 1989, is directed to a method of screening materials for carcinogenic properties by administering those materials to test animals and observing whether the animal produces a 60 kilodalton cancer-associated phosphoprotein. US Patent 6780596 discloses a method by which biological or pharmacological activity of a test material like a plant or herbal material, an extract of a plant or herbal material, a natural or synthetic compound or some combination thereof, can be quantified by observing the pattern of structural changes induced in a eukaryotic cell's proteins. These structural changes may be evidenced by protein phosphorylation, by protein-protein interactions and the like. The amount and nature of protein phosphorylation is qualitatively and quantitatively related to the in vitro concentration of biologically/pharmacologically active components to which mammalian cells are exposed. Additionally, formation or loss of protein-protein complexes may be determined in whole cell homogenates through the use of non-denaturing electrophoresis and staining for proteins or protein phosphorylation.

As is well-known in the pharmaceutical industry, in order to establish the safety and efficacy of a drug, as well as the most therapeutic dosage thereof, it is necessary to establish the specific pharmacokinetics and metabolism associated with such drug when the same is administered to a patient via clinical pharmacology. In this respect, due to the tendencies of different compounds to be absorbed at varying rates, achieve varying peak serum concentrations, and become metabolized and excreted via one or more metabolic pathways, proper formulation is crucial to insure that an administered dosage of a given pharmaceutical composition is neither excessive, so as to cause possible adverse side effects or toxicity, nor below certain threshold concentrations, such that the composition fails to produce the desired therapeutic benefit.

To establish such parameters, detailed and exhaustive in vivo and in vitro studies are conducted to obtain data on pharmaceutical safety and efficacy in order to demonstrate that there will be no unreasonable hazard in initiating trials in human beings. The rate and extent of absorption and excretion of a given compound are usually determined during the course of the subacute toxicity studies by following changes in plasma concentration of the pharmaceutical composition after oral and parenteral administration. Additionally, organs and tissues may have to be analyzed directly for their content of the pharmaceutical composition, as well as any sub-components or metabolites thereof.

To the extent toxicity and efficacy can be sufficiently evaluated, further tests are necessary to determine preferred formulations of the commercial pharmaceutical composition product to be used in treating patients. These formulation considerations are particularly sensitive where pharmaceutical manufacturers strive to produce formulations of pharmaceutical compositions that may be administered in daily doses. In this respect, it is known that pharmaceutical compositions administered daily achieve the highest degree of patient compliance and acceptance, as opposed to pharmaceutical formulations requiring administration two or more times a day or every other day. As is well-known, however, to provide for such precise dosing requires meticulous analysis and formulation such that a particular dosage coincides with a sufficient degree of absorption, distribution and bioavailability within a sufficiently large cross-section of the population so as to produce the desired therapeutic benefit.

While protocols have been established for evaluating the clinical pharmacology of so- called "ethical" pharmaceutical compositions, such standards currently do not apply for herbal remedies or plant extracts that, although not supported by clinical data, are believed to produce significant therapeutic benefits for a variety of conditions. As a consequence, no established standards for clinical testing procedures currently exist, let alone the formulations or dosages of a given composition that are believed to be universally accepted as the preferred dosage ranges. In this regard, most herbal remedy compositions sold in this country merely use powdered whole herb or occasionally standardized extracts obtained from companies that may or may not have completed clinical trials on the extracts, or sell the extracts as prescription drugs in one or more foreign countries. In cases where such standardized extracts are not available, manufacturers tend to obtain extracts from high- quality suppliers and rely solely upon the quality control measures implemented thereby to insure uniform concentrations of such active ingredients. Such manufacturing procedures, however, are ill-suited in pharmaceutical composition manufacturing practices insofar as such practices tend to be unreliable, which consequently results in the production of pharmaceutical compositions that contain too much or not enough of the active ingredient necessary to bring about the therapeutic benefit. Moreover, even to the extent the active ingredient in a given herbal remedy or plant extract is present in an optimal concentration necessary to produce a given therapeutic benefit, how such active ingredient is ultimately formulated with other ingredients, such as an excipient that slows the absorption of such active ingredient, or how such active ingredient reacts to a manufacturing process, such as mixing which potentially destroys the biologically active form of the active ingredient, yet further thwarts the ability of such formulations to uniformly and consistently impart the desired therapeutic benefit.

These threats extend across all lines of pharmaceutical manufacturing practices, whether it be in making tablets, lozenges, liquid suspensions or gel-suspension caplets. Moreover, such threats are compounded further to the extent the active ingredient of such herbal remedy or plant extract comprises a multi-component botanical, the extracts of which are collectively believed to produce a desired therapeutic benefit. However, substantial difficulty occurs when trying to formulate, let alone standardize, herbal extracts that possess the necessary active ingredients, as well as their respective concentrations.

Accordingly, there is a need in the art for a system and method by which pharmaceutical compositions, and in particular herbal remedies and plant extracts, can be evaluated for absorption properties for use in deriving standardized formulations and/or extracts thereof. There is additionally a need in the art for a method for determining the absorption properties of herbal remedies and plant extracts that can reliably reproduce the rate by which the same are absorbed into the human body and the resultant serum concentrations achieved thereby, particularly when the same are administered orally, to thus enable such extract formulations to be standardized. There is yet a further need in the art for a method for determining the absorption rates of herbal remedies and plant extracts that is simple to construct, utilizes relatively inexpensive materials, and is based upon accepted principles and parameters for establishing drug absorption activity using biological absorption models.

For example it is known in the art to use rate of absorption and plasma concentration levels attained thereby, when such compositions are administered orally.

Millions of people in the third world will always use herbal medicines because they believe in them, They also regard them as their own system of medicine. Factors such as easy availability of herbal remedies, accessibility to practitioners at all times and inherent faith in natural things also complement the desire of large sectors of the population to use medicinal herbal products for therapeutic purpose.

Herbal remedies will be with us for a long time. It is therefore important to bring the use of these remedies in to existing framework of rational scientific use of medicines. It will be useful to consider what regulatory and legislative control needs to be exercised on the use of herbal medicines. Linked to this are issues of quality control and the standardization of herbal medicines.

The traditional system of medicine normally advocates the combination of several herbal ingredients to provide the synergistic bioactivity or reducing the side effects if any, which might be associated with the final product.

Due to difficulties in identification of individual herbal ingredients from poly herbal formulation, the products find difficulty in getting registration in developed countries. Due to difficulty of valid quality control methods, we are not able to generate the due faith and confidence of modern medical practioners on herbal medicines. The agency like World Health Organization has recommended the use of TLC fingerprinting as the method of identification of poly herbal formulation but this method is not foolproof for indicating the presence of each herbal ingredient in appropriate amounts.

Lately the concept of marker compound identification and its quantification by various chromatographic techniques in final product is advocated but this method is again full of difficulties such as.

• Marker compounds are not defined from each herbal ingredient. Thus this method cannot be used with every herb, or its extract.

• The marker compound may be present in crude herb but may be lost in the final extract that is used commercially for therapeutic purpose.

• Identification and quantification of marker compound does not indicate the desired activity of said ingredient or formulation because the herbs or its extract are found to work when present in totality, and activity is not linked to marker compound. Thus use of marker compounds as a tool to ascertain quality is not scientifically justifiable. • In some cases the marker compound from more than one plant may be same for example if the formulation advocates the uses of Piper longum and Piper nigrum, both of which contain same marker compound ie. piperine and hence measuring piperine alone will not tell us weather it is coming from P. longum or P. nigrum. Attempts have also been made to use HPTLC/HPLC fingerprinting for identification of herbs from polyherbal formulation. However, such attempts have not been very successful since every individual herb contains several chemical constituents of varying chemistry. Additionally, when several of such multiple ingredient containing herbs are combined as a formulation, the situation is rendered even more difficult.

In general the problem of identification of individual herbal ingredient from poly herbal formulation is the burning problem in front of scientists working on this aspect for which suitable and practically viable methods don't exist till now. Objects of the invention

The main object of the present invention is to provide a practical, commercially viable, reproducible and sensitive method for identifying each individual herbal ingredient present in a poly herbal formulation by simple means.

Another important object of the invention is to provide a method for the identification and analysis of individual ingredients used from animal, marine, semi synthetic, synthetic or biotechnological method, used for formulating the composite formulation. Yet another object of present invention is to identify the individual ingredient from poly herbal formulation and also qualitatively and quantitatively analyse these individual ingredients from formulation by known means.

Still another object of the invention is to analyse the individual ingredients used from animal, marine, semi synthetic, synthetic or biotechnological method, used for formulating the composite formulation. Summary of the invention

Accordingly, the present invention provides a method for identification and analysis of individual ingredients used in composite pharmaceutical formulations, comprising: 1. converting the individual herbal ingredient of the poly herbal formulation into distinctly colored pellets 2. mixing these distinctly colored pellets as per the formulae, packing them as desired;

3. separating these individual colored pellets, for identification and analytical purpose from mixed blend and confirming their identity by comparing the separated colored pellet with their standard respective herb/herbal extract.

4. Further confirmation and quantitative estimation can be done by extracting each individual ingredient thus separated in a conventional manner by any known methods such as gravimetric, titrimetric, spectrophotometric or chromatography. Detailed description of the invention

Pharmaceutical products containing synthetic ingredients are easily identifiable by chemical methods or by spectrophotometric such as UV or chromatographic method such as TLC, HPLC, GC etc. It is therefore relatively simple to determine whether such formulation meets qualitative and quantitative limitations required at the time of actual sale.

However, one of the problems associated with herbal products or other pharmaceutical products of natural origin or containing extracts of natural origin is that a plurality of extracts are contained therein. Available methods of assays, do not provide a solution for identification or quantification of the individual herb/extract in such formulation. The problem is exacerbated since the active principle is often not known in the case of herbal formulations. Theoretically, most herbal formulations or formulations containing extracts of natural origin function on the belief that it is a combination of different extracts which interact and achieve the desired result. Therefore, chromatographic finger printing which is used to ascertain the quality does not guarantee that the finger print represents all the herbs/herbal extract present in the formulation.

The present invention will now be described with reference to the following non- limiting and illustrative examples: EXAMPLES

Example-1: Antihaemorroidal capsule Composition:

Each capsule contains as extract of: Azadirchitra indica 18 mg preferably from 10-50 mg Symplocos racemosa 5 O mg preferably from 20-100 mg

Commiphora mukul 71 mg preferably from 10-150 mg

Terminalia chebula 125 mg preferably from 50-200 mg

Berberis aristata 17 mg preferably from 5-50 mg

Commiphora myrrh 36 mg preferably from 20-100 mg Plumbago zeylanica 7 mg preferably from 12-50 mg

Cyperus rotundus 6 mg preferably from 1.-50 mg

The total batch of 20,000 capsule is taken as follows.

360 gm of Azadirachta indica extract is weighed, passed through 40 mesh and mixed with 360 gm of microcrystalline cellulose in poly bag. The dough of this mixture is prepared with 2% solution of PVPK-30 in DM water. This dough was passed through extruder fitted with 1 mm sieve and needles were collected. These were then put in spheroniser and converted in to pellets/ spansules. These spansules were collected an dried in fluidized bed dryer at 50⁰C . When dried then the spansules were coated with coating blend containing erythrosine color and titanium dioxide dissolved in IPA using fluidized bed coater till the color became bright red. The coated spansules were kept aside.

Similarly pellets of remaining ingredients were prepared using 1:1 mixture of drug and macrocrystalline cellulose. Pellets of these individual ingredients were coated with different colors such as brilliant blue {Symplocos racemosd), Sunset yellow (Terminalia chebula), brilliant black (Plumbago zeylanicά), quinoline yellow (Berberis arϊstatd), titanium dioxide (Cymmiphora myrrh), tartrazine yellow (Commophora mukuϊ) and natural chlorophyll (Commiphora myrrh) so that each ingredient gives different color. Pellets of each ingredient were mixed as a final formulation and filled in 0 sized hard gelatin capsule. For identification the capsule shell was removed and pellets of each color were physically separated with help of magnifying lens. Thus total eight different colored pellets were collected indicating eight different ingredients present in the composite formulation.

These individual colored pellets with the respective active herbal ingredients were analysed using high performance thin layer chromatography as mentioned below Identification of Azadirachta indica from Antihaemorroidal capsule

Pellets of erythrosine color (2 gm) were extracted with acetate (20ml x 3) over steam water bath and filtered. The filtrate was pooled and concentrated to 10ml and kept aside. Similarly standard Azadirachta indica extract was also prepared using lgm amount. 0.5 microlitre of both samples were applied on silica gel 60 F 254 TCL plate (Mfd. By E.Merck., Category No. 5554). The plate was developed in a solvent system consisting of chloroform and methanol in a ratio of 90:10 upto 80 mm. After development, the plate was dried and scanned using Camag TLC scanner III at 254nm under absorbance mode. Chromatogram of standard Azadirachta indica and Azadirachta indica from anti-hemorrhoidal capsule are shown in figure no 1. Identification of Symplocos racemosus from Anti hemorhoidal capsule :

Pellets of brilliant blue color (2gm) were extracted with methanol (20ml x 3) over steam water bath and filtered. The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Symplocos racemosus extract was also extracted using 1 gm amount. 0.5 microlitre of both samples were applied on silica gel 60 F 254 TLC plate (Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of Ethyl acetate, formic acid, acetic acid and water in the ratio of 100: 11: 11: 27 upto 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm. under absorbance mode. The chromatogram of standard Symplocos racemosus and Symplocos racemosus from Anti hermorrhoidal capsule are shown in figure no. 2 Identification of Commiphora mukul from Anti Hemorrhoidal capsule:

Pellets of Tartrazine yellow color (2gm) were extracted with ethyl acetate (20 ml x 3) over steam water bath and filtered. The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Commiphora mukul extract was also extracted using 1 gm amount. 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate ( Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of Chloroform and methanol in the ratio of 90:10 up to 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm under absorbance mode. The chromatogram of standard Commiphora mukul and Commiphora mukul from Anti hemorrhoidal capsule are shown in figure no. 3

Identification of Teminalia Chebula from Antihaemorroida Capsule:

Pellets of sunset yellow color (2gm) were extracted with methanol (20ml x 3) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Teminalia Chebula extract was also extracted using 1 gm amount. 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate ( Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of Ethyl acetate, formic acid, acetic and water in the ratio of 100:11:11:27 upto 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm, under absorbance mode. The chromatogram of standard Teminalia Chebula and Teminalia Chebula from Anti hemorrhoidal capsul are shown in figure no. 4

Identification of Berberis aristata from Antihaemorroidal capsule:

Pellets of quinlline yellow color (2gm) were extracted with methanol (20ml x 3) over steam water bath and filtered. The filtrate were pooled and concentrated to 10ml and kept aside. Similarly standard Berberis aristata extract was also extracted using lgm amount. 0.5 microlitre of both samples were applied on silica gel 60 F 254 TLC plate (Mfg. By E Merck. Category No. 5554). The plate was then developed in a solvent system consisting of Ethyl acetate, formic acid, acetic acid and water in the ratio of 100:11:11:27 upto 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm, under absorbance mode. The chromatogram of standard Berberis aristata and Berberis aristata from Anti hemorrhoidal capsul are shown in figure no.5

Identification of Commiphora myrrh from Anti haemorroidal capsule:

The pellets of chlorophyll color (2gm) were extracted with ethyl acetate (20 ml x 3) over steam water bath and filtered. The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Commiphora myrrh extract was also extracted using 1 gm amount 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate (Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of chloroform and methanol in the ratio of 90 and 10 up to 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm under absorbance mode. Chromatogram of standard Commiphora myrrh and Commiphora myrrh from antihemorrhoidal capsule are shown in figure no.6 Identification of Plumbago zeylanica from Antihaemorroidal capsule:

The pellets of brilliant black color (2gm) were extracted with methanol ( 20 ml x 3 ) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Plumbago zeylanica extract was also extracted using 1 gm amount 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate ( Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of Ethyl acetate, formic acid, acetic and water in the ratio of 100,11,11, and 27 upto 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm under absorbance mode. The chromatogram of standard Plumbago zeylanica and Plumbago zeylanica from antihemorrhoidal capsule are shown in figure no.7 Identification of Cyperus rotundus from Antihaemorroidal capsule:

Pellets of titanium dioxide color (2gm) were extracted with ethyl acetate ( 20 ml x 3 ) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Cyperus rotundus extract was also extracted using 1 gm amount 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate (Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of chloroform, methanol in the ratio of 90: 10 up to 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm. under absorbance mode. The chromatogram of standard Cyperus rotundus and Cyperus rotundus from Anti hemorrhoidal capsule are shown in figure no.8

The HPTCL fingerprint of respective pellets vis-a-vis their active ingredients from which the pellets have been derived are shown in figures 1-8 respectively. The chromatogram clearly show identification of every ingredient present in formulation where the number of peaks and its intensity is perfectly identical and superimposable clearly indicating the suitability of the method for qualitative analysis. ExampIe-2: Immuiiomodulator capsule composition

Each capsule contains: Emblica officinalis extract -100 mg preferably from 50-200 mg Tinospora cordifolia extract -100 mg preferably from 50-200 mg Withania somnifera extract -100 mg preferably from 50-200 mg The total batch of 20,000 capsules is taken as follows.

2 kg each of Emblica officinalis, Tinospora cordifolia and Withania somnifera were weighed, passed through 40 mesh and mixd with 2 kg of microcrystalline cellulose in ploy bag separately. The dough of each blend is prepared with 2 % solution PVPK -30 in DM water. The dough of each blend was passed through extruder fitted with 1 mm sieve and needles were collected. These needles were then put in spheroniser and converted into pellets. These were collected separately and dried in fluidized bed dryer at 50⁰C. The pellets of these individual ingredients were coated with different colors such as brilliant blue ( Emblica officinalis), brilliant black (Tinospora cordifolia ) and erythrosine red ( Withania somnifera ) so that each ingredient gives different color.

Finally the pellets of each ingredient were mixed as per the final formulation and filled in 0 sized hard gelatin capsule. For identification the capsule shell was removed and pellets of each color was physically separated with the help of magnifying lens. Thus a total of three different colored pellets were collected indicating three different ingredients present in composite formulation. These individual colored pellets with the respective active herbal ingredients were analysed using high performance thin layer chromatography by the method given below. Identification of Emblica officinalis from Immunomodulator capsule:

The pellets of brilliant blue color (2gm) were extracted with methanol ( 20 ml x 3 ) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Emblica officinalis extract was also extracted using 1 gm amount 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate ( Mfg. By E Merck. Category No. 5554). The plate is then developed in solvent system consisting of ethyl acetate, formic acid, acetic acid and water in the ratio of 100, 11, 11, and 27 upto 80mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm under absorbance mode. The chromatogram of standard Emblica officinalis and standard Emblica officinalis from Immunomodulator capsule are shown in figure no.9. Identification of Tinospora cordifolia from Immunomodulator capsule:

The pellets of brilliant black color (2gm) were extracted with methanol (20 ml x 3 ) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Tinospora cordifolia extract was also extracted using 1 gm amount 0.5 microlitre of both the samples were applied on silica gel 60 F 254 TLC plate ( Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of chloroform, methanol in the ratio 90 and 10 up to 80 mm After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm under absorbance mode. The chromatogram of standard Tinospora cordifolia and Tinospora cordifolia from Immunomodulator capsule are shown in figure no.10.

Identification of Withania somnifera from Immunomodulator capsule:

The pellets of erythrosine red color (2gm) were extracted with methanol (20 ml x 3 ) over steam water bath and filtered . The filtrate were pooled and concentrated to 10 ml and kept aside. Similarly standard Withania somnifera extract was also extracted using 1 gm amount 0.5 microlitre of both samples were applied on silica gel 60 F 254 TLC plate (Mfg. By E Merck. Category No. 5554). The plate is then developed is solvent system consisting of chloroform, methanol in the ratio 90 and 10 up to 80 mm. After development, the plate is dried and scanned using Camag TLC scanner III at 254 mm uder absorbance mode. The chromatogram of standard Withania somnifera and Withania somnifera from immunomodulator capsule are shown in figure no.11.

The HPTLC fingerprint of respective pellets vis-a-vis their ingredients from which the pellets have been derived have been shown in figure 9-11 respectively . The chromatogram clearly shows the identification of each and every ingredient present in formulation where the number of peaks and its intensity is perfectly identical and superimposable clearly indicating the suitability of the method for qualitative and quantitative analysis.

Similar method can also be applied to any poly herbal formulation containing more than one active herbal ingredient. STABILITY OF FORMULATION The formulation as provided above was packed in PVC /Aluminum foil blisters and also in plastic jar in ambient and accelerated condition of temperature and humidity ( 4OC/ 70% RH ) and the samples analyzed periodically the formulation has been found to be stable for 6 months at accelerated condition and 3 years at ambient condition.

Claims

We claim:

1. A method for identifying and then quantifying individual active ingredients derived from natural source from poly herbal formulation, said method comprising converting the individual active ingredients into pellets/spansules, optionally with pharmaceutically active excipients, coating the pellets of individual ingredients with acceptable food colour distinct from one another, mixing these different color pellets as per the formulation which can be filled either in hard gelatin capsule or for being suspended in syrup, the different color pellets from the formulation being capable of visually separated with the naked eye or with the help of magnifying lens from one another, thus providing physical separation of individual ingredients from poly herbal formulation, capable of being weighed and divided by the factor equivalent to the excipients.

2. A method as claimed in claim 1 wherein further confirmation (qualitative or quantitative) is established by different analytical means as already known in the art such as solvent extractive values, spectrophotometer, chromatography, titrimetry and gravimetric.

3. A method for the identification and analysis of individual ingredients used in composite pharmaceutical formulationa, the method comprising:

(a) converting the individual herbal ingredient of the poly herbal formulation into distinctly colored pellets

(b) mixing these distinctly colored pellets as per the formulae, packing them as desired;

(c) separating these individual colored pellets, for identification and analytical purpose from mixed blend and confirming their identity by comparing the separated colored pellet with their standard respective herb/herbal extract.

4. A method for the identification and analysis of individual ingredients used in composite pharmaceutical formulations substantially as herein described with reference to the foregoing examples.