OA12520A - A novel method chromatographic finger printing andstandardization of single medicines and formulati ons. - Google Patents

Description

012520 ., ±

A NOVEL METHOD FOR CHROMATOGRAPHIC FINGER PRINTING ANDSTANDARDISATION OF SINGLE MEDICINES AND FORMULATIONS

Technical Field 5 The présent invention relates to a novel method of chromatographie finger printing usefulfor Chemical and therapeutic standardization. More particularly, the présent inventionrelates to chromatographie fmgerprinting of organic and organo-metallic molécules whichhâve UV-Visible radiation absorptive property présent in a plant, animal or any othersource capable of being used as a single or formulated medicine. The invention facilitâtes 10 bar coding of one or many constituents présent in the finger print images. It also facilitatecommercial utilization of the fingerprint database having ali the information about the. niedicines, development and using ERP (Enterprise Resource Planning) and CRM(Customer Relationship Management) net work applications like veriding machines etc.

The présent invention employs a novel method of the utilization ofthe Contour and 3D 15 chromatograms of an herbal medicine and formulation developed under standardizedexperimental (chemical and instrumental) conditions proposed as a chromatographie fingerprint of the medicines.

In addition, the présent invention relates to a computer-based method for the analysis of such chromatograms. This novel method is very much used for an authentic identification» 20 of the Chemical constituents'of the single medicines and formulations.

Background Art

History of traditional medicines in the world

The ancient man after many years of évolution started thinking about hirn and tried to 25 understand the nature. He started living under a cover, the caves and in-groups. Theprocess of thought has encouraged him to understand the nature and its inter action withthe living beings. He started using the naturally available flora and fauna for his dailyneeds, in which he used the plant and animal material for his dietary and health needs.Under this process, he explored the properties of various components in the world, like 30 geological, astral and médicinal properties of various biological and plant materials. This started happening from the Stone Age on words. He went on discovering, standardizing and using naturally available materials for his day-to-day living.

This took place in many places of the earth parallel in different parts of the world and h developed more in places where civilization has developed more based on the 012520- 2 intellectuality. Therefore, the history of medicine has a direct relation with the history ofcivilization.

The évidences of an well-organized System of medicine in India were traced in Harappaand Mohanzadaro (History of Medicine in India, Dr Priya Vrit Sharma, Ref.l). In theIndus valley civilization, a System of medicine has prevailed, in which drugs of vegetable,animal and minerai origin were used. The Osadhisukta of the Rigveda is the oldestdocument of the knowledge about plants and herbal medicines. Medicine in India owesmuch to the traditional knowledge of Atharvaveda of which Ayurveda is said as anUpaveda. A large number of disease-syndrome relationships were defined and describedby Charaka andSusruta in their medical treatises ‘The Samhitas’. The treatment was alsoprescribed in a systematic manner and on rational basis.

On the other hand, it was realized that the biological phenomena could not be universallyexplained' by mechanical means as each individual varies in his basic constitution i.e.,Prakruthi that must be kept in mind while prescribing diet or drug to the patient. Thebinary concept like Prakriti-Purusha, Yin-Yang, Normal-Abnormal was seen in almost ailphilosophies.

Diseases are manifestations of humoral imbalance, which hâve to be tacldedcomprehensively on the psychosomatic basis. Health accordingly is equilibriummaintained on physical, mental and spiritual levels. Thus, the Tri-dimensionaî définition ofhealth as propounded by Susrtuta is the idéal one, which has been reflected in thedéfinition, adopted by WHO in modem times.

After going through the ancient literature it was found that, the medicines werestandardized using their physico-chemical properties of the materials. The color, texture,odor and taste were used as a measure of the efficacy of any medicine. Even the shape ofthe medicines was used to understand the médicinal properties of the medicines. Asummary of different philosophies and various factors used in the therapy is given inTables 1-6 which are appearing in the later part of the description. Figure 1 accompanyingthe spécification gives a detailed information on the individual philosophies and conceptsof Indian Ayurveda and Chinese Traditional medicines (Ref.2 H.M.Said, Ref.3 DanielReid) in particular and other philosophies in general. The therapeutic efficacy of amedicine ultimately dépends on the Chemical constituents présent in the medicines underuse and it is the Chemical properties of the constituents that are going to bring a requiredchange in the Chemical constitution of the living animal. ,Oi252o

Many Ayurvedic (one kind of Indian System of medicine) schoiars hâve defined andclassified the medicines based on color and their therapeutic efficacy. A brief summary isgiven in Table 7, which appears in the later part of the description. Thus, the physico-chemical properties of the materials and man is taken in to considération to understand the 5 properties of them and use for the therapy to achieve required therapeutic results. Tables 8-9 which are appearing in the later part of the description gives the information about howthe physical properties (color) and Chemical properties (taste) are used for understandingthe therapeutic efficacy of medicines and their influence on the physiology of the humanbody. One part of the présent work also uses the same methodology, but with instruments. 10 In general, the constituent molécules présent in the drugs and foods can be broadlyclassified in to three categories as polar, medium polar and the non-polar molécules. Thetr total polarity of the molécule dépends on the total Electrophlic and Nucleophilic moieties attached to the molécule along with the unsaturation of the molécules by their conjugation.The living human body, animal body and plants will also contain the same type of 15 molécules wherein different polar molécules will carry out different functions. Diseaseswere cured using the medicines of same polarity as that of the disease causing Chemicalconstituents, i.e. the medicines which can create the disorder can cure the same disorder, assaid by Dr Hanemann. 20 WHO Définition of herbal medicine: WHO has defined herbal medicine as a “Finished, labeled médicinal products that containas active ingrédients aerial or underground parts of plants, or other plant material, orcombinations thereof whether in the crude State or as plant préparations. Plant materialincludes juices, gums, fatty oils, essential oils, and any other substances of this nature. 25 Herbal medicines may contain excipients in addition to the active ingrédients. Medicinescontaining plant material combined with chemically defined active substances, includingchemically defined, isolated constituents of plants, is not considered to be herbalmedicines. Exceptionally, in some countries herbal medicines may also contain, bytradition, natural organic or inorganic active ingrédients which are not of plant origin”. 30 The objective of these guidelines, therefore, is “to define basic criteria for the évaluation ofquality, safety, and efficacy of herbal medicines and thereby to assist national regulatoryauthorities, scientific organizations, and manufacturer to undertake an assessment of thedocumentation/submission/dossiers in respect of such products”. As a general rule in thisassessment, traditional expérience means that long-term use as well as the medical, 35 historical and ethnological background of those products shall be taken into account. The 012520 4 définition of long-term use may vary according to the country but should be at least severaldécades. Therefore, the assessment should take into account a description in themedical/pharmaceutical literature or similar sources, or a documentation of knowledge onthe application of an herbal medicine without a clearly defined time limitation. Marketingauthorizations of similar products should also be taken into account. As per the report, theassessment of quality should be done for the following parameters. WHO guidelines were given for the finished product for which, the assessment of efficacy,activity, evidence required to support indications and combination products. Many herbalremedies consist of a combination of several active ingrédients, and as expérience of theuse of traditional remedies is often based on combination products, assessment shoulddifferentiate between old and new combination products. Identical requirements for theassessment of old and new combinations would resuit in inappropriate assessment ofcertain traditional medicines. In the case of traditionally used combination products, thedocumentation of traditional uses (such as classical texts of Ayurveda, Traditional Chinesemedicine, Unani and Siddha) and expérience may serve as evidence of efficacy.

An explanation of new combination of well known substances including effective doseranges and compatibility should be required in addition to the documentation of traditionalknowledge of each single ingrédient. Each active ingrédient must contribute to the efficacyof the medicine. Clinical studies may be required to justify the efficacy of a new ingrédientand its positive effect on the total combination.

In the report, it was also mentioned that the manufacturing procedure and formula,including the amount of excipients, should be described in detail. A finished productspécification should be defined. A method of identification and yvhere possible,quantification of the plant material in the finished product should also be defined. If the .identification of an active principle is not possible, it should be sujficient to identify acharacteriStic substance or mixture of substances (e.g., "chromatographie fingerprint”) toensure consistent quality of the product. The finished product should comply with generalrequirements for particular dosage forms.

For imported finished products, confirmation of the regulatory status in the country of origin should be provided. The WHO Certification Scheme on the quality of pharmaceutical products moving in international commerce should be applied. More

details about stability, assessment of safety and utilization were given in the said WHO report. 012520 5

The effective régulation of the quality of herbal medicines moving in internationalcommerce also requires close liaison between national institutions that are able to keepunder regular review ail aspects of production and usé of herbal medicines. Also, toconduct or sponsor évaluative studies of their efïicacy, toxicity, safety, acceptability, costand relative value are compared with other drugs used in modem medicine.

Hence, as mentioned above, there is a need for an authentic method of quality control asproposed in this work. It is clearly mentioned that there is a need of a method for ail theabove purposes. The proposed analytical will give answers for almost ail of the needsdescribed above. EXISTING METHODS OF STANDARDIZATION:

Before explaining the method of the invention of standardization, the existing methods ofstandardization (chemical and therapeutic) and chromatographie·, fmger printing arediscussed below. A. PRIOR ART ON CHEMICAL STANDARDIZATION: I) Traditional:

The great sage Charaka explained in his Charaka Samhita that “The widerstanding of thetoïality of an entity does not arisefi-om a fragmentary knowledge of it "(Charaka SamhitaVI. 4.5). This makes it clear that standardization and therapeutic efficacy of any medicinefor which ail the constituents are not taken into considération is futile.

The qualitative and quantitative profile of a herbal medicine will vary due to manygeological, ecological factors, time of collection, place of collection, âge of collection andweather conditions at the time of collection and so on.

Traditional herbalists used to select a medicine based on the organoleptic methodsavailable at that time Iike color, texture, smell and taste by which they used to assess thechemical and therapeutic efficacy of a medicine.

These methods involve intrinsic knowledge and understanding of the inter and intratherapeutic interactions of the medicines and body constituents to cure diseases. Thisknowledge varies from individual to individual and dépends on the individual skill andability. Practically, it will be difficult to provide a rational justification for any mechanismto explain, using this method. Hence, modem science uses instruments for variouspurposes, which éliminâtes the individual factors and facilitâtes reproducibility in data andinformation. 012520’’ 6 ii) Modem:

The therapeutic property of any food or drug will dépend on its Chemical and physicalstatus. Thus, understandîng the Chemical constituents using their physico-chemicalproperties will help to understand the therapeutic efficacy of the medicine.

The physico Chemical properties of the medicines play a major rôle on the therapeuticactivity of the medicine. These properties of molécules can be studied using twoparameters, the polarity and conjugative properties. Polarity is a résultant electrochemicalproperty due to different électron donating (nucleophilic) and électron accepting(electrophilic) moieties attached to the molécules along with the unsaturated double andtriple bonds présent in it. They will influence the rate of activity or reactivity of a moléculein Chemical and biochemical reactions. A thorough estimation of the total polarity of themolécule will give the efficacy of a single or group of molécules as to how active they arechemically and therapeutically. Hence, any standardization, which assesses the aboveproperties, will be useful to know their activity. .

Along with the polarity, which relates mostly to the Electro-chemical property of themolécules the physical structure of the molécule also play an important rôle in thereactivity of the molécules. The more the number of active sites attached to the molécules,the more reactive they will be. The more the molécule is conjugated (having alternativedouble bond and triple bonds) the more it will be reactive chemically and sotherapeutically.

The second parameter that influences the activity of the molécule is the spatialarrangement of atoms in the same molécule, which differs structurally. Dueto this reason,the isomeric (géométrie and chiral) molécules play an important rôle in the biologicalactivity. This stereo-selective nature makes the molécules highly sélective in.their activityin the body where in a large number of biochemical pathways will be working parallelwithout cross interactions and interférence’s. Hence, the chemistry of chiral drugs hasbecome very important. Inotherterms, no key (chiral molécule) will open a different lock(receptor).

Plants usually préparé a plurality/combinatorial library of molécules having the same basicmother structures and varying in the functional groups attached to it. For example,flavones, aurones and chalcones of flavoniods as they exist in nature and a single planthaving such set of molécules will àct like a muîti drug.

Usually molécules having unsaturation and more conjugation absorbs the electromagnetic radiation in the UV-Visible radiation (200-800nm). When the compounds interact with Ί 012520

radiation · they absorb at a spécifie wavelength (absorbance maxima) based on theirChemical, conjugative and structural properties. It is called the characteristic wavelength. Amolécule can hâve more than one-absorbance maxima based on its structural andfunctional properties. When a compound absorbs a particular color from the wholesome of 5 the white light it will express the résultant color of the other colors unabsorbed. Thus, thematerials will express different colors based on their Chemical constituents absorbingvarious colors ffom white light and showing that their color is due to the various functionalgroups attached to it. (Table No 10, which appears later part of the description, explains thesame). The same is taken as a measure of the Chemical and physical properties of the 10 molécules in spectrophotometry.

For example, the red colored medicines absorbs at 500-600nm range. Thus, ail the redcolored medicines will hâve a peak in this wavelength range having spécifie structure andactivity. Hence, the color of the medicines is being used as a measure of its therapeutic andChemical efficacy of the medicines. In ancient times, medicines were classified 15 therapeutically based on the color. The présent method proves the same. The Figure 2which accompanying the spécification depicts relation of colors with humors shows theeffect of different colors on different diseases.

Fig 3, which accompany the spécification, the fingerprints of two Shilajit samples preparedby the method of the invention, shows the différence in the Chemical profile of both 20 samples. Shilajit is a carbonaceous material that forms due to storage of vegetable andanimal matter inside the earth for many years. Mostly due to lava floated over a forestdestroying flora and fauna. This after undergoing many geoîogiçal changes becomescarbonaceous material called Shilajit. It is abundantly available in Russia. It is the mostwidely used as medicine in the world. It is observed that although the general pattern of the 25 molécules is found to be similar in Shilajit samples of different sources, the variation in theconjugative properties of the molécules is found different. This makes these medicinesvary in their therapeutic efficacy hence, this type of fingerprinting is useful.

In the modem Chemical analysis methods, détermination of the percentage of activeprinciples viz., alkaloids, flavonoids, enzymes, vitamins, essential oils, fats, carbohydrates, 30 proteins, ash, acid-insoluble ash, and crude fiber is done by various analytical instruments.Some examples shown below explain how standardization is done in modem science.

It is reported (WWW// Shilajit. Ref.6) that, one of the very important medicines used in

Indian System of medicine Shilajit, is reported to hâve many compoiinds along with fulvic acids, and is claimed to be active principle. As this medicine ffom bituminous source is 35 collected ffom earth stored for many years, it can be seen that the more it is in stored in 012520 δ the earth, the more it will be therapeutically active. Nevertheless, in.the entire globe, thegeologîcal variations may not produce same molécules in ail samples collected fromdifferent parts of the world. Another factor that influences the chemistry of these drugs isthe purification process, which also needs to be standardized.

It is reported (WWWZ/Herbology. Ref. 7) that, mostly standardization is done for theindividual key components, which hâve been empirically, and scientifically proven to bemost advantageous for the human System. So usually, standardization is done for certainmolécules out of ail présent, which are found to hâve activity. However, the synergisticeffect of other compounds présent in the medicine making the total profile of the medicineshould be taken into considération for its efficacy.

It is reported (WWW//Tribulus Terrestrius puncture vine. Ref.8) that, the alcoholic extractof the fruits of Tribulus Terrestrius shows antiurolithiatic activity. In addition to this, theextract also shows a significant diuretic activity. The alkaloid Harman has been reportedfrom the herb and Harmine from the seeds. The plant contains saponins, which onhydrolysis yield steriodal sapogenins. Many molécules of flavonoid in nature are reportedin which the active principle is found saponins. The analytical report is given for the heavymétal analysis and total saponins content (20%w/w)

It is reported (WWW// Charak_com. Ref.9) that, Human life is a synergy of Mental,Physical and Spiritual components, which are related to the Indian Philosophy of Ayurvedawhere Pitta, Kapha and Vata are considered as the basis for the total health of humanbeings. More details of standardization are given in the description of traditional methodsof therapeutic standardization.

It is reported (WWW//Standardized Herbal Extracts. Ref. 10) that, due to EuropeanGuaranteed Potency Law, it becomes compulsory to standardize herbal medicines. Themeaning of herbal standardization is defined as quantification of an active constituent ormarker extract, where in the activity is attributed to be the most.

It is explained (Frank R Stermitz et al, Ref. 11) that, in the plant Berberis Aristata theantimicrobial property of the extract is due to the presence of 5-Hydroxy Hydnocarpin, theberberin acting as anti microbial, and without which it will not. Hence, the synergisticeffect of the entire constituents should be taken into considération while dealing with anherbal medicine but not only an active constituent. WHO in its Régional Publication (Dr Ranjit Roy Choudary. Ref. 12) clearly mentioned what standardization is and what the member countries should do for the better use of its medicines by the people. θ 72520 9

The rôle of acidity and alkalinity can be understood by carefully understanding theextraction process of constituents frotn the medicines at different pH values of theextraction solvents. This heîps to understand the drug release mechanism in the intestinefrom the medicine consumed by the persons having different intestinal pH. The rôle ofacidity and alkalinity was studied and understood carefully in understanding thetherapeutic efficacy of a medicines. Acidity and alkalinity of organic and inorganicmolécules are studied thoroughly to know their properties as shown in Table 11 whichappear later part of the spécification. Of acidity and alkalinity shows the rôle of acidity andalkalinity on health (Ref.13 Devendra Vora).

It is reported (WWW// Chewing. Ref 14.) that, in a study it was observed that people withacidic Systems absorb more pollution than people who had established proper bloodalkalinity. Acid/alkaline (pH) balance is important for normal cell fonction. More detailswere given in the article. Hence, the study of the acidity or alkalinity (organic or inorganic)in terms of ‘polarity’ will give the information of the therapeutic efficacy of the medicines.Hence, the présent method, which can do this job, will be of much use to know thetherapeutic efficacy of the medicines. Using this proposed method, the acidity andalkalinity can be established for the therapeutic standardization of medicines.

References made above will explain the conventional and reported methods ofstandardization, wherein the individual constituents are isolated at a préparative scale andcompared qualitatively and quantitatively with the same compound présent in the samplemedicine under study.

In one of the reports (Khwaja, et al Ref. 15) it was reported that the different individualfractions of ethanolic extract of Saw Palmetto were studied for their bioactivity bymeasuring the détermination of IC 50 in an androgen receptor binding inhibition assay.Measurement of total fatty acid assay of whole extract and individual fractions werediscussed. The fractions containing linolic acid ethyl ester and lauric acid ethyl ester wereidentified. The activity was calculated for each of the fraction for which androgen receptorbinding inhibition has been assayed compared to the total bioactivitty of the sample. Themolecular weight and amount of the individual fatty acids were identified and incorporatedin to the calculation of bioactivity. The total bioactivity of the extract was calculatedcomparing to the total percent activity of linolic and lauric acid ethyl ester fractions.

In the traditional medicine standardization, the total profile should be taken intoconsidération for the therapeutic efficacy of the herbal medicine. Hence, in the présentcomputer-based instrumental method, the total properties of ail the constituents are taken V’ 012520' 10 into considération as suggested in traditional concepts world over. The fingerprints of themedicines were proposed as a visual tool and proof for many purposes of dealing withmedicines particuiarly traditional. Before discussing the method of the invention, theexisting method of analytical method is given below.

Existing analytical methods of Chemical standardization:

Improvement and use of modem analytical methods and instrumentation hâve definitelyled to excellence in quality control methods of medicines. Improvement in analysis has ledto more précisé harvesting of many herbs as explained above and helped to préparéstandardized extracts.

Even though there are traditional methods of identification of médicinal plants likeorganoleptic, microscopie and physical, none of them gives an authentic identification, asgiven by a fmgerprint of the plant material, as far as the Chemical profile is concerned.Hence, it is proposed that the Chromatographie Fingerprint is much useful for qualitycontrol of médicinal plants instead of other organoleptic and microscopie studies. Since,ultimately it is the Chemical constituents that are largely going to participate in thetherapeutic efficacy of the medicine, along with other properties of the herbal medicines;the analytical data of the Chemical constituents should be able to provide· the authenticefficacy of the medicine. It is like fmgerprint of an individual gives the identity of him,

Till now Thin Layer Chromatography (TLC), High Performance Thin LayerChromatography (HPTLC) and High Pressure (Performance) Liquid Chromatography arethe methods commonly used for the analysis of any organic or organometallic compoundsand finger printing. But, ail the methods hâve some merits and demerits for an authenticanalysis of medicines. The enclosed Table 12 appearing later part of the descriptioncompare various commonly existing methods of analysis, provide a general ..idea of themerits and demerits of them.

Figure 4, which is accompanying the spécification, shows how the chromatogramdeveloped from a TLC method is used as a “Chromatographie fingerprint” on the label of acommercial product. Except the assay of constituents no more information was given inthe finger print.

After observing the above Table, it is found that the most suitable technique available forthe analysis of a mixture of compounds is "Chromatography", which gives the profile ofthe mixture after the séparation and identification with a suitable deteetor.

Out of the different types of chromatographie techniques available, the best suitable is'High pressure liquid chromatography '(HPLC). Although thin layer chromatography was V2/46 012520 11 used tïll recent times, avancements brought out in the equipment and séparation columnsof HPLC has revolutionized the analytical field of chromatography.

Most of the pharmaceutical analysis was reported in the form of a chromatogram with thepeaks due to molécules eluted by a mobile phase mostly reported in the official methods 5 and pharmacopoeias. The constituents are analyzed after eluting on a HPLC séparationcolumn detected by using any suitable detectors for analysis.

Usually the chromatographie analysis is done using a référencé standard (internai orexternal). Without a standard reference material, the analysis has no meaning because thepeak of the chromatogram does not provide any kind of Chemical properties of the 10 compound eluted. Hence, the confirmation of the qualitative and quantitative properties(spectral or Chemical) of the components are unclear.

In the qualitative and quantitative analysis of medicines/drugs (Single or Formulation), theemphasis is given mainly on the spectral and Chemical properties of the components elutedafter analyzing the sample. The analysis is done based on the influence of Electre magnetic 15 radiation on the analytes (say the UV-Visible radiation) and their response to it. In theexisting method of chromatography, the analytical report i.e., the chromatogram is notgivingany of the Chemical properties like polarity and UV-Visible absorptive properties ofthe constituents. The chromatogram is not able to show the presence of the moléculeswhich does not absorb at that wave length or hâve a different “Absorbance maxirna” other 20 than the set wavelength (say 225 or 254nm). If the sample is 100% pure and if it is aknown molécule then the analysis at a fixed wavelength is acceptable, but it is highlyimpractical in the case of medicines where in more than one active molécule are présent.Some examples shown at single wavelength are given in Figures 5-12, where in thechromatograms at various wavelengths are given. None of the single chromatogram is able 25 to provide complété information about the Chemical properties of the constituents présentin the medicine particularly in traditional medicines where more than one active principlemay be existing. When the chromatograms and the fmgerprints are compared, the utility ofthe fmgerprints can be under stood.

Hence, any chromatogram presented at a spécifie wavelength is not able to provide the 30 complété Chemical profile of the ingrédients présent in a single medicine and a formulation. So, the chromatogram is partial in its report, and is not acceptable. Any analytical method, which is not giving complété information of ' the analysis, is not scientifically acceptable. 012520 12

In the analysis of herbal medicines, where different types of molécules are présent havingdifferent spectral properties (The absorbance maxima) the chromatogram at a fixed singlewavelength will not be a meaningfol analytical report or the chromatogram.

In the use of herbal medicines, the medicine as a whole is used with some standardtherapeutic conditions prescribed in the ancient literature and scripts. Hence, the concept ofsearching for an active ingrédient is said to be incomplète, because it is the total profilethat is responsible for the médicinal property of the medicine. So, any analytical method,which does not speak about the complété Chemical properties of ail of the constituéesprésent in the medicine under study, will not be useful.

Also the qualitative and quantitative profile of an herbal medicine vary due to manyecological factors like time of collection, place of collection, âge of collection andmonsoon conditions at the part of collection and soon.

It is already mentioned (Frank R Stermirtz et al, Ref. 11) that the synergy of the otherconstituées présent along with the major constituent is equally important because the firstwill not be able to do its fonction without the other constituées présent in the extract asexplained in the beginning. B. PRIOR ART ON THERAPEUTIC STAND ARDIZATION: I) Traditional Method:

The great Indian Medical sages hâve understood and defmed the concept of Indianmedicine by clearly defming the properties, constituées and humors of the living beings.They also understood the inter and intra relations amongst them. In almost ail thetraditional philosophies, the basic concepts include the nature and its rôle on tHe humors ofthe human beings. It is said that the human body is made of seven types of constituées(Saptadhatus). The normal disorders (Tridosha) are of three types. The materialisticproperties of any material in the universe are due to five éléments (Pancha bhutas). Theinteractions of different permutation and combination of these éléments will influence thehealth. Hence, the understanding of these properties will help to understand their physicaland Chemical properties and so, there by their therapeutic efficacy. The philosophers indifferent parts of world hâve also developed such concepts suitable for their tradition andsociety.

In ancient times (pre Samhitic and pre Susrutic period in India),, the physicians used

Nadisastra (Science of reading puise) to know the status of the Tridosha (Vata, Kapha and

Pitta) at the time of diagnosis to know the health status of the patient. The spécifie type of

°1252q 13 puise (not the heart puise) is studied to explain the type of disorder pre-dominant in thepatient (Dr P.V.Sharma, Ref. 1).

It is used to understand the type of dosha(s) prédominant in the patient at the time ofdiagnosis and the respective dosha(s) to be vitiated to cure the disorder. But this art of 5 reading Nadi (Puise) was confined to some people of high caliber, personal skill and abilitywith lot of discipline. Hence, every traditional practitioner was not able to practice it.

To over corne this, the art of understanding the physico-chemical properties of themedicines and the humors of the human being had been developed and standardized. Theinter and intra relations of these properties with nature which influences health had been 10 studied and standardized thus the art of pharmacology and pharmaco-therapeutics wasdeveloped by the physicians.

The therapeutic efficacy of a drug is defined with use of a substance that is capable ofbringing about an (pharmacological) action in the human body (Kriyagimavat) and due tothe collective functioning of many factors, (sama\>ayikaranamf just as a piece of cloth 15 results because from its many component threads actrng togeiher.

In the world, there are two main types of living things, the plants and animais. It is alsosaid that this world is made of five great éléments i.e. Earth, Water, Air, Fire and Space (assaid Panchabhutas in Ayurveda). The basic properties of these materials are of two types,namely Strong (PowerfuI) and Mild (Soft). If we accédé to this highly tenable logic, we 20 can say that in this world, ail actions are due to different permutation and combinationsériés of the above properties, giving a wide range of properties and. materials varying intheir intensity.

S

In the philosophy of most of the traditional medicine world over, the co-inherence of thenature of the five constituents is taken into considération by which the body is made. They 25 will help in understanding the disease or disorder of the patient. This cohérence is calledPurusha in Ayurveda, Yin and Yang in Chinese medicine. Table 6 appearing later part ofthe description gives how the Chinese System has used the above two factors, how theywere classified and defined to standardize the therapy and diseases.

Chinese medicine classifies the status of the human body as Yin and Yang representing 30 sorrow and happiness as mentioned above. These factors are attributed for various properties of the medicines and living beings. The maintenance of these factors is done holisticaily by taking the rôle of Chemical, physiological and social factors in to considération. Most of the time the Chinese medicine has a direct or indirect relation with 012520 various bio energy centers located in the body. The art of acupuncture uses the same. Theother factors that reported in other philosophies hâve resemblance with Chinese medicine.After the panchabhautic concept, the concept of Tridosha (Pitta, Kapha and Vata) plays amajor rôle in the Indian traditional medicine and the seven constituées (Saptadhatus) bywhich the body is made up of.

Ayurveda believes in the holistic philosophy of Iife and emphasis is given for theprévention of diseases rather than curing of diseases. The holistic approach of ayurvédaadvocates that the soûl, mind and the body are the three intégral parts of life and whenthese are in dynamic equilibrium and harmony, the State is called good health (Arogya).When they are in inequilibrium and disharmony, the State is called disease. (Vaishamya).According to Ayurveda, Tridosha maintains the physiological features of various Systemsin dynamic equilibrium status. In other words, harmony of tridoshas bestows good health,disharmony results to disease. Hence, most of the time the tridoshas are dealt with, incuring any disease. The sélection of drug is made for the disease that should be dealt with.A disease is defined as “Any thing that brings a sadness and grief to this person (Purusha)is a disease. They are of four types l.The accidentai (agantavaha) 2.The body born(Sarirah) 3.The Mind born (Manasah) and 4.The natural (Swabhavikah). It is for thisreason, most of the traditional concepts deal with both psychosomatic factors to cure thedisease along with a disciplined and standardized method of life.

The diseases were classified into three classes generally.l, The curable (Sadhya) 2. TheMitigateable or manageable (Yapya) and 3. The incurable (Asadhya).·.

As said above, it is mostîy considered as those bodily diseases having their source arise bythe incompatibilities of the thridoshas viz., Vata, Kapha and Pitta and blood individually orin combination with one another. But, the diseases that arise not due to the above reasonlike psychological are dealt in a different way. That is why any traditional concept is usedto take ail the psychosomatic factors in to considération to deal with a disease. Theindividuaî properties of the doshas are explained as given below.

Broadly it is outlined that the Vata or vayu dosha deals with endocrinological,neuromuscular and nervous activities ail those that cause the major or the gross dynamicsof the life, the foods that cause gas formation can be classified in this category. Pitta dosharefers to digestion and Chemical fonctions or rasa kriya in general and Kapha doshaincludes factors providing form, stability, and cohésion and iubrication factors. As the Firstdosha, ‘Vata’ is considered to influence the other two it is considered as the key factor for

012520 15 any disease. An elaborate description of these humors is given in elsewhere in the body ofthe text.

The decrease of vata Ieads to general duliness in activity. Hence, the drugs, which decreasethis duliness, will be of Vata Hara. The decrease of digestive capacity is calied as pitta 5 dosha. A medicine, which increase the digestive capacity or activate bile mechanism, willbe of Pitta Hara in nature. The decrease of liquidity or mucous will lead to roughness, aninternai burning, an emptiness in stomach, a looseness in the joints, thirst, weakness and acontinuos insomnia. These are the basic symptoms of kapha disorder. Any medicine thatvitiates this disorder will be of Kapha Hara in nature. 10 A detailed description of ail the factors is given for various philosophies in order to understand more generaîly about different traditional medicines world over. Table No 1 andTable No 2 (which are appearing in the later part of the description) gives an elaboratedescription of the Indian Ayurvedic philosophy and various components in it.

Hence, to understand the therapeutic efficacy of a medicine or food, one needs to 15 understand their physical and Chemical properties. In the ancient times people use tounderstand these properties using the organoleptic methods like the taste, the smell and thecolor of the materials. The basic properties cîassifîed were 1.Taste (Rasa), 2.Quality(Guna) S.Potency (Virya) 4.Post assimilative status and effect of the’constituents (Vipaka)and 5. Spécial action (prabhava, geometrical and optical isomer molécules) 20 It is these three factors namely, the Doshas (Disorders), the Dhatus (constituents) and theMalas (excreta) that are mainly to be dealt for curing a disease or a disorder. If the above-mentioned properties of the medicines tally with the dosha, it will be vïtiated orneutralized, thus the disease is cured.

The classification and différentiation of drugs according to Ayurvedic pharmacodynamie 25 and genetic principles vary from one situation to another according to doshicprédominance of the patient. In other words there is a relation between the dravya gunas(médicinal properties) and doshas (disorders). Addition or délétion of one or more drugsmay be necessitated to treat an identical disease with the patients with different individualdoshas or combination of doshas. Hence, Ayurvedic pharmacotherapy is more 30 individualistic according to dosha prédominance of the patient and not generalized as inthe case of modem medical pharmacotherapy. Identification of properties (Rasa, Guna,Veerya, Vipaka and Prabhava) compatible to doshas is unique and more reliable inAyurvedic pharmacotherapy. Û12520 16 ii) Modem method of therapeutic standardization:

The existing pharmacotherapy has not taken the above mentioned concepts intoconsidération. Phytochemists are interested only in isolation, purification and structuralélucidation of the active principles isolated ffom the plants and they passed on them topharmacologists to study their biological activity. The pharmacologists in turn screen themolecule(s) for pharmacological activity, establish its mechanism(s) of action andsubstantiaily rate its eiïicacy in comparison with the existing standard drugs used inmodem medicine.

This concept is in no way going to help the traditional medical practitioners since theisolation of the active principle(s) drastically change the holistic character of the medicinesand their therapeutic efficacy.

Instead of assaying the solvent extraction fractions, active principles etc., obtained fromthe individual plants, the analysis of total extract ffom a medicine using a solventcompatible to the human cells and cell membranes of the body will be of much use toevaluate the pharmacological activity of such medicines.

In the modem clinical trials conducted for the therapeutic standardization, they are done inthree phases (four in the case of international utility), involving large number of people.The information regarding a new medicine to be submitted to Drug Controller generallyconsists of: 1. Chemical structure 2. Pharmacological class 3. Formulation details 4. Data on animais including data on toxicity studies 5. · Data on clinical pharmacology including pharmacokinetics (Behavior of the drug in the human body) 6. Pharmacodynamies (Actions of the drug inside the body) 7. Spécial studies and Status of the drug in the rest of the world. S. Data on Bio-Equivalence studies

The Phase one study is mainly concemed with assessing the drug’s safety to know how the medicine is absorbed, metabolized in the human body and excreted, also it envisages to estimate the side effects and the dosage.

The phase two studies are dedicated to test the efficacy in a randomized way. One group of patients will be given the actual medicine and the second with placebo.

012520 17

In the phase three study, a large scale testing will be taken up to study the effectiveness,benefits and the range of possible adverse reactions of the drug. After successfulcompletion of this step, the industry will go for marketing the drug.

In late phase III and IV studies, pharmaceutical companies will hâve several objectives. 5 Studies will help to know the efficacy of the new drug compared to an existing drug. Thelong-term effectiveness and impact on a patient’s quality of life due to the new drug will beknown. The cost effectiveness of the drug therapy relative to other traditional and newthérapies will be known.

But ail the above studies are costly and time consuming. They will not be taking into 10 account of the rôle of the ecological factors, the genetical disciplineras practiced in theIndian family and marnage relations), the psychoiogical, the social and other variableparameters of the patient in to considération. This will make the effectiveness of the druglimited to a particular group or genetic type of people. 15 C. PRIOR ART OF BARCODING AND ENTERPRISE RESOURCE PLANNING(ERP) CUSTOMER RELATIONSEOP MANAGEMENT APPLICATIONS:

The modem method of making any commercial goods proprietary is Bar-coding. For ailcommercial transactions, the barcode is widely used in many ways. To make the medicinesidentified as proprietary goods, a novel method ofbar coding is proposed in this invention. 20 It is reported (Peernet bar-code store (Java Active X servlet e-business)) that 1800character.and 2700 digits (Even 9,99,999 numbers) can be fed to a commercially availablebar coding software to generate a barcode of any item. When a digital value and/ornumerical number is given to the bar-coding software it generates a spécifie barcodepattern by the îogic specified in the software proprietary for a user. 25 The barcode thiis generated will présent and display the attached ‘display window” fileinformation having ail details of the product/ label, when a barcode readable vendingmachine sees (through electronic eye or sensor) the barcode on the product. The barcodecan be read from any ERP and CRM applications world wide through network.

Presently the catalogue numbers are being used for bar coding the medicines and related 30 products which does not specifically contain any Chemical property of the product asproposed in the method.

OBJECTIVES OF THE PRESENT INVENTION

The main objective of the invention is to propose a novel method of chromatographie 35 finger printing, Chemical and therapeutic standardization and bar coding of organic and 012520 1S . organo-metallic molécules from a plant, animal or a naturally available or man madematerials used as medicines.

Another objective of the présent invention is to provide a novel chromatographie fingerprinting of herbal medicines and formulations which obviâtes the drawbacks detailedabove.

Another objective of the présent invention is to provide a complété Chemical analysis ofthe constituents présent in the medicine under study and their conjugative propertiesindicating the therapeutic efficacy as per the traditional concepts of the medicine usingnew software developed.

Still another objective of the présent invention is to provide a novel method forchromatographie finger printing of herbal medicines useful for the quick identification ofthe actual profile of the compounds présent in the medicine under use along with theirtherapeutic efficacy of the constituents.

In yet another objective of the présent method is to provide a novel chromatographie fingerprinting of herbal medicines and formulations using the contour and 3-D chromatogramsof the herbal medicines and formulations.

In yet another objective of the présent invention is to provide a novel method for ♦ chromatographie finger printing of herbal medicines useful to check the adultération of thecompounds présent in the medicine under use.

Yet another objective of the présent investigation is to préparé a standard analyticalparameters like extraction with same solvent ethyl alcohol, same run time 0-60min, samemobile phase acetonitrile along with phosphate buffer having a pH in the range of 5.5-7.5,and a same UV-Visible Range of 200-S00nm.

Yet another objective of the présent invention is, to categorize and quantify theconstituents of a medicine based on polarity and conjugation from 3-D and contourchromatograms and assess the therapeutic efficacy of the medicine on which humors it isgoing to act (vitiate).

Yet, another objective of the présent invention is to provide a barcode for the selected peakof a molécule given in the image.

Yet another objective of the présent invention is to préparé a database of barcodes for thefingerprints developed useful for ali types of database applications. .

Yet another objective of the présent investigation is to generate display Windows for ail thesamples of the fingerprints having the details of the samples like 3-D and Contourfingerprints, the barcode, details of the origin (Industry or Country), manufacturing date, 19 012520 date of expiry, reported dosha, individual constituents used, their assay, batch number, lotnumber, M.R.P (maximum retail price) etc.

Yet, another objective of the présent invention is to attach the display Windows with therespective barcodes, facilitating to deal with display Windows in ail applications wheneverthey are used as a source of data and information.

Yet another objective of the présent invention is, to préparé a database of display Windowsthus generated and attached to the respective barcodes, to use in the Enterprise ResourcePlanning (ERP) and Customer Relationship Management (CRM) applications for ailcommercial networking transactions of the medicines and samples.

Yet another objective of the présent investigation is to préparé a database of barcode anddisplay Windows and any information, specially required for the regulatory authorities tocontrol the movement of the medicines in and out of the country.

Yet, another objective of the présent invention the UV-Visible spectra of the compoundswill provide the conjugative properties of the molécules and the concentration of theindividual concentrations of the molécules along with the polarity of the molécules.

Yet another objective of the présent invention the use of finger print of contour and 3-Dchromatograms will be the basis for the identification of Chemical constituents to limit thescope of the invention.

Yet another objective of the présent invention is to develop a method of fingerprinting forthe adulterated food and drug samples, substituted and contradictual food and drug samplesand commercial samples of food and drug samples and to identify the pure and adulterated.Yet, another objective of the présent invention is to develop a method of fingerprinting forthe organic and organo-metallic constituents in any type samples to identify the Chemicalconstituents présent in it for various purposes of quality control and processstandardization.

Yet another objective of the présent invention is to develop a method of fingerprinting forthe Allopathie, Ayurvedic, Homoeo, Siddha, Unani, Chinese, Tibetan and ICampo(Japanese) medicine samples for the quality control and Chemical and therapeuticstandardization

Yet, another objective of the présent invention is to develop a method of fingerprinting forthe study of variation of Chemical constituents in naturally occurring or syntheticallyprepared samples and to identify and standardize the Chemical constituents in them.

Yet another objective of the présent invention is to develop a method of fingerprinting forthe study of variation of Chemical constituents in naturally occurring or synthetically 20 01252a prepared samples and to identify and standardize the variation in Chemical constituées inthem due to Geological, Ecological, Genotypic and Phenotypic variation factors.

Yet, another objective of the présent invention is to develop a method of fingerprinting forthe study of Chemical constituées in herbal products of single and formulated medicinesamples and to identify the Chemical constituées in them for Chemical and therapeuticstandardization.

Yet, another objective of the présent invention is to develop a method of fingerprinting forthe study of variation of Chemical constituées in biological samples and to identify andstandardize the Chemical constituées in them.

Yet, another objective of the présent invention is to préparé a large· database, which willgive many generalizations of the therapeutic efïicacy of a particular group of plants,classified as a group for a particular disease or therapeutic classification.

Yet another objective of the présent invention is to provide a method which enables tounderstand and standardize the Physico-Chemical properties of the medicines like colorused in traditionaî method of therapeutic standardization using conjugative and polarityproperties of the individual constituents and the whole medicine.

Yet another objective of the présent invention is to provide a method which enables tounderstand and standardize the Physico-Chemical properties of the medicines like Taste(Rasa) namely Sour, Salty, Pungent, Bitter, and Astringent (Amla, Lavana, Katu, Tikta,and Kashaya as said in Ayurveda respectively) used in traditionaî method of therapeuticstandardization using conjugative and polarity properties of the individual constituents andthe whole medicine.

Yet, another objective of the présent invention is to provide a method, which enables tounderstand and standardize the Physico-Chemical properties of the medicines like Quality,Potency, Métabolite after assimilation or such modifications and Spécifie properties likeChirality of the molécules (Guna, Veerya Vipaka, and Prabhava respectively as said inAyurveda)

Yet another objective of the présent invention is to provide a method which enables tounderstand and standardize the Physico-Chemical properties of the medicines like Heavy,Light, Cold, Hot, Soft Lubricated Supple, Dry, Slow, Sharp (Guru, Laghu, Sheeta, Ushna,Snigdha, Manda, Teekshna respectively as said in Ayurveda) used in-traditional method oftherapeutic standardization using conjugative and polarity properties of the individualconstituents and the whole medicine.

21 °1252Ο

SUMMARY OF THE INVENTION

This invention relates to a method for détection and identification of principles fromextracts of plants or animal, natural or synthetic sources, using chromatographie fingerprinting techniques, said method comprising the steps of: i. ) extracting the organic or organo-metaliic molécules using a suitable solvent; ii. ) subjecting the extract obtained in step (i) to the séparation analysis, using High

Pressure Liquid Chromatography techniques; iii. ) generating contour and 3D chromatograms of the ingrédients eluted based on the pH and polarity; iv. ) converting the 3-D and contour chromatogram obtained into a colored image, analyzing the colored image for its individual colors using the co-ordinatesdenoting ail its 3-dimensional properties of the said image by using an in-builtsoftware; v. ) denoting the concentrations of the various constituents eluted with time; vi. ) generating a chromatogram based on color analyzed, having peaks at various rétention times along with conjugative properties of the molécules; vii. ) identifying the compounds in the said ingrédients by the UV-Vis absorptive properties of the various constituents in the image; viii. ) identifying, determining and classifying the compounds eluted as polar, medium polar and less or non-polar based on the polarity and conjugative properties; ix. ) generating a barcode for a selected peak using the X axis as Rétention Time, Y axis as Wavelength, R as number of Red Pixels, G as number of Green Pixels and B asnumber of Blue Pixels; and x. ) · generating a database of fingerprints and barcodes and identifying the respective compounds in the samples. DETAILED DESCRIPTION OF THE PRESENT INVENTION:

Accordingly, the novel basis of the présent method is, presenting the spectral properties ofthe Chemical constituents displayed in 3-D and contour chromatograms as a novel methodof fingerprinting. The chromatogram generated by this method provides the conjugativeand polarity properties of the individual molécules présent in the medicines giving thetherapeutic efficacy of the medicine.

In a molécule the UV-Visible absorbance capacity of the molécules dépends on thestructure of the molécules. When the double bonds or triple bonds are présent in themolécules altematively in the structure, it is called as conjugated. The more the molécule is 012520 22 . conjugated the more it will be chemically and biologically active. Hence the more themolécule is conjugated the more it will be therapeutically active. Thus, the measurement ofconjugative properties will give the therapeutic efficacy of a medicine. Hence, the use ofconjugate property for the therapeutic standardization is the novelty of the présentinvention. A novel method is proposed for the quality control of herbal medicines and formulationsmostly useful for the fmgerprinting and standardization (Chemical and therapeutic) ofTraditional Medicines unlike a method being used for analyzing only active ingrédient(which is not known in many herbal medicines) for the analysis of medicines at a singlewavelength. It gives the total profile of the Chemical constituents présent in the traditionalmedicines along with physical and Chemical properties of the compounds (Say UV-Visibleabsorptive property and polarity property). In the first part of the method, an image of thefîngerprint of the medicine will be generated. But as image cannot becomes analytical data,a computer-based method is developed to give the qualitative and quantitative data of theingrédients in the form of an analytical chromatographie report. The same is proposed as anovelty of the method of the invention.

The reactivity of any molécule will dépend upon the number of double and triple bondsexisting in the molécules along with the electrophilic and nucleophilic sites on themolécule. The moieties donating électron and accepting électron will create différence inthe total electrical charge of the molécule. This makes the molécule polar. Hence polarityof the molécules will provïde information about the capability of a molécule to donate oraccept the électron with another molécule. This will control the activity of a molécule.Thus, the information of the polarity of a molécule will speak about the reactivity of themolécule. In the présent method, the chromatogram provided by the method will give theconjugative and polarity properties of the constituents présent in a medicine in the fmgerprint. Thus, this method is used for the standardization of the medicines to know thetherapeutic efficacy of a medicine using their conjugative and polarity properties of themedicines. This is the novelty of the proposed method.

As said above the UV-Visible spectra and polarity of the compounds will indicate theconjugative and polarity properties of the compounds and thus indicating the Chemical !médicinal property of the medicines. This profile of spectra of ali'the constituents in asingle picture, “the finger print” as proposed now will become the blue print of theconstituents présent in herbal medicines and formulations. This becomes a superior methodof identification and standardization of herbal medicines than the existing, as the peaks

°Ï252O will express the UV-VIS. Properties or conjugative and polarity properties of theconstituents, unlike in a conventional chromatogram taken at a single wavelength alongwith the quantification of the constituents. 5 As described in the traditional standardization methods the colors of the medicines wereused to know and standardize their therapeutic efficacy. The colors of the molécules areunderstood by their absorptive properties of the radiation of the UV-Visible range ofradiation. Based on the structure, functional groups, conjugation, and the extent ofunsaturation the absorbance of a particular wavelength dépends. The more the molécule is 10 conjugated the longer the wavelength of absorption will be. Hence, the UV-Visibleabsorbance of any molécule is widely used in the qualitative and quantitative properties ofthe constituents. The colors and the therapeutic efficacy of various medicines were given inthe ancient literature. 15 Ultimately the colors of the molécules are due to a spécifie Chemical nature of themolécule. When the same is studied, the Chemical property can also be understood. InAncient times the colors of the fiâmes were used for the quality control of metals andrelated products this, involves the basic spectrophotometric principles. Thus, study andunderstanding of the interaction of the electromagnetic radiation will· be useful to study the 20 Chemical nature and thus the therapeutic efficacy of the medicines. The same principle hasbeen used in the présent spectrophotometric method of fingerprinting and standardization.The main novelty of the présent method involves in the “division of the fingerprint intodifferent therapeutic zones based on the scales of wavelength (Conjugation) and rétentiontime (Polarity) to understand the therapeutic efficacy (in traditional terms) of a single or a 25 formulated medicine” using an instrumental and software based program.

Using the computer-based software developed, a barcode is generated for a selected peakof a molécule given in the image. Where, X is the Rétention Time, Y is the Wave length incontour chromatograms and absorbance in 3-D chromatograms, R the red color indicating 30 the highest concentration of the constituent, G the green color indicating the lesserconcentration of the constituent and B Blue color indicating still lesser concentration of theconstituent are the coordinates provided by the présent software, is feed in anycommercially available re-salable bar coding software, added in the présent softwaregenerates a barcode for a single constituent, or for many constituents. The Image of the 35 fingerprint is viewed on a display window attached to it. This will be displayed whenever the electronic eye of the vending machine reads the barcode. This makes the image (Finger °1252U' 24 print) and barcode proprietary for a product of an industry or a country. This is claimed asanother novelty of the method of the invention.

When the polarity of the column is fixed and the polarity of mobile phase is varied 5 constantly in an increased or decreased order, on a reverse phase column, the constituentsprésent in the sample will elute in the order of high polar constituents eluting first themedium polar constituents next followed by non-polar constituents. Care is taken in elutingthe constituents in the order of increased or decreased order of polarity such that noconstituent of any polarity will be leffc un-eluted from the column achieving total elution. 10 The order and properties of polarity and elution in the case of normal phase columns areapplicable same as in the case of reverse phase column but in reverse. In a normal phasecolumn the non-polar constituents will elute first and followed by polar constituents, basedon order of polarity of the mobile phase used for elution. 15 Thus, a fingerprint developed having the Chemical constituents arranged in the increased ordecreased order of polarity will help to bring therapeutic generalizations about themedicines. This is another novelty of the proposed method.

The image of contour chromatogram developed after the analysis is divided in to three 20 zones on X and Y-axis. The conjugative property (Absorption of a particular wavelength ofradiation) is taken on Y-axis and polarity is taken on the X-axîs, as the elution of theconstituents is controlled using the polarity of the mobile phase composition. Now asreported in literature the Y axis is scaled as per, the therapeutic efficacy based onwavelength (color). The entire image is divided in to six chambers wherein the Chemical 25 constituents hâve spécifie conjugative and polarity properties. This in turn is proportionalto the therapeutic efficacy of the constituents in the chamber. Thus when a medicine isfingerprinted,· based on the color represented for the absorption of a spécifie wavelengthand having a spécifie polarity, the total colors in that zone is calculated and interpreted forthe therapeutic efficacy of the constituents présent in it. Thus, the holistic therapeutic 30 standardization and Chemical standardization is achieved using this method.

Mostly the elution of the samples were done from high polarity mobile phase to lowpolarity mobile phase. Thus in the finger prints the constituents présent in the first zone(Zone-1) will be of high polar in nature. The same pattern applies to the other zones, the 35 medium polar constituents eluted in the medium polar zone (Zone -2) and the low or non- polar constituents eluted in the non-polar zone (Zone-3). This pattern reverse when a normal phase column is used due to its elution property as described above.

°1252Q 25

Most of the high polar molécules are highly reactive chemically, thus biologically. Whenthey enter the first part of the digestive System mouth, they will immediately start acting onthe biological System and the enzymes présent there. Then the constituents will enter thestomach and intestine where they will under go different changes (Post assimilationeffects, Vipaka in Ayurveda) due to the digestive juices and their enzymes présent in thepart. In the process of absorption, the moment the molécules of high activity (high polar)immediately start interacting with the biological System and show their therapeuticpropérties. This is compared thât in Ayurveda, the intestinal part of the human body isclassified as Pitta zone, where the high polar molécules are playing a major rôle. The heatcausing mechanism will play an important rôle in the diseases and biological mechanismsrelated to. It indirectly indicates the molécules of high reactive, the high polar molécules.After the absorption, the blood with ail the absorbed constituents will carry them to heartand the parts related to it. Then the blood will be sent to different parts of the body. InAyurveda, the upper portion of the human body is defmed as the Kapha zone, where the; cold mechanism will be playing an important rôle. Thus, the molécules of medium polarmolécules will play an important rôle in the mechanisms related to this zone.

The low polar and non-polar constituents will be able to enter to the human body onlythrough blood transfer, Thus the body organs where the mechanism of availability of theChemical constituents is only by blood, will be coming in the last category of the polarity.The non-polar oils, fats and other such molécules and mechanisms in the human body areclassified as Vata disorders and ail such disorders are cure using the same type ofmaterials.

The low and non-polar constituents will be eluting in the last zone of the fingerprint. Thus,this zone (zone-3) is considered as Vata zone. Thus, the basic humors of the molécules canbe identified based on their polarity, which facilitâtes to know on what disorder (dosha) itis going to act upon. Thus, the présent method is useful for the therapeutic standardizationof the medicines.

Thus the total constituents présent in the Zone-1 Pitta zone, Zone-2 Kapha zone, Zone-3Vata zone are présent in the form of a pie diagram which represe-nts the ratio of theefficacy of the medicine on each of the disorder. Thus, medicines having constituents inthe order of 50:20:30 will be medicines of tridoshahara of the order'of 50%: 20%: 30%.Thus, the therapeutic efficacy is standardized quantitatively. The increase or decrease ofany one or two of the other doshas is done by formulating medicine by adding othermedicines and préparé a suitable formulation needed to cure a spécifie individual.

°1252Q 26 .

Thus, a fingerprint having the scales of conjugation, absorbance and polarity the 3-Dchromatogram will give information about the therapeutic efficacy of the medicine.Analyzing it using ail its three dimensional properties of the said image will doquantification of 3-D chromatograms of the medicine. For example if the 3 -Dchromatogram is considered as a ‘cap with a hood’ the matching of the entire cap 3dimensionally, with another sample of different qualitative and quantitative properties, theextent it matched will be presented as an analyticaî report qualitatively and quantitatively.Here the hood of the Cap is compared to the peak of the molécule at a particularwavelength. A sample with more number will like a cap with many hoods. Thus thematching of the three dimensional coordinates will provide a foolproof method ofcomparison and analysis. The coordinate it matched will give qualitative and the extent itmatched will give the quantitative data of the sample understudy. This is made possible byspécial software prepared for this purpose. This becomes an ultimate method of qualitycontrol. This is another novelty of the method of the invention.

The invention also relates to a software based data processor of 3 D chromatograms andcolor contour image of an ingrédient, said processor comprising computing means andcapable of: a. an analyzer (extracting colors) for analyzing the colored contour image based on thesélection of various colors (with standards mentioned in release notes, life cycle,Processing) denoting the concentrations of the various constituents eluted with time,and polarity based on rétention time; b. an analyzer for analyzing the 3-D chromatograms of the medicine using ail its 3dimensional properties of the image; c. a means for generating a chromatogram having peaks at various rétention times alongwith conjugative properties of the molécules eluted with time in a specified order ofpolarity; d. an identifier for identifying the compounds in the said molécules by the UV-Visabsorptive properties ofthe various constituents in the image; e. a means for correlating the reported biological, therapeutic activity of the of variousconstituents présent in the medicines understudy based on the polarity and theconjugative properties of the molécules by dividing the fingerprint into therapeuticzones on X and Y axis; 27

01252Q

f. a means for generating a barcode for a selected peak(s) using the image coordinatesviz., X for rétention time, Y for wavelength, R for number of red pixels, G for numberof green pixels and B for number of blue pixels, provided by the proposed software; g. a means for generating a database of fingerprints and barcodes for the samples, 5 facilitating ail kinds of database utilities Iike Enterprise Resource Planning (ERP) and

Customer Resource Management (CRM) applications; and h. a means for generating a database of the ‘display widows’ for ail the samples to beused by the ENTERPRISE RESOURCE PLANNING (ERP) and CUSTOMERRELATIONSHIP MANAGEMENT (CRM) type of business applications.

10 ABBRIVIATIONS USED EN THE PATENT DOCUMENT 1. ERP : Enterprise Resource Planning 2. CRM: Customer Relationship Management 15 3. UV-Visible: Electromagnetic radiation in the range of200nm to SOOnm 4. Organic molécule: A molécule having basic éléments of C, H, N, O, S in its structure. 5. Organo-Metallic molécule: A molécule having a métal along with basic éléments of C,20 H, N, O, S in its structure 6. Contour Chromatogram: A type of chromatogram displayed in the data· generated froma Photo Diode Array detector, which scans the sample with electromagnetic radiationin thé range of 200nm to 800nm. The chromatogram thus generated will provide 25 Rétention time on X-axis, range of absorbance (nm) on Y-axis. Different colors will beused to represent different concentrations of the individual constituées. 7. 3-D chromatogram: This is also generated using the same set of equipment as givenabove. It will be more informative providing the UV-Vis spectra of each of the in-gradients after séparation from a mixture. It helps to identify the constituées using the 30 spectrum. 8. Ayurveda: An Indian philosophy written by Indian sages explaining organized scienceof medicine and health discipline. 9. Oshadisukta: a chapter in Rigveda giving the détails of the properties of the medicinesused as medicines. 35 10. Rasa, Guna, Veerya, Vipaka, and Prabhava: Different Physico-Chêmicaî properties of the medicines and materials used to understand the effîcacy of the medicines used in

Indian System of medicine. 11. Lokapurusha Samanya: Law of Uniformity of Nature 012520" 28 12. Tri doshas: three humors using which the human body is studied viz., Pitta, Kapha andVata used in Indian System of medicine. 13. Prakriti-Purusha: The first one is compared to Mother Nature (woman) and the secondto person (man) used in Indian System of medicine 14. Pitta: A term used in Indian System of medicine for one of the humors in the body inIndian System of medicine to dénoté the personality or a disease which dénotés thedigestion and Chemical fonctions or rasa kriya in general in the human body. 15. Kapha: A term used in Indian System of medicine for one of the humors in the body inIndian System of medicine to dénoté the personality or a disease which dénotés thefactors providing form, stability, and cohésion and lubrication factors in the humanbody. 16. Vata: A term used in Indian System of medicine for one of the humors in the body inIndian System of medicine to dénoté the personality or a disease which dénotés theneurological and endocrinological, and nervous activities in the human body. 17. Geological factors: Global variation in soil nature and ground water constitution etcrelated to earthly components. IS. Ecological Factors: Global variation in tropical régions, monsoon conditions andtempératures. 19. Organoleptic methods: Methods of identification of the properties of medicines like l.Taste (like Sour (Amla), Salty (Lavana), Pungent (Katu), Bittér (Tikta), Astringent(Kashaya)) 2.Color, 3. Odor and 4. Texture etc., using human sensory organs. 20. Tastes (Rasa Physico-Chemical properties) Properties visibly seen (Coior, Size) andfelt (Texture) and ali physical properties and properties like taste and médicinal relatedto the chemistry of the individual constituents présent in the medicines. 21. The medicines were standardized using their properties like Taste (Rasa), Quality(Guna) Potency (Virya), Post assimilative status and effect of the constituents (Vipaka)and Spécial action (Prabhava,) 22. Saptadhatus: The 7 éléments like Rasa (Body fluids), Rakta (Blood), Mamsa (Muscle),Majja (Bone marrow), Asthi (Skeleton System), Medas (Fat) and Shukra(Reproductive) constituents présent in the human body used -in Indian system ofmedicine. 23. Panchbhutas: The 5 natural éléments like Prithivi (Earth), Ap (Water), Teja (Fire),Vayu (Air) and Akash'a (Space) présent in the world used in Indian System of medicine 24. Nadisastra: A science, which explains the health status in the human body, used inIndian system of medicine by studying the puise of the person. 29 °Τ252θ 25. In Indian System of Medicine, Factors causing disease are explained as Agantavaha(accidentai), Sarirah (body born), Manasah (Mind bom) and Swabhavikah (natural) 26. The diseases were classified into three classes as per Indian System of Medicine Theyare Curable (Sadhya), Mitigateable or manageable (Yapya), The incurable (Asadhya) 27. Conjugative property: If a molécule has alternative single and double bond and électrondonating and accepting property it is called conjugative. This is seen in the UV-Visiblespectrum of a molécule. Based on the energy absorbed for the excitation of Sigma andPi électrons in a molécule from the electromagnetic radiation, the molécule will absorba spécifie wavelength of radiation. The absorbance maxima of’a molécule will thusindicate the conjugative property of the molécule under study. 28. Polarity property: If a molécule has différence in its electrochemical property, it iscalled polar. This dépends on the atoms attached to the molécule with électrondonating (nucleophilic) or électron accepting (electrophilic) moieties or fonctionalgroups the molécule will hâve a différence in its electrical charge on its molecularorbital. This makes the molécule to hâve a positive end and négative ends. This type ofmolécules is called polar molécules. The extent and type of electrical charge will makethe molécule polar, medium polar and non-polar in nature. 29. Gradient or temary System of HPLC: A HPLC instrument having two or three liquidpumps to vary the ratio of the aqueous or non-aqueous solvents. This will help tocontrol the total polarity of the mobile phase as per the requirement.

Some abbreviations used in software: 1. JDK: Java Development Kit 2. Con: Contour Chromatogram 3. .3-D: 3 - Dimensional Chromatogram 4. WOS: Without Scale 5. X: Represents the Rétention Time of the chromatogram 6. Y: Represents the absorbance in the 3-D chromatogram and wave length range incontour chromatogram 7. R: Intensity of red color at a particular pixel position S. G: Intensity of green color at a particular pixel position 9. B: Intensity ofblue color at a particular pixel position

Embodiments

One of the présent embodiments of the présent invention relates to a method forchromatographie finger printing, Chemical and therapeutic standardization and bar coding

Wf .. 012520 30 of organic and organo-metallic molécules from a plant, animal or a naturally available orman made materials.

Another embodiment of the présent invention relates to a novel method forchromatographie finger printing of herbal medicines and formulations, which obviâtes thedrawbacks detailed above.

Still another embodiment of the présent invention relates to a method for a complétéChemical analysis of the constituents présent in the medicine under study and theirconjugative properties iridicating thê therapeutic efficacy as per the traditional concepts ofthe medicine using new software developed.

Still another embodiment of the présent invention, relates to a method of novelchromatographie finger printing of medicines that is useful for the quick identification ofthe actual profile of the compounds présent in the medicine under use along with theirtherapeutic efficacy ofthe constituents.

In yet another embodiment of the invention, an embodiment of a novel concept ofchromatographie finger printing of herbal medicines and formulations using the contourand 3-D chromatograms of the herbal medicines and formulations is. proposed. They weredeveloped on a Photo Diode Array detector (PDA) of a High Pressure LiquidChromatograph. This delineates the data of the spectral properties of the constituentsprésent in' the herbal medicines eluted under experimental analytical conditions.

In yet another embodiment of the présent invention relates to a method for thechromatographie finger printing of extract from a medicine of any nature containingmolécules that absorb Ultraviolet and Visible range of radiation (200-S00) or- of any rangeof electromagnetic radiation.

In another embodiment of the présent invention, the UV-Visible spectra of the compoundsprovide the conjugative properties of the molécules and the concentration of the individualconcentrations of the molécules.

In another embodiment of the présent invention the fingerprints developed for a samemedicine extracted under different pH values are helpful to understand the drug release inthe intestine System at different pH values of an individual.

In yet another embodiment of the présent invention, the UV_VIS spectra of ail theconstituents are shown in a single image “The Chromatographie Fingerprint”

In yet another embodiment of the présent invention the finger print becomes the blue printof the constituents présent in a herbal medicine or formulation for an assay and quickidentification of the medicine understudy. 31 072520

In yet another embodiment of the présent invention the finger print using the contour and 3-D chromatogram is the basis for the identification of Chemical constituents existingand/or formed new.

In yet another embodiment the UV-Vis spectra and polarity of the compounds indicates theconjugative and polarity properties of the compounds and thus indicating the Chemical /médicinal property of the medicines. This profile of spectra of ail the constituents in asingle picture, “The Finger Print” as proposed now becomes the blue print of theconstituents présent in herbai medicines and formulations. This becomes a superior methodof identification and standardization of herbai medicines than the existing, as the peaksexpress the UV-VIS. Properties or conjugative and polarity properties of the constituents,unlike in a conventional chromatogram taken at a single wavelength along with thequantification of the constituents.

In yet another embodiment of the présent method the “division of the fmgerprint intodifferent therapeutic zones based on the scales of wavelength (Conjugation) and rétention- time (Polarity) to understand the therapeutic efficacy (in traditional terms) of a single or a• formulated medicine” is done by using an instrumental and software based program.

In yet another embodiment of the présent invention, from a large database prepared usingthis method, it gives many generalizations of the therapeutic efficacy of a particular groupof plants, therapeutically classified as a group for a particular disease..

In yet another embodiment of the présent invention using the X, Y, R, G, B as coordinatesof a selected peak in the fmgerprint, a barcode is generated using a bar coding software,which makes the medicine proprietary for an industry.

In yet another embodiment of the présent invention, 3 -D chromatogram of the medicine isanalyzed using ail its 3 dimensional properties of the said image. If the 3-D chromatogramis considered as a cap with a hood, the matching of the entire cap 3 dimensionally, withanother sample of different qualitative and quantitative properties, the extent it matched ispresented as an analytical report quaütatively and quantitatively. Here the hood of the Capis compared to the peak of the molécule at a particular wavelength. A sample with morenumber will like a cap with many hoods. Thus the matching of the three dimensionalcoordinates will provide a foolproof method of comparison and analysis. The coordinate itmatched will give qualitative and the extent it matched will give the quantitative data ofthe sample understudy. This is made possible by spécial software prepared for thispurpose. This becomes an ultimate method of quality control.

In yet another embodiment of the présent invention relates to a method to provide a novel 012520 32 . chromatographie finger printing of herbai medicines and formulations using the contourand 3-D chromatograms of the herbai medicines and formulations is proposed. They aredeveloped on a Photo Diode Array detector (PDA) of a High Pressure LiquidChromatograph. This delineates the data of the spectral properties of the constituentsprésent in the herbai medicines presented in a spécifie order of polarity under experimentalanalytical conditions.

In yet another embodiment of the présent invention relates to a to use UV-Visible spectraof the compounds which provides the conjugative properties of the molécules and theconcentration of the individual concentrations of the molécules along with the polarity ofthe molécules.

In yet another embodiment of the présent invention relates to a method to provide theUV_VIS spectra of ail the constituents shown in a single image “The ChromatographieFingerprint”. The fmgerprint thus becomes the blue print of the constituents présent in asingle medicine or formulation for an assay and quick identification of the medicineunderstudy.

In yet another embodiment of the présent invention relates to use of fmgerprint of contourand 3-D chromatograms as a basis for the identification of Chemical constituents to limitthe scope of the invention.

In yet another embodiment of the présent invention relates to the method having standardanalytical parameters like Extraction with ethyl alcohol, maintaining same run time 0-60min, using same mobile phase acetonitrile along with phosphate buffer having a pH inthe range of 5.5-7.5, and a same UV-Visible Range of 200-S00nm.

In yet another embodiment of the présent invention, a method uses a standard analyticalparameter like Extraction with same solvent ethyl alcohol for ail samples for the fingerprinting of a particular therapeutic group of samples to make therapeutic general izations.

In yet another embodiment of the présent invention relates to a method of fïngerprintingfor the adulterated food, drug and Chemical samples and to identify the pure andadulterated.

In yet another embodiment of the présent invention relates to a method of fïngerprintingfor the substituted food, drug and Chemical samples and to identify the pure and thesubstituted.

In yet another embodiment of the présent invention relates to a method of fïngerprinting for the contradictual food, drug and Chemical samples and to identify the pure and the substituted. 012520.- 33

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the commercial samples of food and drug and to identify the pure and the substituted.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the organic and organo-metallic constituents in any type samples to identify theChemical constituents présent in it for various purposes of quality control and processstandardization.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the Allopathie, Ayurvédic, Homoeo, Siddha, Unani, Chinese medicine, Tibetan andKampo (Japanese) medicine samples for the quality control and Chemical and therapeuticstandardization

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of variation of Chemical constituents in Naturally occurring samples and toidentify and standardize the Chemical constituents in them.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of variation of Chemical constituents in Naturally occurring samples and toidentify and standardize the variation in Chemical constituents in them due to geologicaland Ecological factors.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of variation of Chemical constituents in Naturally occurring samples and toidentify and standardize the variation in Chemical constituents in them due to Genotypicand Phenotypic variation factors.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of Chemical constituents in Synthetically prepared samples and to identify andstandardize the Chemical constituents in them for Chemical and therapeutic standardizationwhich ever is applicable.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of Chemical constituents in herbaî products of single and formulated medicinesamples and to identify the Chemical constituents in them for Chemical and therapeuticstandardization.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of variation of Chemical constituents in biological samples and to identify andstandardize the Chemical constituents in them.

In yet another embodiment of the présent invention relates to a method of fingerprintingfor the study of variation of Chemical constituents in different brands of products of single 34 012520 and formulated food and medicine samples and to identify the Chemical constituents inthem for Chemical and therapeutic standardization. ïn yet another embodiment of the présent invention relates to a method of preparing adatabase giving many generalizations of the therapeutic efficacy of a particular group ofplants, classified as a group for a particular disease or therapeutic classification.

In yet another embodiment of the présent invention relates to a method to developfingerprinting and to categorize and quantify the constituents of a medicine, based onpolarity and conjugatïon front 3-D and contour chromatograms.

In yet another embodiment of the présent invention relates to a method, which provides abarcode for the selected peak of a molécule given in the image, wherein X- the rétentiontime, Y- the wavelength, R - the number of red pixels, G the nurnber of green pixels and Bthe number of blue pixels are the coordinates provided by the présent computer based(Microchip, Dongle switch, hardware and software locked) software and is feed to anycommercially available re-salable bar-coding software résident in the proposed software,generates a barcode. Some examples of images displaying the X -rétention time, Y -wavelength, R - number of red pixels, G - number of green pixels and B - number of bluepixels the coordinates of a particular peak (s), which is spécifie to the product and thebarcodes thus, generated are enclosed.

In yet another embodiment of the présent invention relates to a method, to préparé adatabase of barcodes for the fmgerprints developed usefiil for ail types of databaseapplications.

In yet another embodiment of the présent invention relates to a method to generate displayWindows for ail the samples the fmgerprints are developed. In the ‘display window’ aildetails of the samples like both 3-D and Contour fmgerprints, the barcode, details of theorigin (Industry or Country), manufacturing date, date of expiry, reported dosha, individualconstituents used, their assay, batch number, lot number, M.R.P and other information onthe label), are displayed. When the barcode on the label is shown to the vending machine,it will display the attached display window. This helps to know the Chemical andtherapeutic authenticity of the medicine being sold/purchased in ail types of regulatory andcommercial applications.

In yet another embodiment of the présent invention relates to a method to attach the display Windows with the respective barcodes, facilitating to deal with display Windows in ail applications whenever they are used as a source of data and information.

In yet another embodiment of the présent invention relates to a method to préparé a database of display Windows thus generated and attached to the respective barcodes, to use 35 01252'ff in the ENTERPRISE RESOURCE PLANNING (ERP) and CUSTOMERRELATIONSHIP MANAGEMENT (CRM) applications for ail commercial networkingtransactions of the medicines and samples for which the database was prepared.

In yet another embodiment of the présent invention relates to a method to préparé adatabase of barcodes and display Windows and any information, specially required for theregulatory authorities to control the movement of the medicines in and out of the country.

In yet another embodiment of the présent invention relates to a method forchromatographie fingerprîhting which enables to understand and standardize the Physico-Chemical properties of the medicines like color for the therapeutic standardisation of themedicines and humors.

In yet another embodiment of the présent invention relates to a method forchromatographie fingerprinting which enables to understand and standardize the Physico-Chemical properties like Taste (Rasa) like Sour (Amla), Salty (Lavana), Pungent (Katu),Bitter (Tikta), Astringent (Kashaya) of the medicines, for the therapeutic stand ardization of. the medicines and humors.

In yet another embodiment of the présent invention relates to a method forchromatographie fingerprinting which enables to understand and standardize the Chemicalproperties of the medicines like Cold, Hot, Slow in action, Sharp in action, Heavy, Light,Soft, Lubricated, Supple, Dry (Guna’s like Sheeta, Ushna, Manda, Teekshna, Guru, Laghu,Snigdha and Rooksha as described in Ayurveda) of the medicines for the therapeuticstandardization of the medicines.

In yet another embodiment of the présent invention relates to a anethod forchromatographie fingerprinting which enables to understand and standardize the Physico-Chemical properties of the medicines like Potency, Métabolite formation after. assimilationand Spécifie properties like Charlotte of the molécules (Described as Veerya, Vipaka andPrabhava) for the therapeutic standardization of the medicines and humors.

In yet another embodiment of the présent invention provides a software based dataprocessor of 3-D chromatograms and contour image of an ingrédient.

The method of the invention is described in steps with reference to the accompanyingtables, drawings, flow charts and examples, which are provided to illustrate some of theembodiments of the invention, and the same should not be construed as limitations on theinventive concept embodied herein.

Description of the accompanying tables and Figures:

The following examples are given by way of illustrations and these should not be construed to Iimit the scope of the invention. 2520 I. Tables 1. Table showing different philosophies and various terminology used in differentmedicines 2. Table showing Relation of Humors, Properties, and different parts of the human body -5 An Ayurvedic approach. 3. Table showing Division in terms of the Macrocosm in Chinese medicine 4. Table showing Division in terms of the Microcosm in Chinese medicine 5. Table showing the relation of five natural éléments and their relation 6. Table showing the meaning of Yin and Yang used in Chinese Medicine 10 7. Table showing the basis of color for the therapeutic classification of the medicines S. Table showing the effect of different colors on different diseases 9. Table showing Properties of the six tastes (Rasas in Ayurveda) and their properties andefficacy 10. Table of Colors and The Relation with Wavelengths. 15 11. Table showing the rôle of acidity and alkalinity in human body 12. Table showing the comparison of different analytical techniques being used for thefinger printing and Chemical standardization. 13. Table showing the parameters used for developing fmgerprints of some of themedicines 20 14. Table showing the therapeutic classification of medicines reported in the proposed invention. 15. Table of medicines shown as thumbnails. 16. Table. showing the division of the fingerprint in to therapeutic zone based on the conjugation and polarity. 25

Π. FIGURES

Figures 1 A&B shows the five basic éléments in Chinese medicine and the relationshipbetween them. Imbalance (excess or deficiency) of any one element leads todisturbance in other éléments and becomes the root cause of a disease. The health 30 of human body is achieved by managing and controîling the above éléments in

Chinese medicine.

Figure 2 shows the effect of colors on the basic humors based on which the medicines ofthe same colors were selected for vitiating the corresponding humor. The color of amedicine is due to the Chemical properties of the constituents présent in it, thus 35 indirectly the Chemical properties were used for the therapeutic standardization.

012520 37

Figure 3 shows the fingerprints of Shilajit of two different brands. The Chemical profile inthe finger print shows the therapeutic efïicacy due to the presence of more numberof molécules with wide conjugative properties. The Chemical profile varies with theâge of the sample, it spent in the earth, the more it is old, the more it will betherapeutically active and may also dépend on the place of collection andpurification process.

Figure 4 shows the existing use of chromatographie fingerprints on a label.

Figure 5 shows chromatogram of Commercial Turmerïc (Food).

Figure 6 shows chromatogram of Furazolidine (Allopathie).

Figure 7 shows chromatogram of Krimikutara Ras (formulation).

Figure S shows chromatogram of Shilajit (good by efïicacy).

Figure 9 shows chromatogram of Shilajit (Poor by efïicacy).

Figure lO.shows chromatogram of Suryavarti (formulation).

Figure 11 shows chromatogram of Tea (food).

Figure 12 shows chromatogram of Trikatu (formulation).

Figure 13 shows the fingerprints of ail yeilow medicines. Here the fingeiprint ofSandigdha Dravyas (a contraversial drug) shows a clear différence in appearance,making the identification more easy.

Figure 14 shows the fingerprints of ail medicines of PITTA HARA in nature. The presenceof constituents in zone-1 indicates the efïïcacy of the medicine.

Figure 15 shows the fingerprints of ail medicines of KAPHA HARA in nature. Thepresence of constituents in zone-2 indicates the efficacy of the medicine.

Figure 16 shows the fingerprints of ail medicines of VATA HARA in nature. The presenceof constituents in zone-3 indicates the efficacy of the medicine.

Figure 17 shows the fingerprints of ail medicines of PITTA KAPHA HARA in nature. Thepresence of constituents in zone-1 and zone-2 indicates the efficacy of themedicine.

Figure 18 shows the fingerprints of ail medicines of KAPHA VATA HARA. in nature. Thepresence of constituents in zone-2 and zone-3 indicates the efficacy of themedicine.

Figure 19 shows the fingerprints of ail medicines of PITTA VATA HARA in nature. The presence of constituents in zone-1 and zone-3 indicates the efficacy of the medicine. 012520

3S

Figure 20 shows the fmgerprints of ail medicines of TRI DOSHA HARA in nature. Thepresence of constituents in ail three zones indicates the efficacy of the medicine.

Figure 21 shows the fmgerprints of Kali musali and Safed musali that are used as tri-5 dushahaia medicine.

Figure 22 shows fmgerprints of different samples of Citrallus Colosynthis. The fingerprintshows the lack of some constituents due to which this method is used forstandardization of extraction process of homoeo mother tinctures from plants. 10

Figure 23 shows fmgerprints of different samples of Holarrena Antidyssentric collectedfrom different places of the country. The fingerprint shows the influence ofecological factors on the Chemical constituent of the plant material. 15 Figure 24 shows the fmgerprints of two samples of Beetle leaves from different places.

The flavonoids présent in the time range of 30-40 min shows the influence ofgenotypic., phenotypic variations and ecological factors on the Chemicalconstituents of the plant material. ' 20 Figure 25 shows the satellite images of India. These satellite images indicates that Indiahas different tropical zones.

Figure 26 shows the fmgerprints of two formulations used as cosmetics like Herbal head

Bath powders. 25

Figure 27 shows the fmgerprints of TRIKATU of two different brands. The différence intheir assay may be due to variations in the constituent éléments of TRIKATU.

Figure 28 shows the fmgerprints of turmeric and its three different commercial products. A 30 common peak occurs at 20 minutes in ail these fmgerprints. (Figures 29 to 92 shows the finger prints of ail the medicines reported in Table 13)

Figure 29 shows both fmgerprints of whole plant of Abel moschus, Moschatus 35 medicum.

Figure 30 shows both fmgerprints of bark of Acacia suma.

Figure 31'shows both fmgerprints of leaflets of Acalypha indica. 40 Figure 32 shows both fmgerprints of leaves of Adhatoda vasakaFigure 33 shows both fmgerprints of leaves of Adiantum caudatumFigure 34 shows both fmgerprints of stem bark of Ailanthus excelsaFigure 35 shows both fmgerprints of rhizome of Acorus calamus 39 °^252θ

Figure 36 shows both fingerprints of big single cloves of Allium porum

Figure 37 shows both fingerprints of small cloves of Allium sativam 5 Figure 38 shows both fingerprints of rhizome of Alpinia galanga

Figure 39 shows both fingerprints of rhizome of Alpinia ofïicinarum

Figure 40 shows both fingerprints of rhizome of Alpinia speciosa

Figure 41 shows both fingerprints of unprocessed raw fruit nut of Areca catechu 10 Figure 42 shows both fingerprints of milk processed nut of Areca catechuFigure 43 shows both fingerprints of stem bark of Areca kateehFigure 44 shows both fingerprints of Homoeo mother tincture of Arnica 15 Figure 45 shows both fingerprints of whole herb of Bacopa monneriFigure 46 shows both fingerprints of stem bark of Berberis aristata ’· 2θ Figure 47 shows both fingerprints of whole plant of Borrhievia diffusa

Figure 48 shows both fingerprints of big, ripped fruit of Capscicum Annum linn.Figure 49 shows both fingerprints of big unriped, fruit of Capscicum annum linn. 25 Figure 50 shows both fingerprints of small, unriped, fruit of Capscicum annum linn.

Figure 51 shows both fingerprints of stem bark of Coscinium fenestratumFigure 52 shows both fingerprints of root and leaf of Coccinidium grandis 30 Figure 53 shows both fingerprints of leaf ofD actlylactinium Aegyptium (erect)

Figure 54 shows both fingerprints of leaf of Dactlylactinium Aegyptium(prostrate)Figure 55 shows both fingerprints of leaf and bark of Diristachis cineraria 35 Figure 56 shows both fingerprints of fruit epicarp of Emblica officinalisFigure 57 shows both fingerprints of formulation of a face packFigure 58 shows both fingerprints of formulation of a face packFigure 59 shows both fingerprints of root bark of Glycerrhzia glabraFigure 60 shows both fingerprints of powder of whole plant of Glycerrhzia glabra 45 Figure 61 shows both fingerprints of whole plant of Gymnema sylvestrae

Figure 62 shows both fingerprints of stem bark of Hollerona AntidysentricaFigure 63 shows both fingerprints of root ofInnula recemosa 50 Figure 64 shows both fingerprints of flower of Michellia champakaFigure 65 shows both fingerprints of Leaf of Moringa olifera

Figure 66 shows both fingerprints of homéopathie mother tincture of Myrica cerefera 55 Figure 67 shows both fingerprints of whole plant of Nahi axillae

Figure 68 shows both fingerprints of stem bark of Oroxylum indicumFigure 69- shows both fingerprints of leaf of Ocimum sanctum 60

Figure 70 shows both fingerprints of leaf of Pluchea lanceolataFigure 71 shows both fingerprints of stem bark of Picrorrhiza kurroh 012520 40

Figure 72 shows both fingerprints of leaf of Piper beetle.

Figure 73 shows both fingerprints of seeds of Psoralia coriiifolia

Figure 74 shows both fingerprints of leaf of Raphanus sativus

Figure 75 shows both fingerprints of root of Ricinus cummunis

Figure 76 shows both fingerprints of stem and root of Rubia cordifolia

Figure 77 shows both fingerprints of root of Saussrea lappa

Figure 78 shows both fingerprints of whole herb of Spheranthus indicus

Figure 79 shows both fingerprints of stem bark of Symplocus racemosus

Figure 80 shows both fingerprints of fruit of Terminalia chebula

Figure 81 shows both fingerprints of fruit of Terminalia bellerica

Figure 82 shows both fingerprints of whole plant Trigonella faenum g.

Figure 83' shows both fingerprints of stem and root of Tribulus terrestrias.

Figure 84 shows both fingerprints of leaves of Tyîophora asthmatica

Figure 85 shows both fingerprints of mother tincture of Homoeo medicine Viburnum

Figure 86 shows both fingerprints of Root of Withinia somnifera

Figure 87 shows both fingerprints of rhizome of processed Zinziber officinalis

Figure 88 shows both fingerprints of powder of Avipattakara churna

Figure 89 shows both fingerprints of an herbal formulation of Kamaduga Ras

Figure 90 shows both fingerprints of a Kumarayasava a herbal medicine by fermentation process

Figure 91 shows both fingerprints of an herbal formulation ofMahalakshmi vilas rasFigure 92 shows both fingerprints of an herbal formulation of Suvarna yogaraja GugguluFigure 93 shows the fingerprint of Anandabhairavi Ras. Right clicking on any particular peak, the image software will display the X, Y, R, G and B coordinates of thepeak, which are used for bar coding. These coordinates are shown inside a box(near the peak) and in the tool bar.

Figure 94 shows the fingerprint of Krimikutara Ras. The image software displays the barcode values of a particular peak.

Figure 95 .shows the barcode generated for Anandabhairavi RasFigure 96 shows the barcode generated for Krimikutara Ras.

Figure 97 shows the display window for Anandabhairavi RasFigure 98 shows the display window for Krimikutara Ras

Figure 99 shows how the network works in an Enterprise Resource Planning and CustomerRelationship Management applications networked using the database preparedby the proposed method.

Figure 100 shows a new chromatogram in the form of a colored bar chart for the tenderleaves of Azadiracta indica collected in February

Figure 101 shows a new chromatogram in the form of a colored bar chart for the medicineof Anandabhairavi Ras an herbal formulation. °^252θ 41

Figure 102 shows a new chromatogram in the form of a colored bar chart for îhe medicineof Krimikutara Ras, an herbal formulation.

Figure 103 shows the Pie diagram for the chromatogram of Azadiracta indica5

Figure 104 shows the Pie diagram for the chromatogram of Anandabhairavi RasFigure 105 shows the Pie diagram for the chromatogram of Krimikutara Ras 10 Figure 106 shows the Pie diagram of vitiation disorders (dosha) quantitative.

Figure 107 shows the Pie diagram of vitiation disorders (dosha) quantitative.

Figure 10S shows the Pie diagram of vitiation disorders (dosha) quantitative. 15

Figure 109 shows the fingerprints as 3D and contour images of several herbal medicines,which are fed into a database and are used for various ERP and CRM ' applications.

Figure 110 shows the fingerprints as 3D and contour images of several herbal medicines,20 which are fed into a database and are used for various ERP and CRM applications.

Figure 111 shows the fmgerprint of adulterated cosmetic samples as 3D and contourimages.

Figure 112 shows the fingerprints of Mother tincture as 3D and contour images these can25 be used to fmd out the dilution of Mother tincture.

Figure 113 shows the fingerprints of isolated medicines and their UV spectra as 3D and.contour images.

Figure 114 shows the fingerprints of allopathie medicines as 3D and contour images.

Figure 115 shows a diagram with operational sequence of the software with various 30 Functionality’s.

The invention is described in detail below with reference to the accompanying drawings,flow charts and examples, which are provided to illustrate some of the embodiments of theinvention, and the same should not be construed as limitations on the inventive conceptembodied herein. 35 METHOD OF CHEMICAL STAND ARDIZATION:

Hence unlike a method currently under use, where in a chromatogram is given at a singlewavelength, a novel method of chromatographie standardization, finger printing and barcoding is proposed, using contour and 3-D chromatograms. It provides the total Chemical 40 profile (properties like polarity and conjugation, there in) of the Chemical constituents 012520 42 . présent in complex medicines like herbal medicines and formulations or any medicine.Further, bar coding the finger prints thus generated will provide many commercial featuresin dealing such medicines using the ERP and CRM applications.

The exist’ng method of TLC fmgerprinting Fig.4 being used as a chromatographie fingerprint, is showing only an assay of the constituents présent in it. It is not providing anyChemical property like conjugation or polarity. Another method of fmgerprinting by HPLCshows a chromatogram at a single wavelength presented as a “Chromatographie FingerPrint” of the medicine. In this, a selected peak is identified chemically, what it is bystructure, using various other analytical techniques like NMR, LC-MS and IR for structuralélucidation. So, the single chromatogram by it self is not able to say what the efficacy ofthe medicine is, without the support of other costlier analytical instruments. It will behighly impractical to use such costly techniques for a complex herbal medicine andformulations prepared by formulating various organic and inorganic medicines for aparticular therapeutic purpose.

The quality of any formulated medicine will dépend on the process with which it wasmade. This will be different for each pharmacy or pharmacist. What actually needed for thequality control of herbal medicines and formulations is a simple analytical method that cangive the number of constituents (qualitative and quantitative) présent in a single medicineor formulation, and the therapeutic efficacy of the medicine under study. Hence, anymethod, which does not provide the above information, is incompletel·

In the method of Chemical standardization of the présent invention, the constituents werefirst extracted in to a suitable solvent. The extract was subjected to séparation intoindividual constituents on a High Pressure Liquid Chromatograph under standardizedanalytical conditions. The 3-D and contour chromatograms given by the instrument wereconverted in to Chromatographie fingerprint images. The images were analyzed usingimage analysis software specially prepared for this work. The out put data is interpreted forthe said standardization. Detailed description of the method is given in experimentaldescription of the method. METHOD OF THERAPEUTIC STANDARDIZATION:

The traditional therapeutic standardization is highly individualistic by ability andperception of the doctor. A general avaîlability of such method will be practically diffieuît.But the existing scientific scénario emphasizes that any method or mechanism needs to bestandardized, and reproducible. Hence, in the présent method of Chemical and therapeuticstandardization an instrumental method is proposed which brings down the human factor. 012520 43

This is made possible by an instrumental analytical technique, which explains about theChemical and therapeutic efficacy of the medicines under study in a simple way. In a mostscientific and organized society of modem science, the knowledge of assessing thetherapeutic efficacy of the medicines should be explained with rational justification rather 5 than individual skills and abilities, as they will differ from individual to individual and arenon-reproducible. The method of the invention envisages the same without deviating fromthe traditional concepts.

As explained above if one can assess the therapeutic efficacy of the medicine by thephysico-chemical properties (Polarity and conjugation), the activity of the medicines is 10 understood thus achieving the therapeutic standardization. In the présent method, theconjugative and polarity properties are taken in to considération to assess the therapeuticefficacy of a medicine.

In the ancient literature a clear classification of soils and plants were given based on theirphysico-chemical nature and therapeutic efficacy. The sélection of medicines for a 15 ,'particular disease was done based on the guidelines like color, texture, odor and physicalappearance. Table 8 of the effect of different colors on different body parts shows how«color was used for this purpose. The soil types and the diversity of the drug action were•also mentioned while selecting a medicine. The effect of climate and its effect in theefficacy on the drug plants were also clearly mentioned. Because the Chemical constituents 20 présent in the plant dépends on these geological and ecological variable factors, guide lineswere laid down for the place of collection, finie (seasonal and daily) of collection, part ofplant for collection and âge of plant for collection, required for a spécifie* therapeuticaction.

Based on the generalia of the plants used for a common type of diseases the plants were 25 classified into 37 groups (Ref. 16 K.H.Krishnamurthy, originally from Susruta Samhitasutrasthana 38). Thus, these plants should contain the Chemical constituents having similartherapeutic efficacy on the reported disease.

When the fingerprints of the different classes were studied, some common features arefound about the therapeutic efîficacies of the medicines. The same efficacy was reported in 30 the traditional literature also. In other words, i.e. the experimental and reported results are equal. Hence, studying different medicines, having different therapeutic efficacy validated the method.

The Figure 13 shows ail medicines, which are yellow in color. In the ancient literature of

Ayurveda ail these medicines were classified as Haridra class, ail the medicines being ο 12520 44 · yellow in color like Haridra (Turmeric). When the fingerprints are studied, it is found thatail these medicines are reported to be used as Kapha Hara, vitiation of disorders related tomucogeneous constituents of the body. Hence, it is understood that the color of themedicines has a direct relation with their therapeutic efficacy. Rationally it is also true thatthe color and the efficacy of the medicines are due to Chemical constituents and theirphysico-chemical properties.

The fingerprints of single medicines like Rubia cordifolia, Saraca Indica, PicrorrhizaKurro, Phyllanthus Niruri and Formulations like Arogya Vardhini and AvipattakaraChurna are presented in Figure 14. The molécules eluted in Zone 1 indicates the presenceof Polar constituents based on the elution pattern due the set analytical conditions. Ageneral tendency of this elution pattern for the medicines reported to be Pitta Hara,confirms that high polar constituents act rnainly as Pitta Hara.

The fingerprints of single medicines like Zinziber officinalis (Processed), innula racimosa,Sausserea Lappa, Ocimum Sanctum, Glycerzia glabra and Shilajit are reported to be KaphaHara. The molécules eluted in Zone 2 indicate the presence of Medium Polar constituents.A general tendency of this elution pattern for the medicines reported to be Kapha Haraconfirms that medium polar constituerits act rnainly as Kapha Hara as shown in Figure 15.The fingerprints of single medicines like Alpinia offinarum, Ricinus communis, andFormulations like Suvarna yogaraja Guggulu, Brihatvata chintamani withswarnamakshkam, Huthasana and Mahayogaraja Guggulu are presented in this Figure. Themolécules eluted in Zone 3 indicate the presence of very low or non-polar constituents,mostly oily in nature, based on the elution pattern due the set analytical conditions. It isobserved that any medicine used for this disordei contain or mixed with oils. Along withoily type of constituents, the herbo minerai organo metallic molécules eluting in this zoneare also found Vata Hara. A general tendency of this elution pattern for the medicinesreported to be Vata Hara, confirms that low or non-polar constituents act rnainly as VataHara as shown in Figure 16.

The fingerprints of single medicines like Azadiracta indica, Curcuma ionga, HollarrheanaAntidyssentrica, Berberis aristata, Psoralia Cordifolia and Citrullus Colosynthis arepresented in this Figure. The molécules eluted in Zone 1 and .2 indicate the presence ofhigh polar and medium polar constituents. Thus, these medicines having the medium polarconstituents are found to be Pitta-Kapha Hara. This confirms that the efficacies of themedicines are under stood by the polarity of the constituents présent in it as shown inFigure 17. 012520 45

The finger prints of the single medicines like Tribulus Terrestrius, Moringa Olifera, PiperBeetle and formulations like Trikatu indicate the presence of constituents in the Zone 2 andZone 3 indicating the efïicacy as Kapha Vata Hara in nature, Figure 1S explain the same. 5 The fîngerprints of the single medicines like Bacopa monneri, Oroxylum Indicum andformulations like Kanchanara Guggulu indicate the presence of constituents in the Zone 1and Zone 3 indicating the efïicacy as Pitta Vata Hara in nature. In a formulation calledAnadabhairavi, even though the efïicacy reported is Pitta Vata Hara it is found Kapha VataHara by finger printing. This indicates the artificial préparation of the medicine was not 10 successful to préparé a medicine of the required efïicacy. Hence, this method is useful inthe process standardization of the préparation of complicated formulations mentioned ”·?> above. Figure 19 indicate the same.

The finger prints of the single medicines like Allium Cepa, Withinia Pubiscence (Red 15 Seeds), Embalika Officinalis and formulation like Mahalakshmi vilas ras indicate thepresence of constituents in ail the three Zones of 1,2 and 3 indicating the presence of Λ molécules of the entire range of polarity. This indicates that they will be of Tri Dosha Haraby efficacy. In the finger print of Mahalakshmi vilas ras the presence of two similar type ofmolécules can be seen like isomeric in nature. The Prabhava effect is understood in these 20 type of medicines when such type of isomeric (Geometrical and Chiral) constituents areprésent. Figure 20 shows the fîngerprints of ail medicines of Tri Dosha Hara.

The fîngerprints of Kalimusali (Curculigo Orchioidis) and Safedmusali (AsparagusAdescendens) indicate how two plants of different family’s were classified under same 25 therapeutic group. The fîngerprints show similar constituents in ail the three zones withlittle différence in assay indicating the tri-doshahara property, as indicated in Figure 21.

The fîngerprints of a single medicine of two different sources like Citrullus Colosynthisused in both Ayurveda and Homoeo are given. On observation of the fîngerprints, it is 30 found to contain constituents of three polarities, but mostly high polar molécules more innumber. On careful observation of the fîngerprints, it is observed that the presence andabsence of molécule at 12 minutes is the only différence between both the images. Thetaste of first medicine was very bitter when compared to the second one. Thus using thetaste, as a measure of the efïicacy of the medicines is also proposed, this was mostly used 35 in the ancient literature, as shown in Figure 22.

The fîngerprints of Hollarrena Antidysentrica, a medicine collected from two differentparts of the country has shown much différence in their Chemical profile. This indicates the 012520 46 · influence of geological, ecological, génotype and phenotypic and other variable factors onthe Chemical constituents of the herbal medicines; this is illustrated by Figure 23, A vast différence was seen in two samples of beetle leaves one from Andhra Pradesh andanother from Calcutta of India Figure 24. This confirms the rôle of ecological, genotypicand phenotypic variations in the Chemical constituents of plant parts.

In the Figure 25 the Eco-regions, précipitation, Température and Climate of India wereshown to understand the rôle of seasons on the ecology of the flora and fauna of it. Thevariations in the seasons will hâve an impact on the Chemical constituents of the herbalplants and thereby medicines produced from them. This applies-.to the entire worldwhenever an herbal plant is collected from different parts of the world.

The fingerprints of two formulations used as cosmetics like Herbal head Bath powders aregiven. The fmgerprint of the pure herbal material is totally different from the adulteratedone. The artificial détergents and foaming agents eluted at 25 to 40 minutes are clearîyseen in the adulterated sample, they are highly basic and soapy in nature. This supports thatthe method of the invention is useful for the regulatory authorities to check the pilferage oftraditional medicines with adultérations and substitutions, as illustrated in Figure 26.

The fingerprints of a formulation namely Trikatu of two different brands shows différencein its assay. This may be due to usage of single medicine(s) were used to préparé theformulations from different sources. The présent method shows.the extent they aredifferent qualitatively and quantitatively, facilitating to préparé standardized medicines andextracts of herbal medicines as shown in Figure 27.

The fingerprints of a single medicine of three different brands of the same food materiallike turmeric is given in figure 28. In the fmger print of the natural turmeric it is observedthat the yellow curcumin molécules eluted at 20 minutes. The same molécules are seen inail the brands commonly. The différence in the profile is the commercial samples arebecause they were prepared processed (Boiled) turmeric rhizomes and the natural is fromunprocessed (Un-boiled) rhizomes.

Figures 29 to 92 gives the fingerprints developed for various medicines and the imageparameters (Elévation and rotation) used for the 3-D and Contour chromatograms of ail themedicines given in table 13. In table no 14, the medicines analyzed were classified on thebasis of therapeutic efficacy. The analysis of fingerprints of the respective medicines usingthe proposed software will support the claim of utility of the method of the invention forthe therapeutic standardization. 47 °1252Ο

This confirms that this method is useful in many purposes of dealing the traditionalmedicines. It is useful for modem medicines also to understand their therapeutic efficacy intraditional ternis.

METHOD OF BAR CODING, ENTERPRISE RESOURCE PLANNING (ERP) AND

5 CUSTOMER RELATIONSHIP MANAGEMENT APPLICATION (CRM)APPLICATION

In the présent method the software analyzes the image and can display the coordinates Xrétention time, Y wavelength, R number of red pixels, G number of green pixels and Bnumber of blue pixels by the présent computer based (Microchip, Dongle switch, hardware 10 and software locked) software for a contour chromatogram, for a particular peak which isspécifie to the product. When this data is transferred/feed to a résident in built re-salablebar-coding software, automatically an optional pixel value of a peak in the image, itgenerates the barcode having attached the ‘display window’ with ail details of the productunder study. 15 -The method of the invention facilitâtes to barcode any number of constituents présent in a.chromatographie finger print of a herbal medicine, using the X rétention time, Y-wavelength, R number of red pixels, G number of green pixels and B number of bluepixels as the coordinates provided by the présent computer based (Microchip, Dongleswitch, hardware and software locked) software and the values of each of the constituents 20 provided by the présent software. These factors will be reprèsenting the Chemical andtherapeutic efficacies of the constituents. Hence instead of a mere catalogue numberpresently used for the bar coding, a novel method of bar coding is proposed where in, thecoordinate values of the constituent will be displayed along with other details. Thus, thebarcode and the coordinates will speak about the Chemical and therapeutic properties of the 25 product.

It becomes a tool for the regulatory authorities like Drug Controller, Public analysts, Foodadultération enforcement authorities, Forensic and Customs and Central excisedepartments for the régulation of the herbal Products. The fingerprint of the medicinesshould be printed .on the label and should be tallied when it is checked. This also helps to 30 monitor the various other brands of a same medicine for an industry to monitor. Figures93-94 shows how the software gives the coordinates for a selected peak of the image.These values will be given for a barcode to be generated. Figures 95,96 are the barcodesthus generated. Figures 97,98 show how a display window will be for an herbal medicinewith ail label details. The barcode will be able to show when the display Windows when 012520 48 they are attached to the barcodes of the respective products. When a large database isprepared for the products and made available in the network, any ERP and CRMapplication can use for any required purpose by network. Figure.99 shows how thenetwork woks in an ERP and CRM applications networked.

VARIOUS STEPSINVOLVED IN THE PRESENT INVENTION

In the présent method of analysis a Validated High Pressure Liquid Chromatographequipped with a Binary Gradient System of pumps, a Photo Diode Array Detector (PDA),and a Software based data processor for présentation of the chromatograms was used. Afterthe complété elution of ail ingrédients, the 3D and contour chromatograms (having theinformation of the UV -Visible Spectra, absorbance and rétention times of ail theconstituents présent in a single medicine or formulation) were converted into an image andproposed as a finger print. This enjoys the merit of not requiring any internai or externalstandard sample for an authentic qualitative and quantitative analysis of ail the ingrédientsprésent in a medicine, unlike in the présent method of analysis of medicines.

Experimental Description of the method:

The proposed method is described in 4 steps with référencé to the accompanying drawings,flow charts and examples, which are provided to illustrate some of the embodiments of theinvention, and the same should not be construed as limitations on the inventive conceptembodied herein.

The entire method is described in the steps mentioned below:

Step 1 : Sélection of medicines and extraction of the constituents

Step2: Séparation of the constituents into individual constituents and generating andconverting the 3-D and Contour Chromatograms in to finger prints.

Step3 : Analysis of the fingerprints using the software developed.

Step4: Interprétation of data

Description of the Présent Method of AnalysisSTEP 1: Sample préparation

Constituents are extracted from the medicines using ethyl alcohol, selected based on theChemical nature (polarity) of the sample. When the pH of the aqueous alcohôl extract isvaried, the extraction of constituents also has varied. The basic pH has extracted morenumber of constituents than acidic pH. Suitable pH was selected for extraction of differentmedicines and the same was maintained using buffers. The rôle of acidity and alkalinitywas taken into considération while selecting the pH for extraction. 012520 49

STEP2

Experimental work done on the instrument:

The extract was subjected to séparation analysis, using High-Pressure LiquidChromatographie (HPLC) instrument. In the présent method of analysis, a Validated High 5 Pressure Liquid Chromatograph equipped with a Binary Gradient System of pumps, a

Photo Diode Array Detector (PDA), and a Software based data processor, for thepréparation of the chromatograms were used. A known amount of the sample (say 20ul) ofextract is injected into rheodyne injector (fitted with 20ul loop). Elution of the sample wasperformed with suitable time programmed gradient System of mobile.phase at a fixed flow 10 (1 ml/min). Care is taken that no part of the sample is left in the column un-eluted. The following analytical conditions set for the analysis. a. A reverse phase column was used along with a time programmed gradient elutionofan aqueous phosphate buffer (In the pH range of 5.5-7.5) and a non-aqueoussolvent (acetonitrile or methanol) is used as eluent based on the Chemical nature of 15 the sample under analysis. b. A wave length range of 200 to SOOnm was used for the PDA detector and the runtime is fixed based on the time program. c. The time program, which changes the concentration of non-aqueous solvent likeAcetonitrile 0-100 % of organic solvent, is used in the instrumental parameters 20 existing in the instrument.

The instrument was triggered for the analysis after injecting the sample into the injector.The run was stopped whenever the analysis is completed or the instrument will stop the runautomatically after the entire time program is completed.

In the three types of display of data, the chromatograms, one window displays25 chromatogram at.a selected waveîength, in another it displays the contour chromatogramwhich displayed the rétention time (run time) of the analysis on X-axis and the waveîengthrange on Y-axis. In another window, it displayed the 3-D chromatogram of the samplewhere in it displayed the rétention time (run time) of the analysis on X-axis, theconcentration range on Y-axis and the waveîength range on Z-axis. The 3-D and contour 30 chromatogram thus developed by the System was converted into an image.

The images thus generated were analyzed by the proposed software, which provides anovel chromatogram and the qualitative and quantitative analytical data of the in-gradientsprésent in the medicines. The pixel values represented by different coîors from Violet,Indigo, Blue, Green, Yellow, Orange and Red attributed as a measure of the concentration 0 ί2520 50 (quantitative) of the constituents proportional to the color. Extracting the individual colorsmentioned above and shows in separate widows for each color. This is the basis ofChemical standardization. Sonie chromatograms thus generated are shown in figures 100-102.

The chromatogram thus provided by the software gives the information of conjugative(shown by the UV-VIS absorbance) and polarity properties of the individual constituentstogether. The image is divided into three zones representing, Zone 1 (High polar zone),Zone 2 (medium polar zone) and Zone 3 (low or non polar zone) scaled by rétention timesbased on the elution pattern, depending on the column used and the mobile phase.Reversing the analytical conditions can reverse the elution pattern.

The 3-D chromatograms of the medicine are analyzed using ail its three dimensionalproperties of the said image. If the 3-D chromatogram is considered as a cap with a hoodthe matching of the entire cap 3 dimensionally, with another sample of different qualitativeand quantitative properties, the extent it matched will be presented as an analytical reportqualitatively and quantitatively. Here the hood of the cap is compared to the peak of themolécule at a particular wavelength. A sample with more number looks like a cap withmany hoods. Thus the matching of the three dimensional coordinates provides a foolproofmethod of comparison and analysis. The coordinate it matched gives qualitative and theextent it matched gives the quantitative data of the sample understudy. This is madepossible by spécial software prepared for this purpose. This becomes an ultimate methodof quality control.

The interaction of the polarity of the molécules being separated, the polarity of thestationary phase used and the polarity of the mobile phase used for the elution of thesample Controls the elution pattern of the molécules. The résultant inter action of ail thethree and other related parameters like température etc., décidés the elution pattern andorder of elution of the constituents based on their polarity. Thus, in a medicine ail the polarmolécules will elute in first ‘Zone 1’ (Polar zone of the image), ail the medium polarmolécules will elute in ‘Zone 2’ (Medium polar zone of the image) and ail the low polar ornon polar molécules will elute in ‘Zone 3’ (Non polar zone of the image). When themolécules eluted in these three zones of many fingerprints, many generalizations weremade regarding the Chemical and therapeutic efïïcacy of the medicines. This is anotherbasis of therapeutic standardization. The zones are shown marked in the Figures 103-105.Thus, the chromatogram gives the information, how it is going to act chemically and sotherapeutically. When the individual constituents présent in each zone and represented 51 072520

graphically or by any means of data présentation, the total constituents of the respectivezone gives the percentage it is going to act on the particular dosha. Thus, the data explainshow it (medicine) is going to act therapeutically on the vitiation of each dosha collectivelybased on the qualitative and quantitative properties of the constituents présent in the 5 medicine. For example if the medicines has 30 % constituents in high polar zone (the pixelquantities of various colors like green, yellow, orange and red of a spécifie zone asquantifies) 70 % in medium polar zone it can be represented as a medicine acts 30% onpitta and 70% on kapha, as the colors represent different concentrations in the fingerprints.Hence a medicine can be assessed as of Pitta- Kapha Hara (30-70%). Thus, the vitiation of 10 doshas is quantified. This helps the doctor to under stand the efficacy of the medicines anddécidé his dosage. Some example Pie diagrams are gîven in the Figures 106-108. 3-D and contour Spectra of the reported herbal medicines were developed using thereported analytical conditions. The thumb nail view of the medicines shows how the fingerprints can be handled by a software as it is done in the software used in handling the 15 .·> human fingerprints. Ail the features like searching the similar and compare the similar> fingerprints etc., can be done by inserting the necessary software features. In Figures 109--;.· 114 the thumbnails of the fingerprints for various medicines are given. The lists of medicines shown as fingerprints were shown in table 15. . STEP 3 20 Analysis of the image using image analysis software:

After the complété elution of ail ingrédients, the 3D and contour chromatograms wereconverted into images and proposed as fingerprints. This enjoys the merit of not requiringany internai or external standard sample for an authentic qualitative and quantitativeanalysis of ail the ingrédients présent in a herbal medicine, unlike in the analysis of a 25 synthetic medicines.

After developing the image of the 3-D and contour chromatograms of the medicine understudy, (Hence forth called as Chromatographie finger print) it is analyzed by the soft wareproposed for the analysis of various colors representing the Qualitative and Quantitativeproperties of the constituents présent in it. 30 Scientifically, an image cannot become an analytical data, hence a computer based imageanalysis software (software and hard ware protected) has been developed to analyze theimage and give proportional concentrations of the ingrédients of the medicine under study.Based on the colors of the constituents présent in various rétention times and pixel valuesof the image. 52 û 1252ο

Now the images of the fingerprints were given to Image Analysis software as said above.The analysis of various colors was done by which the constituents will be represented aspeaks of the chromatogram and thus providing a novel présentation of chromatogram inthe form of a colored bar chart. It shows the number of compounds and their conjugativeproperties UV-VIS absorptive property of ail of the constituents eluted. The detaileddescription of the process involved in the analysis of the image is discussed in the technicalfeatures of the software.

The bar chart type of chromatogram thus developed gives a chromatogram having a scaleof Rétention time (0 - 60) on the X-axis and wavelength in the range of 200-800nm, on theY-axis. It gives the number of pixels occupied by each of the colors of each in-gradient inthe image, facilitating the qualitative and quantitative analysis of the individualconstituents présent in it. Thus, the chromatogram generated is presenting the number ofconstituents présent in a medicine and their UV absorption range with quantity of pixelsproportional to the concentration of the molécules.

When the image is divided into, three zones based on the elution pattern of the moléculesand the changing polarity of the mobile phase. The Zone 1 is polar zone as the columnused is a reverse phase column, the Zone 2 is medium polar zone where in the mediumpolar molécules are eluted and finally the Zone3 is low or non polar zone as the non polarand very low polar molécules will elute in this zone. Thus, the molécules eluted in zone 1will be polar, the molécules eluted in the zone 2 will be of medium polar in nature and themolécules eluted in the zone 3 will be of very low or non polar in nature. Hence, the threezones of the images will give the polarity of ail the constituents eluted.

Based on the polarity of the molécules eluted, the medicines are classified according totraditional System of therapeutic efficacy where in the polar compounds are found to bePitta Hara, the medium polar compounds are Kapha Hara and the low or non polarcompounds are Vata Hara. This is the basis of therapeutic standardization of the medicines.The polarity of the constituents is compared to a continuous spectrum of radiation, wherein the dosha is classified as acute to chronic of each dosha. The starting of the zone will beacute and the end of the zone will represent the chronic. Thus, the compounds présent inthe said zone will act on the said intensity of the diseasg.

Table 16 shows division of the fingerprint in to different therapeutic zone based on the color of absorption and polarity. The scale on X-axis shows the scale of polarity of the molécules based on the polarity of the mobile phase and Y-axis shows the range of wavelength (200-8Q0nm) absorbed. Based on the reported therapeutic efficacy in the 53 012520

literature based on the physico-chemical properties (Color and Chemical properties) andthe experimental the therapeutic efficacy of various medicines was standardized. Somedéviations were found which could be due to the effect of variable environmental factorsinfluencing the Chemical constituents of the medicines. 5 Thus, the method will help to know the therapeutic efficacy of the medicines under study.Hence, the proposed method will become a novel visual proof for the understanding thetherapeutic efficacy of the medicine reported or new, single or formulated.

The analysis of the images was done using software developed for this purpose. The detailsof the software is given in the release notes and Figure. 115 0

Step 4:

Interprétation of the data

The fingerprints generated are analyzed for their Chemical and therapeutic properties. The basic features in a fingerprint are found to be : :5 1) the zone ofthe polarity in which the constituents hâve eiuted; and 2) the conjugative properties of the individual constituents présent. ' The polarity of the column is fixed. It is a normal phase or a reversed phase stationary 20 v phase. In the normal phase column, stationary phase will be polar and in a reverse phasecolumn, the stationary phase will be non-polar. The extent of polarity of the stationaryphase varies front brand to brand even in same type of reverse phase or normal phasecolumn. The polarity of the stationary phase will be controlled using the polarity of themobile phase, additives like buffers and pH. When the polarity of the mobile phase is 25 varied constantly in the increased or decreased order, on a reverse phase column, theconstituents présent in the sample will elute in the same order, i.e., the high polarconstituents will be eiuted by the high polar mobile phase, the medium phase mobile phasewill elute the medium polar constituents and the non-polar constituents will be eiuted bythe' non polar or low polar mobile phase. The most preferred pattern is to change the 30 polarity of the mobile phase either increased or decreased order of polarity such that noconstituent of any polarity will be left un-eluted front the column thus achieving totalelution. Thus by controlling the polarity of the stationary phase, polarity of the mobilephase will be managed to bring a required effect on the polarity of the constituents toachieve séparation of required order of elution. 35 The order and properties of polarity and elution in the case of normal phase columns areapplicable as in the case of reverse phase column but reverse to the reverse phase column. X. 54 012520

The non-polar constituents will elute fîrst, followed by polar constituents, based on theorder of polarity of the mobile phase used for elution.

Thus in the présent elution also the elution of the constituents is controlled and driven inthe required pattern by controlling the polarity of the mobile phase and the order ofchanging it in an orderly way.

Mostly the elution of the samples w.ere done from high polarity mobile phase to lowpolarity mobile phase. Thus in the finger prints the constituents présent in the fîrst zone(Zone-1) will be of high polar in nature. The same pattern applies to the other zones, themedium polar constituents eiuted in the medium polar zone (Zone -2) and the low or non-polar constituents eiuted in the non-polar zone (Zone-3). This pattern reverse when anormal phase column is used due to its elution property as described above.

Most of the high polar molécules will be highly reactive chemically, thus biologically.When they enter the fîrst part of the digestive System mouth, they will immediately startacting on the biological System and the enzymes présent there. Then the constituents willenter the stomach and intestine where they will under go different changes (Postassimilation effects, Vipaka in Ayurveda) due to the digestive juices and their enzymesprésent in the part. In the process of absorption the moment the molécules of high activity(high polar) will immediately start interacting with the biological System and show theirtherapeutic properties. This can be compared that in Ayurveda, the intestinal part of thehuman body 'is classified as Pitta zone, where the high polar molécules are playing a majorrôle. The heat causing mechanism will play an important rôle in the diseases and biologicalmechanisms related to. It indirectly indicates the molécules of high reactive, the high polarmolécules.

After the absorption, the blood with ail the absorbed constituents will carry them to heartand the parts related to it. Then the blood will be sent to different parts of the body. InAyurveda, the upper portion of the human body is defined as the Kapha zone, where thecold mechanism will be playing an important rôle. Thus, the molécules of medium polarmolécules will play an important rôle in the mechanisms related to this zone.

The low polar and non-polar constituents will be able to enter to the human body onlythrough blood transfer, Thus the body organs where the mechanism of availability of theChemical constituents is only by blood will be coming in the last category of the polarity.The non-polar oils, fats and other such molécules and mechanisms in the human body areclassified as Vata disorders and ail such disorders are cure using the same type ofmaterials.

01252Q 55

The low and non-polar constituées will be eluting in the last zone of the fingerprint. Thus,this zone (ZONE-3) is considered as Vata zone. Thus the basic humors of the moléculesare able to be identified as per their polarity which facilitâtes to know on what disorder(dosha) it is going to act upon. Thus, the présent method is useful for the therapeuticstandardization of the medicines.

The image was- divided in to three zones on X and Y-axis. The conjugative property(Absorption of a particular wavelength of radiation) is taken on Y-axis and polarity istaken on the X-axis as the elution of the constituées is controlled using the polarity of themobile phase composition. Now as reported in literature the Y axis is scaled as per, thetherapeutic efficacy based on wavelength (color). The entire image is divided in to sixchambers where in the Chemical constituées hâve a spécifie conjugative and polarityproperty. This in turn is proportional to the therapeutic efficacy of the constituées in thechamber. Thus, when a medicine is fmgerprinted, based on the color represented for theabsorption of a spécifie wavelength and having a spécifie polarity, the total colors in that • zone is calculated and interpreted for the therapeutic efficacy of the constituées présent inv it. Thus, the holistic therapeutic standardization and Chemical standardization is achieved * using this method. A schematic représentation is given in table 15, showing the relation of~ conjugation and polarity to therapeutic efficacy of the different constituées présent in a medicine.

When the 3-D chromatograms of the medicine will be analyzed using ail its 3 dimensionalproperties of the said image. If the 3 -D chromatogram is considered as a cap with a hoodthe matching of the entire cap 3 dimensionally, with another sample of different qualitativeand quantitative properties, the extent it matched will be presented as an analytical reportqualitatively and quantitatively. Here the hood of the Cap is compared to the peak of themolécule at a particular wavelength. A sample with more number will like a cap with manyhoods. Thus the matching of the three dimensional coordinates will provide a foolproofmethod of comparison and analysis. The coordinate it matched will give qualitative and theextent it matched will give the quantitative data of the sample understudy. This is madepossible by spécial software prepared for this purpose. This becomes an ultimate methodof quality control.

But any method without quantification will be of no use. Hence, the total colors of the constituées in the image of a particular zone are considered as a représentation of the amount of the polar constituées présent in the medicine. Thus the total constituées présent in the Zone-1 Pitta zone, Zone-2 Kapha zone, Zone-3 Vata zone are présent in the

°1252Q 56 form of a Pie diagram winch represents the ratio of the efficacy of the medicine on each ofthe disorder. Thus, medicines having constituents in the order of 50:20:30 wiil bemedicines of Tridoshahara of the order of 50%: 20%: 30%. This was done using thesoftware developed. Thus, the therapeutic efficacy is standardized quantitatively. The 5 increase or decrease of any one or two of the other doshas can be done by formulatingmedicine by adding other medicines and préparé a suitable formulation needed to cure aspécifie individual.

The Chemical standardization was done using the software by quantifying the individual 10 constituents based on the colors denoting the concentrations of the in-gradients. The rangeof the wavelength that a molécule absorbed dénotés the conjugative properties.

As described in the traditional standardization methods the colors of the medicines werestandardized based on their colors and their therapeutic efficacy. It applies even in the case 15 of any molécules. The Table 8 of colors and their efficacy will explain how colors wereused to standardize the efficacy of the medicines. The colors of the molécules can beunderstood by their absorptive properties of the radiation of the Uv-Vis range of radiation.In the Table 10 of colors and the relation with wavelengths, the colors of the medicines andtheir characteristic wavelengths are given. Based on the structure, functional groups, 20 conjugation, and the extent of unsaturation will influence the wavelength of absorption(absorbance maxima) of the molécule. The more the molécule is conjugated the longer thewavelength of absorption will be. Hence, the UV-VIS absorbance of any molécule iswidely used in the qualitative and quantitative properties of the constituents. 25 The colors and the therapeutic efficacious of various medicines were given in the ancientliterature. The .colors of the molécules are due to a spécifie Chemical nature of themolécule. The colors of the fiâmes were used for the quality control of metals and relatedproducts, which involves the basic spectrophotometric principles. Thus, study andunderstanding of the interaction of the electromagnetic radiation will be useful to study the 30 Chemical nature and thus the therapeutic efficacy of the medicines. The same principle hasbeen used in the présent spectrophotometric method of fingerprinting and standardization.In other terms an existing concept has been presented in the form of a novel analyticalmethod, removing the error of human factor. Ail the medicines for which fingerprintsdeveloped were given in table of therapeutic efficacy of the medicines were given in table 35 14. The technical details of the software are given in the release notes of the software. °1252() 57

Release Notes forthe Software ProposedI) System requirements (minimum) a. Processor: Pentium II or higher b. OS: Windows 95, Widows 98, Win NT 4.0 and Linux c. RAM: 64 MB or higher d. Monitor: 14” Color Monitor (1024 X 768) or higher e. Software: Java Development Kit (JDK 1.2.X) Π) The operational mechanism of the software:

The various operational mechanisms are deseribed below: operational sequences withvarious functionality are shown in figure 115.

Title of the software: RAJNBOW (An Image analysis software for chromatographiefingerprints)

This software is developed for the chromatographie fingerprints and microscopie images. 1. It is GUI (Graphical User Interface) based software. .2. The software is desïgned to analyze any kind of image particularly for the analysis of chromatographie fingerprints. 3. The reports are given in form of graphs. 4. Life Cycle a. In put: Image b. Processing:

Analysis involves

Extracting Colors (Standard 7 colors and some of their different shades)

Resizing, Deviding in to 3 zones at 20 minutes intervalGraphing (Bar and Pie graphs)

Bar-coding

Standards Followed for extracting the colors:

The software extracts eight colors viz. Red, Green, Blue, Yellow,'· Cyan, Magenta andOrange.

Any color is not absolute. It is mixture of the following shades of the colors présent beforeand after it, they vary between a range of values. The range for the colors used to identifyas the colors given above the respective values are taken from the international standard256-color scale. The values used in the présent software are: 012520 58

For Red color Red Blue Green 200-255 0-64 0-64 and 192-200 0-64 0-32

For Green color

Red Blue Green 0-64 0-4S 200-255 0-65 0-64 65-191 For Blue Color Red Blue Green 0-96 200-255 0-191

Similarly, other colors were taken as standards for the extraction of colors. (Thesestandards are exclusive for the présent software requirements and are modifiable ifrequired).

While image is analyzed, the software reads the image pixel by pixel and reads andextracts the color according to the color standards designated, stores and transfers them forfurther display as bar graphs. c. Output:

Reporting 1. By Graphs 2. By saving data like images, graphs, deviding the image display into three zones 3. By dispîaying the ‘X’ (Rétention time or Pixel value of the image), Y (Wave Iengthor absorbance of the images of contour and 3-D chromatograms respectively), R(Red color), G (Green color) and B (Blue color) coordinates. 4. By transferring these values to an in-buiît bar-coding software to generate a barcode. d. User Interaction: User is allowed to interact with the product in various ways.l.Inputing the desired image (one or more) 2.Resizing the image to desired size and analyzing it. 3.Saving the image, resized image, and graphs to it. 4.Printing the image, resized image, and graphs to it. 012520 59 ΠΙ) Technical features of the software 1. It is software entitled ‘Rainbow’ 2. A software with a facility of opening chromatographie fingerprint images in differentFormats (extensions) like .BMP, JPEG, TEF, GIF from the file folders and analyze it 5 for different colors présent in the image with single pixel sensitivity. 3. A software with a facility of display of the pixel information in the form of l.a graphhaving a scale of X (0-(min. time scale) and Y (200-800nm) coôrdinates and 2. a Piediagram with individual values of each peak (Automatic and Manual) in two separatecolumns beside the graph.

10 4. Software with a facility of printing ail the data generated after analysis using PRINT

Icon. 5. A software with a facility of chariging the page setup for printing using PAGE SETUPIcon

6. A software with a facility of selecting a part of the image and analyze using RESIZE 15 Icon. 7. A software with a facility of opening any number of image analysis Windows fordifferent images, and display of status in WINDOW icon. 8. A software with a facility of dividing the image in to three Zones at 20 min interval,using ZONE icon. 20 9. A software with a facility of inverting the selected image using INVERT icon. 10. A software with a facility of switching over to Notepad, Word pad and MS Word,using EDITOR icon. 11.. A software with a facility of operational information about various features of theSoftware using, the HELP icon.

25 12. Software with a facility of saving the data generated using SAVE AS icon as. JEPG file format. IV) Installation Instructions for the software: a. Installation procedure of Java 1.2.x soft ware platform on which the présent30 Software works.

. Explore the Java CD-ROM • Double click on the jdkl .2.0/ jdkl .2.1/jdkl .2.2 setup icon • The setup will extract the files and conforms from the user whether to load the software inthe System. 35 · On click, ’yes' it asks for the directory into which it should install the files. 60 012520 • By default, c:\jdkl.2 directory will be shown. • If you want to install in "d" drive, change the directory and Install the software. • Once the installation is completed go to c: and open the file named 'autoexec.bat'. • Give the following path in the autoexec.bat file. . Opèn auto exe. Bat and write as follows . Set path = d:\jdkl.2\bin: % path % . Setup class path = d:\jdkl.2\lib\classes.jar; %classpath%

Reboot and use b. Installation of Image Analyzer software proposed 1. Copy the folder of the image analyzer software from the CD on to the System in thedesired directory. 2. Explore the batch file form the software folder into which image analyzer softwarewas copied. 3. Right click on it and click on‘send to desktop as short eut’ 4. A 'MS dos' iconic short eut appears on the desktop. Right click the icon and go toproperties, select program tab and check on the ‘close on exit check box', convertwindow status ‘To minimized’. 5. Apply and close. 6. Now the image analyzer software is ready for use. Double click on the imageanalyzer Icon and it starts working. 7. In the opening window, a box with 'CSIR' will open where in the pass word 'dvk'should be typed. 8. Click the arrow mark (hand) on the right down corner of the opening image to openthe Software. 9. Open the directory of the images of contour images without scale and select theimage to be analyzed. The image will be shown on the image window. 10. Click on the RED analytical window marked with red boarder. A PIE diagram willbe displayed along with a chromatogram with rétention time on X-axis andnanometers on the Y-axis. 11. For constituents of lesser concentration click on the Green, Yellow and Orangecolors. The Other colors are mostly the base line or less than that hence can be'ignored. 12. The details of using the other features of the software are given in the help menu ofthe software including the various features and applications of the software

61 012520 V. Known bugs: Not found VI . Abbreviations used: a. JDK: Java Development Kit 5 b. Con: Contour Chromatogram c. 3-D: 3 - Dimensional Chromatogram d. WOS: Without Scale e. X: Represents the Rétention Time of the chromatogram f. Y: Représente the absorbance in the 3-D chromatogram and wave length 10 range in contour chromatogram g· R: Intensity of red color at a particular pixel position h. G: Intensity of green color at a particular pixel position i. B: Intensity of blue color at a particular pixel position

15 VIL Meaning of the Various Icons and fonctions a. PRINT icon will facilitate in printing ail the data generated after analysis. b. PAGE SETUP icon will facilitate in changing the page setup for printing. c. RESIZE icon will facilitate the sélection of a part of the image and analyze theselected part of the image. 20 d. WINDOW icon will facilitate in opening any number of image analysis Windowsfor different images, and display of status in e. ZONE icon will facilitate in dividing the image in to three Zones at 20-mininterval. f. INVERT icon will facilitate in inverting the selected image. 25 g. EDITOR icon will facilitate in switching over to Notepad, Word pad and MSWord. · h. HELP icon will facilitate in the operational information about various features ofthe Software usage. i. SAVE AS icon will facilitate in saving the data generated in a *. JPEG file format. 35 62 01252.0

Main advantages of the présent invention are: 1. The contour chromatogram of the medicine becomes a Fingerprint of it. Because itcontains the UV-Vis spectrum band with concentration of the ingrédients alongwith the polarity of the molécule. The fmgerprints developed for a same medicine 5 extracted under different pH value helps to understand the drug release in the intestine System at different pH values thus facilitating the pharmacodynamies ofthe medicines under study. 2. The spectral bands of ali the constituents are given in a single picture assessing themedicine about its therapeutic properties and nature, very easy. 10 3. The 3-D Chromatogram becomes a photo of ail the UV-spectra of ail wavelengths of each constituent in a single picture indicating the Chemical (conjugative andpolarity) property of the molécule eluted. 4. A database of the fmgerprints of various herbal medicines available in the countryuseful for Quality control, Forensic and customs departments'to control the use and 15 misuse of the herbal medicines at the public interest. 5. The database also gives information about the médicinal value of the variousmédicinal plants of the country (therapeutically classified) in the country and therôle of the ecological factors on the Chemical constituents· of the same plantavailable in various tropical Zones of the country. This facilitâtes to select a plant 20 for collection of the herbal medicines suitable to be used for the therapeutic usage for a médicinal professional or an herbal trader. 6. Analysis of the fmgerprints using this software gives rôle of écologies! factors onvarious herbal medicines available in the country and it is useful for Quality 25 Control, Forensic and Customs Departments to control use and misuse of the herbal medicines at the national interest 7. The analysis of the fmgerprints is useful to understand the therapeutic efficacy ofthé medicines using the physico-chemical properties of the medicines as reported inthe ancient literature. 30 8. The analysis also gives information about the médicinal value of the various médicinal plants in the country and the rôle of the ecological factors on theChemical ingrédients of the same medicine available in various parts of the country. 9. The therapeutic and ethano-botanical classification of the fmgerprints helps to bringsome generalizations useful for the doctors and researchers for a complété 35 understanding of the traditional medicines by analyzing the fmgerprints.

012520 63 10. By bar coding the image properties the medicines/plant extracts/plants are savedfrom piracy as the facility to create a barcode using the properties of the imagethrough note pad facility.

11. The barcodes are utilized in ail commercial transactions of modem ERP and CRM 5 applications.

APPLICATION UTILITIES OFTHE PRESENT INVENTIONINTERNATIONAL

It is usefol for any country for fmgerprinting and patenting the traditional medicines of thatcountry. Because the finger print of a single médicinal plant is not similar to a finger print 10 of the same plant· in another place or country due to the variations in its Chemical profile.

The variations in Chemical profile is due to the influence of Ecological factors like thetropical région variations, soil, water quality and the genotypic and phenotypic variationsfactors on the chemistry of the plant.

This method helps the countiy to fulfill one of the régulations màde by WHO, for the15 ... member countries to standardize methods for the utility and quality control of herbal , medicines and their régulation.

NATIONAL

This is useful as a tool to prevent international piracy of traditional medicines by Passing aLaw that the “The Médicinal plants for which the finger prints are developed are national 20 property”. If a medicine is applied for any type of patent, in any place of the world and ifthe finger print, tallies with the finger print of the medicine available in the challengingcountry, the patent could be objected.

STRATEGIC

Bar coding the fingerprints of the medicines helps in the authentic régulation and 25 protection of the médicinal plants.

By converting the barcode of the fingerprint of a médicinal plant into a machine-readablelanguage,· commercial and regulatory work becomes easy.

Fingerprints of the medicines helps the Food and Drug controllers, Customs and CentralExcise departments to regulate, check the use, misuse and pilferage of the herbal medicines 30 inside the country and while allowing importing of such medicines.

INDUSTRIAL A fingerprint developed for a medicine or a formulation helps the industry to protect theirprocess technology by comparing the fingerprint of the same medicine of other brands.Thus, it helps to implement the patent law more efificiently. 012520 64

The fingerprint helps to monitor how the medicines are changing its médicinal propertiesby the addition of another medicine at different stages of process of preparing aformulation.

Industry can use the database of fingerprints developed for ail the native plants available in 5 the country, for their sélection of the place of collection of a medicine. It helps theindustry, which part of the country, and in which season is suitable for the collection ofherbal medicines, as the ecological factor changes the therapeutic efficacy of themedicines.

SCIENTIFIC 10 This method helps the researchers to understand the traditional formulations prepared. Italso helps to monitor a new formulation under préparation.

It helps to know how new molécules are formed when a complicated traditionalformulation is prepared.

The fingerprints developed for a same medicine extracted under different pH value helps to 15 understand the drug release in the intestine System at different pH values of an individual.

The contour chromatogram of the medicine becomes a Fingerprint of it. Because itcontains the UV-Vis spectrum band with concentration of the ingrédients along with thepolarity of the molécule.

The spectral bands of ail the constituents are given in a single picture, assess the medicine 20 about its therapeutic properties and nature, very easily.

The 3-D Chromatogram becomes a photo of ail the UV-spectra of ail wavelengths of eachconstituent in a single picture indicating the Chemical (conjugative and polarity) property @of the molécule eluted

The database also gives information about the médicinal value of the various médicinal 25 plants of the country (therapeutically classified) in the country and the rôle of theecological factors on the Chemical constituents of the same plant available in varioustropical Zones of the country. This facilitâtes to select a plant for collection of the herbalmedicines suitable to be used for the therapeutic usage for a medical professional or anherbal trader. 30 The therapeutic and ethano-botanical classification of the fingerprints helps to bring some generalizations useful for the doctors and researchers for complété understanding of the traditional medicines by analyzing the fingerprints.

The présent method facilitâtes to préparé chromatographie finger printing of herbal medicines and formulations, which is useful for many quality control and régulation 35 purposes. 65 012520

The présent method facilitâtes Chemical standardization, (qualitative and quantitative) ofthe said medicines by providing the conjugative and polarity properties of the individualmolécules présent in the medicines or any organic or organo-metallic compound which hasUV -VIS absorptive property. This kind of analysis is of much use in the chromatographie 5 analysis of Herbal medicines of Single and Formulations, where the use of extemal orinternai standards are practically not possible.

The invention facilitâtes to study, understand and monitor the therapeutic efficacy of thesaid medicine under study. It helps to understand the therapeutic actions and properties oftraditional medicines reported in the ancient literature and confirm the same in the form of 10 a reproducible analytical data. Thus, it provides therapeutic standardization of themedicines under study. It shows the polarity zones like Polar, Medium polar and Non polarmolécules présent in the sample, thus facilitating to understand the efficacy of the medicineas a whole.

This method facilitâtes to re-standardize the reported medicines to the présent therapeutic L5 « needs. It helps to monitor and study formation of new organic and organo-metallic> molécules, which has UV-VÏS absorptive property, in the process of preparing a reported? or a new formulation. This also helps to standardize the process technology of preparing areported or new formulation by monitoring the constituents and their changing Chemical and therapeutic properties. 20 It facilitâtes to geiierate a barcode by an in built bar coding software, wherein the Xrétention time, Y wavelength, R number of red pixels, G number of green pixels and Bnumber of blue pixels are the coordinates given by the présent software. Some examples ofthe.barcode for chromatograms are given. The invention also facilitâtes bar coding one ormore of the constituents présent in the fingerprint thus facilitating the commercial 25 transactions easy.by ENTERPRISE RESOURCE PLANNING (ERP) and CUSTOMERRELATIONSHIP MANAGEMENT (CRM) applications. A database thus prepared helpsthe regulatory authorities to monitor the movement of the said medicines inside or fromoutside the country, from production to the consumer. The database of the barcodes thusprepared, becomes the resource for the ERP vending machines or of any of such kind. The 30 machine will display ail details of the medicines like company, its Chemical fingerprint,and the therapeutic efficacy of the medicines the said medicine. This makes theidentification of the medicines more authentic than the présent. A data base of the fmgerprints thus generated using this method helps to bring manygeneralizations of the therapeutic efficacy of a particular therapeutic class of plants. Thus, °1252& 66 one can under stand why a particular plant is added in that class. This is explained in the

Table 14 enclosed.

The fingerprints printed on the label of the medicine helps the doctors to understand the therapeutic efficacy of the medicine just before use and confirm the quality control of the 5 medicines for every batch.

The Image analysis of Chromatographie Finger Print Images (Contour Chromatograms) ofvarious Medicines of any philosophies (Single and Formulations) developed, are usefùlfor many purposes as described in various steps of this application.

SOCIAL 10 This is useful to know for a consumer, the therapeutic efficacy of medicines single orformulated claimed on the label and confirais to contain the same.

This helps the consumer-act to monitor the quality control of herbal medicines sold in themarket and protect the interests of the consumer.

ADULTERATION 15 The Image analysis of Chromatographie Finger Print Images (Contour Chromatograms) ofvarious Medicines of any philosophies (Single and Formulations) developed is useful fordétection of any adultération of the medicines. 012520 67

Table 1: Table of Different Philosophies And Valions Terminologÿ Used in Mcdicine TRI MALAS 1 .Purisha 2.Mutra 3,S\veda 1. Malam 2. Mu tram 3. Vervai Information not available * Dri Ma 1. Bsan 2. Gcin 3. Rnul ce m! CÛ CO eu—· e4 rô SAPTA DHATU a ·§ £ -3 'a «as e<22«2w»— <s ro ’T *n vo t-* Z“\ • g ε οι « -S 1î*Iîï s| §&amp;« 5 ® 2 *2 1=§·9 §1ce s o ,2 «aCS M ;2 « ><s H wô l.Sinuses 2. Blood vessels 3. Muscles 4. hair 5. Bones Lus Zuns Bdun 1. DansMa(Rasa) 2. Khrg (Rakta) 3.Sa (Mamsa) 4. TsiI (Medas) 5. Rvs (Asthi) ô.Rkan (Majja) 7.Kliu Ba (Shukra) lPrimary :Bloôd, Phlegm, Bile, Saudai Secondary: LMahsoora(Inlravascular) 2. TaIliya (Pericellular) 3. Qureeba (Intercellular) 4. Munviya (Cellular) Body organs Simple, Compound M 2 Æ 2 eo -30 3 2 c 0 o 0p # x q<S *O rf PROPERTIES 1. Rasa-Tasle-6 2. Guna- Property-Basically 20 3. Veeiya -Potency-2 4. Vipaka- MetaboIite-3 5. Prabhava- Spécifie properties-Innumerable 6. karma-Action | ε ε ! ! li &amp; c > > ·—« CM <*% *3* Sa « 8 b >roP ïS-aw a « m w—’ rô TT wô Information not available * Z—S I p a -ο 2s ΐ B 5 0 m 2— ri n v _ - ·β o ^4; ôS Q > OO» fô "’T PANCIIA BHUTA 1. Prithivi 2. Ap 3. Teja ! 4. Vayu 5. Akasha z—* ’> '-s'a S 33 ^‘2 >*.3X c- ob^ < I fc 8 5.1 i ί A >J — ci en TT «n 1. Wood 2. Fire 3. Earth 4. Mêlai 5. Water X“>. z-s ? » Z? â «o22- fs rn ’T z—s V. z—«s •r* S ·=> .î< 1! U, O s^ S3 '-'j- s « « ‘Il £ < X c£ ω< —’ fs’ cô T* U· .. V «CÎ ♦ 0 S H.h5c2E< — «N fi 'T TRI DOSIlA(Hara) Vata, Pitta, Kapha. 1. Yang 2. Yin z**\ Z—> 5 £ {Λ z—s L·. C3« ra ra > a- « iS «f vi à" —' —t 2 § > g -g § Σ CC £X—- CM rn .2 g — 'Z-'n J s ës > £ S > ï- oo,2 Ί2a — es a,2 C3 « O Ω C η φ— CM en ΤΓ 1. Yellow Bile 2. Black Bile 3. Phlegm 4.131ood ë O tO O ES eu >·. 3 3? .2 il*ïl § « S J 4g a. 03 g i ! 2 SS" 1 C/3 S Chinese Binary : Yin-Yang ufixhi 2 E .. 0 iïï 0 Z. Gteek Contrary medicine SI.No - CM -r Ό 68 ÛÎ2520

Table 2: Relation Of Humors,Properties,A»d Different Parts OfTheHuman Body - An Ayurvedic Approach Approach RELATION ON VEPAKA (POST ASSIMILAT! VE EFFECT) 1 | ' «g «g jj 2 3 «5 ud y Ό <5 RELATION ON GUNA « Je 3 2 « U-ses3a «r 2 3 s 4 =r J j 5 J jow «f g « sf £SJ!8 8 « Λ ô Ό w EFFECT ON DOSHAS(DECREASING THEDOSHA)DUETODHATUS a. Pilla Vata Hara b. Vala Hara c. Vata Hara d. KapIia Hara e. Kapha Pitta Hara f. KapIia Pitta Hara 1 MAHABHUTARELATIONSW1TH DHATUS > 5-= £. 1 + ? -s 5 S> ·§ .1 S 5 > g£ 5 t » « S C ao-S ü e .3 « xi d Ό W u CHEMICAL PROPERTIES .. 2? "3 O C- P- /*·* « f*T ha 53 ” 2 ο,, 3 S, ω •a g ffl c υ ï 7BS = M-C o 52. i ” 2 y 3 _ g >> •.'ô 8 | E □ U I g O « n 2 ÎFg.^S^saejf.sJi.g'lÊg.^Eg, s b à 1 p f s 1 Ji g sîÎsë g rt Λ ο *c o \U es q bû-ci m 4. C u W □ 7) w-î Ci 4’r* Ή - SAPTA DHATUS ra § 13 -g « § il (§ 3, S 2 « 2 55 —« c»i m" vS ό r* PANCHA BHUTA (PHYSICAL PROPERTIES) y a '·£> « 5» g C S· ,37.« -3a. < μ > i—' <N fn tr <ri TRI MALAS fil CL Σ ci—- CM H < -B Q a £ £ £ SI. No 1 i —* CM* ΡΠ

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Structural eluents [ Sinuses Blood Vessels Muscle U ’3 E Bones | Emotional Anger > £ Anxity en en y e Ό CS 00 Fear Tsangs Liver Heart Spleen ec es 3 U Kidney Fus Bile Small intestine | Stomach y c en y •Ξ y Ef ce U Bladder j Sense organs I Eye Tongue Mouth V) O U, ω Eléments . -σ O O £ Fire | [ Earth S s Water SL No. CM ΓΊ Ό· νΊ 012520 70

Table 5: Table Showing the Relation of Five Natural Eléments and Their Relation, in Chinese Medicine

Yin &amp; Yang | Heart &amp; Small intestine | Lung &amp; Large intestine I Kidney &amp; Bladder I | Liver &amp; Gall bladder ( Spleen &amp; Stomach 4) 0 4) 0 X! «j JZ Æ 4-* x: o th O «♦"J s to s to O ondst îpuods puods spond iponds O- O υ 0 0 fc e/3 V fc t b c 0 ε O t g g *3 O O U 4) O o TJ o 0 x: O o w ce o t w ÏÏ s £ £ W T5 Ό <υ <u X3 TJ TJ Lm U, 0 o 4> u 4> 4—» 4-4 TJ TJ <L> 4> 0 c Q. Έ. Ή en or E E E E o o o 0 <υ O O O >> >> oc C£ eu oc O£ U- l— U U. O <u <n 0) û> c c c c C ω w W w w O c c s 5 s o CS *^î" Ό 00 »—« c <L> U *n | Earth ε 4> V *s IOO 4) ce ·*-* <u « £ £ £' S TJ TJ TJ lü <L> 4) X3 TJ 1_4 <υ 4) <υ 4> 4> 4—4 XI X3 TJ o 4> c <υ CE E 0 or c 0 or "Ü E Ή E c c c o o <υ 4> 4> O O c c C c a 4) <u 4> 4> <u > > > > > ci cd <d «J «υ <L> 4» 4> 4> X X X X X rt ï 3 3 5 m tr> r* 0 e •w

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°1252O 71

Table 7

Table Showing The Basis Of Coior For The Therapeutic Standardization Of Medicines

The naines of the medicines were given as slolta

01252Q 72

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The rôle of colors and their influence on different body parts,The basis of coïor is used for selecting a tnedicine.For eg. A. plant with Indigo flowers will cure ENT problems 012520 73

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This table shows how tastes and medicines are related to vitiation of diseases. 03 012520

Table 10: Table of Colors and The Relation witli Wavelengths.

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012520 75

Figure 11 : Rôle of Acidîty and Alkalinity in Human Body

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Table 13 PARAMETERS USED FOR FINGER PRINTING OF MEDICINES

Page 1 of table 13 FIG NO. BOTANICAL NAMEOFTHE PLANT VERNACULAR NAME PART USED 3-D PARAMETERS* ELEVATION DEGREES ROTATION DEGREES 29 ABELMOSCHUSMOSCHATUS MEDICUM KA’STURI BENDA WHOLE PLANT 20 15 30 ACACIA SUMA SWETHAKHADIRA BARK 15 65 31 ACALYPHAINDICA KUPPINTA LEAFLETS 10 60 32 ADHATODA VASAKA VASA LEAVES 25 45 . 33 ADIANTUM CAUDATUM MAYURASHKHI LEAVES 20 40 34 AILANTHUS EXCELSA ARALU STEM BARK 10 65 35 ACORUS CALAMUS VACHA RHIZOME 10 130 ’ 36 ALLIUMPORUM MAHALASUNA LASSAN BIGSINGLE CLOVES 20 130 37 ALLIUM SATIVAM LASUNA LASSAN, SMALL .CLOVES 20 130 · 38 ALPINIA GALANGA GREATER GALANGA RHIZOME 20 75 39 ALPINIA OFFICINARUM LESSER GALANGA RHIZOME 15 75 40 ALPINIA SPECIOSA LIGHTER GALANGA RHIZOME 10 60 41 ARECA CATECHU BEETLE NUT UN PROCESSEDFRUIT NUT 15 40 42 ARECA CATECHU BEETLE NUT MILK PROCESSED NUTS 15 40 43 ARECA KATEEH RAKTHA KHADIRA STEM BARK 15 65 44 ARNICA ARNICA MOTHERTINCTURE OFWHOLE PLANT 10 55 45 BACOPA MONNERI BRAHMI 1 WHOLE HERB 15 45 46 BERBERIS ARISTATA DARUHARIDRA STEM AND BARK 15 170 012520 79

Page 2 of Table 13 g 47 BORRHEE VIA DIFFUSA PUNARNAVA WHOLEPLANT 15 « 48 CAPSCICUM ANNUML MIRCH BIGJUPED FRUIT 10 70 49 CAPSCICUMANNUML MIRCH BIG UNRIPEDFRUIT 10 70 50 CAPSCICUM ANNUM L MIRCH SMALL, UNRIPED, FRUIT 10 70 51 COSCINIUM FENESTRATIUM LATADARVI STEMBARK 15 125 ' 52 COCCINIDIUM GRANDIS DONDA ROOT AND LEAF 25 30 53 DACTLYLACTINIUM AEGYPTIUM(ERECT) GRASS LEAF 25 40 54 DACTLYLACTINIUM AEGYPTIUM(PROSTRAT E) GRASS LEAF 25 40 55 DERISTACHIS CINERAR1A TUMMA LEAF AND BARK 20 15 56 EMBLICA OFFICINALIS AMALAKI FRUIT EPICARP 5 50 57 FACE PACK BRAND 1 FORMULATION 20 25 · 5S FACE PACK BRAND 2 FORMULATION 20 25 59 GLYCERRHZIA GLABRA YASHTI MADHU ROOT BARK 15 130 60 GLYCERRHZIA GLABRA YASHTIMADHU POWDER OFWHOLE PALNT 15 130 ' 61 GYMNEMA SYLVESTRAE PODAPATRI WHOLE PLANT 25 15 62 HOLLERONA ANTEDYSENTRICA KUTAJA STEMBARK 10 60 63 INNULA RECEMOSA PUSHKARAMULA ROOT 5 45 64 MICHELLIA CHAMP AKA MANU SAMPENGA FLOWER 20 40 65 MORTNGA OLIFERA MUNAGA LEAF 25 40 . 66 MYRICA CEREFERA BAY BERRY MOMEOPATHIC MOTHER TINCTURE 20 35 012520 80

Page 3 of table 13 67 NAHI AXILLAE NAHI WHOLEPLANT 10 130 68 OROXYLUMINDICUM SYONAKA STEMBARK 10 170 69 OCIMUM SANCTUM RAMATULASI LEAF 15 130 70 PLUCHEA LANCEOLATA PATRARASNA LEAF 10 65 71 PICRORRHIZA KURROH KATUKIROHINI STEMBARK 15 125 72 PIPER BEETLE BEETLE LEAF 25 160 73 PSORALIA CORILEFOLIA BAKUCHI SEEDS 25 60 74 RAPHANUS SATIVUS MULLANGI,WHITE LEAF 15 25 75 RICINUS CUMMUNIS ERANDAMULA ROOT 10 135 76 RUBIA CORDIFOLIA MANJISTA STEM ANDROOT 10 40 77 SAUSSREA LAPPA KUSHTA ROOT 5 80 · 78 SPHERANTHUSINDICUS MUNDI WHOLE HERB 15 70 79 SYMPLOCUS RACEMOSUS LODHRA STEMBARK 15 65 SO TERMINALIA CHEBULA HARITAKI FRUIT 10 40 . 81 TERMINALIA 'BELLERICA - VIBHITAKI FRUIT 20 35 82 TRIGONELLA FAENUM G. MENTHI WHOLE PLANT 15 160 83 TRIBULUS TERRE STRIAS GOSHURA STEM AND ROOT 25 45 84 1 TYLOPHORA ASTHMATICA LEAVES 10 65 85 VIBURNUM MOTHER TINCTURE MOTHERTINCTURE OFHOMOEOMEDICINE 20 15 012520 81

Page 4 of table 13 86 WIIHINIA SOMNIFERA ASWAGANDHA ROOT 5 50 I 87 ZINZIBER OFFICINALE» SHUNTI PROCESSED ZINGER, RHIZOME 15 130 88 AVIPATTAKARA CHURNA AYURVEDA FORMULATION POWDER 25 60 89 KAMADUGA SIDDHA FORMULATION POWDER 10 25 90 KUMARAYASAVA AYURVEDICMEDDICINEBYFERMENTATIONPROCESS ' LIQUID 10 35 91 MAHALAKSHMIVILASRAS SIDDHA FORMULATION POWDER 20 35 92 SUVARNA YOGARAJAGUGGULU SIDDHA FORMULATION POWDER 10 40

ALL THE OTHER PARAMETERS OF RANGE OF WAVE LENGTH, ABSORBANCES CALE AND RETENTION TIMES ARE SHOWN IN INDIVIDUAL FIGURE 012520 82

Table 14: MEDICINES USED FOR FINGER PRINTING

2 2 2 2 O O A O O WM M MM k J WM Q S 8 S § H H b b mE b b a « < K <1 £m < GO n S P P P < P P 8 S w · P P b P Q « P ë 51 w Q g fe bo O S ω 52 wω ω O £ O b < b S CS O O P o s S CS O WM P O WM s b b O Cm w3 b b b P b b b S g O > §3 M g JS < ê f .u w< Uw HA HARAirder on wlIFIES) Cm â y | 5 -> <- 55 S U b §a2 WM Pw -> -» -> -> -> -> — -> <υ en m£ > çn u o U o en 5 •c 5m O < « MM < U 41* U irotectiti eases, disordei ce U ce sorders, orders c U 41 u s XJ U O en χ· 41 « O b > S s· .SS u *J5 55 U a w U ci o Ξ • bb b b Laxati Peptîc Piles HepatSkin dGynea Pitta j U ~ « e·£ 3P ua Jaund u c. 4) Cm ·- 'S B. » 41 w S O "O es υ WM MM Ci < 2 O WH P ë P S CO P 2 O 2 O WM H P s P < H a b S H < y P <J 2Ë Q <b p 2 Pi S <3 b i 2 WM S O 2 WM S O .LANT ARIA < P P 2 M S o 2 2 IM S o CS 222 P CS> ° < b 5 2 ÏÏ s HERE ω P —4 Tr > SX SCm P O b 2 ob es b Cm o WM MM M < b MM MM fcd S 2 b < g £ < 2 MM *r« WM 2 < > < 2 « Q ai G < < o P < 2 MM < < mJ < <Z) < 5 b CS < > < U e-=? ω < s < < MM -b eu mLt ca < Q > < b < > O P Q > é 2 MM U h ί-ο b CK CU ς£ x P < 2 < O O 2 P ί ê £ b ca O U 2 CS X P < 5 < υ < « < O b b V5 <ZJ 012520 83 (Page 2 of Table 14)

KAPHA H ARA ROOT ROOT WHOLE HERB | SMALL CLOVES | j BÏG SINGLE CLOVE STEM BARK BIG UNRIPED, FRUIT POWDER ROOT, BARK WHOLE PLANT WITH FLOWERS AND SEEDS ROOT FORMULATION LEAF. -> -> -> -> -> -> -> -> -» i-> -> Digestive disorders Aphrodisiac, impotency Respiratory disorders Piles Cougli Diarrhoea | Swasa | VJ « * en Diabetis Digestive disorders Etnitic . | Venerial, Skin diseases Panduroga, Rasayana Sllieshma roga,Prameha,urinaryblader &amp;kidneydisorders Ci P* 5« ··* C3 CZ5 «3 £ en ** <UC3 « «C3 ·— 6 Ή Worm infestation Cough, . Fever ARALU i SAFED MUSALI VASA MAYURASHIKHI | LASUNA | i MAHALASUNA SWETHA KHADIRA KATUVEERA BIMBI | HERBAL FORMULATION YASHTI MADHU LATA KASTURI ! PUSHKARAMULA § 2 WM S © ea a fcâ >-r* >M RAMATULASI AILANTHUS EXCELSA ASPARAGUS ADESCENDENTUM ADHATODA VASICA ADIANTUM CAUDATUM | ALLIUM SATIVAM | ALLIUM PORUM ACACIA SUMA i CAPSCICUM ANNUM L COCCINIDIUM GRANDIS | CHOPACHINYADI CHURNAM GLYCERRHZ1A GLABRA HIBISCUS ABELMOSCHUS 1 INNULA RECEMOSA Z é < H § WM S 2 Sd OCIMUM SANCTUM 84 <

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ROOT POWDER [FORMULATION 1 FORMULATION FORMULATION â s £ iS H>—< Ah STEMBARK, EXUDATE BARK I fi W en CZ3 ω u o a CU fcc J TENDERLEAVES ROOTBARK HOMOEO MOTHER TINCTURE COMMERCIAL POWDER-1 i_ -> -» -» -> -> -> -> -» —> -> -> -» -> -> Constipation, Rlieuinatoid disorders «Λ O «5 O ’-5 O C5 S 3 5 | Jwara Arthrites ! _____î Sciatica Skin diseases diabetis Digestive disorders | ! Diabetis, l Skin disorders ! Skin diseases, Infective conditions l Obesity Skin disorders Purgative, juandice,aborttificient &amp; "o? oa _ « «S Ο Ό.2 ~ gS U g-2 .5 S,4» ^5 \ .£ l? 2·“ -£ «£ B Όr ο ί-ο « o£ Ή/-5 ERANDA MULA SIDDHA FORMULATION j HERBO-MINERAL HERBAL FORMULATION ω Z »-< s t O fcd K ! RAKTHA t KHADIRA [ ARALU 1 KRAMUKA NIMBA 1 DARUHARIDRA ! INDRAVARUNI TURMERIC t — . --------- -- RICINUS COMMUNIS suvarna YOGARAJA | GUGGULU j SH1TAMSU RAS S < 0 O >. <3 » 5 o > ο =3<S3 ü VATA GAJANKUSA RAS ACACIA CATECHU " , AILANTHUS EXCELSA j ARECA CATECHU AZARIDICTA INDICA | BERBERIS ARISTATA <Λ E g H 3 > en ggt OO U 0 Z O ►J i 0 U a, U 012520 86 (Page 5 ofTable 14) COMMERCIAL POWDER-2 COMMERCIAL POWDER-3 STEM RAW RHIZOME i , WIIOLE PLANT LEAVES FRUIT I STEM BARK FROM ANDHRA I PREADESH | STEM BARK FROM KERALA I STEM,ROOT SEEDS,SEED OIL ROOT -> -> -> -> -» -» -> -» -> -» -> -> -> -> > —} -> -> Worni infestation,dysentry,diarrhoea,skindisorders, wounds Worm infestation dysentry,diarrhoea,skin disorders Wounds Diabetis Obesity, Skin disorders Skin , Allegic, Diabetic Diuretic, improvescomplexion *-» CI Λ .Ξ ‘•3 £ £ S "S C V C <s> O > £3 | Diarrhoea, Ilaemorroids Diarrhoea, AU gi tract disorders Skin disorders Leukemia Blood purifier Leucoderma, Skin diseases Laxative [ Liver disorders ‘ TURMERIC TURMERIC ! i LATA DARVI HARIDRA GRASS JAMBU KUTAJA j KUTAJA MANJISTA t L_________ BAKUCHI ' 3g§ § CURCUMA LONGA < O Z O -J S «t 3 O cd => O COSCINIUM FENESTRATIUM CURCUMA LONGA DACTYLACTINIUMAEGIPTIUM(PROSTRATE ANDERECT) EUGENIA! JAMBOLONA SIZYGIUM CUMINI HOLLERNA ' ANTIDYSENTRICA HOLLERNA ANTIDYSENTRICA RUBIA CORDIFOLIA PSORALIACORYLIF Ί OLIA PICRORRHIZA i KURROA < 012520 87 (Page 6 of Table 14)

WHOLE HERB BARK WHOLE HERB RHIZOME | LEAF,LEAF JUICE I FORMULATION [ FLOWER I LEAF ! LEAVES FROM I COASTAL ANDHRA t PRADESH LEAVES CULCATTA FORMULATION FORMULATION « -> -> -> -» -» -> -» -> -» -> -> -» -> Diabetis.colic «e ü O "u U CS *5 ¢/3 U >c O U '•5 Έ cS 2 «ca ? ό Krimihara, i Migraîn,vrishya, j lymphatic disorders ’HA VATA HARA Medhya Speech disorders I Gyneac disorders, hepatomegaly, spleen 0 inegaly,burns,uterine disorders Indigestion, Skin disorders Cosmetic, Skin diseascs Abcess, oedema Kasa, svvasa Digestive disorders Kasa, swasa Digestive disorders Indigestion | Indigestion METHIKA LODIIRA MUNDI >-< VACHA 2 =3 bd HERBAL FORMULATION CHAMPAKA SIGRU NAGA VALLI NAGA VALLI HERBAL FORMULATION HERBAL FORMULATION ï TRIGONELLA , FENUM G. SYMPLOCOS RACEMOSA [ SPERANTHUS INDICUS - ACORUS CALAMUS ALOEVERA AGN1TUNDINA MICHELLA CHAMPAKA [ MORINGA OLEIFERA PIPER BEETLE I PIPER BEETLE I i TRIKATU CHURNA-1 1 00 <! X O 3 H 2 H 012520 88 (Page 7 of table 14)

FORMULATION STEM AND ROOT LEAVES PITTA VATA IIARA STEM BARK FORMULATION WHOLEHERB t LEAF AND BARK 1 § ta S S O s £ FORMULATION STEM BARK ë K s CO O O H ai i- ROOT BIG.RIPED FRUIT ROOT -> -> -> -> -> -» -» -> —> -> -» -> 4r- -> -> — -> -> -» -» -> Indigestion Urinary disorders,Oedema Diabetis, asthama Prameha Medhya.skin disorders Hridya, Obescity Malaria Inflammatory conditions 1 Odema , Digestive disorders Apbrodisiac G.I tract disorders Apbrodisiac HERBAL i FORMULATION i GOKSHURA AJADWESHI SWETHA KHADIRA IIERBO- MINERAL BRAHMI ! VEERATARU, TUMMA K < 2 HERBAL FORMULATION SYONAKA SWETHAl MUSALI MIRCH, KATIVEERÀ KALI MUSALI 1 O X U 3 H 3 a t—1 n on ci H CO fcj ai ai ω H CO j o · n ai H TYLOPHORA ASTHMATICA ACACIA S UMA ANANDABUAIRAVI BACOPA MONNERI DICROSTACHYS CINERA ENICOSTEMMA AXILLAE KANCHANARA' GUGGULU | OROXYLUM INDICUM i_ ASPARAGUS| ABSCENDENSES CAPSCICUM ANNUM L j CURCULIGO ORCHIOIDES 012520 89 (Page 8 ofTable 14) FRUIT EPICARP FORMULATION FORMULATION « J P « » P P « FRUIT FRUIT H O O CS Z O *-< H J P O fa FORMULATION -> -» -> -» -» -> -» -> -» -» ·_» -» "S s - 8 >> >. 2•ne*1 • x V) Su ea tiK CS Z Diarrhoea AU types of levers j l | Haemorrhoïds Haemorrhoïds ) Laxative Rasayana Kasa,swasa, Skin diseases, urinarycalliculus j General debilily, rejuvenatîon [ [ ! rERATIONS | Good by efficacy j Absence ofsomeimportantconstituents likekushta (sausserealappa) and manjista(rubia cordifolia), making tlie formulation less effective AMALAKI HAEMORRHOÏDS HERBO-MINERAL ! PALANDU PALANDU j HARITAKI VÏBHITAKI ASWAGANDHARED SEEDS PLANT ROOT !É Q < - EMBLICA OFFICINALES KARPURADI RAS MAHALAKSHMIVILAS i RAS | ONOIN BIG ONIONSMALL J TERMINALIA CHEBULA TERMINALIA BELLERICA 1 , WITHINIA PUB. I ! i_ i FACE PACK (G) | FACE PACK (B) !_:_ 012520 90 (Page 9 οί Table 14)

FORMULATION FORMULATION HOMOEO MEDICINES HOMOEO MOTHERTINCTURE HOMOEO MOTHERTINCTURE BRAND-1 HOMOEO MOTHER 1 TINCTURE BRAND-2 HOMOEO MEDICINE 1 (ZI Q 2 P O O U Q W H < J O .(ZI A FLAVONOID 7-IIYDROXY QUERCETIN , SINGLE ISOLATED 1 COMPOUND ADULTERATED WITH ADIURETIC ALLOPATHIC AMIDE CONSTITUENT *o c £ y S ex C3 ex •S Ξ « £ o z; Adulterated withfoannng agents Pain reliever, aftereffects of injury Hepatoprotective O O w O U c, O es /·> ST Gyneacologîcal disorders ! Hepatoprotective Prameha BAY BERRY 1 BAY BERRY MOTHER TINCTURE SINGLE COMPOUND ! SINGLE COMPOUND 1 HERBAL HEAD ΒΑΤΗPOWDER (G) HERBAL HEAD ΒΑΤΗPOWDER (B) ARNICA 1 M Y RICA CEREFERA 3 « a g a U O 2 > 5 S a a ? VIT EX NEGUNDO 1 AZAMALYCIN -a < a a £ O , E ωaSa a <5q ω-< S 012520 91 (Page 10 of Table 14)_____ __

BERBERIS ARISTATA SINGLE I BERBARINSTD ISOLATED (FLUKA)

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Claims (45)

1. 012520 Claims

   1. A method for détection and identification of constituents of extracts of plants or animal, natural or synthetic sources possessing medicinH vnb'- using chromatographie finger printing techniques, the said method comprising steps of: i) extracting organic or organo-metallic compounds from plants or animal,natural or synthetic sources using a suitable solvent; ii) subjecting the extract obtained in step (i) to séparation based on pH andpolarity, using High Pressure Liquid Chromatography techniques; x. generating contour and 3D chromatograms of the constituents eluted instep (ii); xi. converting the 3-D and contour chromatogram obtained into a coloredimage, analyzing the colored image for its individual colors using theco-ordinates denoting ail its 3-dimensional properties of the said imageby using an in-built software; xii. denoting the concentrations of the various constituents eluted with time; xiii. generating a chromatogram based on color analyzed, having peaks atvarious rétention times along with conjugative properties of theconstituents; xiv. identifying the compounds in the said eluted constituents by the UV-VISabsorptive properties of the various compounds in the - image,identifying, determining and classifying the compounds eluted as polar,medium polar and less or non-polar based on the polarity andconjugative properties; xv. generating a barcode for a selected peak using X axis as RétentionTime, Y axis as Wavelength, R as number of Red Pixels, G as numberof Green Pixels and B as number of Blue Pixels; and xvi. generating a database of fingerprints and barcodes and identifying therespective compounds of the extract.
2. 2. A method as claimed in claim 1 wherein, the solvents with different polaritiesare used based on the hydrophilic and hydrophobie nature of the constituents of -qî- 012520 sample under study, and ethyl alcohol as solvent being used for standardizationof médicinal extract.
3. 3. A method as claimed in claim 1 wherein, the fingerprints are developed for thesame médicinal extract under different pH ranges.
4. 4. A method as claimed in claim 1 wherein, the HPLC techniques used is byemploying any commercially available HPLC apparatus with the Photo DiodeArray detector, preferably with a gradient or temary System of pumps.
5. 5. A method as claimed in claim l wherein, the pH and polarity of the mobilephase is controlled by varying the ratio of the mixture 0 to 100% of an aqueoussolvent, water or a buffer at a required pH by using a sait such as Potassium Di-Hydrogen orthophosphate or Di potassium hydrogen orthophosphate andphosphoric acid maintaining the required pH, with a non-aqueous solvent.
6. 6. A method as claimed in claim 1 wherein, the solvent used in step (iii) is selectedfrom a group consisting of non-aqueous, organic and aqueous, water or bufferat a known pH, and are selected based on the range of polarity.
7. 7. A method as claimed in claim 1 wherein, convert the contour chromatogramsinto a colored image consisting of conjugative and polarity properties of theconstituents of the médicinal extract under study.
8. 8. A method as claimed in claim lwherein, the therapeutic efficacy of a médicinalextract (single or formulated) is assessed using the quality of the constituentsprésent in a particular polarity and UV-VIS absorptive zone.
9. 9. A method as claimed in claim 1 wherein, the software generates a barcode for aselected peak or peaks or image using the X axis as Rétention Time, Y axis asWavelength, R as number of Red Pixels, G as number of Green Pixels and B asnumber of Blue Pixels as the coordinates, provided by the software, whichmakes the product propriety for an industry.
10. 10. A software called “Rainbow” for détection and identification of extracts ofplants or animais, natural or synthetic sources possessing médicinal valuesobtained as claimed in claim l has the following features: (a) a software with a facility of opening chromatographie fingerprint imagesin different Formats (extensions) like .BMP, JPEG, TIF, GIF from the 012520 file folders and analyze it for different colors présent in the image withsingle pixel sensitivity; (b) a software with a facility of display of the pixel information in the formof (l).a graph having a scale of X (0-(min. time seule) and Y (200-800nm) coordinates and (2). a Pie diagram with individual values ofeach peak (Automatic and Manual) in two separate columns beside thegraph; (c) a software with a facility of printing ail the data generated after analysisusing PRINT Icon; (d) a software with a facility of changing the page setup for printing usingPAGE SETUP Icon; (e) a software with a facility of seîecting a part of the image and analyzeusing RESIZE Icon; (f) a software with a facility of opening any number of image analysisWindows for different images, and display of status in WINDOW icon; (g) a software with a facility of dividing the image in to three Zones at 20min interval, using ZONE icon; (h) a software with a facility of inverting the selected image using INVERTicon; (i) a software with a facility of switching over to Notepad, Word pad andMS Word, using EDITOR icon; (j) a software with a facility of operational information about .variousfeatures of the Software using, the HELP icon; and (k) a software with a facility of saving the data generated using SAVE ASicon as. JEPG file format.
11. 11. A software as claimed in claim 10, is used for processing of 3 D chromatogramsand color contour image of extracts of plants or animais, natural or syntheticsources possessing médicinal values, the said software execution processcomprising steps of: a. generating a coloured contour image of extract of step (a), analyzing thecolored contour image based on the sélection of various colors (withstandards mentioned in rciease notes, life cycle, processing) denoting the 012520 concentrations of the various constituents eluted with time, and polarity based on rétention time; b. analyzing the 3-D chromatograms of the eluted constituents using ail its3 dimensional properties of the image; c. generating a chromatogram having peaks at various rétention timesalong with conjugative properties of the constituent compounds elutedwith time in a specified order of polarity; d. identifying compounds in the said eluted constituents by the UV-VISabsorptive properties of the various constituents in the image; e. correlating the reported biological, therapeutic activity of the of variouseluted constituents présent in the extract understudy based on thepolarity and the conjugative properties of the eluted constituents bydividing the fingerprint into therapeutic zones on X and Y axis; f. generating a barcode for a selected peak(s) using the imagecoordinates viz., X for rétention time, Y for wavelength, R for numberof red pixels, G for number of green pixels and B for number of bîuepixels, provided by the software; g. generating a database of fingerprints and barcodes for the elutedconstituents, facilitating ail kinds of database utilities like EnterpriseResource Planning (ERP) and Customer Resource Management (CRM)applications; and h. generating a database of the ‘display widows’ for ail the_ elutedconstituents to be used by the ENTERPRISE RESOURCE PLANNING(ERP) and CUSTOMER RELATIONSHIP MANAGEMENT (CRM)type of business applications.
12. 12. A software as claimed in daim 11 uses solvent for extraction, which is selectedbased on the polarity, hydrophilic and hydrophobie nature of the constituentsprésent in the plant, plant or animal, natural or synthetic in origin havingmédicinal value.
13. 13. A software as claimed as claimed in claim 11 wherein, the HPLC apparatusused is selected from any commercially available HPLC apparatus with the - 012520 Photo Diode Array detector, preferably with a gradient or temary System ofpumps.
14. 14. A software as claimed in claim 11 wherein, the polarity of the mobile phase of anon-aqueous and an aqueous solvent of a spécifie pH is controlled by varyingthe ratio of the mobile phase from 0% to 100% of an aqueous solvents likewater or a buffer of a known pH, along with a non-aqueous solvent or vice-versa.
15. 15. A software as claimed in claim 11 wherein, on analysis of 3-D and contourchromatograms provide a chromatogram with rétention time and wavelength onits X and Y-axis.
16. 16. A software as claimed in claim 11, wherein, on analysis of 3-D and contourchromatograms provide a data indicating the vitiation of doses quantitatively inpercentage ratio.
17. 17. A software as claimed in claim 11, uses a single solvent éthanol for extractingthe constituents; analytical conditions and instrumental parameters for ailextracted constituents to bring the therapeutic generalizations there by achievingthe therapeutic standardization. ,
18. 18. A computational method of chromatographie finger printing, Chemical andtherapeutic standardization and bar coding of organic and organo-metallicmolécules from a plant, animal or a naturally available or man made materialsused as medicines, said method comprising: a) selecting plant, animal or a naturally available or man madematerials which possess médicinal value, and extracting theconstituents, b) separating the constituents into individual compounds, generatingand converting the 3-D and contour chromatograms into fingerprints, c) analyzing the fingerprints using the software developed, and d) interpreting the data.
19. 19. A method as claimed in claim 1 wherein, in step (iv) provides an in-builtsoftware for the Chemical analysis of constituents présent in the extract understudy and their conjugative and polarity properties indicating the therapeutic - IC? s 012520 efficacy as per the traditional concepts of the medicine using the new softwaredeveloped.
20. 20. A method as claimed in claim 1 wherein, in step (iv), the software provides anovel concept for obtaining chromatographie finger printing of material havingmédicinal value for the quick identification of the actual profile of thecompounds présent in the medicine under use along with their therapeuticefficacy of the constituents.
21. 21. A method as claimed in claim 1 wherein, in step (iv), the software provides anovel chromatographie finger printing developed on a Photo Diode ArrayDetector (PDA) of a High Pressure Liquid Chromatograph, which, delineatesthe data of the spectral properties of the constituents présent in the materialhaving médicinal value presented in a spécifie order of polarity under similarexperimental analytical conditions.
22. 22. A method as claimed in claim 1 wherein, in step (vii) “The ChromatographieFingerprint”, obtained provides the blue print of the constituents présent in amaterial for an assay and quick identification of the médicinal extractunderstudy.
23. 23. A method as claimed in claim 1 wherein, same standard analytical parameterslike extraction with same solvent ethyl alcohol, same run time Q-60min, samemobile phase acetonitrile along with phosphate, buffer having a pH in the rangeof 5.5-7.5, and a same UV-Visible Range of 200-800nm for fingerprinting andChemical and therapeutic standardization.
24. 24. A method as claimed in claim 1 wherein, in step (ix), the fingerprint. chromatograph obtained is used for the study of adulterated, substituted, contradictual and commercial food and drug samples and to identify the pureand impure.
25. 25. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for identifying the Chemical constituentsprésent in it for the purpose of process standardization, quality control activitiesand therapeutic standardization of Allopathie, Ayurvedic, Homoeo, Siddha,Unani, Chinese, Tibetan, Kampo (Japanese) medicines. -loa- 012520
26. 26. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for the study of variation of Chemicalconstituents due to various ecoiogical factors, geological factors, genotypic andphenotypic variations (in plants) in naturally occurring samples and to identifyand standardize the Chemical constituents in them.
27. 27. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for the study of Chemical constituents insynthetically prepared samples and to identify and standardize the Chemicalconstituents in them for Chemical and therapeutic standardization which ever isapplicable.
28. 28. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for the study of Chemical constituents in herbalproducts of single medicine samples and to identify the Chemical constituents inthem for Chemical and therapeutic standardization.
29. 29. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained fingerprinting is used for the study of Chemicalconstituents in herbal products of formulated medicine samples and to identifythe Chemical constituents in them for Chemical and therapeutic standardization.
30. 30. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for the study of variation of Chemicalconstituents in biological samples and to identify and standardize the Chemicalconstituents in them.
31. 31. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained is used for the study of variation of Chemicalconstituents in different brands of products of single and formulated food andmedicine samples and to identify the Chemical constituents in them for Chemicaland therapeutic standardization.
32. 32. A method as claimed in claim 1 wherein, préparation of a database of a largenumber samples gives many generalizations of the therapeutic efficacy of aparticular group of plants, classtfied as a group for a particular disease ortherapeutic classification. 012520
33. 33. A method as claimed in claim 1 wherein, in step (ix), the fingerprintchromatograph obtained for medicines facilitâtes to categorize and quantify theconstituents of a medicine based on polarity and conjugation from 3-D andcontour chromatograms and assess the therapeutic efficacy of the medicine onwhich humors it is going to act (vitiate).
34. 34. A method as claimed in claim 1 wherein, in step (ix), the Fingerprintchromatograph obtained enables to understand and standardise the Physico-Chemical properties of the medicines like color for the use of therapeuticstandardization of medicines and humors using conjugative and polarityproperties given in the chromatographie fingerprints.
35. 35. A method as claimed in claim 1 wherein, in step (ix), the Fingerprintchromatograph obtained enables to understand and standardize the Physico-Chemical properties of the medicines like Tastes (Rasa) like Sour, Salty,Pungent, Bitter, Astringent (Amla, Lavana, Katu, Tikta, Kashaya as described inAyurveda) used for therapeutic standardization using conjugative and polarityproperties shown in the chromatographie fingerprints.
36. 36. A method as claimed in clçrim 1 wherein, in step (ix), the fingerprintchromatograph obtained enables to understand and standardize the Physico-Chemical properties of the medicines like Property, Potency, Métabolite,Spécifie properties like Chirality of the molécules (Guna, Veerya Vipaka,Prabhava) used for the therapeutic standardization using conjugative andpolarity properties of the individual constituents and the whole medicine shownin the chromatographie fingerprints.
37. 37. . A method as claimed in claim 1 wherein, in step (ix), the fingerprint chromatograph obtained enables to understand and standardize the Physico-Chemical properties (Gunas) of the medicines like Cold, Hot, Slow in action,Sharp in action, Héavy, Light, Soft Lubricated Supple, Dry (Sheeta, Ushna,Manda, Teekshna, Guru, Laghu, Snigdha, Rooksha as described in Ayurveda)used for the therapeutic standardization using conjugative and polarityproperties of the médicinal extract shown in chromatographie fingerprints. '^012520.
38. 38. A software as claimed in claim 10 is used as a data processor of 3 Dchromatograms and color contour image of an ingrédient, said processorcomprising computing means and; i) an analyzer (extracting colors) for anaiyzing the colored contour imagebased on the sélection of various colors (with standards mentioned inrelease notes, life cycle, processing) denoting the concentrations of thevarious constituents eîuted with time, and polarity based on rétentiontime; il) an analyzer for anaiyzing the 3-D chromatograms of the médicinalextract using ail its 3 dimensional properties of the image; iii) a means for generating a chromatogram having peaks at variousrétention times along with conjugative properties of the compoundseluted with time in a specified order of polarity; iv) an identifier for identifying the compounds in the said molécules by theUV-VIS absorptive properties of the various eluted constituents in theimage; v) a means for correlating the reported biological, therapeutic activity ofthe of various eluted constituents présent in the médicinal sampleunderstudy based on the polarity and the conjugative properties of themolécules by dividing the fingerprint into therapeutic zones on X and Yaxis;. vi) a means for generating a barcode for a selected peak(s) using the imagecoordinates viz., X for rétention time, Y for wavelength, R for numberof red pixels, G for number of green pixels and B for number. of bluepixels, provided by the proposed software; vii) a means for generating a database of fingerprints and barcodes for thesamples, facilitating ail kinds of database utilities like EnterpriseResource Planning (ERP) and Customer Resource Management (CRM)applications; and viii) a means for generating a database of the ‘display widows’ for ail thesamples to be used by the ENTERPRISE RESOURCE PLANNING - 106 - 012520 (ERP) and CUSTOMER RELATIONSHIP MANAGEMENT (CRM) type of business applications
39. 39. A processor as claimed in claim 38 wherein, the HPLC apparatus used isselected from any commercially available HPLC apparatus with the PhotoDiode Amy detector, preferably with a gradient or temary System of pumps.
40. 40. A processor as claimed in claim 38 wherein, the polarity of the mobile phase ofa non-aqueous and an aqueous solvent of a spécifie pH is controlled by varyingthe ratio of the mobile phase from 0% to 100% of an aqueous solvents likewater or a buffer of a known pH, along with a non-aqueous solvent or vice-versa.
41. 41. A processor as claimed in claim 38 wherein, on analysis of 3-D and contourchromatograms that gives a chromatogram with rétention time and wavelengthon its X and Y-axis.
42. 42. A processor as claimed in claim 38 wherein, on analysis of 3-D and contourchromatograms using new software, which gives a data having indicated thevitiation of doshas quantitative!y in percentage ratio.
43. 43. A processor as claimed in claim 38 wherein, a single solvent éthanol is used forextraction of the constituents; same analytical conditions and instrumentalparameters were used for ail samples to bring the therapeutic generalizations toachieve the therapeutic standardization
44. 44. A processor as claimed in claim 38 wherein, the software Rainbow is havingfollowing features: (a) a software with a facility of opening chromatographie fingerprint imagesin.different Formats (extensions) like .BMP, JPEG, TIF, GrF from thefile folders and analyze it for different colors présent in the image withsingle pixel sensitivity; (b) a software with a facility of display of the pixel information in the formof l.a graph having a scale of X (0-(min. time scale) and Y (200-800nm)coordinates and 2. a Pie diagram with individual values of each peak(Automatic and Manual) in two separate columns beside the graph; (c) software with a facility of printing ail the data generated after analysisusing PRINT Icon; 012520 — iOG (d) a software with a facility of changing the page setup for printing usingPAGE SETUP Icon; (e) a software with a facility of selecting a part of the image and analyzeusing RESIZE Icon; (f) a software with a facility of opening any number of image analysisWindows for different images, and display of status in WINDOW icon; (g) a software with a facility of dividing the image in to three Zones at 20min interval, using ZONE icon; (h) a software with a facility of inverting the selected image using INVERTicon; (i) a software with a facility of switching over to Notepad, Word pad andMS Word, using EDITOR icon; (j) a software with a facility of operational information about variousfeatures of the Software using, the HELP icon; and (k) software with a facility of saving the data generated using SAVE ASicon as JEPG file format.
45. 45. Use of fingerprints of contour and 3 -D chromatograms of the constituents asclaimed in any of the proceeding daims are the basis for identification ofChemical constituents.