1. 1. A novel scheme and method for the detection identification & differentiation of Giloy satwa with most conmen adulterant of the satwa of potato & the satwa of maize comprising: a. Extraction procedure of satwa from Potato, Maize & stem of Tinospora Cordifolia; b. Pre treatment onto stationery phase before the application of satwa from Giloy Potato and Maize; c. Development of innovative proportion of loading solution for sampling of satwa from Giloy Potato and Maize onto stationery phase ; d. Development of newly designed proportion of solvent system for the separation of satwa from Giloy Potato and Maize onto stationery phase; e. Detection of the specific marker for the differentiation of Giloy satwa and satwa of potato & maize; f. Identification of specific marker present in the Giloy satwa and absent in satwa of potato & maize; g. Identification of specific marker presents in satwa of potato & maize and absent in the Giloy satwa. h. Densitometric scanning of separated peaks for the confirmation of pure & fake samples by spectra pattern.

2. The process as claimed in Claim 1, wherein the stationery phase systems for separation of Giloy satwa with the satwa of potato & maize, in the vapors of iodine for five minutes before loading the sample.

3. The process as claimed in Claim 1, wherein the mobile phase for solvent system is selected from Acetonirtile Phosphate buffer Water Ethanol, Acetone.

4. The process as claimed in Claim 1-H, wherein the Spetom-of each component is considered as individualization character of compound which differentiates between pure & fake giloy satwa samples

[0001] Technical Field:
The present invention relates to novel scheme and method for the development of marker for the detection of Giloy satwa obtained from the stem of Tinospora Cordifolia and its differentiation from conmen adulterants such as potato, maize corn starches in order to prevent the adulteration in Giloy satwa. Giloy satwa used as key therapeutic medicine in Ayurvedic System of Medicine. The present invention is also relates to a process of extraction of Giloy satwa (polysaccharide fraction P.V.Leyon, G.Kutton 2003) from the stem of Tinospora Cordifolia and extraction of starch from the potato, maize and corn, for the purposes of detection of specific marker in order to differentiate between Giloy satwa and starch from the potato, maize and corn, which are the most conmen adulterant in Giloy satwa.
The focus of the invention is to highlight the value of the Giloy Satwa (Tinospora Cordifolia) during recent time and our invention is keen to provide the right way for adopting the pure Giloy Satwa from suppliers of Giloy satwa by Ayurvedic Medicines Manufactures, Practicing Vadiyas and other common users of Giloy Satwa by using my newly invented scheme and method expressed in this invention which was never available earlier any where in the universe.
A scheme and method of development of marker for identification ,detection and differentiation of pure giloy satwa with conmen adulterants is obtained via a process of stated in the "METHOD AND MATERIAL" section of specification.
Background & prior art:
[0002] The common habitat of plant grows throughout India from kumaon to kanyakumari. The Synonyms are in Sanskrit is Amratavalli, Amrita, Madhuparin, Guduchi, Chinnodbhava,in Assam is Siddhilata, Amariata, in Beng. is Gulancha, in Guj. is Galac, Garo,in Hindi is Giloe, Gur, in Kan is Amrutabal, in Mha is Chittamrittu, Amri, in Mar Gulvel., in Ori is Gulu, in Tam is Seendal, Seendil ko,in Telis Thipateega and in Urdu is Gilo.
[0003] Giloy is very widely used in ayurvedic preparations and considered as tridosanashk in since ancient Ayurved. The whole plant is considered as therapeutic but the Giloy satwa is considered as most important therapeutic constituent and considered as tridosh-nashk. All the diagnostic and prescription principles in the Ayurvedic and Siddha system of medicine is entirely based on the three words Vata, Pitta & Kapha.The tendency to act, rest and balance in the humans is within Vata, Pitta & Kapha. The cause of all the disease is the imbalance of Vata, Pitta & Kapha .In ancient ayurveda Giloy has been considered as Tridosha pacifying agent, which means it has specific pharmacological property for balancing the Vata, Pitta & Kapha at the same time, which results as curing of many diseases by pacifying activity. In present scenario Giloy Satwa is a most conmen ingredient of most of the ayurvedic formulation for total nutrition, nourishment, diabetic, cancer, hypertension, antipyretic and analgesic ayurvedic formulations.
[0004] In the review of modern research Giloy exhibits the anti tumors and anti-inflammatory properties. Giloy satwa used as bactericides, emetic, tonic, stimulant, diuretic, aphrodisiac, and sedative, stomachic and in asthma, erysipelas, fever, gout, inflammation, jaundice, leprosy, rheumatism, sores, tuberculosis, tumors, liver disorders and malaria.
[0005] Review of modern research on Phytochemistry, Pharmacological & Biological Activities of Giloy
Phytochemistry: Although a large number of chemical compounds such as Furanoid diterpene.clerodane furano - diterpene i.e. Columbin, Tinosporaside.Sesquiterpene glucosidetinocordifolioside.Sesquiterpenetinocordifolin.Tinosponone.Tinocordioside.Cor dioside.Jatrorrhizine, Berberine.Phenyl propene disaccharides Cordifolioside A, B and C.ß - Sitosterol.20 - ß - hydroxyl ecdysone.Tinosporon.Tinosporic acidDiterpenoid furanolactones are reported in plant.
Therapeutically Satwa fraction is known to be responsible for the maximum pharmacologically activity (Mathew S, Kutton G 1997). The source of pharmacologic active constituents is the Satwa fraction extracted from the stem of the plants Tinospora Cordifolia. The solid starchy extract is known by the name of Guduchi satva, Giloy ka sat or polo. According to the Deshmukh et al (1957), Giloy satwa is cream colourd, biteer having starch grains, crystal fiber vessel portion, and parenchyma and cork cells.
Pharmacological & Biological Activities : Satwa is consists of specific polysaccharides that are made up of a high number of molecular components up to millions of atoms and this makes it difficult to be absorbed. The Polysaccharides of the Giloy satwa is considered as easily assimilated and absorbed in the body due to the specific action of a substance already present in the Giloy satwa ,which enables it for easily incorporate. This property of Giloy satwa makes it different from other plant polysaccharides & starch.
[0006] It is considered that the bitter extract obtained pharmacological activity.
[0007] It causes reduction in fasting blood sugar. Aqueous extract shows anti inflammatory, analgesic & antipyretic action & immunosuppressive effect.
[0008] It also exhibits protective effect in carbon tetra chloride induced hepatotoxicity. It significantly inhibited antibody formation by typhoid 'H' antigen. It also potentiated morphine analgesia.
[0009] Intravenous exposure to aqueous extract in dose of 5, 10 & 15 mg/kg. body weight produces a temporary, but marked fall in blood pressure, bradycardia in anaesthetic dogs. This drug also decreased blood urea levels in uraemic dogs and human subjects.
[0010] The starch substances present in Guduchi, stems and roots is well known in Ayurvedic medicine and is considered as nutrient, useful in chronic diarrhea, chronic dysentery and is treating intermittent fevers and various other ailments.
[0011] The alkaloids berberine, protoberberine palmitine and jatrarrhizine have been shown to possess invitro activity against Plasmodium falciparum comparable to Quinine.
[0012] None of these alkaloids were active invivo against Plasmodium berghii. The clerodone diterpenes have not been studied pharmacologically in detail probably due to their low contents, however, they do contain interesting pharmacopharic features besides having a bitter taste.
[0013] In addition to above, Guduchi has following pharmacological activities:
[0014] Adaptogenic Activity: The alcoholic extract exhibits significant antistress activity at l000mg/kg compared with diazepam at 2.5mg/kg. Sharma & Khosa have reported that alcoholic against stress induced changes in nor epinephrine, dopamine, 5 -hydroxyl triptamine, & 5 - hydroxyl indole acetic acid & levels of experimental rats. The extracts prepared by extraction of Guduchi stems with aqueous alcohol, acetone and petroleum ether have shown antistress activity in mice at a dose of l00mg/kg body weight. A significant anabolic activity was observed in petroleum extract as control group.
[0015] Antiallergic Activity: An aqueous extract of T.cordifolia decreased bronchospasm in guinea pigs, decreased capillary permeability in mice and reduced the number of disrupted mast cells in rats.
[0016] Anticancer Activity : A formulation containing Tinospora cordifolia, Asparagus racemosus, Withania somnifera and Picrorrhiza kurroa markedly inhibited the suppression of chemotactic activity and production of interleukin - 1 and tumour necrosis factor induced by the carcinogen ochratoxin in mouse macrophages.
[0017] Antineoplastic activity: Aqueous, methanolic and dichloromethane extracts of Tinospora cordifolia showed dose - dependent increases in lethality to HeLa cells in vitro. The most potent activity was found in the dichloromethane extract.
[0018] Antioxidant Activity :An extract of Tinospora cordifolia reduced the toxicity induced by free radicals and inhibited lipid peroxidation and the generation of superoxide and hydroxyl radicals in vitro. It reduced the toxic side effects of cyclophosphamide in mice, as shown by total white blood cell count, bone marrow cellularity and esterase -positive cells. It also partially reduced elevated lipid peroxides hi serum and liver, as well as alkaline phosphatase and glutamine pyruvate transaminase.
[0019] Anti inflammatory & Hepatoprotective activity: The water - soluble portion of alcoholic extract has been shown to provide protection to liver damage induced by administration of carbon tetrachloride in mice, rats & rabbits. The above observations were supported by other investigators also. The stem extract has also found to improve surgical outcome in patients with malignant obstructive jaundice by strengthening the host defences.
[0020] Immuno - modulating Activity: Syringin, Cordioside and Cordifolioside were found to possess immunopotentiating activity. The possible mechanism of immuno -modulatory activity of Tinospora cordifolia stem extract was elucidated as activation of macrophages leading to increase in GM - CSF which in turn leads to leucocytosis and improved neutrophil function. The ethanolic extract was also found to possess immune -modulating activity
[0021] Detail description of present analytical method of detection of Giloy (Tinospora Cordifolia).
[0022 ] Botanical Descriptions
Large glabarous twiner with terete stem with long filiform, fleshy, aerial roots. The stem has a loose grayish bark when mature. The leaves are simple, alternate, long petioled and possess a characteristic heart shape, giving the name cordifolia to the plant. Flowers are green, unisexual, dioecious spikes. The fruits are ovoid, glossy, red, drupes, containing curved seeds. [0023] Macroscopic
Stem rather succulent, soft, possessing long, filiform, aerial root arising from branches. Bark warty, creamish white or grey brown, wood, soft, perforated. Dried sample consists of 5 to 10cm long, conical pieces, light in weight, bark light and papery, brittle, dark brown, wood with longitudinal surface ridges, and radially divided into wedge shaped pieces in cross sections. Pieces difficult to fracture when fully dried and can be torn only twisting, odourless, taste bitter.
[0024] Microscopic
TS show cork, cortex and vasculature. The cork comprises of an outer zone of thick walled brownish compressed cells and an inner zone of thin - walled colourless, tangentially arranged cells. The cork tissue is broken at some places due to lenticels. Cortex is wide. The outer zone of cortex consists of 3 - 5 rows of irregularly arranged tangentially elongated chlorenchymatous cells and the cells situated towards inner side are polygonal in shape and filled with abundant starch grains. Starch grains are simple, ovoid or ovoidelliptical, occasionally compound of 2 to 4 components. Several secretary cells found scattered in the cortex.
Pericycle fibers containing a single prism in each chamber. Vascular zone composed of discrete vascular strands with 10 - 12 or more wedge - shaped strips of xylem, externally surrounded by semi - circular strips of phloem, alternating with wide medullary rays. Phloem consists of sieve tube, companion cells and phloem parenchyma of polygonal and tangentially elongated cells. Some of the cells of phloem parenchyma contain calcium oxalate crystals, cambium is of 1 to 2 layers, xylem consists of vessel elements, trachieds, parenchyma and fibers. Vessel elements cylindrical in shape bearing bordered pits. Medullary rays 15 to 20 cells wide. Pith mostly made up of large thin - walled cells containing starch grains. The presence of discrete vascular strands in the mature stem of Tinospora cordifolia is one of the different anomalous secondary structures found in Menispermaceae.
[0025] Determination of Total Ash: Weigh accurately 2 to 3g of the air - dried crude in a tared platinum or silica dish and incinerate at a temperature not exceeding 450° until free from carbon cool and weigh. If a carbon free ash cannot be obtained in this way, exhaust the charred mass with hot water, collect the residue on an ash less filter paper, incinerate the residue and filter paper until the ash is white or nearly so, add filtrate, evaporate to dryness and ignite at a temperature not exceeding 450°. Calculate the percentage of ash with reference to the air - dried drug.
[0026] Determination of Alcohol Soluble Extractive:Macerate 5g of the dried drug, coarsely powdered with 100ml of ethanol of the specified strength in a closed flask for 24hrs, shaking frequently during the first 6hrs and allowing to stand for 18hrs. Thereafter, filter rapidly taking precautions against loss of ethanol, evaporate 25ml of the filtrate to dryness in tared flat - bottomed shallow dish, dry at 105° and weigh. Calculate the percentage of ethanol - soluble extractive with reference to the air - dried drug.
[0027] Determination of Water Soluble Extractive: Add 5g to 80° in a stoppered flask. Shake well and allow standing for lOminutes, cool, add 2g of kieselguhr and filter. Transfer 5ml of the filtrate to a tared evaporating dish, 7.5cm in diameter, evaporate the solvent on a water bath, continue drying for 30minutes, finally dry in a steam oven for 2hrs and weigh the residue. Calculate the percentage of water - soluble extractive with reference to the air - dried drug.
[0028] Present format for analysis: Then necessity of invention is further illustrated by following presently existing non specific and non reproducible analysis methods.
(Table Removed)
The Gilov satwa possesses characteristic of normal starch and so can not be differtiated. (Pharmacognosv of Indian Drugs, Central Council for research in Avurveda and Siddha India. Vol-I. Page 348-349)
[0029] Description of specification of present Chromatography method of detection of Giloy satwa (Tinospora Cordifolia)
[0030] Chromatography: The following markers are specific only for plant whereas pretreated adulterated satwa, with plant juice of Tinospora Cordifolia also exhibits the marker of plant of Tinospora Cordifolia plant and always mistaken as pure Giloy satwa by most of the Ayurvedic formulation manufactures.
[0031] Objectives:
1. An objective of the present invention is to provide an efficient extraction scheme and
method for preparation of Giloy satwa & adulterated satwa.
2. Another objective of the present invention is to provide specific, accurate &
reproducible scheme and method in order to Identify, Detect & Differentiate the pure
Giloy satwa & adulterated satwa by the specific marker present and express in of Giloy
satwa and absent in fake Giloy satwa.
3. A further objective of the present invention is to provide a robust scheme and
method for the determination of the concentration of marker
[0032] Need of My Invention: -
In present boom in ayurvedic and herbal products the alarming and emerging problem for manufacture of herbal preparation is to preserve the actually pharmacologically active substance for healing properties of herbal medicines. Without the presence of actual pharmacologically active substance or presence of other pharmacologically active substance in the herbal preparation would not act accurately and even can harm the user. The actual pharmacologically active substance is responsible for specific therapeutic effects on the various systems of humans.
[0033] The large market & heavy demand of raw herbs the adulteration imitation, duplication, immature form and substandard raw herbs are providing by raw herbs suppliers. Giloy is very widely used in Ayurvedic preparations.
[0034] .The commercialization in ayurvedic manufacturing made the supply of Giloy satwa vulnerable an adulterated or fake, impure Giloy satwa is being supplied by the suppliers very frequently due to heavy demand of Giloy satwa and lack of non specific analytical method of identification of Giloy satwa. The other cause of adulterer supply is the preparation of Giloy satwa is very hard and time tacking and yield is uncertain many tunes. Now the practice of procurement of Giloy satwa by big herbal manufactures becomes very common from local venders. Due to the high demand and good pries the adulteration in satwa is very conmen. The pries difference between pure Giloy satwa and fake adulterated satwa is more then two thousand rupees per kg.One kg of potato satwa of can be prepared in hundred rupees whereas pure Giloy satwa cost about two thousands rupees per kg after tedious and very time tacking process. The common source of adulteration as we identified is the starch of potato, maize and rice. The adulterants are cheap and easily available.
[0035] After the review of modern research and ancient's literature the term Giloy refers to a heavenly elixir which was reputed to protect the people from all deices and keep them eternally young. Giloy exhibits anti tumors and anti-inflammatory properties. Giloy satwa used as bactericides, emetic, tonic, stimulant, diuretic, aphrodisiac, and sedative, stomachic and in asthma, erysipelas, fever, gout, inflammation, jaundice, leprosy, rheumatism, sores, tuberculosis, tumors, liver disorders and malaria.
[0036] The impact of my invention is very broad.
Firstly our scheme and method will assure the identity of pure Giloy satwa, which yields the overall improvement in the quality of therapeutic activity of herbal medicines and becomes a highly valuable in order to keep the preparations for accurate therapeutic affect on the humans which are declared and claimed according to the pharmacology of Giloy satwa.
[0037] Secondly the detection of impure or adulterated satwa, which does not act according to established pharmacology of Giloy satwa and or exhibits the harmful pharmacological effects on the herbal medicine users, can be prevented by our scheme and method. Patanjali Ayurved Ltd. invention has observe this threatening problem and come out with the novel scheme & method for the development of marker for the identification , detection and differentiation of Giloy satwa obtained from the stem of Tinospora Cordifolia from conmen adulterants such as potato, maize corn starches in order to prevent the adulteration in Giloy satwa to overcome this problem.
[0038] The existing method of the identification of Giloy satwa are insufficient and not capable to specifically detect & differentiate the adulterants .The suppliers prepared the starch from other sources and mix it with the juice of Giloy plant stem, by this clever mixing it taste as Giloy satwa and it appears as Giloy satwa and the wrong and adulterant material passe as Giloy satwa, the adulterant material is very badly affect quality and therapeutic efficacy of medicine preparation during the entire process of ayurvedic medicine manufacturing.
[0039] In Ayurved & natural products industries the detection & differentiation between pure Giloy satwa & fake satwa by reliable, robust, swift scientific scheme and method is essential for the safety of humans. We have invented the present scheme & method for present need, our method is very specific accurate & reproducible in order to detect & differentiate the pure Giloy satwa & adulterated satwa.
[0040] Examples:
[0041] Example 1:
1. Collection of Potato, Maize & fresh Tinospora Cordifolia stems. (After proper
identification by botanical & food department of Patanjali Ayurveda)
2. Prepration of starch from Potato, Maize & fresh Tinospora Cordifolia stems
3. Pre treatment of extracted material from Potato, Maize & satwa of Tinospora
Cordifolia
4. HPTLC analysis of pretreated extracted material from Potato, Maize & satwa of
Tinospora Cordifolia
[0042] Example 2:
Experimental Materials
1. Potato, Maize & fresh Tinospora Cordifolia stems.
2. Organic solvents
3. Solvent extraction apparatus
4. Glass wares, Plastic wares and DM water.
The glasswares, plasticwares used in the present study were procured from M/s Borosil (Mumbai) and M/s Axygen. The chemicals were procured from Merck (Mumbai), Qualigens (Mumbai) and Sigma (USA).The materials/reagents used in various methods are mentioned in the relevant section of methodology.
[0043] Example 3:
EXTRACTION PROCESS & RECOVERY OF POLLYSACCRIDE FRACTION FROM THE STEMS OF Tinospora Cordifolia (Pendse, G. P and Dutt, S.K 1932)
A - The well matured fresh Tinospora Cordifolia stems, which comply with
the minimum parameters given the Indian Herbal Pharmacopoeia,
was crushed in a disintegrator to coarse powder. B - It was then extracted with 14 % v/v alcohol followed with
Dimineralised water. C - The extracted solution received from hydro - alcoholic and
aqueous extractions is concentrated separately and then, mixed in a
homogenizer to make a homogenous solution before final
concentrate. D - The solution when attains a concentrate of 30 - 50% solids was be
spray dried or it can concentrated to a thick paste of 65 - 70%
solids.
E - The thick paste is then dried in vacuum tray dryer without using any heat F - The dried flakes are pulverized to 80 - 100 mesh and packed under hygienic condition. The other interfering materials was removed by the treatment of TCA
G - The powder before analysis was lyophilized at room temperature .packing can be sterilized by passing through a stream.
[0044] Example 4:
EXTRACTION PROCESS & RECOVERY OF STARCH FRACTION FROM
POTATO AND MAIZE.
A - The well matured POTATO AND MAIZE was taken, which comply with the minimum parameters given in raw vegetable & cereals manual disintegrator to very small pieces.
B - It is then extracted with mix with the Dimineralised water.
C - The mixture was kept for one hour for the sedimentation of starch fraction for over night.
D - The supernatant of solution then discarded slowly when attains a concentrate. E - The thick paste is then dried in vacuum tray dryer without temperature. F - The dried flakes are pulverized to 80-100 mesh and packed under
G- Kept the powder for next step of analysis.
[0045] Example 5:
HPTLC analysis of pretreated extracted material from Potato, Maize & satwa of Tinospora Cordifolia for the identification, detection & differentiation scheme for Giloy satwa with the satwa of potato & maize has been done. As we review the literature we come to narrow down our search for identification, detection specific marker present exclusively in Polysaccharide fraction of the giloy satwa to differentiate the pure satwa with the fake satwa. The Polysaccharide fraction Giloy satwa has high molecular weight linked with the glycosidic bonds, to another sugar component. The sample preparation is the most critical aspect of chromatographic analysis. We have reviewed the maximum available literature for the separation of high molecular weight plant polysaccharide fraction by capillary & adsorption phenomena but unfortunately we does not find any relevant and direct literature for the separation of Giloy satwa by capillary, adsorption phenomena for the identification, detection and differentiation of the presence of predetermined levels of certain compounds principle present in giloy satwa used as a marker.
[0046] Example 6:
Novel method & scheme for loading of sample We have attempted the different method and mechanism for stationery phase systems for separation of Giloy satwa with the satwa of potato & maize. We used the cellulose, silica, silica 50,000 and amino acid bond silica layers for the separation. Cellulose, silica, silica 50,000 and amino acid bond silica layers were not give the clear separation of samples. Finally we have developed a novel mechanism for stationery phase systems to eliminate the possibility of reducing the layer activity due to polar solvent and viscosity of the sample. We have taken commercially available plates with uniform thickness coating of silica gel-G or silica gel GF254 of uniform particle sizes on aluminum supports. We put the plates in vapors of iodine for few minutes before loading the sample. The pretreatment of the stationery phase give the clear separation due the partition, adsoroption and new anion exchange phenomena.

The treatment with iodine fumes formed the layer between the sample and adsorption media by which we eliminated the possibility of reducing of the layer activity and the sample binding onto the stationery phase due to the viscosity of the sample can be reduced and clear and be which allows the sample to run smoothly in solvent system and the clear and better resolution has been achieved. However use of non treated plates is not recommended because our treatment forms the layer between the sample and stationery phase by which the binding of the sample can be reduced and clear and better separation can be achieved.
[0047] Example 7:
We have tried the conventional method of lodging of sample in methanol which has not given separation of the sample due to the adherence of sample onto the stationery phase .we assume that every sample has it oven character and one uniform lading solvent as methanol, could not be applicable on all samples as used conventionally in HPTLC spotting. Our new scheme and method of lodging the sample is novel and different from conventional sample loading methods. We have taken the dry 50 mg of the extracted sample of giloy satwa with fake satwa. Further we dissolved all the samples in 1ml DM water & Ethanol (50:50) in separate test tubes and hold the test tube for hour in test tube shaker. Now the sample is ready for injection, we take the supernatant solution from test tube and spotted the 0.200 micro liter sample spot of each sample by automatic applicator with nitrogen flushing. Alternatively, samples can also be applied as narrow bands using a band applicator. Application of unknown volumes of test and reference solutions can be done using calibrated capillaries.
[0048] Example 8:
Design for the separation of sample is novel and first used by me for the Detection & Differentiation for Giloy satwa with the satwa of potato & maize. We have tried various solvent systems for separation of Giloy satwa as it was the hydrophilic substance. Since excess of water in solvent system decrease the layer activity which is the main cause of the failure of sample separation by the other conventional solvent system .The other possible cause of the failure of sample separation by the conventional solvent system is slow migration of sample. We used number of proportion for developing the best solvent system with water as part of the solvent system. Finally we have developed a novel proportion of three solvents, which exhibits best separation of Giloy satwa with the satwa of potato & maize.
[0049] Example 9:
The novel proportion of three solvents is Acetonirtile: Phosphate buffer: Water. The innovated proportion is 80:15:2 at ph 5.6 The chamber was saturated for ISminutes for saturation after pouring the solvent system and then placed the plate in the chamber and developed in the solvent system up to a distance of 8cm
[0050] Example 10:
Finally the combination of different stationery phase and novel proportion of three solvents express the novel marker for the identification & differentiation of Giloy satwa with most conmen adulterant of the satwa of potato & maize.
[00511 Example 11: The RESULTS of the present study from Fig 1 to 16 shows that there is a clear separation of Giloy satwa with satwa of potato and maize by the combination of our scheme and method
HPTLC PROTOCOL DETAILS
(Table Removed)
(Figure Removed) 3D representation of all the tracks
Details of the sample applications
(Figure Removed)
(Figure Removed)
COMPARISON OF SPECTRA OF PURE GOLOY SATWA
COMPARISON OF SPECTRA OF ADULTERATED GOLOY SATWA
(Figure Removed)
a. After separation sample of each satwa exhibits the characteristic spot at differed Rfs.
Giloy satwa represent Rf value 0.55 whereas adulterated satwa of potato exhibits the Rf
value 0.15 and adulterated satwa of maize exhibits the Rf value 0.20.(Fig 1 to 8)
b. We have also estimated the amount of marker present in the samples by comparing the
peak area. We record the peak area and prepare the calibration curve by plotting peak vs
concentration of marker.
c. Further we have done the densitometry scans performed on CAMAG - 3 scanners of
related peaks present in pure giloy satwa which exhibits the reproducible spectra.
Analytical procedure of my method and scheme was validated in the official method of
analysis of the Association of Chemist (AOAC international).For repeatability we have
done this process in three replicates and the accuracy of scheme and method procedure
express the closeness of agreement between the value which is accepted as reference and
true value of pure sample and confirmed by superimposing the reference UV spectra
giloy satwa (Fig. 8 to 16).
[0052]I Example 12:
Traditional method of Giloy satwa identification is appearance & taste, which is easily imitated by the suppliers by mixing of original plant juice in potato and maize satwa. Present prescribed method for quality control is not very specific , accurate & reproducible due to the range of base line that is from .005 to 5 (%w/w)
[0053] Example 13
Our scheme and method is new due to it was not used by any one in the universe before we use to for the detection identification & differentiation of Giloy satwa with most conmen adulterant of the satwa of potato & maize. We are the first user of scheme for preparation for the satwa of adulterants whereas other is even not aware about the most of adulterants.
[0054] Example 14:
We use the new stationery phase, new sample loading solution and new proportion of
solvent system for the complete analysis of Giloy satwa along with most frequent
adulterant, the satwa of potato, the satwa of maize by using the all new anion exchange
phenomena along with partition, adsorption, capillary phenomenon first time in the
history of TLC for the identification, detection and differentiation of pure and fake giloy
satwa.
By my scheme and method I have developed the procedure for the identification, detection and differentiation in Giloy satwa and satwa of potato & maize by presence and absence of the specific marker. This Specific marker is never reported earlier by any one in the universe for the detection identification & differentiation of Giloy satwa with most conmen adulterant of the satwa of potato & maize.
The presence of specific identification and detection marker allowed the analyst to compare the presence and absence of marker at specific Rf 0.53 value and further confirmation by superimposing spectra of marker peak. The absence of peak of Rf value 0.53 indicates that sample does not originate from plant of Tinospora Cordifolia.Whereas presence of peak at Rf value of 0.39 with peak of Rf value 0.53 result as, that sample of pure giloy satwa is mixed with potato which is further confirmed by the spectra comparison of both the peaks. Fake Giloy satwa sample was not exhibits the peak at 0.53 and the spectrum of pure giloy satwa peak is entirely different then that of other adulterant peaks.

We Claim:
1. 1. A novel scheme and method for the detection identification & differentiation of Giloy satwa with most conmen adulterant of the satwa of potato & the satwa of maize comprising:
a. Extraction procedure of satwa from Potato, Maize & stem of Tinospora
Cordifolia;
b. Pre treatment onto stationery phase before the application of satwa from
Giloy Potato and Maize;
c. Development of innovative proportion of loading solution for sampling of
satwa from Giloy Potato and Maize onto stationery phase ;
d. Development of newly designed proportion of solvent system for the
separation of satwa from Giloy Potato and Maize onto stationery phase;
e. Detection of the specific marker for the differentiation of Giloy satwa and
satwa of potato & maize;
f. Identification of specific marker present in the Giloy satwa and absent in
satwa of potato & maize;
g. Identification of specific marker presents in satwa of potato & maize and
absent in the Giloy satwa.
h. Densitometric scanning of separated peaks for the confirmation of pure & fake samples by spectra pattern.
2. The process as claimed in Claim 1, wherein the stationery phase systems for separation of Giloy satwa with the satwa of potato & maize, in the vapors of iodine for five minutes before loading the sample.
3. The process as claimed in Claim 1, wherein the mobile phase for solvent system is selected from Acetonirtile Phosphate buffer Water Ethanol, Acetone.
4. The process as claimed in Claim 1-H, wherein the Spetom-of each component is considered as individualization character of compound which differentiates between pure & fake giloy satwa samples