|Title of Invention||
A NOVEL KIT FOR AUTHENTICATION OF NATURALLY OCCURRING RAW DRUGS AND FINISHED PRODUCTS AND SEMI-QUANTIFICATION OF BIOACTIVE MARKERS
|Abstract||This invention thus provides a process of providing a product, namely Diagnostic kit, based on Thin Layer Chromatography (TLC) used for authentication of naturally occurnng raw drugs and finished products prepared from the same and semiquantification of bioactive markers. The natural products are whole or part of plant, animal or microbes. The kit contains the necessary components and instructions to carry out the test. The kit can be used by non-technical personnel in a non-laboratory environment. The stepwise processes leading to the product are disclosed.|
|Full Text||Field of Invention:
The present invention relates to providing a field kit for authentication of naturally occurring raw drugs and finished products as well as semi-quantification of bioactive markers in the raw drugs and formulations. The kit can be used in a non-laboratory environment by non-technical personnel.
Background of the invention:
Identification of naturally occurring raw drugs and finished products has become a matter of controversy in herbal sector since raw drugs sold in the market are dried plant materials and therefore difficult to identify based on morphology. The samples may be substituted or adulterated with other plants during trade. Substitution or omission of important raw materials in the Ayurvedic formulations has become a concern for the sector. There are complaints from the traders in the sector that materials are being rejected by the buyers on the reason that the 'active principle' is not at the required level.
In India, there are numerous small-scale and cottage industries, which use herbs collected from local fields, forests or obtain from markets and formulate medicine based on traditional specifications. According to AYUSH Department, Government of India , greater than 90% of the Traditional Medicine industry falls under the micro to small-scale level with a turn over of a few lacs. GMP (Good Manufacturing Practice) License is a mandatory requirement stipulated by Government of India (Schedule T of the Drugs & Cosmetics Act, 2000) that needs to be maintained by any manufacturing company in this sector. The awareness of the majority of the Traditional Medicine sector in terms of modern Quality Control requirements is minimal and their financial background not sufficient to set up modern laboratories for testing. In such circumstances, cost effective kits that can authenticate the quality of raw drugs and finished products would not only be useful but would also be adopted easily by the sector.
It is well known that modern chemical tools such as TLC (Thin Layer Chromatography) can help to authenticate the true identity of a natural product and distinguish from adulterated or substituted material. The first English publication on TLC was done by Stahl,E. in 1965 (Egon Stahl,Thin Layer Chromatography, A Laboratory hand book, second edition, Springer,2005, p:5). Successful attempt was made to reproduce the Thin Layer chromatography separation of 170 medicinal plant drugs by H.Wagner and S. Baldt.( H.Wagner, S.Balt, Plant Drug Analysis, A Thin Layer Chromatography Atlas, Second edition,Springer,2001). The TLC identity tests are explained for more than 50 plants in Indian Herbal Pharmacopoeia Eg: Acacia catachu, Andrographis paniculata, Curcuma longa, Withania somnifera etc. ( Indian Herbal Pharmacopoeia, revised new edition, Indian Drug Manufacturers Association,Mumbai,2002). Limitations of prior art that exists is that TLC testing of extracts and products requires laboratory facilities such as soxhlet apparatus, water bath, oven etc. The procedures cannot be done in non-lab conditions by non-technical people. Quantification of bioactive markers requires equipment such as the densitometer and on an average, a simple TLC may take at least 3 hours to complete. All the components required to do the tests will have to be individually assembled. Diagnostic kit based on TLC has been developed that can be used in non-laboratory environments by non technical personnel to check the quality of the raw drugs as well as formulations. The kit helps in authentication of naturally occurring raw drugs and formulations. The raw drugs are of plant, animal or microbial origin. These kits come complete with materials and instructions on how to use it. Semi-quantification of the bioactive reference marker can be done visually by following the instructions. The instructions are simple with illustrations so that any non technical person can also follow it easily.
Summary of the Invention:
The present invention comprises of series of steps leading to the product (Kit) that can be used to authenticate naturally occurring raw drugs and finished products in a non-laboratory environment by non-technical personnel. The kit can also be used to semi-
quantify bioactive chemical marker. The kit contains materials and instructions required for authentication.
Detailed description of the invention :
Stepwise process leading to product is as follows:
Step 1: Sizing of raw materials:
Milling of raw material using mixer followed by sieving through sieves of BIS (Beaureu of Indian Standards). Sieving of the material through tea strainers or through muslin cloth. The particle size of the material sieved through the sieves and through the tea strainers are compared.
Leaf of Andrographis paniculata was taken, milled, sieved and used for the experiments
Step 2: Extraction of raw materials and finished products.
Extraction was done using suitable solvent ranging from nonpolar to polar solvents (Eg: n-heptane, n-hexane, pentane, cyclohexane, isooctane, Trichloroethylene, 1.1.2-Trichloro-trifluoroethane, Di-isopropyl ether, Toluene, Xylene, Benzene, Diethyl ether, Tert.butyl methyl ether, dichloromethane, dichloro ethane, 1-butanol, butyl acetate, n-propanol, tetrahydrofuran, 2-propanol, ethyl acetate, chloroform, methyl ethyl ketone, 1,4-dioxane, acetone, methanol, ethanol, acetonitrilr, acetic acid, dimethyl formamide, dimethyl sulphoxide, water and combination of any of these solvents including acids and bases) using raw drugs /finished products by refluxing, maceration or simple boiling (Fig 1).
Repeated extractions were tested by doing the extraction 1, 2 and 3 times. The time required for each extraction was standardised after testing for 5min, l0min, lhour, 2hours, 24 hours etc.
The efficiency of extraction for each raw drug or formulation was decided based on the bioactive marker quantified using densitometer. Eg: Andrographohde extracted from
Andrographis paniculata from different extarcts were compared using densitometer (CAMAG TLC Scanner 3)and the best solvent and method of extraction was decided based on highest yield of andrographolide..
A. Extraction is performed using refluxing of material. 1g of drug is refluxed with each of the selected solvents (like Methanol, Acetone, Ether etc.) for 2hour on water bath, filtered and concentrated. All samples were spotted on TLC plates along with the standard Andrographolide so as to compare the content of Andrographolide extracted.
B. Extraction is performed using maceration of material.1g of drug is taken and
macerated with the selected solvents for 24h, then filtered and concentrated. All
samples were spotted on TLC plate along with the standard Andrographolide so
as to compare the content of Andrographolide extracted.
C. Extraction is performed using refluxing of material.1g of powdered drug is
refluxed on water bath at 60 °C with 25ml of methanol for 2h and filtered. The
filtrate obtained was concentrated. Similarly 2 times reflux, 3 times reflux and 4
times reflux were done in order to see the variation in the content of
D. Extraction is performed using maceration of material. Four batches of 1g each
were kept for extraction with 25ml of methanol with time period of 8h, 16h, 24h
and 32h. After the defined period of time the samples were filtered and the
filtrates were concentrated.
E. Extraction was performed using simple boiling of material, 1g of powdered drug
was taken in 25ml methanol and boiled on water bath for 2minutes.The mixture
was filtered and filtrate concentrated. Similar procedure was adopted for
5minutes and 10 minutes.
All samples extracted by different methods of extraction were spotted on a TLC plate and observed for the yield of Andrographolide. Three times reflux and 24h maceration had given maximum yield of Andrographolide (Refer Fig:2) whereas l0minutes boiling (Kit method) was found to be effective technique as a simplified and faster method of extraction in a non-laboratory environment (Refer fig:2). Thus 10 minutes boiling (Kit method) was selected as suitable method for extraction.
Step 3: The comparison is made between samples prepared by 3 times reflux (Method C) with 10 min boiling (Method E) by taking five different samples (from different regions) and two replicates for each sample for each method.
Step 4: Selection of Stationary phase.
The selection of stationary phase was done by considering the resolution of bands in samples and markers. Both normal and reversed phase plates were tried.
For Andrographis paniculata kit Stationary phase silica gel F254 was selected.
Step 5: Selection of suitable mobile phase for the elution of compounds in the medicinal plant extracts or in the formulation.
The mobile phase are solvents from non-polar to polar Eg: n-heptane, n-hexane, pentane, cyclohexane, isooctane, Trichloroethylene, 1.1.2-Trichloro-trifluoroethane, Di-isopropyl ether, Toluene, Xylene, Benzene, Diethyl ether, Tert.butyl methyl ether, dichloromethane, dichloro ethane, 1-butanol, butyl acetate, n-propanol, tetrahydrofuran, 2-propanol, ethyl acetate, chloroform, methyl ethyl ketone, 1,4-dioxane, acetone, methanol, ethanol, acetonitrilr, acetic acid, dimethyl formamide, dimethyl sulphoxide, water and combination of any of these solvents including acids and bases.
Step 6: Spotting of extracts on TLC plate
The standard marker, reference and sample extracts were applied on TLC plate using Linomat V applicator.
The samples as above were applied using pre marked capillary tubes and the amount per track of bioactive marker compared.
Step 7: Selection of suitable visualizing agent:
The plates were observed for the number of bands, Rf values and colour of bands visually in daylight and under UV (254 and 366 nm wavelength) before treating with visualizing agents. Selection of visualizing agent was done by considering colour and clarity of spots after treating the plate and also the ease of preparation of reagent. Reagents tested were of the general nature such as Anisaldehyde sulphuric acid, Vanillin Sulphuric acid, 10% Methanolic Sulphuric acid, Iodine vapours or specific ones such as Dragendorff s, 5% Ferric chloride in Methanol, Antimony-III-Chloride reagent, Bartons reagent, Hydrochloric acid-glacial acetic acid reagent, Iodine-chloroform reagent, Iodine-hydrochloric acid reagent, Iodoplatinate reagent, Liebermann-Burchard reagent, Millon reagent, Ninhydrine Reagent, Potassium Hydroxide reagent (5% & 10%), Potassium permagnate- Sulphuric acid reagent, Vanillin-glacial acid reagent, Vanillin-hydrocloric acid reagent, Vanillin-phosphoric acid reagent.
For visualization of Andrographis paniculata plant extract on TLC plate, 10% Methanolic sulphuric acid and Anisaldehyde sulphuric acid were selected. Both the reagents had given good colour and clarity for spots but based on the ease in the method of preparation 10% Methanolic sulphuric acid was selected as visualizing agent for Diagnostic kit.
10% Methanolic sulphuric acid reagent was prepared by adding sulphuric acid dropwise to methanol with shaking.
Anisaldehyde sulphuric acid reagent was prepared by mixing 0.5ml anisaldehyde with 10ml glacial acetic acid, followed by 85ml methanol and 5ml of concentrated sulphuric acid.
Step 8: Derivatization of the plate
The plates were derivatised by dipping in the visualizing agent and followed by drying in oven.
The plates were derivatised by dipping the plate in visualizing agent followed by drying on a hot cast-iron pan.
Step 9: Preparation of standard instruction sheet and kit components
Instructions and components of the kit were prepared and standardised based on the feasibility of tests being performed in non-lab environment by non- technical personnel. The instructions were translated from English to other languages based on the region and requirement.
Description of Drawings :
Fig:l Illustrates yield of Andrographolide in various solvent extracts.
Fig:2 Illustrates the % yield of the Andrographolide in various extracts prepared by different methods.
Fig-3 Illustrates the % yield of the Andrographolide obtained using 3 times reflux and Kit method.
1. Indian Herbal Pharmacopoeia, volume -1, A joint publication of Regional Research Laboratory and Indian Drug Manufacturers Association, p- 18-29 & 165-173.
2. Dr. Pulok K. Mukherjee, Quality Control of Herbal Drugs, An approach to evaluation of botanicals, First edition, Bussiness horizons, New Delhi, 2002, p-701-704 & 764-766.
3. Indian Herbal Pharmacopoeia, Revised new edition, published by Indian Drug Manufacturers Association, Mumbai, 2002,p- 57-69 & 467-478.
4. Dr.V. Rajpal, Standardization of Botanicals, volume-1, Estern publishers, New Delhi, 2002, p- 29-37 & 253-259.
5. C.K.Kokate, A.P.Purohit, S.B. Gokhale, Pharmacognosy, Thirtieth edition, Nirali prakashan, 2005, p-251-253 & 518-520.
6. The Ayurvedic Pharmacopoeia of India, part-1,volume-l,first edition, Government of India, Ministry of Health and Family Welfare, Department of Indian system of Medicine & Homoeopathy, New Delhi, p-15-16.
7. Trease and Evans, Pharmacognosy, 14th edition,1996, p- 320, 436,437, 491, 502.
8. H.Wagner, S.Balt, Plant Drug Analysis, A Thin Layer Chromatography Atlas, 2nd edition, Springer, Germany, 1996.
9. Anonymous, Quality control methods for medicinal plant materials, WHO, Geneva 1998,p-8, 9,28 & 30.
10. Instruction manual, Equipment Infra Red Moisture Balance, Advance Research Instruments co., New Delhi.
11. Indian Pharmacopoeia, Ministry of Health and Family Welfare, Government of India, 1996.
12. Camag Bibliography Services, CD- ROM version 1.06,2002.
13. The Gazette of India, Schedule T, Good Manufacturing Practices for Ayurvedic, Siddha and Unani medicines, Ministry of Health and Family Welfare, Notification, 23rd June 2000, p-23-46.
Application of Invention, explored in various sectors
• Small, Medium and large scale industries of Traditional medicine (Ayurveda, Unani, Siddha, Homeopathy)
• Traders of medicinal raw drugs
• Farmers who are cultivating the herbs
• Herbal extract industry
• Regulatory authorities from Government Departments (Drug Control)
• Forest officers at the forest check point to control pilferage of precious forest species
• Customs officers at the port of export to control pilferage
• Foreign countries that are importing the medicinal raw drugs
• Researchers from universities and institutions and companies
• Common consumers to check the quality of natural materials and finsished products purchased
1. The invention relates to a process of providing a field kit for authentication of naturally occurring raw drugs and finished products as well as semi-quantification of bioactive markers in the raw drugs and formulations, used in non-laboratory environment by non-technical personnel.
2. The process as claimed in claim 1 is for semi-quantification of bioactive chemical marker.
3. The process as claimed in preceding claims, wherein extraction process is simple, standardized and is done using house hold utensils and facility and the instructions for sampling is standardized using the given applicator to obtain the exact amount each time.
4. The process as claimed in preceding claims, wherein the standardized reference material is pre-spotted on TLC (Thin Layer Chromatography) plate that runs alongside the sample spot and the development of the plate can be done in a suitable tank like a beaker.
5. The process as claimed in preceding claims, wherein the solvent system has been standardized for the extracts to provide the best results, and visualizing the bands, standardized instructions have been provided and the heating of the plate can be done on kitchen utensils such as a cast- iron pan.
6. The invention relating to a process of providing a field kit for authentication of naturally occurring raw drugs and finished products as well as semi-quantification of bioactive markers in the raw drugs and formulations, used in non-laboratory environment by non-technical personnel has been described in preceding claims with reference to a preferred embodiments and drawings
|Indian Patent Application Number||368/CHE/2007|
|PG Journal Number||19/2010|
|Date of Filing||23-Feb-2007|
|Name of Patentee||M/S. FOUNDATION FOR REVITALISATION OF LOCAL HEALTH TRADITIONS|
|Applicant Address||74/2, JARAKABANDE KAVAL, POST ATTUR, VIA YELAHANKA, BANGALORE - 560 064, KARNATAKA|
|PCT International Classification Number||C12Q1/18|
|PCT International Application Number||N/A|
|PCT International Filing date|