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Compendial herbal

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Compendial herbal

  1. 1. COMPENDIAL METHODS FOR EVALUATION OF CRUDE DRUG AND 5/25/2011 HERBAL FORMULATION Prepared by: Jagani Nayan M. M. Pharm (sem-II) Q.A. Guided by : Mr. Jignesh S. Shah, Asst. Professor, Q. A. department.1 Department of Quality Assurance SJTPC - RAJKOT
  2. 2.  INTRODUCTION Definition: A crude drug is naturally occurring, unrefined substance derived from organic or inorganic sources such as plant, 5/25/2011 animal, bacteria, organs or whole organisms intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals. The term “herbal drugs” denotes plants or plant parts that have been converted into phytopharmaceuticals by means of simple processes involving harvesting, drying, and storage. Herbalism is a traditional medicinal or folk medicine practice based on use of plant and plant extracts known as herbal medicine, phytotherapy, herbology etc. Following are the Q. C. Parameter for evaluation of crude drug and herbal formulation : 2
  3. 3.  PARAMETERS FOR EVALUATION OF HERBAL DRUGS 5/25/2011(A) PHYSICAL PARAMETERS Viscosity,  Solubility, Volatile matter,  Moisture content, Density,  Specific gravity, Refractive index,  Optical rotation, Hemolytic activity,  Bitterness value, Foaming index,  Swelling index, Melting point,  Ash value. 3
  4. 4. (B) CHEMICAL PARAMETER 5/25/2011 Detection of alkaloids. Detection of carbohydrates and glycosides. Detection of phytosterols. Detection of fixed oils and fats. Detection of saponins. Detection of phenolic compounds and tannins. Detection of protein and free amino acids. Detection of gums and mucilage. Detection of volatile oils. 4
  5. 5. (C) BOTANICAL PARAMETER Microscopical parameters. Macroscopical parameters. 5/25/2011(D) BIOLOGICAL / TOXICOLOGICAL PARAMETER Determination of pesticides. Determination of arsenic and heavy metals. Determination radioactive contamination. Determination of aflatoxins. 5
  6. 6.  General considerations: The metric system is used throughout. All temperatures are 5/25/2011 expressed in degrees Celsius (°C). Tests are normally carried out at room temperature (between 15 and 25°C, or up to 30°C in some climatic zones). Any glassware used in the tests should be of suitable quality. Graduated and volumetric vessels should be calibrated at room temperature. When a water-bath is referred, a bath containing boiling water (about 100°C) is to be used, unless a specific water temperature is given. Unless otherwise specified, all solutions indicated in the tests are prepared with distilled or demineralized water of adequate purity. 6
  7. 7.  Determination of Foreign Matter: Any organism, part or product of an organism, other than that named in 5/25/2011 the specification and description of the plant material concerned. Material not adhering to the medicinal plant materials, such as soil, stones, sand, and dust. Sample size:o Plant material Sample sizeo roots, rhizomes and bark 500 go leaves, flowers, seeds 250 go cut medicinal plant materialso (average weight of each fragment less than 0.5 g) 50 g 7
  8. 8.  Recommended procedure: Weigh 100 –500 g of the drug sample to be examined, or the 5/25/2011 minimum quantity prescribed in the monograph, and spread it out in a thin layer. The foreign matter should be detected by inspection with the unaided eye or by the use of a lens (6x). Separate and weigh it and calculate the percentage present. 8
  9. 9.  Determination of Pesticides: 5/25/2011 Pesticides are chemicals derived from synthetic and natural sources which are effective in small concentrations against pest. Even though there are no serious reports of toxicity due to the presence of pesticides and fumigants, it is important that herbs and herbal products are free of these chemicals or at least are controlled for the absence of unsafe levels. Herbal drugs are liable to contain pesticide residues, which accumulate from agricultural practices, such as spraying, treatment of soils during cultivation, and administering of fumigants during storage. However, it may be desirable to test herbal drugs for broad groups in general, rather than for individual pesticides. 9
  10. 10.  Classification of pesticides (according to pest they control):A) Fungicides: prevents plant from diseases caused by phyto pathogenic fungi and used as soil or seed fungicidal disinfectants. Types: 5/25/2011 a. disinfectants for seed: parathion, carboxins. b. disinfectants for soil: c. Leaf fungicides: colloidal sulphur, barium sulphide, dithiocarbamates.B) Herbicides: ‘weed killers’ which are used for destroying the unwanted plants. Types: a. Total herbicides b. Selective herbicides (widely used) c. Water weed killers d. Harvesting aids Eg.: carbamates, urea derivatives, triazines , quarternary ammonium compounds. 10
  11. 11. C) Insecticides: used to protect the crop from insects. Types: 5/25/2011 a. Inorganic insecticides: arsenic compounds (lead arsenate) flourine compounds (sodium floride). b. Orgenic insecticides: nicotine, pyrethrumD) Others like nematocides, rodenticides, bactericides. 11
  12. 12.  Method: 20-50 gm powder + acetonitrile –water mixture blend for 5min at high speed & filter take filtrate & transferred in 1000ml searating funnel & add 100ml of light petroleum ether. shake, 1-2min. Add 10ml NaCL(40%) +600ml water shake vigorously for 30-45sec. Petroleum ether layer is collected & washed with water thrice. 12 5/25/2011
  13. 13. Cont… This is treated with anh. Sodium sulphate. extract is subjected to column chromatography. i. column packing: activated fluorosil ii. eluted with: petroleum ether iii. 3 fractions of 200ml are collected. a.1st elute contains chlorinated pesticides. b. 2nd elute contains dieldrein. c. 3rd elute contains malathion. 5/25/2011
  14. 14. cont…iv. The elutes are concentrated to 10ml & used for TLC or HPTLC.v. Particulars for TLC: - standard samples: in petroleum ether. - adsorbent: pre-coated silica gel GF254 plate (10*20cm) of uniform thickness. - solvent system: N-hexane – acetone. - detection: under UV chamber. 5/25/2011
  15. 15.  Maximum limit of pesticide residues for herbal drugs: According to EP(1997) limits for pesticides calculated using following formula: 5/25/2011 MRL= ADI * W____ MDI * (100 * safety factor) Where, MRL: maximum residue limits(mg/kg) ADI: acceptable daily intake =NOEL *(safety factor ) NOEL: non-observable effect level. W: body weight(kg) MDI: mean daily intake of drug. Safety factor: 1/100 to 1/2000. 15
  16. 16.  When the herbal crude drug is used to prepare the extracts, tinctures or other phytopharmaceutical formulations in which there is difference in concentration of pesticide residues, so the MRL is calculated as : 5/25/2011 MRL= ADI * W * E____ MDI * (100 * safety factor) where, E: the extraction co-efficient of the pesticide which depend on method of preparation. The quantity of the pesticide transferred from raw material to the final formulations depends on several factors like:  Types and quantity of the solvents.  Temperature.  Duration of heating.  Degree of contamination. 16
  17. 17.  DETERMINATION OF HEAVY METAL: (AS PER WHO) Limit test for Arsenic Limit test for cadmium and lead 17 5/25/2011
  18. 18.  Metal: Characterized by luster, malleability, high electric and thermal conductivity; chemically form bases which can react with acids Heavy Metal: Metal of High Specific Gravity: - Cadmium 112 - Lead 207 - Mercury 200 - Zinc 65 (Not so heavy) - Aluminum 27 (Light) -Arsenic 75 - Selenium 79 Heavy Metals – Sources: In general: Worldwide: associated with mining industry. Others: - Environmental pollution, - Accidental inclusion in processing, - Contamination from containers. 18 5/25/2011
  19. 19.  Excess heavy metal accumulation in soil is toxic to humans and other animals. Exposure to heavy metals is normally chronic. (exposure over a longer period of time), due to food chain transfer. Acute (immediate) poisoning from heavy metals is rare through ingestion or dermal contact, but is possible. Chronic problems associated with long-term heavy metal exposures are: Lead – mental lapse. Cadmium – affects kidney, liver, and GI tract. Arsenic – skin poisoning, affects kidneys and CNS. 19 5/25/2011
  20. 20.  LIMIT TEST FOR ARSENIC: Principle: The amount of arsenic in the medicinal plant material is estimated by matching the depth colour with that of a standard stain. Reactions: H2AsO4 KI H3AsO3 Arsenic acid arsenious acid H3AsO3 + 3H2 AsH3 + H2O Arsenious acid arsine gas AsH3 + mercuric bromide yellow stain 20 5/25/2011
  21. 21.  Preparation of the sample (by acid digestion): Place35-70 g of coarsely ground material, accurately weighed, in a Kjeldahl flask, capacity 800-1000 ml. +(10-25 ml) water + (25-50 ml) nitric acid + carefully add (20 ml) sulphuric acid. Heat Gradually add nitric acid (~1000 g/l), drop by drop, until all the organic matter is destroyed. a clear solution with copious vapours of sulfur trioxide is obtained. Cool add (75 ml) water + (25 ml) ammonium oxalate. Heat again until sulfur trioxide vapours develop. Cool, transfer to a 250-ml volumetric flask and dilute to volume with water. 21 5/25/2011
  22. 22.  Preparation of standard stain: Add (10 ml) hydrochloric acid + 1 ml dilute arsenic As to 50 ml water. The resulting solution, when treated as described in the general test yields a stain on mercuric bromide paper. standard stain (10 μg of As). 22 5/25/2011
  23. 23.  Method: Take an aliquot (25-50 ml) of the test solution +1 g of potassium iodide + l0 g of granulated zinc in the widemouthed bottle place the prepared glass tube assembly quickly in position. reaction 40 minutes,40’c temp. Compare any yellow stain that is produced on the mercuric bromide paper with a standard stain produced in a similar manner with a known quantity of dilute arsenic. The contents of lead and cadmium may be determined by atomic absorption spectrophotometry Maximum amount in dried plant materials: -lead: 10mg/kg. -cadmium: 0.3mg/kg. 23 5/25/2011
  24. 24.  Determination of Ash: The ash remaining following ignition of medicinal plant materials is determined by three different methods which measure total ash, acid- 5/25/2011 insoluble ash and water-soluble ash. The total ash method is designed to measure the total amount of material remaining after ignition. This includes both "physiological ash", which is derived from the plant tissue itself, and "non- physiological" ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water. 24
  25. 25.  Recommended procedures: Determination of Total Ash Incinerate about 2 to 3 g accurately weighed, of the ground drug in a silica 5/25/2011 dish at a temperature not exceeding 450º 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 ashless filter paper, incinerate the residue and filter paper, add the filtrate, evaporate to dryness, and ignite at a temperature not exceeding 450º. Calculate the percentage of ash with reference to the air-dried drug. Determination of Acid Insoluble Ash Boil the ash obtained above for 5 minutes with 25 ml of dilute hydrochloric acid; collect the insoluble matter in a Gooch crucible, or on an ashless filter paper, wash with hot water and ignite to constant weight. Calculate the percentage of acid-insoluble ash with reference to the air dried drug. Determination of Water Soluble Ash Boil the ash for 5 minutes with 25 ml of water; collect insoluble matter in a Gooch crucible, or on an ashless filter paper, wash with hot water, and ignite for 15 minutes at a temprature not exceeding 450º. 25
  26. 26.  Determination of swelling index: Many medicinal plant materials are of specific therapeutic or pharmaceutical utility because of their swelling properties, especially 5/25/2011 gums and those containing an appreciable amount of mucilage, pectin or hemicellulose. The swelling index is the volume in ml taken up by the swelling of 1 g of plant material under specified conditions. Its determination is based on the addition of water or a swelling agent as specified in the test procedure for each individual plant material (either whole, cut or pulverized). Using a glass-stoppered measuring cylinder, the material is shaken repeatedly for 1 hour and then allowed to stand for a required period of time. The volume of the mixture (in ml) is then read. The mixture of whole plant material with the swelling agent is easy to achieve, but cut or pulverized material requires vigorous shaking at specified intervals to ensure even distribution of the material in the swelling agent. 26
  27. 27.  MACROSCOPIC EXAMINATION Organoleptic evaluation of drugs refers to the evaluation of a drug by colour, odour, size, shape, taste and special features including touch, texture etc. Since the majority of information on the identity, purity and quality of the material can be drawn from these observations, they are of primary importance before any further testing can be carried out. For this purpose authentic specimen of the material under study and samples of pharmacopoeial quality should be available to serve as a reference. This evaluation procedure provides the simplest and quickest means to establish the identity and purity and thereby ensure quality of a particular sample. 27
  28. 28.  If it is found to be devoid of or significantly different from the specified sensory characters like colour, consistency, odour, etc., it is considered as not fulfilling the requirements. However judgment based on the sensory characteristics like odour, taste etc., may vary from person to person and time to time based on individuals nature. So the description of this features are very difficult so that often the characteristic like odour and taste can only described as characteristic and reference made to the analysts memory. No preliminary treatment is necessary for evaluating the sample in this manner excepting the softening and stretching of the wrinkled and contracted leaves and flowers etc.
  29. 29.  MICROSCOPIC EXAMINATION This allows more detailed examination of a drug and it can be used to identify the organised drugs by their known histological character. This mostly used for qualitative evaluation of organised crude drugs in entire and powdered forms and can be used to distinguish cellular structure. For the effective results, various reagents can be used to distinguish cellular structure. eg. A drop of phloroglucinol and conc. Hcl gives red stain with lignin. Mucilage is stained with rhuthenium red to give pink color. Following are the parameter evaluated by histological examination: 29
  30. 30. a) Stomatal index : is the percentage which give the number of stomata to total number of epidermal cells. Procedure : Place leaf fragments of about 5 × 5 mm in size in a test tube containing about 5 ml of chloral hydrate solution and heat in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer a fragment to a microscopic slide and prepare the mount, the lower epidermis uppermost, in chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side of the cover- glass to prevent the preparation from drying. Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is attached. Mark on the drawing paper a cross (x) for each epidermal cell and a circle (o) for each stoma. It is calculated by following equation : S. I. = S / E + S * 100 where, S = no. of stomata per unit area, 30 E = no. of epidernal cells in unit area.
  31. 31. b) Stomatal no.: is the average no. of stomata present per sq mm of epidermal cell.c) Palisade ratio : is the average no. of palisade cells beneath each epidermal cell.d) Vein islet & veinlet termination number : is the no. of vein islets & veinlet termination per sq mm of the leaf surface midway between midrib and margin. 31
  32. 32.  Determination of Volatile Oil in Drugs Volatile oils are characterized by their odour, oil-like appearance and ability to volatilize at room temperature. Chemically, they are usually composed of mixtures of monoterpenes, sesquiterpenes and their 5/25/2011 oxygenated derivatives. Volatile oil is considered to be the "essence" of the plant material, and are often biologically active, they are also known as "essential oils". The determination of volatile oil in a drug is made by distilling the drug with a mixture of water and glycerin, collecting the distillate in a graduated tube in which the aqueous portion of the distillate is automatically separated and returned to the distilling flask, and measuring the volume of the oil. The content of the volatile oil is expressed as a percentage v/w. 32
  33. 33. 5/25/2011 Apparatus used for determination of Volatile OilFig. 1 33
  34. 34.  Determination of Tannins Tannins are substances capable of turning animal hides into leather by binding proteins to form water-insoluble substances that are resistant to proteolytic enzymes. This process, when applied to living tissue, is 5/25/2011 known as an "astringent" action and is the reason for the therapeutic application of tannins. Chemically, tannins are complex substances; they usually occur as mixtures of polyphenols that are difficult to separate and crystallize. They are easily oxidized, polymerized in solution, form colloidal solution with water & non crystalline compound. Types of tannins:a) Hydrolysable : Eg. Gallic acid, pyrogallol, ellagic acid etc.b) Condensed : Eg. Catechutannic acid, catechin etc. 34
  35. 35. o Procedure : Weighed accurately a quantity of powder into a conical flask. Add 150 ml of water and heat over a boiling water-bath for 30 minutes. Cool, transfer the mixture to a 250-ml volumetric flask and dilute to volume with water. Allow the solid material to settle and filter the liquid through a filter-paper, discarding the first 50 ml of the filtrate. 5/25/2011 To determine the total amount of material that is extractable into water, evaporate 50.0 ml of the plant material extract to dryness, dry the residue in an oven at 105°C for 4 hours and weigh (W1). To determine the amount of plant material not bound to hide powder that is extractable into water, take 80.0ml of the plant material extract, add 6.0g of hide powder R and shake well for 60 minutes. Filter and evaporate 50.0ml of the clear filtrate to dryness. Dry the residue in an oven at 105°C and weigh (W2). To determine the solubility of hide powder, take 6.0 g of hide powder R, add 80.0ml of water and shake well for 60 minutes. Filter and evaporate 50.0ml of the clear filtrate to dryness. Dry the residue in an oven at 105°C and weigh (W3). Calculate the quantity of tannins as a percentage using the following formula: W1 - ( W2 - W3 ) / w * 500 where w = the weight of the plant material in grams. 35
  36. 36.  Determination of foaming index Many medicinal plant materials contain saponins that can cause a persistent foam when an aqueous decoction is shaken. 5/25/2011 The foaming ability of an aqueous decoction of plant materials and their extracts is measured in terms of a foaming index.o Recommended Procedure: Weigh accurately 1 gm coarse powder, transferred to 5ooml conical flask containing 100ml boiling water. Boil for 30 min Cool, filter to 100 ml volumetric flask & make up volume to 100ml. 36
  37. 37. Take 10 T. T., Mark with 1 to 10, add 1ml, 2ml, 3ml etc. solution to T. T. 5/25/2011 Make up volume with water to 10 ml in each T. T. Shake them in a lengthwise motion for 15 seconds, two shakes per second. Allow to stand for 15 minutes and measure the height of the foam. Result given as :a) If height of foam is less than 1cm, foaming index is less than 1000.b) If height of foam is more than 1cm, foaming index is more than 1000.c) If a height of foam is 1 cm in any tube, then according to volume of filterate added in tube is used to determine index.d) Foaming index is calculated by formula : 1000 / a Where, a = volume of filterate added to test tube 37
  38. 38.  Determination of Extractable matter Determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material and herbal formulation. 5/25/2011 Extractive value of crude drug are useful for their evaluation, when the constituents of a drug can not be readily estimated by any other means.o Recommended procedure: Method 1 : Hot extraction Take accurately weighed coarse powdered air dried material + 100 ml solvent in a glass stoppered conical flask. Shake, stand for 1 hr Boil for 1 hr, cool. Adjust to original volume. Shake & filter. 38
  39. 39. transfer 25 ml of filterate to a tarred flat bottomed dish and evaporate todryness at 105 C for 6 hr & weigh.Calculate content of extractable matter in mg per gm of air dried material. 5/25/2011Method 2 : Cold Maceration :Take accurately weighed coarse powdered air dried material + 100 mlsolvent in a glass stoppered conical flask. Macerate for 6 hr Allow to stand for 18 hr, Filter.transfer 25 ml of filterate to a tarred flat bottomed dish and evaporate todryness at 105 C for 6 hr & weigh. 39Calculate content of extractable matter in mg per gm of air dried material.
  40. 40.  Determination of Haemolytic activity: Many medicinal plant material, especially those derive from families 5/25/2011 Carryophyllaceae, Araliaceae, Primulaceae and Dioscoreaceae contains saponins. The most characteristic property of saponin is ability to cause haemolysis. The haemolytic activity of plant material is determined by comparison with reference material, saponin R, which has haemolytic activity of 1000 units per gm. A suspension of erythrocyte is mixed with equal volume of serial dilution of plant material extract. The lowest concentration cause complete haemolysis is determined after allowing mixtures to stand for a given period of time. A similar test is carried out simultaneously with saponin R. 40
  41. 41. o Method:a) Preliminary test: Prepare serial dilution of plant material extract with phosphate buffer 7.4 and blood suspension (2%) using four Test tube as follow: 5/25/2011 Tube No. 1 2 3 4 Plant material extract (ml) 0.1 0.2 0.5 1 Phosphate buffer ph 7.4 0.9 0.8 0.5 - Blood suspension (ml) 1 1 1 1 Mix, avoiding formation of foam, allow to stand for 6 hr at room temperature. Examine tube & record dilution for total haemolysis & proceed as follows :a) If total haemolysis is observed only in tube no. 4, use original extract.b) If observed in 3, 4, prepare 2 fold dilution of original extract.c) If observed in 2, 3, 4, prepare 5 fold dilution of original extract.d) If all tubes contain, clear, red solution, prepare 10 fold dilution of 41 original extract.
  42. 42. b) Main test: Prepare serial dilution of plant material extract diluted / undiluted with phosphate 5/25/2011 buffer 7.4 and blood suspension (2%) using 13 Test tube as follow: Tube no. 1 2 3 4 5 6 7 8 9 10 11 12 13 Plant 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 material extract Phosphate 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 - buffer pH 7.4 Blood 1 1 1 1 1 1 1 1 1 1 1 1 1 suspension Mix, observe result after 24 hr. Calculate amount material in gm that produce complete haemolysis. Prepare similar dilutions of saponin R & calculate quantity of saponin R in gm that produce complete haemolysis. Calculate haemolytic activity of material using following formula: 1000 * a / b where, 1000 = defined haemolytic activity of saponin R on ox blood, a = Quantity of saponin R that produce total haemolysis, 42 b = Quantity of material that produce total haemolysis.
  43. 43.  Determination of Radio active contamination: There are many sources of ionization radiation, including radionuclides, occurring in the environment. Hence a certain degree of exposure is inevitable. Dangerous contamination, however, may be the consequence of a nuclear accident. The WHO, in close cooperation with several other international organizations (IAEA), has developed guidelines in the event of a widespread contamination by radionuclides resulting from major nuclear accidents. These publications emphasize that the health risk, in general, due to radioactive contamination from naturally occurring radio nuclides is not a real concern, but those arising from major nuclear accidents such as the nuclear accident in Chernobyl, may be serious and depend on the specific radionuclide, the level of contamination, and the quantity of the contaminant consumed. Taking into account the quantity of herbal medicine normally consumed by an individual, they are unlikely to be a health risk. Therefore, at present, 43 no limits are proposed for radioactive contamination.
  44. 44.  List of Q. C. methods for medicinal plant material as per W. H. O. Determination of foreign matter Macroscopic and microscopic examination Thin-layer chromatography Determination of ash Determination of extractable matter Determination of water and volatile matter Determination of volatile oils Determination of bitterness value Determination of haemolytic activity Determination of tannins Determination of swelling index Determination of foaming index Determination of pesticide residues Determination of arsenic and heavy metals Determination of microorganisms 44 Radioactive contamination
  45. 45.  References:1. Quality control methods for medicinal plant material (WHO).2. “Quality standard of indian medicinal plants” ,Indian council of medicinal research , new delhi-2003 , volume-I.3. The ayurvedic pharmacopoiea for india, volume-I.4. “Quality control of herbal drugs-an approach to evaluation of botanicals ” , mukherjee p.k. , bussiness horizons pharmaceutical publishers , new delhi-2002.5. Indian pharamacopoiea 2007, volume – 3.6. Pharamacognosy by C. K. Kokate 36th edition, Nirali prakashan.7. Experimental pharmacognosy by Biren N. Shah. 458.
  46. 46.  Questions:-1. Describe quality control methods for medicinal plant material as per WHO.2. Enlist quality control methods for medicinal plant material as per WHO. Describe determination of –a) Pesticide residue in plant material,b) Extractable matter,c) Ash value.3. Enlist quality control methods for medicinal plant material as per WHO. How extractable matter from plant material is determined?4. Define compendial method. Enlist standardisation parameters for evaluation of herbal drugs. Add a note on determination of Ash value. 46
  47. 47. 5/25/2011 47
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