Hybridoma Technology ( Production , Purification , and Application )
Characterization of Herbal crude drugs according to WHO (NIPER Hyderabad)
1. Presented by:
Medtiya Pravin Pokarlal
Reg.no. PE/2019/314
Department of Pharmaceutics
National Institute of Pharmaceutical Education and Research, Hyderabad
Department of Pharmaceuticals, Ministry of Chemicals & Fertilizers, Govt. of India.
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2. Introduction to herbal medicines
Classification of herbal medicines
Characterization of herbal medicines
Stability of herbal medicines
Case study
Conclusion
Bibliography
FLOW OF PRESENTATION
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3. INTRODUCTION
• Herbal medicines are plant derived materials and preparations with therapeutic or
other human health benefits, which contain either raw or processed ingredients
from one or more plants.
• Of the 2,50,000 higher plant species on earth, more than 80,000 are medicinal.
India is one of the world's 12 biodiversity centers with the presence of over 45000
different plant species.
• Of these, about 15000-20000 plants have good medicinal value. However, only
7000-7500 species are used for their medicinal values by traditional communities
• The use of herbs as medicine is the oldest form of healthcare known to humanity
and has been used in all cultures throughout history
• immense increase in sales of herbal OTC (Over the counter drugs. )
• This is growing to a billion dollar industry. The need for safety and efficacy has also
escalated since the western interest has grown. Thus the need for standardization
has come into view.
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4. Classification of herbal medicines
Herbal
medicines
Traditional
medicine
Apamarg kshar
Ayurvedic
medicine
Himalaya
vasaka
Phytomedicine
Nano silymarin
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6. MACROSCOPIC AND MICROSCOPIC
Macroscopic examination:-
Visual inspection provides the simplest and quickest means by which to establish identity, purity
and quality.
Size:-measured by graduated ruler.
Colour:-sample Vs reference – colour comparison.
Surface characteristics, texture & fracture characteristics:-
Examined by using a magnifying lens.
Odour:-characteristic & strength of the odour (weak, none, distinct, strong)
and odour sensation (aromatic, fruity, rancid, mouldy etc.)
Taste:-applied only if required specifically.
12
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7. Microscopic examination:-
Full and accurate characterization of plant material. invaluable for assuring the identity of
the material and as an initial screening test for impurities.
Preliminary treatment.
Preparation of specimens
Classification of microscopic particles
Histochemical detection of cell walls & contents(starch grain, aleurone grain, etc.)
Measurement of specimens
Leaf surface data(stomatal no., stomatal index, palisade ratio, vein-islet no.)
e.g. Atropa acuminate: vein islet no:8-11, stomata no. 5-14, stomata index: 2.5-4.7
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8. POWDER FINENESS & SIEVE SIZE
• The coarseness or fineness of a powder is classed according to the nominal aperture size
expressed in hum of the mesh of the sieve through which the powder will pass.
• To check degree of fragmentation of samples
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9. LOSS ON DRYING
• It is the determination of water & volatile oil in crude drug.
• Gravimetric method mostly used. Dessication method for materials that form sticky mass at high
temp.
• LOD can be expressed as %W/W & can be determined by following procedure:
Weigh the quantity of crude drug according to monograph in tared dish
Evaporated at low temperature & solvent removes & heat on water bath until residue is dry
Transfer to oven & dry to constant Weight at 1050C or stated in monograph
Owing to hygroscopic nature of some residue it is necessary to use dishes with well fitted cover &
cool in dessicator
𝑙𝑜𝑠𝑠 𝑜𝑛 𝑑𝑟𝑦𝑖𝑛𝑔 =
(𝑤−𝑑)×100
(𝑤)
where , w = wet weight , d = weight after drying.
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10. Moisture content
• An excess of water in herbal materials will encourage microbial
growth, the presence of fungi or insects, and deterioration following
hydrolysis.
• Limits for water content should therefore be set for every given
herbal material. This is especially important for materials that absorb
moisture easily or deteriorate quickly in the presence of water.
• The moisture content of the drug should be minimized in order to
prevent decomposition of crude drug either due to chemical change
or microbial contamination.
Procedure:
Karl Fischer apparatus to be performed only In dry powder drug formulations
Azeotropic method(toluene distillation method): direct measurement of water
present
%Water content =
100(𝑛1 −𝑛)
𝑤
Where w = weight in gram of material being examined
n = number of ml of water obtained in 1st distillation
n1 = total no.of ml of water obtained in both distillations
E.g. – Aloe should have moisture content not more than 10% w/w
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11. Volatile content
• Volatile oils are characterized by their odor, oil-like appearance and ability to
volatilize at room temperature. Chemically, they are usually composed of
mixtures of, for example, monoterpenes, sesquiterpenes and their oxygenated
derivatives.
• For the drugs containing volatile constituents, toluene distillation method/steam
distillation method is used to determine the volatile oil contents
• Determination by Steam distillation
• Significance:
Because they are considered to be the “essence” of the herbal material, and are
often biologically active, they are also known as “essential oils”. The term “volatile
oil” is preferred because it is more specific and describes the physical properties.
e.g. caraway not less than 2.5% & clove not less than 15%
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12. Ash value
• Determine the quality and purity of the drug and to establish its identity.
• Ash contains inorganic radicals lie phosphates, carbonates, and silicates of
sodium, potassium, magnesium, calcium, etc.
• Procedure: take 2-3 gm of dried crude drug in fused silica dish incinerate at
temp 500-6000c until white. collect residue on ash less filter paper which is free
from carbon & cool & weigh. Calculate %ash with reference to dried drug
• % 𝑇𝑜𝑡𝑎𝑙 𝐴𝑠ℎ =
𝑓𝑖𝑛𝑎𝑙 𝑊𝑡.
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑡.
*100
• Significance: Present in definite amount in a particular crude Drug,hence
quantitative determination in terms of various ash values helps in their
standardization. Ash value is used to determine foreign inorganic matter
present as impurity.
•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,
•Is the residue of the extraneous matter (e.g. Sand and soil) adhering to the plant surface, determination of
excess calcium oxalate or calcium carbonate crystals present
Total ash
•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. Sometimes, inorganic
variables like calcium oxalate, silica, and carbonate content of the crude drug affects ‘Total cash value’. Such
variables are removed by treating with acid (as they are soluble in hydrochloric acid) and acid insoluble ash
value
Acid-
insoluble ash
•Difference in weight between the total ash and the residue after treatment of the total ash with water
•Good indicator of either previous extraction of water soluble salts in drugs or incorrect preparation
Water-
soluble ash01-06-2020 12
13. Extractable value
Useful for the evaluation of a crude drug and at the same time give
idea about the nature of the chemical constituents present, which is
helpful for the estimation of specific constituents, soluble in that
particular solvent used for extraction.
Significance:
This method determines the amount of active constituents extracted
with solvents from a given amount of herbal material. It is employed
for materials for which as yet no suitable chemical or biological assay
exists.
Water soluble
extractive
Applies to drug
containing water
soluble
constituents
Gives idea about
tannins, sugar, plant
acid, mucilage,
glycosides
Alcohol soluble
extractive
Ideal solvent for
extraction of resins
& generally 95%
ethanol is used
Official method for
Myrrh & asafoetida
Hot-extraction method:-
4.0g of coarsely powdered air-dried material, accurately weighed, in a glass-stoppered conical flask.
Add 100ml of water and weigh to obtain the total weight including the flask.
Shake well and allow to stand for 1 hour.
Reflux condenser to the flask and boil gently for 1 hour; cool and weigh.
Shake well and filter rapidly through a dry filter.
Transfer 25 ml of the filtrate to a tarred flat-bottomed dish
Dry at 105°C for 6 hours, cool in a desiccator for 30 minutes,
Weigh & calculate the content of extract in mg per g of air-dried material.
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14. Cold-Extraction:
4.0g of coarsely powderedair-dried material, accurately weighed,in a glass-stoppered conical flask.
Macerate with 100ml of the solvent specified for the plant material concerned for 6 hours,
Shaking frequently, then allow to stand for 18 hours.
Transfer 25 ml of the filtrate to a tared flat-bottomed dish
Dry at 105°C for 6 hours, cool in a desiccator for 30 minutes
Weigh &calculate the content of extract in mg per g of air-dried material.
Methods of extraction of extractable matter
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15. DETERMINATION OF FOREIGN MATTER
• Herbal materials should be entirely free from visible signs of contamination by
moulds or insects, and other animal contamination, including animal excreta.
• No abnormal odour, discoloration, slime or signs of deterioration should be
detected.
• No poisonous, dangerous or otherwise harmful foreign matter or residue should be
allowed.
• Special care should be taken to avoid formation of moulds, since they may produce
aflatoxins.
Definition: foreign material contain any of following:
• parts of the herbal material or materials,
• any organism, part or product of an organism,
• mineral admixtures not adhering to the herbal materials,
Sampling:
Herbal material Sample size
Roots, rhizomes and bark 500g
Leaves, flowers, seeds and fruit 250g
Cut herbal materials
(Average weight of each fragment less than0.5g)
50g
Procedure:
weigh sample as mentioned in sampling or otherwise specified, spread out in thin layer magnifying lens (6×
or 10×), or with the help of a suitable sieve, according to the requirements for the specific herbal material
Sift the remainder of the sample through a No. 250 sieve; dust is regarded as mineral admixture. Weigh the
portions of this sorted foreign matter to within 0.05 g.
% of Foreign Organic Matter = N × W × 94,100 × 100/S ×M × P
Where;
• N = No. of chart particles in 25 field
• S = No. of spores in the same area of 25 fields
• W = Weight in mg of lycopodium taken
• M= weight in mg of the sample
• P= number of characteristics particles per mg of the pure foreign
matter
• 94,000= number of spores per mg of lycopodium
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16. Chromatographic fingerprint
• Separation, identification, impurity detection and assay of herbal drug
in the formulation or in the extract are carried out by following
methods: -HPTLC, HPLC/Densitometric chromatography, GLC, TLC
• Importance-The herbal drug shows variability in its chemical
constituents according to various locations/weather. To avoid any
erroneous identification chromatographic fingerprint remains the
assessment of choice.
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17. Who guideline for potential contamination
• Determination of heavy metals – e.g. cadmium, lead, arsenic, etc WHO, (1998) mentions
maximum permissible limits in raw materials only for arsenic, cadmium, and lead, which
amount to 1.0, 0.3, and 10 ppm, respectively..
• Pesticide residue – WHO and FAO (Food and Agricultural Organization) set limits of
pesticides, which are usually present in the herbs. These pesticides are mixed with the
herbs during the time of cultivation. Mainly pesticides like DDT, BHC, toxaphene, aldrin,
and endosulfan cause serious side-effects in human beings if the crude drugs are mixed
with these agents
• Microbial contamination – usually medicinal plants containing bacteria and molds are
coming from soil and atmosphere. Analysis of the limits of E. coli and molds clearly throws
light towards the harvesting and production practices. Aflatoxins should be completely
removed or should not be present.
• Radioactive contamination – Microbial growth in herbals are usually avoided by
irradiation. This process may sterilize the plant material but the radioactivity hazard
should be taken into account. The radioactivity of the plant samples should be checked
accordingly to the guidelines of International Atomic Energy (IAE) in Vienna and that of
WHO.
Type of contaminant examples
microorganisms Staphylococcus aureus, Escherichia coli (certain strains), Salmonella,
Shigella, Pseudomonas aeruginosa
Microbial toxins Bacterial endotoxins, aflatoxins
Pesticides Chlorinated pesticides (e.g., DDT, dieldrin), organic phosphates,
carbamate insecticides and herbicides, dithiocarbamate fungicides,
triazin herbicides
Fumigation agents Ethylene oxide, methyl bromide, phosphine
Radioactivity Cs-134, Cs-137, Ru-103, I-131, Sr-90
Metals Lead, cadmium, mercury, arsenic
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18. Microbial contamination
• Contamination either at source or during processing is possible.
• Maximum possible limits of each organism are given in various texts. WHO limit for number of
micro-organisms per gram of material
• Alfatoxins- Aflatoxins are naturally occurring mycotoxins produced mainly by Aspergillus flavus
and Aspergillus parasiticus. The presence of aflatoxins can be determined by chromatographic
methods using standard aflatoxins B1, B2, G1, G2 mixtures.
• Determination- IP method: NMT 2 μg/kg of aflatoxins B1& Total aflatoxins 4 μg/kg. USP method:
NMT 5ppb of aflatoxins B1& Total aflatoxins 20ppb.
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20. Bitterness value
• Medicinal plants having strong bitter taste are therapeutically used as appetizing agents .Their
bitterness stimulates secretions in the gastrointestinal tract, especially of gastric juice.
• The bitter sensation is not felt by the whole surface of the tongue, but is limited to the middle
section of the upper surface of the tongue. A certain amount of training is required to perform
this test.
• A person who does not appreciate a bitter sensation when tasting a solution of 0.058 mg of
quinine hydrochloride R in 10 ml of water is not suitable to undertake this determination
• Determined by comparing the threshold bitter concentration of an extract material with that of
quinine hydrochloride.The bitterness value is expressed as unit’s equivalent to the bitterness of a
solution containing 1gm of quinine hydrochloride in 2000ml.
• Method for determination- 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water
and the stock solution(0.01mg/ml) is prepared. Then it is diluted in 9 testtubes(10ml) and tested
and compared with drug.
• Caution: This test should not be carried out until the identity of the herbal material has been
confirmed.
• E.g. acceptance limit for gentianae radix is 10,000 bitterness value
Procedure:
taste 10 ml of the most dilute solution swirling it in the mouth mainly near the base of the tongue for 30 seconds
After 30 sec if no bitter sensation
Spit out sol. & wait for 1 min(delayed sensitivity)
Rinse with safe drinking water
Next highest conc. Not tasted until 10 min
threshold bitter conc. is lowest conc. at which a material continues to provoke a bitter sensation after 30 seconds.
After first series of tests, rinse the mouth thoroughly with safe drinking-water until no bitter sensation remains.
Wait at least 10 minutes before carrying out second test.
find the threshold bitter concentration of the material by tasting the dilutions in tubes 1−4.
If the solution in tube no. 5 does not give a bitter sensation, find the threshold bitter concentration by tasting the
dilutions in tubes 6−10.
Formula:
Bitterness value in unit per gm =
2000∗𝐶
𝐴∗𝐵
Where, A = concentration of stock solution
B = volume of test solution in tube with threshold bitter concentration
C = quantity of quinine hydrochloride in the tube with threshold bitter concentration
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21. Haemolytic property
• Many medicinal plant materials, of the families Caryophyllaceae, Araliaceae,
Sapindaceae, Primulaceae, and Dioscoreaceae contain saponins.
• The most characteristic property of saponins is their ability to cause haemolysis; when
added to a suspension of blood, saponins produce changes in erythrocyte membranes,
causing haemoglobin to diffuse into the surrounding medium.
• The haemolytic activity of plant materials, or a preparation containing saponins, is
determined by comparison with that of a reference material, saponin R, which has a
haemolytic activity of 1000 units per g.
• Determination- Calculate the haemolytic activity of the medicinal plant material using
the following formula: 1000 ×a/b
• Where, 1000 = the defined haemolytic activity of saponin R in relation to ox blood,
• a = quantity of saponin R that produces total haemolysis (g)
• b = quantity of plant material that produces total haemolysis (g)
• E.g. Min conc. of Dioscorea bulbifera is 27.5(µg/ml)
Procedure:
1) Preparation of erythrocyte suspension: 2% ox blood suspension
2) Preparation of reference sol.: Weigh & transfer 10mg saponin to 100ml volumetric flask & make up with
phosphate buffer pH7.4
3)Preparation of test sol:
The extract of herbal material and dilutions should be prepared as specified in the test procedure for the herbal
material concerned, using phosphate buffer pH 7.4 TS.
Preliminary test: serial dilution of the herbal material extract ml(0.1, 0.2, 0.5, 1.0) with phosphate buffer pH 7.4
and blood suspension (2%)(1ml) using four test-tubes & Gently invert testtube to mix, After 30 min shake & keep
for 6 hours at RT
Examine test-tubes hemolysis(clear red solution)
Total hemolysis in 1.0ml herbal extract main test
Total hemolysis in 0.5ml & 1.0ml 2x dilution of original herbal extract
Total hemolysis in 0.2, 0.5 & 1.0 5x dilution of original herbal extract
After 6 hr all testtube remain clear 10x dilution of original herbal extract
No hemolysis repeat test
Main test: Prepare a serial dilution of the herbal material extract, undiluted or diluted as determined by the
preliminary test, with phosphate buffer pH 7.4 TS and blood suspension (2%) using 13 test-tubes(0.45- 1.0ml)
Carry dilution & observe after 24 hr
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22. Swelling index
• Many herbal materials are of specific therapeutic because of their swelling properties −
especially 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).
• Significance: It gives an idea about the mucilage content of the drug; hence it is useful in
the evaluation of crude drugs containing mucilage.
• E.g. swelling index of Agar is 10
Procedure:
take specific qty. (fine & weighed) to 25ml glass stoppered measuring cylinder(inner dia. 16mm, length
125mm, 0.2ml division from 0-25ml) ad 25 ml water
Shake every 10 min for 1 hour
Stand for 3 hour at RT
Measure vol in ml occupied by material 7 any sticky mucilage
Calculate the mean value of the individual determinations, related to 1 g of herbal material.
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23. Foaming index
• Medicinal plant contain saponins that can cause persistent foam when an
aqueous decoction is shaken. The foaming ability of an aqueous decoction and
extracts is measured in terms of a foaming index.
• Calculate the foaming index using the following formula 1000/a :
where a = the volume in ml of the decoction used for preparing the dilution in the
tube where foaming to a height of 1 cm is observed.
Procedure:
Weigh 1g herbal coarse(sieve size no. 1250) & transfer to 500ml conical flask having 100 ml boiled water&
maintain for 30 min.
Cool & filter to 100ml vol. flask & make up with water
Purt decoction to 10 stoppered testtubes(1ml,2ml,3ml,…10ml decoction)& adjust to 10 ml water
Shake for 15 sec(2shake/sec)
Allow stand for 30 min
Measure height of foam
If foam height less than 1 cm foam index<100
If foam height in any test-tube is 1cm vol of material give index
If foam height > 1cm foam index >1000
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24. Toxicological
• Arsenic and heavy metals- Contamination of medicinal plant materials
with arsenic and heavy metals can be attributed to many causes including
environmental pollution and traces of pesticides.
• There are different methods to identify the amount and concentration of
heavy metals in herbal drugs. Limit test for arsenic and Limit test for
cadmium and lead are few of them.
• The contents of lead and cadmium may be determined by inverse
voltametry or by atomic emission spectrophotometry.
• Determination-The following maximum amounts in dried plant materials,
which are based on the ADI values, are proposed: ▫ lead, 10 mg/kg; ▫
cadmium, 0.3 mg/kg. Stain produced on HgBr2 paper in comparison to
standard stain.
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25. Stability testing
Stability testing: It is a challenging task, because the entire herb or herbal product is
regarded as the active substance, regardless of whether constituents with defined
therapeutic activity are known.
Objective: is to provide evidence on how the quality of the herbal products varies with
the time under the influence of environmental factors such as-temperature, light,
oxygen, moisture, excipient in the dosage form, particle size of drug, microbial
contamination, trace metal contamination,leaching from the container, etc. and to
establish a recommended storage condition, retest period and shelf-life.
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26. CONCLUSION
• New formulation of medicinal drugs can emerge only if they are
evaluated & analysed using sophisticated modern techniques of
characterization
• Limit for parameters of herbal products help in production of pure
drugs with less side effects of final products.
• The advancement of analytical techniques will serve as a rapid and
specific tool in the herbal research, thereby, allowing the
manufacturers to set quality standards and specifications so as to
seek marketing approval from regulatory authorities for therapeutic
efficacy, safety and shelf- life of herbal drugs
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27. Case study from monograph
• Tulsi leaves: fresh or dried leaves of Ocimum sanctum L. (Lamiaceae)
• Organoleptic properties: Odor: characteristic, aromatic; taste: slightly pungent
• Macroscopic: Leaves green to greenish-brown, 2.5–7.5 cm long, 1–3 cm wide, oblong,
ovate.
• Microscopic characteristics: upper epidermis consists of a layer of small, quadrangular
transparent cells with thin walls, and thin smooth cuticle. Stomata diacytic
• Physical: Total ash: Not more than 13%
Acid-insoluble ash: Not more than 1%
Sulfated ash: Not more than 20%
Water-soluble extractive: Not less than 5%
Alcohol-soluble extractive: Not less than 5.0%
Loss on drying: Not more than 14%
• Chemical: identity test by thin layer chromatography, Contains not less than 0.5%
essential oil
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Pesticide residues: The recommended maximum limit of aldrin and dieldrin is not
more than 0.05 mg/kg
Heavy metals: For maximum limits and analysis of heavy metals, consult the WHO
guidelines on quality control methods for medicinal plants
Radioactive residues: consult the WHO guidelines on quality control methods for
medicinal plants
Other purity test : foreign organic matter
Pharmacological: Analgesic activity, Antispasmodic activity, Antimicrobial activity,
Anti-inflammatory activity, Antipyretic activity , Immunostimulatory activity,
Endocrinological effects,
Alamgir, A.N.M, . In Therapeutic Use of Medicinal Plants and Their Extracts(1) (295-353)
28. References
• https://www.who.int/medicines/publications/qas_herbalmed/en/
• Alamgir, A.N.M., 2017. Pharmacopoeia and herbal monograph, the aim and use of WHO’s herbal
monograph, WHO’s guide lines for herbal monograph, pharmacognostical research and
monographs of organized, unorganized drugs and drugs from animal sources. In Therapeutic Use
of Medicinal Plants and Their Extracts: Volume 1 (pp. 295-353). Springer, Cham.
• Pradhan, N., Gavali, J. and Waghmare, N., 2015. WHO (World Health Organization)
guidelines for standardization of herbal drugs. International Ayurvedic Medical Journal, 3,
pp.2238-43.
• Bijauliya, R.K., Alok, S., Chanchal, D.K. and Kumar, M., 2017. A comprehensive review on
standardization of herbal drugs. International Journal of Pharmaceutical Sciences and
Research, 8(9), pp.3663-3667.
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Ash value is used to determine the quality and purity of the drug and to establish its identity. Ash contains inorganic radicals lie phosphates, carbonates, and silicates of sodium, potassium, magnesium, calcium, etc. These are present in definite amount in a particular crude drug, hence quantitative determination in terms of various ash values helps in their standardization. Ash value is used to determine foreign inorganic matter present as impurity.
Total ash value: It is carried out at low temperatures possibly because alkali chlorides, which are volatile at low temperatures, may be lost.The total ash consists of carbonates, phosphates, silicates and silica.
Acid insoluble ash- is determined. Acid insoluble Ash, Water soluble ash and sulphated ash are also evaluated.
It is useful for evaluation of a crude drug. It gives an idea about the nature of the chemical constituents present in a crude drug. Useful for estimation of constituents extracted with the solvent used for extraction. Employed for material for which as yet no suitable chemical or biological assay exists. It can be done by following methods: Cold maceration, hot extraction and ethanol