Analytical, Toxicity, Experimental and Clinical Validation of Ayurvedic Formulations /Herbal Crude Drugs

K V GOPINATH M Pharm PhD,CPhTTirumala Tirupati Devasthanams

TIRUPATIe-mail:gopinath.karnam@gmail.com

Analytical, Chemical, & Biological Methodology of Validation/Standardization

Analytical methods should be scientifically sound (e.g., specific, sensitive, and accurate) and provide results that are reliable and reproducible

Standardization of drug means confirmation of its identity, quality and purity throughout all phases of its cycle ie shelf-life, storage, distribution & use by various parameters.

Different techniques involved in standardization of crude drugs

-Macroscopic methods

-Microscopic methods

-Physical methods

-Chemical methods

-Biological methods

PHYSICAL METHODS STANDARDIZATION OF HERBAL DRUGS

Viscosity Melting point Solubility Moisture content and volatile

matter Specific gravity Density Optical rotation Refractive index Particle size determination Bulk density

Bitterness value Hemolytic activity Swelling index Foaming index Ash value Astringency Organoleptic testing Determination of Ash value pH estimation Estimation of foreign

materials

CHEMICAL METHODS OF STANDARDIZATION - HERBAL DRUGS

Detection of carbohydrates and glycosides Detection of fixed oils and fats Detection of protein and free amino acids Detection of alkaloids Detection of phytosterols Detection of saponins Detection of phenolic compounds and tannins Detection of gums and mucilage Detection of volatile oils Chemical Assays Limit test for heavy metals Determination of Acid Value, Saponification value, & Iodine value

BIOLOGIAL/ MICROBIOLOGICAL /TOXICOLOGIAL STANDARDIZATION

Bio Assays

-Matching bioassay

-Interpolation Method

-Bracketing Method

-Multiple point

TOXICOLOGICAL STANDARDIZATION

-Determination of pesticides.

-Determination of arsenic and heavy metals

-Determination radioactive contamination

-Determination of aflatoxins.

PESTICIDES

-Fungicides

-Herbicides

-Insecticides

-Acarcicides

-Nematocides

-Rodenticides

-Bactericides

BIOLOGIAL/ MICROBIOLOGICAL /TOXICOLOGIAL STANDARDIZATION

Toxic residues may arise in crude drugs, foods, spices and cosmetics as a result of pesticide application and fumigation during cultivation and storage .

TLC and GC are available for determination of organochlorine and urea derivatives, enzymatic methods for organophosporus compounds, colorimetric methods for urea derivatives and spectroscopic techniques for paraquat, triazines and heavy metals.

Toxic residues may be substantially removed or reduced by infusion process of the dried plant material and storage at 30ᶿ has been seen to reduce rapidly the ethylene oxide residues in senna pods.

In certain instances, necessary test for aflatoxins (poisonous substance in the spores of the fungus Aspergillus flavus induces cancer) , and radioactive impurities

BIOLOGIAL/ MICROBIOLOGICAL /TOXICOLOGIAL STANDARDIZATION

Acceptable residue level (ARL) :

ARL = ADI*E*60

MUI*100

Where

ADI=maximum acceptable daily intake of pesticides (mg/kg of body weight),

E= extraction factor, which determines the transition rate of the pesticides from the plant material into the dosage form,

MDI=Mean Daily Intake of medicinal plant products,

60 in numerator=adult body weight,

100 in denominator=consumption factor.

viscosity

Viscosity is an expression of the resistance to flow of a system under an applied stress.

Eg. Castor Oil 1000 cp; Glycerin, 93% 400 cp ;Water 1.0019 cp The unit of viscosity is the poise/centipoise/stokes/centistokes Kinematic viscosity = n/p Choice of Viscometer: Capillary viscometer, Falling-Sphere

Viscometer, Cup-and- Bob Viscometer, Cone-and plate Viscometer Viscosity play a role:

-mixing and flow materials, their packaging container and their removal prior to use

-Consistency from fluid to semisolid to solid, can affect its patient acceptability, physical stability, and even bioavailability

- Play a role in selection equipment of processing used in the manufacture

Melting Point

The freezing point or melting point of a pure crystalline solid is strictly defined as the temperature at which pure liquid and solid exit in equilibrium at 1 atm.

A crystal that is bound together by weak forces generally has a low heat of fusion and a low mp, whereas one bound together by strong forces has a high heat of fusion and a high mp.

The mp of saturated hydrocarbons increases with increase in molecular weight ; it is used as a identity, and purity of a compound.

The mp and solubilities of the xanthenes of pharmaceutical interest. E.g. The methylation of theophylline to form caffeine and the lengthening of the side chain from methyl(caffeine) to propyl in the 7 position result in a decrease of the mp and an increase in solubility

Theophylline R=H 270-274 o C 4.5 mole/L X 102

Caffeine (R=CH3) 238 oC 13.3 mole/L X 102

Solubility

Definition: quantitatively as the concentration of solute in a saturated solution at a certain temperature, and in qualitatively as the spontaneous interaction of two or more substances to form a homogeneous molecular dispersion

Concentration Expressions: Molarity, Normality, Molality, Mole Fraction, Mole percent, %w/w, % w/v,%v/v, milligram percent, ppm

Solubility depends on chemical, electrical, and structural effects that lead to mutual interactions between the solute and the solvent.

Solubility of insoluble substances can improved by Co-solvency, Solubilisation, pH Modification, Conversion to its salt, Hydro trophy

Solubility can determined by gravimetric and volumetric method The presence of adulterant could be indicated by solubility studies

-E.g. pure Asafoetida is soluble in carbon disulphide

Descriptive terms used to express solubility in Indian Pharmacopoeia

Descriptive terms Relative quantities of solvent

Very soluble Less than 1 part Freely soluble From 1 to 10 parts Soluble From 10 to 30 parts Sparingly soluble From 30 to 100 parts Slightly soluble From 100 to 1000 parts Very slightly soluble From 1000 to 10,000 parts Practically insoluble More than 10,000 parts

Bulk Density

Definition: Mass of powder divided by the bulk volume Three-tap method has been found to give most consistent results Bulk density of powder depends primarily on particle-size

distribution, particle shape, and the tendency of the particle to adhere to one another.

Name of the chemical Bulk density (g/cm3)

True density (g/cm3)

Magnesium Carbonate Heavy 0.39 3.0

Magnesium Carbonate Light 0.07 3.0

Talc 0.48 2.7

Density

Density can be defined as mass per unit volume and is expressed as g/cm3 in cgs system and kg/m3.

Specific Gravity: Also known as relative density defined as the ratio of the mass of a substance to the mass an equal volume of water at 40 C or some other specified temperature.

Specific gravity may be determined by the use of various types of pycnometers, Mohr-Westphal balance, hydrometers etc

Important in interconverting between mass and volume

Determination of Moisture Content & Volatile Matter

It is the amount of volatile matter i.e., water drying off from the drug is known as Moisture content.

Method: Place about 10 g of drug in a tared evaporating dish. Dry at 105º for 5 hours, and to a constant weight (two successive weighing should not exceed 0.25%).

Distillation method

Importance: Purchase of drugs( aloes, gelatin ,gums, digitalis, hamamelis leaf, yarrow, hawthorn berries, starch,) which contain excessive moisture - uneconomical.

-It promotes bacterial growth and product decomposition

- It promotes even enzyme activation along with temperature

DETERMINATION OF FOREIGN MATTER

Foreign matter is a material consisting of moulds, insects, animal faecal matter and other contaminations such as earth, stones and extraneous material.

The amount of foreign matter shall not be more than the percentage prescribed in the monograph.

DETERMINATION OF FOREIGN MATTER

Weigh 100 –500 g of the drug sample to be examined, or the 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 .

Drugs Foreign matter limit

Quillaia Bark 2%

Cascara bark 1%

Valerian root 5% stem bases, 2% other foreign matter

Optical Rotation

It is the angle through which the plane of polarization of light is rotated(clockwise or anticlockwise) when the polarized light is passed through a sample of the liquid.

It can detected by mean of Polarimeter. The observed rotation is a function of thickness of the layer(1 cm), its temperature (200 C) and nature of the lamp (sodium vapor lamp) .

It is quick test to check the identity and purity of the compound, especially volatile oils.

The BP distinguishes between nutmeg oils from the East Indies (+10 0 to 250) and west Indies (+250 to 450).

Caraway oil +74 0 to 80 0 Peppermint oil -100 to -300

Lemon Oil +570 to +70 0 Spearmint oil -450 to -600

Terpene less lemon oil -50 to +20 Citronella oil, ceylon -90 to -180

Cinnamon oil 00 to -20 Citronella oil, Java -50 to +20

Refractive index

It is the ratio between the velocity of light in air and the velocity in the substance under test.

n = sin i/sin r = velocity of light in air/ velocity of light in the test substance.

It can be detected by Abbe refractometer or Leico Auto Abbe refractometer.

– They measure the n with precision to the fifth decimal place

– The dark and colored samples are easily detected.

– The shadow-line location is determined by the instrument software

– No mechanical components are involved, thus reducing wear with time.

• It is particularly valuable for purity assessment of volatile oils and fixed oils.

-Oils of Cassia 1.61; Lemon oil 1.474-1.476

-Cinnamon 1.573 – 1.600; Terpeneless lemon oil 1.475-1.485

-Cinnamon leaf 1.53;

Particle size determination

Micromeritics Microscopy, sieving and Sedimentation-Andreasen apparatus Applications

- Size and surface area of a particle can be related in a significant way to the physical, chemical, and pharmacological properties of a drug.

-Clinically, the particle size of a drug can affect its release from dosage forms that are administered orally, parenterally, rectally and topically.

-The successful formulation of suspensions, emulsions,and tablets, from the viewpoints of both physical stability and pharmacological response also depend on particle size.

-In the area of tablet and capsule manufacture, control of the particle size is essential in achieving the necessary flow properties and proper mixingof granules and powders.

- Extraction procedures

ASH VALUES AND EXTRACTIVES

The residue remaining after incineration of a vegetable drug is the ash (inorganic) content of drug

Total ash method is used to measure the total amount of material remaining after incineration.

-Importance and indicates to some extent the amount of extra care taken in the preparation of the drug

Acid insoluble ash is the residue obtained after boiling the total ash with dil. HCl and igniting the remaining insoluble matter.

-This usually consists mainly of silica and a high acid-insoluble ash in drugs such as Senna, cloves, liquorice, Valerian and tragacanth indicates contamination with earthy material.

- In case of ginger a minimum % of water-soluble ash is demanded.

Water soluble ash is the difference in weight between total ash and residue after treatment of total ash with water.

Sulphated Ash involves treatment with H2SO4 & ignition at 600oC

DETERMINATION OF EXTRACTABLE MATTER-Hot extraction

place 4 gms powdered material in a conical flask. Add water and weigh to obtain total weight.

Shake and allowed to stand for 1hr. attach the reflux condenser and boil for 1hr.

Readjust to the original weight with solvent. Shake and filter. Transfer the filter to a flat bottomed disk and evaporate to

dryness on a water bath. Dry at 105˚ c for 6hrs, cool and weigh immediately. Calculate the content of extractable matter in mg per g of air

dried material. E.g.

Gentian drug, liquorice Evaluated by % Water soluble extractives

Valerian drug, asafoetida Evaluated by % of ethanol 70% extractive

ORGANOLEPTIC EVALUATION -BITTERNESS VALUE

The bitterness value is determined organoleptically by comparison with a standard quinineHCl solution .

This standard is relevant to Centaury, Gentian and Wormwood This drugs are used for their bitter effect (appetizer). The bitterness value is expressed as units equivalent to the

bitterness of a solution containing 1gm of quinine hydrochloride in 2000ml.

0.1gm of quinine hydrochloride is dissolved in 100ml drinking water and the stock solution is prepared. Then it is diluted and tested and compared with drug.

Bitterness value in unit per gm = 2000*c

A*BWhere, 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

HAEMOLYTIC ACTIVITY

Hemolytic activity of plant material is determined by comparison with that of reference material, Saponin R, having hemolytic activity of 1000units/g.

  Method of preparation of standard: Fill a glass stopper flask to 1/10 of its volume with sodium citrate. Add sufficient volume of blood freshly collected from healthy ox and shake. This can be stored for about 8 days at 2- c. place 1ml of citrated blood in a 4̊ 4̊(volumetric flask with phosphate buffer pH 7.4.

Haemolytic activity = 1000* a/b

Where, 1000 = defined haemolytic activity of Saponin standard

a = quantity of saponin standard that produce total haemolysis (g)

b = quantity of plant material that produce total haemolysis (g)

SWELLING INDEX

Swelling index is defined as the volume in milliliters occupied by 1 g of a drug, including any adhering mucilage, after it has swollen in an aqueous liquid for 4 h.

Method: The 1 g of a drug is treated with 1 ml ethanol (96%) and 25 ml water in a graduated cylinder, shaken every 10 min for 1hr and allowed to stand as specified.

Agar 10

Cetraria 4.5

Fenugreek 6

Fucus 6

Ispaghula husk (0.1gm)

40

Ispaghula seed 9

FOAMING INDEX

The foaming ability of an aqueous decoction of plant material and their extracts is measured in terms of foaming index.

Cosmetic interest

Astringency/Tannin content– Tannins(eg. Tannic acid),Krameria, catechu, galls,

Aspidosperma, hamamelis, pomegranate rind, kinos

The method refers to those polyphenols adsorbed by hide powder and giving a color reaction with sodium phosphomolybdotungstate reagent

pH Determination

pH = -log (H3O+)

The determination of pH can be made by means of any electrode whose potential depends on the hydrogen ion activity

The hydrogen and glass electrodes are typical pH electrodes. Two hydrogen electrodes can be combined to measure pH, one

serving as the indicating electrode and the other as the reference electrode, although this arrangement is not generally used in practice.

The calomel and the silver chloride electrode are more convenient as reference electrodes and are typically used with commercial pH meters.

Importance:

-Physical, chemical and biological stability of crude drugs are function of pH

-Qualitative and quantitative analysis

Microscopic Method of Analysis – Crude Fibres

Cell wall, cell shape and cell contents- which are of value in identification and in the detection of adulteration

Parenchymatous tissue, the epidermis and stomata, epidermal trichomes, the endodermis, cork tissue, collenchyma, sclereids, fibres(more resistant cellular material), xylem, phloem, secretory tissues are important in identity and detection of purity.

E.g. -wavy walled epidermal cells –stramonium;

-papillose epidermis- coca leaf;

-The lignified, thick walled pericyclic fibres, accompanied by a parenchymatous sheath of cells containing prisms of calcium oxalate, constitute an important diagnostic character of senna.

-Ginger rhizome which contain relatively large amounts of oleoresin and starch

Microscopic method of Analysis

Types of Stomata: There are several types of stomata, distinguished by the form and arrangement of the surrounding cells.

- Anomocytic (irregular-celled)

- Anisocytic (unequal-celled)

- Diacytic (cross-celled)

- Paracytic (parallel-celled)

Microscopic method of Analysis: Determination of Stomatal Index

The stomatal index is the percentage of the number of stomata formed by the total number of epidermal cells, including the stomata, each stoma being counted as one cell.

S x 100

Stomatal index = ————

E + S

Where S = the number of stomata in a given area of leaf ; and E = the number of epidermal cells (including trichomes) in the same

area of leaf.

- For each sample of leaf make not fewer than ten determinations and calculate the average index.

Microscopic method of Analysis: Determination of Palisade Ratio

Palisade ratio is the average number of palisade cells under one epidermal cell.

For each sample of leaf make not fewer than ten determinations and calculate the average number

18.4

Palisade ratio —— = 4.5

4

Microscopic method of Analysis: Determination of Vein-Islet Number

The mesophyll of a leaf is divided into small portions of photosynthetic tissue by anastomosis of the veins and veinlets; such small portions or areas are termed “Vein-Islets”.

The number of vein-islets per square millimeter is termed the “Vein-Islet number”.

This value has been shown to be constant for any given species and, for full-grown leaves, to be unaffected by the age of the plant or the size of the leaves.

The vein-islet number has proved useful for the critical distinction of certain nearly related species.

Macroscopic, Microscopic and Chemical test of Analysis With an Example

Alexandrian senna

-Macroscopic: Seldom exceed 40 mm in length, grayish – green; more asymmetric at base;

-Microscopic: Hairs are more numerous; most of the stomata have 2 subsidiary cells only; Vein-islet number 25-29.5; stomatal index usually 12.5

-Chemical Tests: Ether extract of hydrolysed acid solution of drug gives with methanolic magnesium acetate solution:

A pink color in day light, a pale greenish-orange in filtered UV light

Tinnevelly senna

-Macroscopic: Seldom exceed 50 mm in length; yellowish – green, less asymmetric at base;

Microscopic: Hairs less numerous; the stomata having 2 or 3 subsidiary cells; Vein-islet number 19.5-22.5; Stomatal index usually 17.5

-Chemical Test: Same as in A.senna

An orange color in daylight, a yellowish-green in filtered UV light

Chemical Method of Analysis

Water determination by Karl Fischer procedure Mechanism:

The reagent consists of a solution of iodine, Sulfur dioxide, and pyridine in dry methanol. This is titrated against a sample

containing water , which causes a loss of the dark brown color. The basic reaction is a reduction of iodine by sulfur dioxide in the presence of water.

This test is particularly applicable to expensive drugs and chemicals containing small quantities of moisture.

E.g. Dry extracts of alkaloid containg drugs, fixed oils ( arachis, castor oil, olive oil, and sesame oil for parentaral use.

For crude drugs such as digitalis and ipecacuanha moisture contentis important.

Assays

Drugs which cannot be assayed by chemical, or physical means are evaluated by biological methods.

A crude drug may be assayed for a particular group of constituents(e.g. The total alkaloids in belladonna or the total glycosides of digitalis) or specific components ( e.g. reserpine content of Rauwolfia spp.)

INDICATION FOR BIOLOGICAL EVALUATION This is true for the substances having an Interfering obstacles When quantity is too small. No specific chemical test is available When the action of drug is due to a mixture of substance Purification of drug is not possible

Assays

Types of assay employed for crude drugs

Type of Assay Examples

Physical *Cineole in eucalyptus oil (f.p. of o-cresol complex)*Separation and weighing of active constituents(e.g. Colchicine in colchicum corm; total balsamic esters of peru balsam

Chemical ‘Total alkaloids’ of many drugs (e.g. acid-base titrations);Non-aqueous titrations of alkaloid salts(e.g strychnine in nux vomica. Morphine in opium)

Biological Cardio active drugs, antibiotics, vitamins, anthroquinone derivatives, mydriatic drugs, saponins, antitumour drugs, antiamoebic drugs, ginkgolides(anti-PAF activity

Radioimmunoassay(RIA)

Cardinolides (Digitalis lanata); Sennosides(Cassia angustifolia) Morphine (poppy capsules)

Enzyme -Immunoassay

Quassia, tropane alkaloids (solanaaceae) ;ergot alkaloids

Bio - Assays

When the estimation of crude drug or its preparation is done by means of its effect on living organism like bacteria, fungi, or animal tissue or entire animal it is known as BIOASSAY.

TYPES OF BIOASSAY

QUANTAL:-It is all or none phenomenon GRADED:-Based on observation that there is a proportionate

increase in the observed response with increase in concentration or dose.

-Matching bioassay

-Interpolation Method

-Bracketing Method

-Multiple point bioassay

Process Validation and Drug Quality

FDA has the authority and responsibility to inspect and evaluate process validation

The cGMP regulations for validating pharmaceutical (drug) manufacturing require that drug products be produced with a high degree of assurance of meeting all the attributes they are intended to possess .

Clinical Validation involve principles of Clinical Trials

Effective process validation contributes significantly to assuring drug quality

The basic principle of quality assurance is that a drug should be produced that is fit for its intended use

Process Validation and Drug Quality

Quality, safety, and efficacy are built into the product

Quality cannot be adequately assured merely by in-process and finished-product inspection or testing

Each step of a manufacturing process is controlled to assure that the finished product meets all quality

Classification of Pharmaceutical validation

Class A: Tests designed to establish identity, whether of bulk drug substance or of a particular ingredient in a finished dosage form.

Class B: Method designed to detect and quantititative impurities in bulk drug substances or finished dosage forms.

Class C: Methods used to determine quantitatively the concentration of the bulk drug substance or of major ingredient in a finished dosage form.

Class D: Method used to assess the characteristic of finished dosage forms such as dissolution profile and content uniformity

Analytical Parameters:

-Accuracy; -Precision; -Robustness

-Linearity & Range -Selectivity -Limit of detection

-Limit of quantification -Ruggedness

Approach to Process Validation

Process Validation is defined as a scientific evidence that a process is capable of consistently delivering quality product

• Stage 1 – Process Design: The commercial manufacturing process is defined during this stage based on knowledge gained through development and scale-up activities.

• Stage 2 – Process Qualification: During this stage, the process design is evaluated to determine if the process is capable of reproducible commercial manufacturing.

• Stage 3 – Continued Process Verification: Ongoing assurance is gained during routine production that the process remains in a state of control.

Approach to Process Validation

A successful validation program depends upon information and knowledge from product and process development

Understand the sources of variation

– Detect the presence and degree of variation

– Understand the impact of variation on the process and ultimately on product attributes

– Control the variation in a manner commensurate with the risk it represents to the process and product

General Considerations for Process Validation

An integrated team approach - expertise from a variety of disciplines Throughout the product lifecycle, various scientific studies can be

initiated to discover, observe, correlate, or confirm information about the product and process

Validation that employs risk based decision making throughout that lifecycle

Consistency between batches are goals of process validation activities The plan should identify the following items:

-the studies or tests to use,

-the criteria appropriate to assess outcomes,

-the timing of qualification activities,

-the responsibilities of relevant departments and the quality unit, &

- the procedures for documenting and approving the qualification.

General Considerations for Process Validation

A written protocol that specifies the manufacturing conditions, controls, testing, and expected outcomes is essential for this stage of process validation

-The manufacturing conditions, including operating parameters, processing limits, and component (raw material) inputs.

-The data to be collected and when and how it will be evaluated.

-Tests to be performed (in-process, release, characterization) and acceptance criteria for each significant processing step.

-The sampling plan, including sampling points, number of samples, and the frequency of sampling for each unit operation and attribute

-Criteria and process performance indicators that allow for a science- and risk-based decision about the ability of the process to consistently produce quality products

General Considerations for Process Validation

Design of facilities and the qualification of utilities and equipment, personnel training and qualification, and verification of material sources (components and container/closures), if not previously accomplished.

Status of the validation of analytical methods used in measuring the process, in-process materials, and the product.

Review and approval of the protocol by appropriate departments and the quality unit.

Documentation at each stage of the process validation lifecycle is essential for effective communication in complex, lengthy, and multidisciplinary projects