9. Determination of Bitterness value Bitters are the medicinal plant materials that have a strong bitter taste and employed therapeutically, mostly as appetizing agent. Their bitterness stimulate secretion in the GIT especially of gastric juice. The total bitterness need to be measured by taste, which is main physiological parameters.

As prescribed by WHO the bitter properties of plant material are determined by comparing the threshold bitter concentration of an extract of the materials with that of a dilute solution of quinine HCl.

10. Determination of Hemolytic activity Saponin have characteristic of frothing property and have ability to cause haemolysis when added to a suspension of blood. Many medicinal plant material especially derived from family: Caryophyllacea, Aralacae, Sapindacea, Primulaceae & Dioscoreacea contain saponin. Method of determination: by comparing with reference material Saponin which have hemolytic activity 1000 unit per g.

11. Determination of Swelling index Swelling factor: measuring the volume of mucilage produced in 24 hours for 1 g of seed. Many medicinal plant materials are of specific therapeutic utility because of their swelling properties. The swelling index is the volume in ml taken up by the swelling of 1 g of plant material under specified condition.

12. Determination of foaming index The saponin are high Mwt containing phytoconstituents having detergent activity. Many medicinal plant materials contain saponin that can cause a persistent foam when an aqueous decoction is shaken. The foaming ability of an aqueous decoction of plant material is measured in term of foaming index.

13. Determination of Tannins Tannins are widely distributed in plants and occur in solution in cell sap, often in the distinct vacuoles. Tanins are substance capable of turning animal hides into leather by binding proteins to form water insoluble substance that are resistant to proteolytic enzyme.

14. Determination of Arsenic and Heavy metals The medicinal plant material can be contaminated with arsenic and heavy metals which can be attributed to many causes including environmental pollution and traces of pesticides. As these components even in trace amounts are dangerous, they have to be removed from the herbal drugs. Limit tests for these material have been prescribed in almost all the pharmacopoeia.

15. Determination of pesticides Pesticides: simple substances or mixtures used to eliminate undesirable vegetable and animal life in agricultural and upon ecosystem. Chemical pesticide mainly organochlorine, organophosphorous, carbamate & triazine have pronounced physiological activity and cause unpleasant S/E, such as disturbance of biological equilibrium. e.g. Organochlorine: over stimulation of CNS Organophosphorous: potent cholinesterase inhibitors (CNS symptoms such as restlessness, depression of respiratory and cardivascular system. Standardized phytopharmaceuticals must have a known content of active characteristics substance, exogenous substances must be below specified limits recommended.

Methods for determination of pesticide residues Chromatography (GC/HPLC) is the principle method for determination of pesticide residues. Other method also available: Measuring total organic phosphorous and total arsenic or total lead. As prescribed by WHO if the pesticide to which the plant are exposed are known or cab be identified by suitable method, a well established method for determination of this pesticide residue should be employed. When pesticide are unknown , it may be necessary to measure total organic chlorine, phosphorous, arsenic and lead.

Removal of pesticide from phytopharmaceuitcals Many alternative methodology have to be developed for the removal of pesticide from crude drug. DDT in Cassia angustifolia leaves and podscan be removed by supercritical fluid extraction with CO2 at 100 bar. Preserve herbal drug using CO2 pressurized up to 40 bar at R.T for 1.2 hour, this is harmless and effective.

Analysis of crude drug extracts and isolates Quantitative chemical analysis by determine the purity of the isolated substance or the concentration of a single substances or a group of substances in a mixture by finger printing and different other techniques. The nature of the substance (chemical group, specific identity, polarity,..etc) in the mixture can be determined by a number of means including chromatographic and wet chemical test. For isolated compound whose structure is less certain , additional methods such as MS, UV, NMR can be applied to elucidate unambiguously the molecular structure of compound.

Analytical Methods The quantitative determination of constituents has been made easy by recent developments in analytical instrumentation. Recent advances in the isolation, purification, and structure elucidation of naturally occurring metabolites have made it possible to establish appropriate strategies for the determination and analysis of quality and the process of standardization of herbal preparations. Classification of plants and organisms by their chemical constituents is referred to as chemotaxonomy. TLC, HPLC, GC, quantitative TLC (QTLC), and high-performance TLC (HPTLC) can determine the homogeneity of a plant extract. Over-pressured layer chromatography (OPLC), infrared and UV-VIS spectrometry, MS, GC, liquid chromatography (LC) used alone, or in combinations such as GC/MS, LC/MS, and MS/MS, and nuclear magnetic resonance (NMR), are powerful tools, often used for standardization and to control the quality of both the raw material and the finished product. The results from these sophisticated techniques provide a chemical fingerprint as to the nature of chemicals or impurities present in the plant or extract.

Parameters required for quality evaluation of herbal drugs

QUALITY EVALUATION OF HERBAL DRUGS CHEMICAL BIOLOGICAL ORGANOLEPTIC BOTANICAL PHYSICAL Moist. Cont. Extrac. Values Ash Values Fluores. Analy. Macroscopic Microscopic Qualitative Quantitative SEM Studies Powder Studies Shape External Marking Colour Odour Taste Texture Fracture Antagonistic Microbial Contamination Toxicological Pharmacological Other specific activities Bacterial Fungal Chromatography Heavy metal Pesticide residue Mycotoxin HPTLC GLC HPLC HPTLC Finger printing Sec. Metabolites DNA Finger printing Standardization & Quality Evaluation of Herbal drugs

Example on standardization Paronychia argentea Paronychia argentea has many folk names: Rijl El Hmameh in Jordan,Mountain Knot Grass in England, Bessat Al- Ard in North Africa, and other common names as Silver nailroot, and Silvery whitlow Wort. Edible Uses: Tea. An infusion of the flowers is used as a tea. Folkloric uses: Paronychia argentea has many uses in folk medicine, infusion of entire plant, except the roots, is used to expel renal stones, it has also been used as a diuretic, febrifuge, appetizer, or as mild aphrodisiac. It is also used in the treatment of TB. Medicinal uses: Many studies showed that Paronychia argentea has hypoglycemic activity, and it has been proved to be useful as gastric analgesic, bladder, prostate, abdominal ailments treatment, and stomach ulcers treatment, it also showed significant alpha amylase and acetyl cholinesterase (AChE) enzyme inhibitory activity, the plant extract of Paronychia argentea showed antioxidant activity. Other in vivo and invitro studies on different extracts from Paronychia argentea revealed the immunomodulating activity of the plant.

Chemical Constituents: Many chemical compounds have been identified and isolated from Paronychia argentea as: jaceosidin, tricin, nepetin, octadecanoic acid, 1-docosanol, glycerol octadecanoate monoester, β- sitosteryl glucoside, β- sitosterol, luteolin-3- methyl ether, ethyl-α- D- galacto- pyranoside, Dpinitol and vanillic acid, luteolin

Standardized using vanillic acid: Mobile phase: Methanol : Water 1%Acetic acid (30:70) , flow rate =1.0 ml/min, at 260 nm

Example 2: Artemisia dracunulus

Example 3: Ginkgo biloba dosage forms High-performance liquid chromatographic (HPLC) - photodiode array (PDA) detection method to quantitate five flavonol components as markers; rutin, quercitrin, quercetin, kaempferol and isorhamnetin for use in the quality control of Ginkgo biloba dosage forms.

Figure 1: HPLC chromatogram of reference standards, rutin, quercitrin, quercetin, kaempferol and isorhamnetin with corresponding retention times at l = 350nm. Separation of the flavonols was achieved at 45°C on a minibore Phenomenex Luna 5mm C18 (2) column with dimensions 250 x 2.00mm using a one step linear gradient and flow rate of 0.4 ml/min. Mobile phase A (acetonitrile) and B (0.3% formic acid) ratios where changed after 15 minutes from 15:85 to 25:75 and total run time was 33 minutes.

Figure 2: Overlaid HPLC chromatograms of the reference standards including the profiles of the analysed Ginkgo biloba dosage forms (λ = 350nm).

Adulteration Adulteration: Substitution of the original crude drugs partially or fully with other substances which is either free from or inferior in therapeutic and chemical properties, may be deliberate or accidental Admixture: addition of one article to another through accident, ignorance or carelessness, e.g. soil in underground organ, co-collection of two similar species. Sophistication: deliberate addition of superior or inferior material with intent to defraud, e.g. powder ginger may be diluted with starch with addition of little coloring material to give yellow color. Substitution: addition of an entirely different article in place of that which required, e.g. cotton seed oil in place of olive oil.

Types of adulteration or substitution of herbal drug 1. Substitution with inferior commercial verities due to morphological resemblance to authentic drugs, e.g. capsicum annum fruits and Japanese chilies for fruit of capsicum minimum. 2. Adulteration by artificially manufactured substitutes, e.g. artificial invert sugar for honey. 3. Substitution by Exhausted drugs, e.g. volatile oil contain clove, fennel etc. 4. Substitution by superficially similar but cheaper natural substance, e.g. leaves of species-Ailanthus are substituted for belladonna, senna. 5. Adulteration by addition of worthless heavy materials, e.g. pieces of limestone are found in asafetida. 6. Addition of synthetic principles, e.g. add citral to oil of lemon. 7. Use of vegetative matter from the same plant.

Causes and measures of adulteration Adulteration have caused a major problem in the research on commercial natural products. Adulteration is implicitly by manufacturers who purchase only low priced ingredient by accepting certificate of purity as sole proof of quality and by common practice of employing “Label claims” in cosmetic industry. Detection of adulteration using many different means of detection which will establish (1) identity of adulterant, (2) determine quality of drug. Guarantee purity & quality of natural product: Appropriate level of testing must be carefully assessed before using raw material based on the monographs available in different official books as will as regulatory guidelines.

Deterioration Deterioration: impairment of the quality on value of an article due to destruction or abstraction of valuable constituents by bad treatment or aging or to the deliberate extraction of the constituents and the sale of the residue as the original drug. Crude drug are prone to deterioration on storage. The shelf life of crude drug influenced by many factors, include not only the quality of storage condition but also the stability of the secondary metabolite. Primary factors: 1. Light 2. Moisture/Humidity 3. Temperature 4. Airic oxidation, e.g. volatile oil with terpenoid resinified & fixed oil rancidification

Secondary factors Living organism usually develop in stored drugs where the condition are satisfactory for them. 1. Bacteria & Moulds: dried herbs are liable to contamination by spore of bacteria and moulds present in air. 2. Mites & Nematode worms: If found, mits are present in counless number up to 1.0 mm length, examined by microscope. 3. Insects & Moths: The moths involved are unspectacular in appearance , 22-30 mm with off-white wings, besides some insects , cockroaches, ants are found cause deterioration of product. 4. Coleoptera or Beetles: These are the insects that constitutes the largest order of the animal kingdom, e.g. stegobion paniceum in liquarice.

Control measure for Deterioration The container used for storage and its closure must not interact physically or chemically with material within in any way would alter its composition. A well closed container must protect the contents from extraneous matter or from loss of material while handling and tightly closed container must protect the material from deliquescence or evaporation under normal condition. Storage area should be kept clean and spillages not allowed to enter cracks or accessible crevices. Periodic spraying of the premises with insecticide prevent the spread of infestation. Elimination effect of deterioration due to both the primary & secondary factors are desirable. Cool & dry condition is most suitable condition of living organism.

Crude drugs purchased by the herbalist may already have been sterilized by treatment of bulk consignment with ethylene oxide or methyl bromide under controlled conditions, dry treatment like this should comply with acceptable limit of toxic residues, e.g. senna pods 50ppm ethylene oxide