PHG434. Evaluation of herbal products Lec 3. Analytical evaluation. In general, quality control is based on three important pharmacopoeias definitions: Identity : Is the herb, the one it should be?
Evaluation of herbal products
In general, quality control is based on three important pharmacopoeias definitions:
Identity: Is the herb, the one it should be?
Purity: Are there contaminants, e.g., in the form of other herbs which should not be there?
Content or assay: Is the content of active constituents within the defined limits.
I- Detection of foreign matters:
Foreign organic matter refers to any other part of the plant or animals except that constituting the drug, and to any other vegetable or animal tissues or substances.
The Permissible percentage of foreign matter in a drug is usually specified in its official monograph.
A- determination of ash:
Ash is inorganic residue left when vegetable drug is incinerated. Analysis of plant ash shows that it contains Ca, Mg, K, Na, Si, P and Fe.
The amount and composition of ash varies according to some factors:
1- The part and age of the plant: in the young leaves, the ash may constitute about 5% while in mature leaves reaches about 15%. The ash content of the wood (5%) is usually much lower than that of the bark (20%).
2- Culture treatment: type of soil and elements present in it.
3- The constitution of the ash varies with the time of collection.
In the Pharmacopeia a maximum of 2% of acid insoluble ash is permitted unless otherwise stated in the monograph.
The total ash and acid insoluble ash are determined by the official method.
This consists of incinerating and weighting the total ash, then boiling the total ash with dilute hydrochloric acid, filtering, igniting and weighting the acid insoluble ash.
Acid insoluble ash consists of sand, silicates and other dirties and is an indication of the amount of dirty present in the sample. It is called foreign inorganic matter.
B- Moisture is normally present to the extent of 5-10% in all dried drugs. An excess of moisture is considered as an adulterant.
Excessive moisture is considered an adulterant because
a- Its added weight.
b- Excess moisture is conductive to the promotion of mold and bacterial growth and subsequently to deterioration of the drug.
Moisture is usually determined in one of four ways, the specific method often being stated in the drug monograph.
i- Gravimetric method:
a- If the drug contains no volatile material, a weighed sample is heated at 100◦c to constant weight, the loss in weight being the water content.
b- If volatile constituents are present, these must first be determined by the volatile ether-extractive method and their weight deduced from the loss in weight by drying, before the water content can be determined.
ii- Volumetric (Toluene distillation) method:
It is applies in either of the above cases. The water is distilled from the drug with toluene in a continuous distillation apparatus. The water is caught in the trap and is determined by direct measurement of the volume.
iii- Titrimetric (Karl Fischer) method:
It is applicable for expensive drugs and chemicals containing small quantities of moisture. This method of water determination is based on the stoichiometric reactionof the solution of sulfur dioxide, pyridine in dry methanol and iodine. this is titrated against a sample containing water which cause loss of brown color until the end point where the color of iodine persist.
iv- Determination of moisture by spectroscopic methods:
Watercan absorb energy at various wavelength throughout the electromagnetic spectrum and this can be made a basis for the quantitative determination. Measurements can be made in both the infrared and ultraviolet regions.
5- Detection of filth:
In quality control procedures the pharmacopoeias stated: drugs containing appreciable quantities of foreign organic matter, animal excreta, insects or mould should however be rejected even though the percentage of such substances be insufficient to cause the rejection of the drug.
1- Insects, insect parts, rodent hairs and feces may be separated from the drug by means of liquids of varying specific gravity.
Most drug material will float on chloroform, but rodent pellets will sink in this liquid.
2- Insect parts can be separated by boiling the drug with water, cooling and then vigorously stirring in a small amount of mineral oil.
The mineral oil rises to the top carrying with it the insect fragments. Floating strata may be separated by mean of Wildman trap flask and sinking strata by means of a percolator equipped with two corks at either end of the stem.
The recovered impurities are then identified microscopically.
Methods of controlling insect in crude vegetable drugs:
Most common methods are:
1- Heat treatment, which consists of exposing the drug to a temperature of 60-65◦C is often effective especially for insect eggs that are not penetrated rapidly by insecticides.
The duration of the exposure is determined by the volume of drug to be treated. It may vary from a few minutes to several days. Prolonged exposure to high temperature may inactivate enzymes.
2- Drugs may be fumigated by the use of various volatile insecticidal agents in closed areas e.g., CCl4, CH3Br or mixture of ethylene oxide and carbon dioxide.
- Most fumigants do not kill the eggs of insects and therefore it is common to repeat fumigation at intervals of two week, to give better chance of eliminating all insects, since time is permitted for eggs to hatch and larvae are killed.
- Stored drugs that are susceptible to insect attacks should be fumigated routinely every 3or 4 months.
If medicinal plants are to be fumigated in greenhouse, it is desirable to fumigate at night, since the plant are more sensitive to such poisonous in the daytime then their stomata are fully open than at night when the stomata are partially closed.
3- liming, a procedure used for a few drugs e.g., ginger.
4- Low temperature storage is preferred than above methods. Exposure to subfreezing temperatures of 10-15◦C for 12-18hrs will destroy even eggs.
5- Exposure to alternate periods of low and high temperature frequently is more effective for killing insects than a prolonged period of low temperature exposure.
Some drugs have specific biological and pharmacological activity which is utilized for their evaluation. Actually this activity is due to specific type of constituents present in the plant extract. For evaluation the experiments were carried out on both intact and isolated organs of living animals. With the help of bioassays (testing the drugs on living animals), strength of drug in its preparation can also be evaluated. Some important biological evaluations are as follow:
* Antibiotic activity
Some bacteria such as Salmonella typhi, Styphylococcusaureusand E. coliare used to determine the antiseptic value (the degree of antiseptic activity e.g. phenol co-efficient of certain drugs). The activity of antibiotics is also determined by using Klebsiella pneumonia, Micrococcus flavus, Sarciraluteaetc. living bacteria, yeast and molds are used to evaluate certain vitamins. Microbiological assays by cylinder plate method and turbidimetric method are used in evaluation.
* Antifertility activity
Antifertility drugs include contraceptives and abortificients. Contraceptive drugs are used to prevent pregnancy and abortificient to terminate pregnancy. Female rats are used for antifertility activity i.e. measure the pregnancy rate (antiovulation and anti-implantation) and male rats are used for anti-spermatogenicactivity (inhibition of spermatogenesis) and spermicidal activity (sperm motility) of herbal drugs.
* Hypoglycemic activity
Rabbits, rats or mice are used to test hypoglycemic activity of plant extract. Radio-immune assay (RIA) or Enzyme linked immunosorbate assay (ELISA) are done for measurement of insulin levels.
* Neuro-pharmacological activity
Testing the herbal drugs with effects on central and autonomic nervous system. CNS acting drugs like cocaine (Erythroxylum coca), morphine (Papaversomniferum), cannabinol (Cannabis sativa) are tested using rodents. For testing the herbal drugs for their effects on guinea pig ileum for antispasmodic activity.
Chromatography and chemical fingerprints of herbal medicines
Several problems influence the quality of herbal drugs:
• Herbal drugs are usually mixtures of many constituents.
• The active principle(s) is (are), in most cases unknown.
• Selective analytical methods or reference compounds may not be available commercially.
• Plant materials are chemically and naturally variable.
• Chemo-varieties and chemo cultivars exist.
• The source and quality of the raw material are variable.
• The methods of harvesting, drying, storage, transportation, and processing (for example, mode of extraction and polarity of the extracting solvent, instability of constituent, etc.) have an effet.
Thin layer chromatography
TLC was the most common, versatile method of choice for herbal analysis before instrumental chromatography methods like GC and HPLC were established. Even nowadays, TLC is still frequently used for the analysis of herbal medicines since various pharmacopoeias such as Indian herbal pharmacopoeia, Ayurvedicpharmacopoeia; American Herbal Pharmacopoeia (AHP), Chinese drug monographs and analysis, Pharmacopoeia of the People’s Republic of China, etc. Rather, TLC is used as an easier method of initial screening with a semi quantitative evaluation together with other chromatographic techniques as there is relatively less change in the simple TLC separation of herbal medicines than with instrumental chromatography.
Gas chromatography (GC), also known as gas liquid chromatography (GLC), is a technique for separation of mixtures into components by a process which depends on the redistribution of the components between a stationary phase or support material in the form of a liquid, solid or combination of both and a gaseous mobile phase. It is well-known that many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by gas chromatography is very important in the analysis of herbal medicines. The GC analysis of the volatile oils has a number of advantages. Ex. GC of the volatile oil gives a reasonable “fingerprint” which can be used to identify the plant.
High-performance liquid chromatography
High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of small particle (3-50μm) packing contained in a column with a small bore (2-5mm), one end of which is attached to a source of pressurized liquid eluent (mobile phase). The three forms of high performance liquid chromatography most often used are ion exchange, partition and adsorption. HPLC is a popular method for the analysis of herbal medicines because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. In general, HPLC can be used to analyze almost all the compounds in the herbal medicines. Thus, over the past decades, HPLC has received the most extensive application in the analysis of herbal medicines. Reversed-phase (RP) columns may be the most popular columns used in the analytical separation of herbal medicines.
Capillary electrophoresis was introduced in early 1980s as a powerful analytical and separation technique and has since been developed almost explosively. It allows an efficient way to document the purity/complexity of a sample and can handle virtually every kind of charged sample components ranging from simple inorganic ions to DNA. Thus, there was an obvious increase of electrophoretic methods, especially capillary electrophoresis, used in the analysis of herbal medicines in last decades.
Most of the used techniques are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF). CE is promising for the separation and analysis of active ingredients in herbal medicines, since it needs only small amounts of standards and can analyze samples rapidly with very good separation ability.
Also, it is a good tool for producing the chemical fingerprints of the herbal medicines, since it has similar technical characteristics of liquid chromatography.
LC-IR, LC-MS, LC-NMR
The hyphenated technique developed from the coupling of liquid chromatography and infrared spectroscopy is known as LC-IR. LC-IR is an important technique as it shows absorption peaks of functional groups in mid IR region which helps in structural identification of compounds present in a sample. The detection technique of IR is comparatively slow than other techniques like MS or NMR.
Mass spectrometry is the most sensitive and selective method for molecular analysis and can yield information on the molecular weight as well as the structure of the molecule. Combining chromatography with mass spectrometry provides the advantage of both chromatography as a separation method and mass spectrometry as an identification method. In mass spectrometry, there is a range of methods to ionize compounds and then separate the ions. Common methods of ionization used in conjunction with gas chromatography are electron impact (EI) and electron capture ionization (ECI). EI is primarily configured to select positive ions, whereas ECI is usually configured for negative
ions (ECNI). EI is particularly useful for routine analysis and provides reproducible mass spectra with structural information which allows library searching. GC–MS was the first successful online combination of chromatography with mass spectrometry, and is widely used in the analysis of essential oil in herbal medicines.
HPLC–DAD, HPLC–MS and others
HPLC–DAD (Diode-Array Detection) has become a common technique in most analytical laboratories in the world now. With the additional UV spectral information, the qualitative analysis of complex samples in herbal medicines turns out to be much easier than before. For instance, checking peak purity and comparing with the available standard spectrum of the known compound to the one in the investigated sample. Especially, with the introduction of electrospray mass spectrometry, the coupling of liquid chromatography and mass spectrometry has opened the new way to widely and routinely applied to the analysis of herbal medicines.
Superimposed HPLC-UV chromatogram of Ginkgo bilobain commercial products.