Introduction
Microscopical examination and pharmacognostic evaluation of phyto drug may not apparently bear any direct co-relation with pharmacological and phytochemical evaluations. One should always remember that botanical identity of the phyto drug is an essential pre-requisite for undertaking the analysis of medicinal properties of any plant. If botanical identity of drug happens to be doubtful the entire phytochemical and pharmacological work on the plant becomes invalid. Thus the botanical identity of a crude drug threshold in the process of pharmacological investigations.
Pharmacognostic Evaluation
A systematic pharmacognostic study was carried out on the herbal drugs selected, to describe them more scientifically and to identify specific characteristics, if any, which will be helpful in the quality assurance and standardization of these plant drugs.
Leaf Constants
Determination of Stomatal Index
Stomatal index is the percentage which the number of stomata form to the total number of epidermal cells, each stoma being counted as one cell. Stomatal index was be calculated by using the following equation.
I = S X 100
E+S
I=Stomatal index,
S=No. of stomata per unit area,
E=No. of epidermal cells in the same unit area.
Middle part of the leaf was cleared by boiling with chloral hydrate solution. The lower epidermis was peeled by means of forceps and mounted on the slide with glycerine water. Camera lucida and drawing board were arranged for making drawings to scale. A square of 1mm was drawn by means of stage micrometer. The slide with cleared leaf (epidermis) was placed on the stage. The epidermal cells and stomata were traced out. The number of stomata present in 1sq mm area was counted. (Stomatal Number). The result for each of the ten fields was recorded and the average number of stomata per sq.mm was calculated.
The stomatal index was determined using the above formula.
The slides were prepared for Gynandropsis gynandra,(fig.2). The Stomatal number and Stomatal index values are given in Table.2.
Determination of Vein-Islet Number
Vein-islet is the small area of green tissue surrounded by the veinlets. The vein-islet number is the average number of vein-islet per square millimeter of a leaf surface. It is determined by counting the number of vein-islets in an area of 4 sq.mm of the central part of the leaf between the midrib and the margin.
A portion of leaf was cleaned by boiling in chloral hydrate solution for about thirty minutes and slide was prepared. Camera lucida and drawing board were arranged for making drawings to scale. Stage micrometer was arranged on the microscope and using 16 mm objective, a line was drawn equivalent to 1 mm as seen through the microscope. A square was constructed on this line. The patter was moved so that the square is seen in the eye piece, in the centre of the field. The slide with the cleared leaf epidermis was placed on the stage. The veins which are included within the square were traced off, completing the outlines of those islets which overlap adjacent sides of the square. The number of vein islets in 1sq mm was counted. (The slides were prepared for Gynandropsis gynandra,(fig.5)). The average number of vein islets in the four adjoining squares gave. The Vein islet number.(Table -3)
Determination of Palisade Ratio
Palisade ratio is the average number of palisade cells beneath one epidermal cell of a leaf. It is determined by counting the palisade cells beneath four continuous epidermal cells.
A piece of the leaf was cleared by boiling in chloral hydrate solution for about thirty minutes and slide was prepared. Camera lucida and drawing board were arranged for making drawings to scale. Using 4mm objective, the outlines of four cells of the epidermis were traced off. The palisade layer was focussed and sufficient cells were traced off to cover the tracings of the epidermal cells. The outlines of those palisade cells which are intersected by the epidermal walls, were completed. The palisade cells under the four epidermal cells were counted. The average number of cells beneath a single epidermal cell was calculated. (The slides were prepared for Gynandropsis gynandra,(fig.8). The determination was repeated for five groups of four epidermal cells from different parts of the leaf. The average of the results gave the palisade ratio. (Table-4)
Histology of Gynandropsis gynandra:
Midrib of Leaf:
The transverse section of midrib of Gynandropsis gynandra Linn comprises of the epidermis, cortex, endodermis and vascular bundles. (fig.13)
Upper epidermis:
Comprises of barrel shaped cells which are closely packed, devoid of chloroplast and possess glandular trichomes.
Cortex:
Below the epidermis layers of cortical cells are present which are made up of polygonal parenchymatous cells.
Endodermis:
Endodermis is made up of rectangular barrel shaped cells with casparian thickenings.
Pericycle:
Below the endodermis three layered pericycle is present which is made up of parenchymatous cells.
Vascular Bundles:
A four to five layered phloem tissue is present that is made up of thinwalled phloem parenchymatous cells and phloem companion cells.
Xylem tissue is made up of xylem elements, xylem parenchyma and xylem companion cells.
Lower Epidermis:
Is made up of polygonal cells which are closely packed together.
2.4.2 Stem:
Transverse section of Gynandropsis gynandra Linn stem comprises of epidermis, exodermis, cortex, endodermis and vascular bundles. (fig.14)
Epidermis:
External layer with tightly joined cells that are devoid of stomata. This layer is usually termed as rhizodermis. It is also known as epiblema. This layer with covering trichomes dries and its place is taken by typical secondary boundary tissue called exodermis having glandular trichomes.
Exodermis:
This layer is present below the epidermis and is often regarded as a protective layer. The walls of the cells become suberized.
Eames, in 1947, regarded this as hypodermis; Foster and Guttenberg, in 1943, gave it the name exodermis because of the presence of suberin in its walls. The suberin lamella develop on the inner side of the primary wall. They differ from cork cells since they contain protoplasmic contents.
Cortex:
The cortex is comparatively simple in histology and is generally composed of thin walled cells with lots of intercellular spaces. The cells are arranged in concentric layers with cells in each layer alternating with others.
Endodermis:
It is a distinct layer of cells differentiated from the innermost layer of cortex. The layer is uniseriate, made up of barrel shaped cells. Casparian strips are present radially.
Pericycle:
Below the endodermis, a few layers of parenchymatous cells are present which make up the pericycle.
Vascular Bundles:
The stem exhibits secondary growth, hence a complete ring of cambium is formed. A distinct secondary phloem is visible on the outer side. There is outer fascicular cambium which is made of parenchymatous cells. The phloem consists of phloem fibres, sieve tubes and companion cells. The secondary xylem shows distinct vessels and forms a continuous band interrupted here and there by narrow rays which are uniseriate.
The secondary xylem constitutes a large portion of the bundles; it is present on the inner side and consists of vessels with simple perforated tracheids with a few simple pits on radial walls and some xylem parenchyma.
Pith:
Thin walled or thick walled cells filled with tannin and crystals of gypsum constitute the small pith.
Stomata:
Anisocytic or cruciferous (unequal) type of stomata which occurs in Capparadaceae family. The stoma is usually surrounded by three or four subsidiary cells, one of which is markedly smaller than the others. (fig.15)
Physico Chemical Evaluation of Crude Drugs
Extractive Values
Extractive values are useful for evaluation of crude drugs and give an idea about the nature of chemical constituents present in them. The amount of extractive a drug yields to a given solvent is often an approximate measure of a certain constituent or group of related constituents the drug contains. In some cases the amount of a certain constituent or group of related constituents the drug contains, in some cases the amount of drug soluble in a given solvent is an index of its purity. The solvent used for extraction should be in a position to dissolve quantities of substances desired.
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Determination of Alcohol Soluble Extractive
5 g of macerated and air-dried coarse powder of drug was mixed with 100 ml of 95% alcohol in a closed flask and kept for 24 hours, shaking frequently during the first 6 hours and then allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precautions against loss of the solvent. About 25 ml of the filtrate was evaporated to dryness in a tared, flat-bottomed shallow dish, dried at 105o C and weighed. The percentage of alcohol-soluble extractive was calculated with reference to the air-dried drug.
Determination of Water Soluble Extractive
Proceeded as directed for the determination of alcohol soluble extractive, using chloroform water I.P. as a solvent.
Determination of Chloroform Soluble Extractive
Proceeded as directed for the determination of alcohol soluble extractive, using chloroform as solvent.
Determination of Petroleum Ether Soluble Extractive
Proceeded as directed for the determination of alcohol soluble extractive, using petroleum ether as a solvent. (Table 6)
Loss On Drying
About 5 g of powder was accurately weighed, placed in a petri-dish and dried in hot-air oven at 110° C for four hours. After cooling, it was placed in a desiccator. The loss in weight was recorded. This was repeated till constant weight was obtained and % Loss on Drying was calculated with reference to the air-dried drug. (Table 7)
Determination of Ash Values
Ash values are helpful in determining the quality and purity of crude drugs in powdered form. Ashing involves an oxidation of the components of the product. The total ash usually consists of inorganic radicals like carbonates, phosphates, silicates and silica of sodium, potassium, magnesium and calcium. A high ash value is indicative of contamination, substitution or adulteration.
Sometimes, inorganic variables like calcium oxalate, silica, carbonate content of crude drug affects “total ash” values; such variables are then removed by treating with acid (as they are soluble in hydrochloric acid) and then acid-insoluble ash value is determined. Ash insoluble in hydrochloric acid is the residue obtained after extracting the total ash with hydrochloric acid. This acid-insoluble ash value particularly indicates contamination with silicious materials like earth or sand. Water-soluble ash is that part of the total ash content which is soluble in water. It is a good indicator of either previous extraction of water soluble salts in the drug or incorrect preparation.
For the determination of various ash values viz. total ash, acid-insoluble ash, water-soluble ash, the shade dried parts of the selected plant materials were powdered and passed through sieve no:40 and studies were carried out. The values vary within fairly wide limits and is therefore an important parameter for the purpose of evaluation of crude drugs.
Determination of Total Ash
A flat, thin porcelain crucible was weighed and ignited. About 2 g of the powdered drug was taken into the crucible. The crucible was incinerated at temperatures not exceeding 4500C, until free from carbon.The crucible was cooled in a desiccator and weighed. The procedure was repeated to get constant weight.The percentage of total ash was calculated with reference to the air dried drug. (Table No.8)
Determination of Acid-insoluble Ash
The total ash obtained was boiled with 25 ml of 2 M hydrochloric acid for 5mins. The insoluble ash was collected on an ashless filter paper and washed with hot water. The insoluble ash was transferred to a pre-weighed silica crucible, ignited, cooled, weighed and procedure was repeated to get constant weight. The percentage of Acid-insoluble ash of the crude drug was calculated with reference to the air-dried sample of the crude drug. (Table No.9)
Determination of Water-soluble Ash
The total ash obtained was boiled in 25 ml chloroform water for five minutes. The insoluble ash was collected on an ashless filter paper and washed with hot water. The insoluble ash was transferred into pre-weighed silica crucible, ignited for 15 minutes at a temperature not exceeding 450o C. The crucible was cooled, weighed and the procedure was repeated to get constant weight .Weight of the insoluble matter was subtracted from the weight of the total ash. The difference of weight was considered as the water-soluble ash. The percentage of water-soluble ash was determined with reference to the air-dried drug. (Table No.10)
Fluorescence analysis of the crude drugs:
Many crude drugs show fluorescence when the sample is exposed to ultraviolet radiation. Evaluation of crude drugs based on fluorescence in daylight is not much used, as it is usually unreliable due to the weakness of the fluorescence effect. Fluorescence lamps (366 nm) are fitted with suitable filters, which eliminates visible radiation from the lamp and transmits ultraviolet radiation of definite wavelength. Several crude drugs show characteristic fluorescence useful for their evaluation. (Table No.11)
Total Solid Content
About 5 g of extract was accurately weighed in a petri-dish and kept in a hot-air oven and maintained at 110°C for four hours. After cooling, the loss in weight was recorded. This procedure was repeated till constant weight was obtained. (Table No. 12)
Total solid content (%) = Loss in weight x 100/W
W = Weight of the extract in grams
Extraction
Maceration
The powdered materials were extracted with alcohol (95%) by cold maceration method.
Weighed quantity of powdered crude drugs were taken into round bottom flasks with alcohol, in the drug to solvent ratio 1:3 and kept for maceration for a period of 7 days. Finally the flask was left undisturbed for 12 hrs and then the contents were shaker and filtered through Whatman filter paper No.1. The marc was re-extracted with drug solvent ratio of 1:2. The extracts were combined and concentrated in a rotary flash evaporator, till free from solvent. The extracts, thus obtained were stored in a refrigerator at 40C until used. (Table No.13)
Qualitative Phytochemical Screening
A spectrum of natural compounds like alkaloids, glycosides, tannis, essential oils and other similar secondary metabolites which exert physiological activity are synthesized in the plant, in addition to the carbohydrates, proteins and lipids utilized by man as food articles.
A systematic and complete study of crude drugs should include a thorough investigation of both primary and secondary metabolites derived as a result of plant metabolism. The different qualitative chemical tests are to be performed for establishing profile of a given extract/fraction for its nature of chemical composition.
The following tests were carried out on the extracts to detect various phytoconstituents present in them.
Detection of Alkaloids
About 50 mg of solvent – free extract was stirred with little quantity of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloid tests viz., Mayer’s Test, Wagner’s Test, Hager’s Test, Dragendroff’s Test
Detection of Carbohydrates
About 100mg of the extract was dissolved in 5 ml of distilled water and filtered. The presence of carbohydrates were tested by Molisch’s Test, Fehling’s Test, Barfoed’s Test and Benedict’s Test
Detection of Glycosides
For detection of glycosides, about 50 mg of extract was hydrolyzed with concentrated hydrochloric acid for 2 hrs on a water bath, filtered and the hydrolysate was subjected to the Glycoside testa viz., Borntrager’s Test, Legal’s Test,
Detection of Saponins
Foam or Froth Test
Detection of Proteins and Amino Acids
About 100 mg of extract was dissolved in 10 ml of distilled water and filtered through Whatmann No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. Viz., Millon’s Test, Biuret Test, Ninhydrin Test
Detection of Phytosterols and triterpenoids:
Tested by Libermann – Burchard’s and Salkwoski test
Detection of Phenolic Compounds and Tannins
Tested by Ferric chloride test, Gelatin test, Lead acetate test, Alkaline reagents, and Shinoda test or Magnesium – Hydrochloric acid reduction
Thin Layer Chromatography
Thin Layer Chromatography of extracts was done by using standard procedures and is mainly used for the detection of the nature of phytoconstituents present.
Thin Layer Chromatography is a very effective technique for the separation of chemical constituents of an extract and for their identification. The history of TLC has been reviewed by various authors. A major breakthrough in this field was the commercial availability of convenient precoated plates in the early 70’s Pharmacopoeias are increasingly employing this technique for assessing the quality and purity of compounds of both synthetic and natural origin. TLC profiles developed for an extract from a define solvent system and other parameters could be used as a fingerprint in comparative qualitative evaluation of herbal drugs. The trend of evaluation by this method is becoming popular in view of its simplicity and reproducibility.
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TLC is an important analytical tool in the separation, identification and estimation of different classes of natural products. In this technique, the different components are separated by the differential migration of solute between two phases – a stationary phase and a mobile phase. Here, the principle of separation is adsorption and the stationary phase acts as an adsorbent. Depending on the particular type of stationary phase, its preparation and use with different solvents, separation can be achieved on the basis of partition or a combination of partition and adsorption.
Preparation of Plates
100 g of Silica gel-G was weighed and made into a homogenous suspension with 200 ml of distilled water to form aslurry. The slurry was poured into a TLC applicator, which was adjusted to 0.25 mm thickness on flat glass plate of different dimensions (10 x 2, 10 x 5, 20 x 5, 20 x 10 cm etc.). The coated plates were allowed to dry in air, followed by heating at 100 – 105o C for 1 hour, cooled and stored in a dry atmosphere to protect from moisture. Before using, the plates were activated by heating at 100o C for 10 minutes.
Detection of Steroids / Triterpenoids and their Glycosides
Solvent systems used:
Ethyl acetate: Methanol : Water 81 : 11 : 8
Ethyl acetate: Methanol : Water 75 : 15 : 10
Chloroform : Methanol : Water 70 : 30 : 4
Chloroform : Methanol : Water 64 : 50 : 10
n-Butanol :Acetic acid: Water 4 : 1 : 5 (upper phase)
Benzene : Ethyl acetate 90 : 10, 80 : 20, 50 : 50
Chloroform : Methanol 95 : 5, 90 : 10, 80 : 20
Ethyl acetate: Methanol 90 : 10, 80 : 20, 50 : 50
Spray Reagents:
1) Vanillin – Sulphuric acid (VS) reagent
Solution I : 5% ethanolic sulphuric acid
Solution II : 1% ethanolic vanillin
The developed TLC plate was sprayed with 10 ml of solution I, followed immediately by 5-10 ml of solution II, then heate for 5-10 minutes at 100o C under observation. steroids / triterpenoids and their glycosides give blue, blue – violet or pink colored spots.
2) Vanillin – Phosphoric acid (VPA) reagent
Solution a: 1 gm vanillin dissolved in 100 ml of 50% phosphoric acid
Solution b: 2 parts 24 % phosphoric acid and 8 parts 2% ethanolic Vanillic acid
After spraying with either solution a or b, the plate was heated for 10 minutes at 100o C Red – Violet colour indicates the presence of steroids / triterpenoids and their glycosides.
3) Antimony (III) chloride reagent
20% solution of antimony (III) chloride
The developed TLC plate was sprayed with reagent and then heated for 5-6 minutes at 100o C
Red – violet color in visible light; red – violet, blue and green fluorescence in UV at 365 nm indicates the presence of steroids / triterpenoids and their glycosides.
4) Anisaldehyde – sulphuric acid reagent
0.5 ml of anisaldehyde was mixed with 10 ml glacial acetic acid, followed by 85 ml of methanol and 5 ml of concentrated sulphuric acid, in that order. The developed TLC plate was sprayed with reagent, heated at 100o C for 5 – 10 minutes. steroids / triterpenoids and their glycosides give blue, blue – violet or pink coloured spots.
Detection of Flavonoids and their Glycosides
Solvent systems used:
Chloroform : Methanol 80:20, 70:30, 50:50
Ethyl acetate : Methanol: Water 81:11:8
n- Butanol : Acetic acid : Water 4 : 1 : 5 (upper phase)
Ethyl acetate: Formic acid: Glacial acetic acid: water
100:11:11:27
Ethyl acetate: Formic acid: Glacial acetic acid: Ethyl methyl ketone: Water 50:7:3:30:10
Detection
The developed TLC plate was observed in visible light and in UV at 365 nm. Flavonoids and their glycosides appear as yellow, dark blue, orange zones / spots. The color gets intensified on exposure of the plates to ammonia vapors.
Detection of Alkaloids
Solvent systems used
Benzene : Ethyl acetate : Diethylamine 6:3:1
Toluene: Ethyl acetate: Formic acid 5:4:1
Detection:
Dragendorff’s reagent
The developed TLC plate was sprayed with reagent and then heated for 5-6 minutes at 1000C, spot will be developed.
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