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PHARMACOGNOSY LAB REPORT PHARMACOGNOSY Practical 1 : Macroscopical and chemical analysis of crude drugs Topics: 1. Macroscopy Foreign Matter In Crude Drugs 2. Colour Tests Thin Layer Chromatography Aim: 1. To examine the macroscopical features and sensory character of a few crude drugs of different ...

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PHARMACOGNOSY LAB REPORT PHARMACOGNOSY Practical 1

: Macroscopical and chemical analysis of crude drugs

Topics: 1. Macroscopy & Foreign Matter In Crude Drugs 2. Colour Tests & Thin Layer Chromatography Aim: 1. To examine the macroscopical features and sensory character of a few crude drugs of different parts of plants. 2. To evaluate the foreign matter in adulterated specimen. 3. To determine the colour produced by the plant materials provided after treatment with various chemical reagents. 4. To perform TLC analysis on the powdered plant material and, therefore, obtaining its fingerprint. Introduction: Evaluation of drug means confirmation of its identity, determination of its quality and purity and detection of nature of adulteration. The evaluation of a crude drug is necessary because of these three main reasons which are biochemical variation in the drugs, deterioration due to treatment or storage, and substitution or adulteration which a result of carelessness, ignorance or fraud. Over the years the nature and degree of evaluation of crude drugs has undergone a systematic changes. Identification of crude drugs may be examined by comparison macroscopical characteristic with the standard description available. Macroscopic evaluation involves the investigation of external appearance, such as shape and size, and also sensory characters, for instance, colour, odour and taste. The descriptions usually refer to the features that can be seen by unaided eyes or with the aid of hand lens. The percentage of certain impurities that included in the crude drugs must be evaluated to maintain the quality and purity of crude drug. The impurities may takes place either by direct or intentional adulteration and indirect or unintentional adulteration. Direct or intentional adulteration is done intentionally which usually includes practices in which an herbal drug is substituted partially or fully with other inferior products. Due to morphological resemblance to the authentic herb, many different inferior commercial varieties are used as adulterants. Indirect or unintentional adulteration sometimes occurs without bad intention of the manufacturer or

supplier. Sometimes in the absence of proper means of evaluation, an authentic drug partially or fully devoid of the active ingredients may enter the market. Factors such as geographical sources, growing conditions, processing, and storage are all factors that influence the quality of the drug. A standard limit commonly prescribed for this kind of impurity is about 2%. If no limit is given for foreign matter in the monograph for certain drug, this means that the drug should consist only 100% of the substances specified. Chemical tests are useful for the qualitative chemical evaluation of a crude drug. These tests are either general or specific and are usually performed for identification of the powdered drugs. The Malaysian Herbal Monograph employs colour test and thin layer chromatography (TLC) for the identification of powdered crude drugs. The colour tests are general and preliminary test of the crude drug. When treated plant material or its extract with chemical reagents, colour reactions will be produced by the plants material. The colour may be specific to a particular part of plant material or a particular type of species and varieties. TLC is another chemical identification test that can be used in identification and authentication of crude drugs. TLC is also used to detect impurities and adulterants that present in the plant material. The sample of crude drugs or herbal preparations is identified based on their chemical constituent profiles. A standard which normally the active ingredient of the sample and a marker (not necessary the active ingredient) are needed for the TLC process. The use of a marker is to determine whether the system is working well or vice versa. Otherwise a fingerprint of the separated constituents is determined. The positions of the major and distinctive bands in the chromatogram are described relative to a non-constituent marker or to the solvent front. The bands are compared to a reference compound or a reference extract. TLC is a chromatographic technique used to separate the components of a mixture using a thin stationary phase supported by inert backing. TLC functions in the same principle as other chromatography where a compound has different affinities for mobile and stationary phases, and this affects the speed of migration. A stationary phase is usually a thin sorbent layer supported on glass plate or aluminium sheet. The main sorbent layers used are silica gel G, aluminium oxide G, kieselguhr G and cellulose powder. The solution of the sample is spotted onto the plate by using a micropipette in a form of band but as narrow as possible to achieve a good separation. The solution should be applied in stages and the spots dried in a stream or warm air. The plate is developed in a suitable mobile phase in a saturated developing chamber. As the mobile phase move through the plate, the sample will also move but in a different rate based on their chemical profile. The detection of the separated zone can be determined by either visual examination in daylight or under ultra-violet light (254 and 366 nm). The zone can also be detected by spraying it with a suitable location reagent which results in the development of various colours. The distance of a particular zone moved relative to the solvent front is calculated as Rf value. The visual appearance (usually colour and intensity of colour) is also observed.

Procedures: A. Macroscopical Examination of Crude Drugs 1. The morphological group of the plant specimen provided was being observed regarding the macroscopical features (e.g. shape, size, external characters,etc.) and sensory characters. 2. The specimen was drawn and observations were recorded. B. Evaluation of Foreign Matter in Crude Drugs 1. The specimen was weighed. The weight of the specimen was recorded. 2. Then, the specimen was spread in a thin layer on the white paper. 3. Using naked eyes or with the aid of a hand lens, the specimen was examined. 4. The adulterants were separated individually from the part of the plant specified. 5. The adulterants were weighed and the percentage was recorded. C. Colour Test for Powdered Plant Material 1. The colour of powder was recorded 2. 2mg of the specimen was placed into a sample well on a plate. 3. Five drops of distilled water were added using a narrow-tipped Pasteur pipette. 4. Steps (2) and (3) were repeated with each chemical reagents: concentrated sulphuric acid, concentrated hydrochloric acid, 5% sodium hydroxide, 5% potassium hydroxide, 25% ammonium hydroxide and 5% ferric chloride. 5. The colors of the mixture containing water and chemical reagents were observed and recorded. D. Thin Layer Chromatography for Powdered Plant Material Extraction of Plant Material 1. 0.5 g of the powdered plant material provided was weighed and placed in a conical flask. 2. 10 mL of 96% ethanol was added and the mixture was heated over a boiling water bath for 2 minutes. 3. The mixture was allowed to cool and filter.

4. The filtrate was evaporated to dryness over a boiling water bath. 5. The extract was reconstituted with 0.5 mL of a mixture of chloroform-methanol (1:1) TLC Analysis of Plant Extract 1. 10 µ L of the extract was applied using a micropipette in a form of a narrow band (about 1 cm long) onto a normal phase silica gel GF 254 TLC plate (0.25 mm thick). The solvent was allowed to evaporate completely. 2. The plate was developed in a mobile phase consisting a mixture of a chloroformmethanol (9:1) for a length of 8 cm from the baseline. 3. After removal of the plate from the TLC tank, the solvent was allowed to evaporate in a fume cupboard. 4. Observation of the separated spots in daylight was recorded, indicating the appearance and Rf values. 5. The TLC plate was examined under the ultraviolet light of 254 nm and the observation regarding the appearance and Rf values of the spots that quench UV light were recorded. 6. The plate was placed in TLC tank containing saturated iodine vapour until distinct colours of the separated bands were seen. The observation of the colours developed in daylight was quickly recorded and the Rf values of all spots, noting the colour and intensity were determined.

Results and Discussion:

Fig 1.1: Dry cremocarp of Foeniculum vulgare Mill. ssp. vulgare var. vulgare. (Bitter fennel)

Stem

Fig 1.2: Adulterants of Bitter fennel

A. Macroscopical Examination of Crude Drugs

(a) Refer Figure 1.1 (b) Macroscopical features Shape Size External character

cylindrical shape with a rounded base 3-12 mm long and 3-4 mm wide It bears 5 prominent, slightly carenated ridges

Sensory characters Colours Odour Taste

greenish-brown, brown or green slightly bitter sweet anise-licorire aroma

B. Evaluation of Foreign Matter in Crude Drugs

(a) Refer to Figure 1.2 (b) Percentage of adulterants: Weight of the specimen: 23.3287 g Weight of adulterants: 7.4668 g Percentage of adulterants in the specimen =

𝟕.𝟒𝟔𝟔𝟖 𝒈 𝟐𝟑.𝟑𝟐𝟖𝟕 𝒈

× 𝟏𝟎𝟎%

= 32 % adulterants. (c) There are a lot of adulterants can be found in the bitter fennel specimen. Types of adulterants contain in the specimens which are devoid of or significantly different from specific characters include different shape, size and colour compare to the crude bitter fennel, present of holes which may be due to insects, stalks, and stone. (d) According to British Pharmacopoeia, the percentage of foreign matter that may include in the drug must be not more than 2% w/w. The percentage of adulterants present in the specimens is 32% which is over the limit allowed. About 68% of crude drugs left in the specimens are considered the not significant amount to be used in the industrial field and it should be rejected.

C. Colour Test for Powdered Plant Material (a) Chemical Reagent D. TBlank (water) hconcentrated sulphuric acid i concentrated nhydrochloric acid 5% sodium hydroxide L 5% potassium hydroxide a 25% ammonium yhydroxide e5% ferric chloride

Authentic Powdered Plant Material colourless Light yellow Light yellow yellow yellow Light yellow Brownish yellow

r Chromatography for Powdered Plant Material (a) Chromatogram after exposure to iodine

Adulterant Powdered Plant Material colourless Light yellow, lighter than authentic plant material Light yellow, lighter than authentic plant material Yellow, lighter than authentic plant material Yellow, lighter than authentic plant material Light yellow, lighter than authentic plant material Brownish yellow, lighter than authentic plant material

(b) Rf value =

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑝𝑜𝑡 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

Under ultraviolet light B (Authentic sample) Point 1 =

7,1 𝑐𝑚 8.0𝑐𝑚

= 0.8875 Point 4 =

4.0𝑐𝑚 8.0𝑐𝑚

= 0.5000 Point 7 =

6.3𝑐𝑚

Point 2 = 8.0𝑐𝑚 = 0.7875 3.3𝑐𝑚

Point 5 = 8.0𝑐𝑚

= 0.4125

Point 3 =

5.2𝑐𝑚 8.0𝑐𝑚

= 0.6500 Point 6 =

1.4𝑐𝑚 8.0𝑐𝑚

= 0.1750

0.9𝑐𝑚 8.0𝑐𝑚

= 0.1125 B1 (Adulterated sample) Point 1 =

7.1𝑐𝑚 8.0𝑐𝑚

= 0.8875 Point 4 =

5.8𝑐𝑚 8.0𝑐𝑚

= 0.7250 Point 7 =

2.9𝑐𝑚 8.0𝑐𝑚

= 0.3625

6.8𝑐𝑚

Point 2 = 8.0𝑐𝑚

= 0.8500 4.9𝑐𝑚

Point 5 = 8.0𝑐𝑚

= 0.6125 1.4𝑐𝑚

Point 8 = 8.0𝑐𝑚

= 0.1750

Point 3 =

6.3𝑐𝑚 8.0𝑐𝑚

= 0.7875 3.5𝑐𝑚

Point 6 = 8.0𝑐𝑚 = 0.4375 Point 9 =

0.9𝑐𝑚 8.0𝑐𝑚

= 0.1125

Daylight after exposure to iodine vapour B (Authentic sample) Point 1 =

7.1𝑐𝑚 8.0𝑐𝑚

= 0.8875 Point 4 =

1.6𝑐𝑚 8.0𝑐𝑚

= 0.2000

Point 2 =

6.5𝑐𝑚 8.0𝑐𝑚

= 0.8125 0.9𝑐𝑚

Point 5 = 8.0𝑐𝑚

= 0.1125

Point 3 =

4.0𝑐𝑚 8.0𝑐𝑚

= 0.5000

B1 (Adulterated sample) Point 1 =

7.2𝑐𝑚 8.0𝑐𝑚

= 0.9000 Point 4 =

3.7𝑐𝑚 8.0𝑐𝑚

= 0.4625

7.1𝑐𝑚

Point 2 = 8.0𝑐𝑚

= 0.8875 1.6𝑐𝑚

Point 5 = 8.0𝑐𝑚

= 0.2000

Point 3 =

6.5𝑐𝑚 8.0𝑐𝑚

= 0.8125 0.9𝑐𝑚

Point 6 = 8.0𝑐𝑚 = 0.1125

(3) Colour test is one of the identification test for crude drugs. Colour change during a reaction is due to the chemical composition in the plant. Malaysian Herbal Monograph (MHM) reagents such as, concentrated sulphuric acid, concentrated hydrochloric acid, 5% sodium hydroxide, 5% potassium hydroxide, 25% ammonium hydroxide and 5% ferric chloride were used in the colour test. These reagents were useful to chemically characterise the powdered bitter fennel. The variation in colour intensity of tested sample showed that there are differences in chemical composition between the 2 samples which are authentic powdered plant material and adulterated powdered plant material. Besides, environmental factor is one of the common cause of variation within plants of the same species grow in different geographical location. Based on the observations, the colour intensity of the adulterant powdered plant material in different chemical reagents are much lighter compared to authentic powdered plant material. In thin layer chromatography (TLC), the number of major and distinct minor spots seen on the chromatogram, Rf values and the intensity of the spots are the important parameters for the identification of the medicinal plants as well as the compound present in the sample. In this experiment, the spots are hardly to be seen when it is observed under daylight without the aid of the reagent. The observation showed a trace of pale yellow colour and there are no distinct spots on the chromatogram. When observed under ultraviolet light, the spots appear in the blue greenish colour. The appearance of these spots are due to the presence of the fluorescent substances that is sensitive to ultraviolet light. Besides that, iodine had been used as an indicator for the visualization of chromatogram of TLC as iodine will react with the organic compounds of the sample, thus producing distinct brownish spots. In the identification of medicinal plant, one reference sample labelled as B had been used to compare with interested sample labelled as B1.

By comparing the number of spots appeared in the two samples, B1 sample showed additional spots when compare with the reference sample, B. In addition, there are few spots that showed different Rf value to that of the reference sample. From these, we can conclude that there are presence of different compounds in the interested sample. Apart from these, the intensity of the spots can be assessed to determine the amount of the each compounds present in the sample. Higher intensity shows greater quantity of the particular compound. By comparing to the standard reference sample, low intensity showed by the interested sample may indicates instability of the sample causing the deterioration of the specific compound, meanwhile, greater intensity may indicate the amount of the particular compound has exceed the standard quantity. (4) Color test is relatively cheaper and simpler compared to other identification test. However, there are disadvantages and flaws for this test too. The result is highly affected by the test condition, amount of the substances present, presence of extraneous material in test sample and operator discrepancy. In order to reduce errors made during the experiment, the operator should weigh every sample equally or as close as possible. Besides, the test should be carried out in fume hood when concentrated sulphuric acid, concentrated hydrochloric acid were used as they are highly corrosive. One of the problem we encounter while performing the TLC is during the extraction of plant material. While boiling the mixture of the powdered plant material and ethanol, we directly immersed the mixture into the boiling water bath. By right it should be heated over the boiling water bath. This may be due to the steam produced by the boiling water can ensure a more constant temperature for uniform heating compare to immersing into the water bath. Secondly, is forming the uniform narrow bands of the two samples on the TLC plate. Due to lack of experience and practice, the width of the two bands produced are not uniform. This more or least may affect the separation result. Hence, in order to minimise it, the band can be formed by dipping the extract drop by drop at one time until a length of 1cm, allow it to dry first, then only repeat the steps until finish the required amount of extract. Thirdly will be the staining of iodine with iodine vapour, the time of staining should be appropriate as long period of staining may cause the background of the TLC plate to be stained as well. Hence, the plate should be taken out once the spots can be seen obviously.

Conclusion: The macroscopical features of a crude drug obtained from plant can be examined through its shape, size and the external character. On the other hand, the sensory characters can be determined are the drug’s colour, odour and taste. These macroscopical features and sensory characters are important when it comes to identification of the crude drug. The presence of any foreign matters like different parts of the plant beside the part of interest as well as distorted drug in the crude drug are considered as adulterated. The percentage of adulterants should not exceed the amount stated in the British Pharmacopoeia, if it is so, the final product should be rejected. Colour test is one of the identification test. The differences in colour changes between the two samples when added with specific chemical reagent show there are different composition between both the samples. The colour intensity can also be used as an important parameter in identifying the crude drugs. TLC analysis is an advance method for identification of crude drugs. The number of major and distinct minor spots seen on the chromatogram, Rf values and the intensity of the spots are the important parameters for the identification of the crude drugs as well as the composition of the samples. Any changes in these parameter between the sample of interest and reference sample show there are different composition in the samples, thus can conclude the sample had been adulterated.

Reference: 1) British Pharmacopoeia: https://www.pharmacopoeia.com/ 2) http://www.koop-phyto.org/en/medicinal-plants/pharmacopoeia-europaea.php https://chem.libretexts.org/Core/Analytical_Chemistry/Lab_Techniques/Thin_Layer_Chromatog raphy 3) M. Rezvanian, Z. T. Ooi, Jamia A. Jamal*, K. Husain, J. Jalil, Z. Yaacob, H. F. Mohamad and A. A. Ghani Pharmacognostic and Chromatographic Analysis of Malaysian Piper Nigrum Linn. Fruits Indian J Pharm Sci 2016; 78(3): 334-343 4) http://www.uspbpep.com/bp2008/data/7647.asp 5) Recitation Notes For Experiment Thin Layer Chromatography Website : https://www.utdallas.edu/~scortes/ochem/OChem_Lab1/recit_notes/recit_exp5_tlc.pdf...