Introduction
Ayurveda has grouped the stem bark of five plants viz. Nyagrodha (Ficus bengalensis Linn.), Shirish (Albizzia lebbeck Benth.), Ashwatha (Ficus religiosa Linn) Vetasa (Salix caprea Linn.) and Plaksha (Ficus infectoria Roxb.) as ingredients in Himavan agada (the stem bark of five trees).1
The Himavan agada are indicated as Varnya (improve complexion), Stanyashodhana (purifies breast milk), Vranaghna (wound healing), Visarpa (for treating Herpes zooster) and Shophaghna (anti-inflammatory) identified by Arundatta the commentator of Ashtanga Hrudaya. 2 Also Himavan agada (anti-poisonous formulation) used to treat the Mandali Sarpa Visha.
Therefore the present study has been planned to explore and analyze the poly herbal combination were subjected to Physico-Phytochemical analysis of Panchvalkal mentioned in Himvan agada.
Aims and Objective
To analyze the Physico-chemical, phyto- chemical properties of Himvan Agada by using HPTLC fingerprinting.
Materials and Methods
1. Preparation of Himavan agada
Raw drugs required for preparation were procured form authenticated market dealer. Procured drugs were authenticated in Department of Dravyaguna, Gaur Brahman Ayurvedic College, Rohtak. Drugs were pounded individualy in Khalwa Yantra to get Yawakuta form. After pounding drugs are mixed (I part each) to prepare Himavan agada. Ingredient of Himavan agada is mentioned in table.
Table 1
Ingredients of panchvalkl mentioned in Himvan agada
Determination of ash value/total ash 3
Equipment and glassware used
Muffle-furnace, Silica Crucible, Desiccator, Ash less filter paper, Beaker.
Reagent/ chemicals used
NA
Procedure
Incinerate about 2 to 3 gm of drug (W1) accurately weighed and ground in a tarred silica crucible (W2). Keeps the crucible in a muffle-furnace at a temperature not exceeding 450˚C- 600˚C (According to the physical property of product) for 4 hrs. Cool in desicator and weigh (W3). If a carbon free ash cannot be obtained in this way. After cooling reignite the crucible at same temp. for 01 hrs. and calculated the weight difference. Collect the residue on an ash less filter paper. Incinerate the residue and filter paper. Add the filtrate, evaporate to dryness. Ignite at a temperature not exceeding 450˚C - 600˚C. Calculate the percentage of ash with reference to the air-dried drug.
Determination of acid insoluble ash 4
Equipment and glassware used
Muffle-furnace, Silica Crucible, Desiccator, Ash less filter paper, Beaker.
Reagent/ chemicals used
Dil. hydrochloric acid
Procedure
Prepare ash. Transfer the ash in a 250 ml beaker without loss of ash and add 100 ml of dil. hydrochloric acid. Wash the crucible with 10 ml of acid and transfer the washings to the beaker. Heat the beaker till the liquid boils. Filter the solution and collect the insoluble matter on ash less filter paper (Whatman). Wash with hot water until the filtrate is neutral. Transfer the filter paper containing the insoluble matter to the original crucible. Dry on a hot plate and ignite at 600˚C in a muffle furnace (until become white ash). Allow the residue to cool in suitable desiccators for 30 minutes and weigh without delay. Repeat the process until constant weight (W3) is obtained. Calculate the acid insoluble ash with reference to the air dried drug.
Determination of water soluble ash 5
Equipment and glassware used
Muffle-furnace, Silica dish, Desiccators, Ash less filter paper, Beaker.
Reagent/ chemicals used
Purified Water
Procedure
Prepare ash. Boil the total ash for 5 minutes with 25 ml of water. Collect insoluble matter in an ash less filter paper. Wash with hot water and ignite for 15 minutes at a temperature not exceeding 450°-600˚C. Subtract the weight (W3) of the insoluble matter from the weight of the ash; the difference in weight represents the water-soluble ash. Calculate the percentage of water- soluble ash with reference to the air-dried drug.
Determination of alcohol soluble extractive6
Description
The extracts obtained by exhausting crude drugs are indicative of approximate measures of their chemical constituents. Taking into consideration the diversity in chemical nature and properties of contents of drugs, various solvents are used for determination of extractives. The solvent used for extraction is in a position to dissolve appreciable quantities of substances desired.
Reagent/ chemicals used
Alcohol
Procedure
Macerate 5 gm (W1) of the coarsely powdered drug with 100 ml of Alcohol of specified strength in a closed flask for 24 hours. Shake frequently during six hours. Allow to stand for eighteen hours. Filter rapidly taking precautions against loss of solvent. Evaporate 25 ml of the filtrate to dryness in a tarred flat bottom shallow dish (W2). Dry at 1050C to constant weight and weigh (W3). Calculate the percentage of Alcohol– soluble extractive with reference to the air-dried drug.
Determination of water soluble extractive7
Description
The extracts obtained by exhausting crude drugs are indicative of approximate measures of their chemical constituents. Taking into consideration the diversity in chemical nature and properties of contents of drugs, various solvents are used for determination of extractives. The solvent used for extraction is in a position to dissolve appreciable quantities of substances desired.
Reagent/ chemicals used
DM. Water
Procedure
Macerate 5 gm (W1) of the coarsely powdered drug with 100 ml of distilled water in a closed flask for 24 hours. Shake frequently during six hours. Allow to stand for eighteen hours. Filter rapidly taking precautions against loss of water. Evaporate 25 ml of the filtrate to dryness in a tarred flat bottom shallow dish (W2). Dry at 1050C to constant weight and weigh (W3). Calculate the percentage of water – soluble extractive with reference to the air-dried drug.
Loss on drying (Oven Method)8
Reagent/ chemicals used
NA
Procedure
(Oven Method)
Sample is prepared by cutting shredding so that the parts are about 3 mm in thickness. Place about 10 gm (W1) accurately weighed up to third decimal place of drug (Without preliminary drying) in a tarred evaporating dish + drug weight (W2). Dry at 105˚C for 5 hours and weigh (W3). Continue the drying and weighing at one hour interval until difference between two successive weighing corresponds to not more than 0.25 percent. (Constant weight is reached when two consecutive weighing after drying for 30 minutes). Cool for 30 minutes in desiccator, show not more than 0.001 g difference.
Table 2
Qualitative Phyto-chemical investigation in himavan agada formulations9
Thin layer chromatography (TLC) test10
Description
TLC is the technique in which a solute undergoes distribution between two phases, stationary phase and a mobile phase. The stationary phase acts as an adsorbent in a relatively thin uniform layer of a dry finely powdered material applied to a glass, plastic or metal sheet.
Separation may be achieved on the basis of partition a or a combination of partition and adsorption, depending on the particular type of stationary phase, its preparation and uses of different solvents.
Equipment and glassware used
Oven, TLC developing chamber, Visualizing chamber, Pre-coated aluminium plates with silica gel 60 F254, Beaker, measuring cylinder with stoppered and glass pipettes.
HIGH performace thin layer chromatography (TLC) test11
HPTLC is the technique in which a solute undergoes distribution between two phases, stationary phase and a mobile phase. The stationary phase acts as an adsorbent in a relatively thin uniform layer of a dry finely powdered material applied to a glass, plastic or metal sheet.
Separation may be achieved on the basis of partition a or a combination of partition and adsorption, depending on the particular type of stationary phase, its preparation and uses of different solvents.
Instrument contains different parts to applicate, scan and visualize the elution of different spots after development of TLC plate. So, we can easily develop a fingerprint profile for the same.
Observation and Results
Table 3
Results of preliminary physicochemical and phytochemical analysis
|
Sl.No. |
Parameters |
Sample |
|
1. |
Alcohol Soluble Extractive |
7.62% |
|
2. |
Water Soluble Extractive |
15.26% |
|
3. |
LOD |
10.29% |
|
4. |
Total Ash |
8.16% |
|
5. |
Water Soluble Ash |
2.55% |
|
6. |
Acid Insoluble Ash |
0.91% |
Qualitative parameters
Table 5
Results of phytochemical profile
|
Sl. no |
Qualitative chemical Test |
Results |
Method |
|
1. |
Alkeloids |
Negative |
API 2008 |
|
2. |
Glycosides |
Positive |
|
|
3. |
Tannins |
Positive |
|
|
4. |
Flavonides |
Positive |
|
|
5. |
Saponins |
Negative |
|
|
6. |
Steroids |
Negative |
Table 6
Results of TLC analysis
|
Test Parameter |
Limits |
Result |
Test Method |
|
TLC Profile Sample |
254nm |
0.68 |
API 2008 |
|
366nm |
0.93,0.82,0.62,0.4,0.2 |
||
|
Derivatized |
0.93, 0.82, 0.75, 0.70, 0.5, 0.31 |
|
Discussion & conclusion
HPTLC fingerprinting profile
The different concentration of Methanolic extract of samples was applied on HPTLC plates in 6, 8 and 10 µl. Plate was developed and elution of different spots visualized under 254, 366nm and white light after derivatization with Anisaldehyde sulphuric acid reagent.
Physico-phytochemical analysis
The quantitative tests e.g. total ash, acid-insoluble ash, water-soluble ash, alcohol-soluble extractive, water- soluble extractive, ether-soluble extractive, moisture content, volatile oil content and assays are the methods upon which the standards of Pharmacopoeia depend. So it is necessary to analyze Ayurvedic formulation on these parameters.
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