INTRODUCTION:

Acorus calamus is perennial, semi-aquatic and smelly plant found in both temperate and subtemperate zones⁵. Acorus calamus is very popular for the remedies of cough and cold and for other respiratory disorders like bronchitis¹⁴. It also provides aid to the digestive system and act against flatulent⁶ colic, dyspepsia and vomiting⁶. Acorus calamus depresses central nervous system and well-known ingredient in formulation for psycho-somatic disorder like epilepsy¹⁰.

Standardisation of the herbal drug is essential to assess quality of drugs. The quality assessment of the drug is important to justify their acceptability and safety. The literature survey revealed that the rhizomes of acorus calamus has been used for the wide range of disease further the survey revealed that the degree of research in standardisation of rhizome extract of Acorus calamus linn was very less. So, it was felt that there is a scope in standardising the rhizome extract of Acorus calamus linn. This paper focuses on the evaluation of physiochemical parameter and thin layer chromatography of rhizome of Acorus calamus linn.

MATERIALS AND METHODS:

The mature rhizomes of Acorus calamus were procured from the local market. The dried rhizomes of Acorus calamus linn was grounded by electrical grinder. Till the fine powder is formed in a mixer grinder. The powdered materialwas subjectedto solventextractionwith water for 48hrs for maceration. The resulting mixture is filtered. The obtained extract was stored in refrigerator until further use. Physiochemical standardization methods including determination of total ash and acid insoluble ash, extractive values were carried out as per WHO recommendations and authentic procedures mention in Ayurvedic pharmacopoeia of India. Whereas total tannins were determined by using calibration curve method.

ORANOLEPTIC PARAMETER

The organoleptic characters of the herbal drugs are very much essential for its genuinity and to prevent the adulteration. The characters of rhizome such as colour, odour and taste etc was given in the table.1

Table-1

S.NO	PARAMETER	RHIZOME
1	Colour	Externally- Light Brown Internally- Buff
2	Odour	Sweet aromatic
3	Taste	Pungent & bitter
4	Size	5.0-12.0 cm long; 0.8-2.0 cm thick
5	Shape	Creeping
6	Touch	Rough
7	Fracture	Short

PHYSIOCHEMICAL PARAMETER

FOREIGN MATTER

Herbal drugs should be prepared from the confirmed part of the plant. They should be totally free from insects or moulds, including visible and excreta contaminant such as stones, sand, harmful and poisonous foreign matter and chemical residues. Animal objects such as insects and invisible microbial contaminants, which produces toxins, as well as the potential contaminants of herbal medicines.

Dried crude drug was weighed about 100g and spreaded over on white platform/tray.The sample was inspected for foreign matter.The foreign matter was separated from the crude drug and weighed. From this weight the foreign matter was derived with reference to the dried crude drug and expressed as % w/w.Foreign matter should not be more than 2%.

ASH VALUE

The residue after incineration is the total ash content of the crude drug, which simply represents inorganic salts, naturally found in drug or adhering to it or deliberately added to it, in the form of adulteration. Accurately weighed 4g of air dried material was taken in a previously weighed and tarred crucible and it was ignited at 600ºC to obtain the residue. The residue was allowed to cool for 30 mins in a desiccator and then weighed immediately without delay. From the weight of the ash, the ash value was derived with reference to the air-dried drug. It was calculated and expressed as % w/w.

ACID INSOLUBLE ASH

The ash obtained was further analysed for acid insoluble particles in ash. To a crucible containing total ash 25ml of Hcl was added and closed with watch glass then boiled gently for 5mins and then the watch glass was washed with 5ml water this liquid was added to the crucible. Acid insoluble matter was collected separately and ignite to the constant weight then the residue was allowed to cool for 30mins and weighed without delay. From the weight of the ash, the acid insoluble ash value was derived with reference to the air-dried drug. It was calculated and expressed as % w/w.

WATER SOLUBLE EXTRACTIVE

COLD Maceration 4.0g of coarsely powdered air-dried material, accurately weighed, in a glass-stoppered conical flask. Macerated with 100ml of the Water for 6 hours. Shaking frequently, then allow to stand for 18 hours. The solution was filtered rapidly taking ‘care not to lose any solvent, 25ml filtrate was transferred to a flat-bottomed dish and evaporated to dryness on a water bath. The content was dried at 105 ˚c for 6 hours, then cooled in a desiccator for 30mins and weighed without delay. The content of extractable matter in mg per g of air-dried material was calculated.

VOLATILE OIL CONTENT

Volatile oils are the liquid component of the plant cells immiscible with water, volatile at ordinary temperature that can be steam distilled at ordinary pressure. Many herbal drugs are containing volatile oil as flavouring agent. For the drug containing volatile constituents steam distillation method is used to determine volatile oil content.

10g of powdered drug was added to 250ml water in a distillation flask few pieces of porcelain were added in to it then the apparatus was set for the distillation. During the distillation the content of the flask was shaken at interval for steady boiling. Distillation was continued till no more liquid collects. The result was expressed in volume by weight percentage.

TOTAL TANNIN CONTENT

The polyhydroxy phenolic compounds of tannins present in plant extract reacts with folins phenol reagent to produce a blue colour which absorbs maximally at 640nm Which is the basis of this determination of the total tannin content of plant extract.

REQUIREMENTS

Folins phenol reagent (1:2) (commercially available) It is prepared in 1:2 ratio with distilled water.

Stock tannic acid solution -Accurately weighed 10mg of tannic acid was dissolved in distilled water and made up to 10ml in a standard flask.

Working standard solution -1ml of the stock solution was diluted to 10ml with distilled water in a standard flask. 1ml of this solution contains 100µg of tannic acid.

Preparation of extract - 10mg of extract was weighed and dissolved in 10ml of methanol: water (70:30). From this 1ml was used for estimation.

Instrument-Photoelectric colorimetry 114.

From the working standard solution 0.2, 0.4, 0.6, 0.8 and 1ml of solutions were pipette out into a series of test tubes. 1ml of extract was pipette out in the other test tube the volume in all the test tubes was made up to 1ml with distilled water.1ml of distilled water was used as blank. To all the test tubes 0.5ml of Folins Phenol Reagent (1:2) followed by 3ml of 35% sodium carbonate solution was added and kept at room temperature for 5minutes. A blue colour was formed, and its colour intensity was read at 640nm.A standard graph of tannic acid was plotted, from which the Total Tannin content of extract was determined in terms of Tannic Acid equivalents. The observation is tabulated, and result is represented in the table-2 and graph - 1 respectively. The total tannin content from the graph was found to be 20µg/ml.

Table-2

Solution	Absorbance
Blank	0.09
STD 0.2ml (20µg)	0.18
STD 0.4ml (40µg)	0.18
STD 0.6ml (60µg)	0.23
STD 0.8ml (80µg)	0.28
STD 1ml (100µg)	0.35
Sample	0.70

Total Tannin Content by Calibration Curve Method

Thin Layer Chromatographic Study

Many trials after solvent system was selected and prepared (mobile phase) my mixing n-butanol, glacial acetic acid and water in the ratio of 4:1:5 the slurry is prepared by mixing the adsorbent in water in the ratio of 1:2 solvent system added to the chromatographic chamber the slurry is spreaded on the TLC plates and it is dried in an oven. After that draw, a baseline above 2 cm on the glass plate the sample was spotted on the TLC plate then the plate was introduced in to the chromatographic chamber, when the solvent travels the 3/4th of the plate. The plate was removed from the chamber and it was dried then the detecting agent was sprayed on the plate then it was kept in an oven for 10min to get a spot. The Rf value was calculated. The Rf value was found to be 0.69.

RESULT AND DISCUSSION:

Rhizome of the Acorus calamus Linn was collected and analysed as per various standardization parameters. The analytical data was given in table in table 3.

TABLE.3

S.NO	PARAMETER	REPORT
1.	FOREIGN MATTER	0.18%
2.	WATER SOLUBLE MATTER	3.25%
3.	TOTAL ASH	8.7%
4.	ACID INSOLUBLE ASH	1.5%
6.	VOLATILE OIL CONTENT	1%

Quantitative standards revealed total ash content 8.7% which indicate the presence of inorganic salt, Acid insoluble ash was 1.5%. Water soluble extractive value was 3.25% which indicate the presence of sugar, inorganic compounds.TLC of the rhizome extract developed in the mobile phase n-butanol: acetic acid: water in the ratio of 4:1:5 the Rf value was found to be 0.69 which indicate the presence of terpenoid.Volatile oil content was about 1%. Total tannin was found to be 20µg from the calibration curve. The foreign organic matter is 0.18% which is within the limit.

CONCLUSION:

Standardisation of herbal drug is a matter of great concern. Standardisation is very much essential for assessment of purity and identification of any sample. Physiochemical parameter was carried out on selected sample as per WHO guidelines in order to establish appropriate data that can be used in identifying crude drug. The study revealed specific identities for the plant, which will be useful in identification, as a control to abet adulterants and for future standardization work. The present work can be used as one of the tool for standardisation of this crude drug to identify and decide the authenticity of this drug in herbal industry/ trade.

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Received on 18.12.2017 Modified on 15.01.2018

Asian J. Research Chem. 2018; 11(2):423-426.

DOI:10.5958/0974-4150.2018.00077.9