Botanical identification to assess the standards through the comprehensive pharmacognostical study of Acorus calamus Linn. leaves

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INTRODUCTION
corus calamus Linn. is a hydrophyte with stolons and ensiform leaf, found near wetlands, riparian zone and lacustrine (1).It has swordshaped, fragrant leaves that may grow up to 6 feet tall, little yellow/green blooms and a branching rhizome.It is extensively dispersed across India and Ceylon in farmed and natural marshes, as well as in marshy areas of Kashmir and Sirmoor in Manipur, Naga Hills, and Sikkim at altitudes of up to 6000 feet in the Himalayas (2).The roots and rhizomes have been used medicinally for centuries.Due to their antispasmodic, carminative, and anthelmintic properties, they are also used to treat seizure psychological conditions, chronic diarrhoea, dysentery, bronchial catarrh, a high body temperature and glandular and abdomen cancers (3,4).They are also used for rheumatism, sinusitis, and eczema (5).Recent research has shown that rhizomes can increase insulin sensitivity and have antibacterial effects against a fish pathogen (6).When chopped up and kept alongside dry foods, mature green leaves can act as an insect repellant as well as perform other functions (7), lipid-lowering activity, hypoglycemic activity (8,9), neuroleptic activity (10), microorganism killing and reduce pain actions (11), Acorus calamus leaf extracts in methanolic and acetone have recently been demonstrated to exhibit CNS depressing properties, which can be used in the future for antiepileptic activity (12).Acorus calamus leaf aqueous extract has demonstrated antiinflammatory properties (13).

MATERIALS AND METHODS
The leaves of the Acorus calamus were obtained from the medicinal garden of S.D. College of Pharmacy & Vocational Studies, MZN.And Patanjali Yogpeeth, Haridwar after a brief study of research articles.The authentication of the plant was done by Dr Sunita Garg from NISCAIR, New Delhi.The authentication no is NIScPR/RHMD/Consult/2022/4251-52.

Identification and pharmacognostical studies of A. calamus
The plant was identified based on organoleptic, macroscopic, and microscopic (anatomy) observations as summarized below:

Macroscopic assessment
Fresh leaves of A. calamus were examined for several macroscopic characteristics, including duration, type of leaf base, petiole presence or absence, and lamina characteristics.The composition, incision, form, venation, edge, apex, base, surface, and texture of lamina are among its distinguishing characteristics (14).

Qualitative microscopy
Transverse slices of a fresh leaf sample were cut by hand, coated with glycerin, and seen under a compound microscope at a 10X power of magnification.(METZER M. India Pvt.Ltd).

Identifying foreign matter
The tested drug sample, 50 g, was weighed and thinly layered.Inspection with the unassisted eye revealed the alien object.estimated the percent present after separating, weighing, and sorting it.The drugs used in the study were devoid of mildew, insects, animal feces, and other contaminants including dirt, stones, and other objects (15).

Moisture content determination (Hot air oven method)
To ascertain the moisture content, 1 gram of leaf powder was weighed in a silica crucible and heated to 105°C until the weight remained constant.Moisture content (w/w%) was used to measure weight loss (16).

Assessment of total ash
A silicon crucible that had already been lit and weighed was filled with around 2.0gm of finely ground leaves.On the bottom of the crucible, a thin, equal coating of powder was applied.Gradually raising the temperature to get the crucible dull red hot until it was carbon-free caused it to burn.The crucible was weighed after cooling.To get a consistent weight, the process was repeated.

Ash insoluble in acid
The ash prepared in the manner previously described was heated for five minutes with 25 cc of 2N HCl.On an ash-free filter paper, the insoluble ash was gathered and thoroughly cleaned with hot water.Transferred into a silica crucible, the insoluble ash was burned, weighed, and then removed.To get a consistent weight, the process was repeated.

Water soluble ash
With 25 ml of water, the ash produced in the manner described in the determination of total ash was heated for 5 minutes.On ash-free filter paper, the insoluble material was gathered, and hot water was used to wash it away.The insoluble ash was put into a silica crucible, lit, and weighed after 15 minutes.To get a consistent weight, the process was repeated.The weight of the total ash was divided by the weight of the insoluble material.The weight difference was interpreted as a water-soluble ash (17).

Hot extraction method
Acorus calamus leaves were collected and given a thorough water wash.They were then pounded into a powder after drying in the shade.25g of the dried leaf powder was extracted sequentially with n-hexane, C4H8O2, chloroform, ethanol, and H2O using a Soxhlet apparatus.Each extract's yield was noted, and it was then kept for future use in a 40°C refrigerator.After being filtered using Whatman filter paper no.1 and dried in a water bath until they formed a semisolid mass, each extract produced from the subsequent extractions was combined (18).

Cold extraction
In a closed flask, 2.0g of coarsely ground plant material was extracted for 24 hours with 100 ml of the appropriate solvent, shaking regularly for the first 6 hours, and then letting it stand for 18 hours (19,20).

Determination of swelling index
The WHO states that the swelling index is a measurement of the swelling of 1 g of crude medication in volume (ml).Put 1.0g of leaf powder into a 25 ml measuring cylinder with a stopper.then mix in 25 cc of water.For 24 hours, gently stir the mixture every now and then.Without tampering, this combination was left at ambient temperature.The amount of space the powdered medication and gooey mucilage took up in milliliters.

Determination of foaming index
The capacity of an herbal drug's aqueous decoction to produce foam is used to calculate the foaming index.A 100 ml conical flask was filled with precisely weighed 1.0g of leaf powder, boiling water was added, and the mixture was then allowed to cool.After filtering, the volume is increased to 100 ml in a volumetric flask.One to ten milliliters of the decoction were placed in each of the ten test tubes in turn.Water was added to the remaining capacity to bring it up to 10 ml, and the tubes were then corked shut.The test tubes were forcefully shaken before being set aside for 15 minutes.Each test tube's foam was measured for height.after measuring the froth, the foaming index was calculated as, Foaming Index: 1000\a, Where "a" stands for the amount of decoction in milliliters that was used to make the dilution in the tube, where foaming was seen to build to a height of 1 cm (21).

Chemical (Qualitative) evaluation of plant drug
Qualitative tests were carried out to determine if phytoconstituents were present in the extracts produced by the cold maceration procedure using petroleum ether, chloroform, ethyl acetate, ethanol, methanol, and water.These experiments sought to % of moisture content = Weight of powder sample Loss in weight of powder sample X 10 pinpoint the presence of specific phytochemicals in each extract, revealing important details about their chemical make-up and potential biological effects (22,23).

Analysis of fluorescence
Utilizing a common procedure, the whole plant powder underwent fluorescence analysis.The plant powder was subjected to several solvent treatments, including both acidic and basic ones, to conduct the analysis.They were monitored in daylight and subjected to UV radiation (short and long wavelengths) following treatment.An essential tool for screening substances with the ability to display various colors under UV light is fluorescence analysis.Some substances can be transformed into fluorescent derivatives even if they are not fluorescent in and of themselves when they are exposed to solvents.This examination revealed the shift in the color (24).

Analysis using thin layer chromatography
To analyze all the extracts, thin layer chromatography (TLC) was employed with silica gel G as an adsorbent.The components on the TLC plates were divided using several mobile phases.Following derivatization in iodine vapor, the plates were processed, and the Rf (retention factor) value for each spot was determined.The numerous components contained in each extract may be recognized and compared thanks to this thorough examination (25).

Macroscopic evaluation
To identify the plant drug its necessary to check the fresh leaves of plant drug and confirm the texture color and odor which was reported in several literature.Results are shown in Table 1.This data is very informative regarding the selection of actual plant drugs.

Microscopic evaluation
The microscopy of A. calamus Linn fresh leaves showed that it was a monocot plant.T.S. of leaves shows epidermis with randomly arranged vascular bundles and shows the thick-walled parenchyma and aerenchyma.The result of the microscopy of the plant is given in Figs. 1 and 2.    Powder drug with petroleum ether Yellowish Yellowish Yellowish

Physicochemical parameters
Results were computed after the powdered drug's physicochemical properties, such as foreign matter, ash values, extractive values, moisture content, etc., were analyzed (Table 2).

Chemical evaluation of plant drug (Qualitative examination)
Distinct qualitative assays used to analyze the chemical composition of Acorus calamus L. leaves revealed the occurrence or non-occurrence of distinct plant chemical compounds in various extracts.Table no 3 includes the comprehensive outcome.

Analysis of fluorescence
The powdered leaf medication treated by cold maceration and variously viewed in daylight, 254 nm for short UV light and 365 nm for long UV light yielded the results listed in Table 4.

TLC analysis
The leaf oil showed the spots with Rf values of 0.71 with characteristic blue florescence under UV 365 nm.

DISCUSSION
Establishing the identification, purity, safety, and quality of herbal medicines through standardization is essential.To standardize a drug, numerous macroscopic, microscopic, physiochemical, qualitative, fluorescence, and TLC investigations are performed.The microscopic technique is one of the easiest and most economical ways to start figuring out the precise identification of the source material.Identification of the unprocessed drug will be achievable through research on the morphology and microscopy of the leaf.Physicochemical constant features, such as extractive values, moisture content, total ash, water-soluble ash, acid-insoluble ash, foaming index, and swelling index, which are listed in Table 2, can be used to assess the quality and purity of Acorus calamus Linn.leaf pharmaceuticals.
Standardization is a crucial method for establishing the identification, purity, safety, and quality of herbal medicines.Various macroscopic, microscopic, physiochemical, qualitative, fluorescence and TLC analyses are carried out to standardize a medicine.One of the most affordable and straightforward ways to begin determining the accurate identification of the source material is the microscopic approach.Studies of the leaf's morphology and microscopy will make it possible to recognize the unprocessed medication (26).The quality and purity of Acorus calamus Linn.leaf drugs may be determined using physicochemical constant characteristics such as extractive values, moisture content, total ash, water-soluble ash, acid insoluble ash, foaming index, and swelling index, which are mentioned in Table 2. Most of the carbonates, phosphates, and silicates in crude medicine are either obtained from plants or are leftovers, such as soil pollutants that may stick to plant surfaces.The Acorus calamus Linn.leaf's total ash value of 0.233 showed that it contains considerable amounts of carbonates, phosphates, silicates, or a combination of those three substances.The acid insoluble ash value (0.066), which offers an estimate of the quantity of siliceous material contamination, demonstrated that the leaf had a minimal level of siliceous pollutants.Of the total ash, the amount of ash that is soluble in water is 0.2.It aids in identifying improper preparation and adulterants in crude drugs (27).Secondary metabolites were found in Acorus calamus Linn.leaf during a phytochemical screening, as indicated in Table 3.

CONCLUSION
The identification of distinct plant sections and the detection of adulterants may be possible using the TLC chromatogram of Acorus calamus Linn.The goal of the current study was to develop pharmacognostic guidelines for identifying and authenticating Acorus calamus Linn.and identifying adulterants.This study's identification of the key diagnostic characteristics of Acorus calamus Linn.may serve as a partial monograph.

Table 1 :
Botanical and sensory characteristics of the Acorus calamus Linn.

Table 3 :
Chemicals found in the leaf extract of Acorus calamus Linn.

Table 4 :
Fluorescence analysis of Acorus calamus Linn.leaf powder