INTRODUCTION:

Churna is a famous classical Ayurvedic dosage form consisting of coarse powder of an herb or combination of herbs. Churnas are administration orally for therapeutic purposes as well as for production of other Ayurvedic formulations like Ghana, Ghrita, Vati etc. Nagarmotha Churnais one of such Ayurvedic churna which is made from the rhizome of nagarmotha plant (Cyperus rotundus L.). Ayurvedic texts mentioned Nagarmotha as a detoxifying herb and a very efficient ama-pachaka (eliminator of endotoxins). It is prescribed mainly in arthritis, ameobiasis and digestive disorders for improving gastrointestinal health and as anti-inflammatory agent^1,2. To the best of our knowledge, there is no documentation about the characterisation of Nagarmotha Churna to assess its identity, quality and purity. The present work was therefore carried out to develop the quality control parameter of Nagarmotha Churna as per the guidelines of Ayurvedic Pharmacopoeia of India (API) and World Health Organization (WHO).

EXPERIMENTAL:

Churna Preparation:

Cyperus rotundus rhizomes were purchased from an herbal retail shop Bhubaneswar, Odisha, India in September, 2017. The herb was identified by Prof (Dr.) Debajyoti Das, Department of Pharmacognosy, School of Pharmaceutical Sciences, Bhubaneswar, India. Rhizomes were cleaned properly and spread evenly and dried in shady condition for 3 to 4 days. Churna was prepared according to the general method of churna preparation by hammering grinding and proper sieving through 80# sieve³

Determination of foreign matter:

Foreign matter content was determined as per the standard procedure. Briefly, 100g of the churna was spread to form a thin layer. Foreign matter content was detected either through visual inspection using naked eye or by the use of a lens (6x). After separation, the foreign matter was weighed and the percentage was calculated.^4,5

Organoleptic parameters:

The organoleptic characteristics of the churna such as colour, taste, odour, appearance and texture were evaluated as reported as per standard methods.^5,6

Fluorescence analysis:

For analysis of fluorescence characters, Nagarmotha Churna samples were separately treated with different standard reagents and then examined in ordinary visible light as well as under long (365 nm) and short (254 nm) wave ultra violet light.^7,8

Physicochemical investigation:

Different physicochemical properties of the churna such as alcohol soluble extractive, water soluble extractive, total ash content, acid insoluble ash content, percentage loss on drying and pH measurement of 1% suspension were performed as per the standard protocols.^9-12

Phytochemical Tests:

Qualitative phytochemical tests were performed for aqueous, methanolic and chloroform extracts of Nagarmotha Churna. The tested phytoconstituents include alkaloids, terpenoids, steroids, glycosides, flavonoids, saponins, tannins, carbohydrates, and proteins.^13-15

Thin Layer Chromatographic (TLC) Study:

One gram of churna sample was dissolved in 20ml of methanol and refluxed on a water bath at 90-100°C for 15 min. The resultant methanolic extract was filtered and concentrated up to 5ml for applying in TLC plates. The solvent system selected, showing best separation by trial and error method, was found to be Ethyl acetate: Toluene: Formic acid in the ratio of 3:2:0.2. After development of the spots on 0.2 mm thick precoated Silica Gel 60 F₂₅₄ plates (Merck KGaA), the plates were observed under ultraviolet lights (254 nm and 366 nm) and in an iodine chamber. The movements of the analyses were expressed by its retention factor (R_f) values.^16-18

Fourier transforms infrared (FT-IR) spectroscopic profiling:

FTIR spectrum of the methanolic extract of Nagarmotha Churna was recorded in the range of 4000-400 cm^-1spectrum (4cm^-1resolution) using a Bruker alpha FTIR spectrophotometer equipped with a deuterated triglycerine sulphate (DTGS) detector. After putting a few drops of the sample extract intothe contact with attenuated total reflectance (ATR) plate of Zinc Selenide (ZnSe), FTIR spectra was recorded as average of 24 scans with the background correction for air spectrum. Data acquisition and interpretation was done with the OPUS software (Windows-based).

RESULTS AND DISCUSSION:

Pharmacognostical study:

Foreign matter content:

Herbal drugs should be free from soil, insects or moulds, animal excreta and contaminant such as stones, sand etc. Foreign matter content indicates the amount of extraneous material which does not originally belong to the original drug. Accordingly, the foreign matter contents of Nagarmotha Churna was found to be well below the maximum permissible limit as per Ayurvedic Pharmacopoeia of India (API), i.e., less than 0.5% (w/w).

Organoleptic characteristics:

Organoleptic evaluation is simple, economic and rapid method for identification of herbal drugs. It refers to the evaluation of a drug by colour, odour, taste, size and shape, as well as special futures including touch, texture, etc. In our study, the prepared Nagarmotha Churna was brownish black in colour with pleasant and fragrant odour and tasted starchy. Detailed organoleptic charecteristics of the Nagarmotha Churna are presented in Table 1.

Table 1. Organoleptic characteristic of Nagarmotha Churna

Organoleptic parameter	Observation
Appearance	Ovoid shaped powder
Texture	fine
Colour	Brownish black externally.
Odour	Fragrant
Taste	Starchy

Fluorescence behaviour:

Fluorescence is the phenomenon exhibited by various fluorescent constituents present in the herbal material in presence of daylight or UV light. It is one of the important pharmacognostic parameter utilised in the identification of authentic samples and recognizing adulterants.¹⁹ Nagarmotha Churna, after treating with standard reagent solutions, exhibited characteristic fluorescence behaviour under both ordinary and ultraviolet light (Table 2).

Physicochemical parameters:

Physicochemical parameters of an herbal product play a significant role in assuring its quality and consistency during commercial production. Loss on drying determines percentage of water and volatile matter. Ash values are very important for judging the degree of purity and in evaluating the quality of crude drugs.²⁰ The total ash of a crude drug reflects the amount of inorganic materials existing after complete combustion. It consists mainly of impurities like carbonates, phosphates, and silicates of sodium, potassium, calcium, and magnesium. Acid insoluble ash is a measure of silica present such as sand and siliceous earth matter. On the other hand, water soluble ash measures the water soluble fraction of total ash. Nagarmotha Churna was found to contain 8.7% w/w total ash, 3.2% w/w acid insoluble ash and 4.6% w/w water soluble ash. This high percentage of total ash revealed presence of appreciable amount of inorganic constituents in the churna.

Extractive values are useful for evaluating the type as well as the quantity of the chemical constituents present in a crude drug. The amount of extractive obtained in a given solvent is often ascertains the phytoconstituents present in that crude drug. The amount of water soluble, methanol soluble and chloroform soluble extractive in Nagarmotha Churna was found to be 9.92%, 6.96 % and 2.54%, respectively. This observed variation in the extractive values may be due to the difference in phytoconstituents extracted with the change in polarity of the extracting solvent.

High moisture content can result in decomposition of crude drugs due to enzymetic activation. In this respect, Nagarmotha Churna registered only 3.52% w/w loss on drying at 105°C indicating its expected long self-life on storage. Moreover, the pH of the 1% aqueous suspension of the churna was found to be 5.22, i.e. mild acidic in character.

Phytochemical screening:

Depending upon physiological and climate conditions secondary metabolites varies even in the same plant species. The different factors contributing to these variations include latitude, longitude, rainfall, climatic conditions, soil quality, age of the plant, time of collection etc. Hence it is essential to perform the phytochemical screening of an authentic sample of an herbal material. However, the phytoconstituents vary with the nature of extracting solvent. Hence, we performed phytochemical screening of Nagarmotha Churna in three different solvent extracts i.e. water, methanol and chloroform. The results of phytochemical screening of Nagarmotha Churna extracts indicated presence of phytoconstituets like carbohydrate, glycoside, phenolic, alkaloids, steroids, and terpinoids (Table 3). Presence of these constituents in the Churna may be essential for its therapeutic applications.^21,22

Table 3. Phytochemical screening of the churna extract:

Sl No.	Plant constituent	Water extract	Methanol extract	Chloroform extract
1	Carbohydrate	+	+	-
2	Glycoside	+	+	-
3	Phenolic	+	+	+
4	Alkaloid	-	+	-
5	Flavonoids	-	-	-
6	Saponins	-	-	-
7	Steroids	-	-	+
8	Terpinoids	+	+	-

Where; (+) = Present; (-) = Absent

Thin layer chromatography (TLC) study:

TLC is the method of choice for rapid identification of plant secondary metabolites. In TLC, the different retention factor (R_f) values indicate the presence of different phytoconstituents in a single extracts. Moreover, difference in R_f values of the components separated also reflects idea about their polarities. Combined results of the TLC profile of the methanolic extract of Nagarmotha Churna, visualised at short UV (254 nm), Long UV (366 nm) and under iodine vapour, revealed the presence of 19 distinct spots (Figure 1, Table 4). This indicate presence of at least 19 phytoconstituents in the methanolic extract of the churna. Among these 19 sports, the number of spots visible under at short UV, long UV and iodine vapour were respectively 11, 10 and 11 in number. Some of the spots were found to be unique under a detection condition, while other spots were common among themselves.


(a)	(b)	(c)

Figure 1. Photographs of TLC plates of Nagarmotha Churna methanolic extract visualised at a) 254 nm, b) 366 nm and c) under iodine vapour

FT-IR spectrophotometric profiling:

Fourier Transform Infrared Spectrophotometer (FT-IR) is a reliable and sensitive for identification of the nature of chemical bonds and the functional groups present in the sample.²³

Table 4. TLC profile of NagarmothaChurna

Spot No.	Detection R_fValue
Spot No.	UV light (254 nm)	UV light (366 nm)	Iodine vapour
1	0.08	-	-
2	0.13	-	0.13
3	-	0.19	-
4	0.23	-	0.22
5	0.28	0.27	0.28
6	0.33	-	-
7	0.36	-	0.35
8	-	0.39	-
9	0.42	-	0.44
10	-	0.49	-
11	0.52	-	0.53
12	-	0.55	-
13	0.58	0.59	0.58
14	-	0.64	0.63
15	-	0.68	-
16	-	0.74	0.75
17	0.86	-	0.84
18	-	0.92	-
19	0.96	-	0.96
	11	10	11

Table 5. FT-IR peak values and the functional groups of Nagarmotha Churna.

Sl. No.	Peak Value (cm^-1)	Bond type	Functional group
1	3354.31	O-H Stretching	Alcohol/Phenol
2	2971.07	C-H Stretching	Alkane
3	2824.46	C-H Stretching	Aldehyde
4	2230.14	-C=C- Stretch	Alkynes
5	2045.53	C=-C Stretching	Alkynes
6	1754.69	C=O Stretch	Aldehyde/ ketone
7	1658.44	C=C Stretch	Arometic
8	1448.99	N-O asymmetric stretch	Nitro group
9	1415.63	C-H Bending	Alkanes
10	1114.47	C-N Stretch	Aliphatic amines
11	1021.08	C-O Stretch	Alcohols/ Carboxilic acid/ Ester/ Ether

FT-IR fingerprint of a plant extract or powder is a useful and rapid method for its authentication and quality assurance.²⁴ The FT-IR absorption spectra of methanolic extract of Nagarmotha Churna showed some prominent peaks (Figure 2). The assignments of functional groups for these peaks are summarized in Table 5. This spectrum revealed the functional constituents present in the methanolic extract of Nagarmotha Churna. Further, the spectrum can be useful as the FT-IR fingerprint for methanolic extract of Nagarmotha Churna.

CONCLUSION:

In the present work, the Nagarmotha Churna was characterized on the basis of the pharmacognostic, physicochemical and toxicological parameters. More FT-IR spectroscopic and TLC fingerprint of the churna were developed. The parameters presented in this paper may be used for the preparation of the monograph on quality standards for Nagarmotha Churna to maintain its batch-to-batch consistency. This document could also be utilised for FTIR spectroscopic and TLC based rapid authentication of this churna.

CONFLICT OF INTEREST:

Authors declare no conﬂict of interests.

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Received on 26.04.2019 Modified on 21.05.2019

Research J. Pharm. and Tech. 2019; 12(12): 5967-5971.

DOI: 10.5958/0974-360X.2019.01036.9

Treatment	Visible (Ordinary) light	UV light
Treatment	Visible (Ordinary) light	254 nm	366 nm
Powder as such	Brown	Dark green	Brown
Powder+1N HNO3	Reddish brown	Greenish black	Greenish black
Powder+1N HCL	Yellowish brown	Deep black	Brown
Powder+50%NaOH	Deep brown	Green	Dark brown
Powder+H₂SO₄	Blackish brown	Greenish black	Black
Powder+Methanol	Brown	Brown	Black
Powder+Iodine	Reddish brown	Dark brown	Greenish black
Powder+FeCl₃	Yellowish brown	Yellowish brown	Black
Powder+10%NaOH	Brown	Greenish Brown	Black