INTRODUCTION:

Traditional medicines and/or herbal medicines are currently in high demand with its increasing popularity and equally herbal medicines require the quality evaluation for the purity of the finished product for its desired efficacy [1-2]. Quality evaluation and standardization of an ayurvedic polyherbal formulation is always challenging task to verify the presence of the raw botanical ingredients as it is the complex mixture of many ingredients. Most of the herbal preparations, especially the classical formulations of Ayurveda, Unani and Siddha, are polyherbal.

In addition to that the processing methods are unique for the preparations of formulations and made the raw ingredients into very complex mixtures, from which the identification and estimation of chemical components is very difficult, and it includes comparison of the phytochemical profile of the formulations with that of individual ingredients by co-chromatography technique of herbal extracts and formulations [3].

Ingredients may be the any part of the plant materials and they have its own characteristic features. Among them, one of the key features is essential oil/volatile oil content. Some of the plants hold characteristic odours and volatile substances which occur as essential oils, gum exudates and oleo-resin in one or more parts such as root, wood, bark, flower, leaf and fruit. Volatile content of essential oils are mixture of the many complex compounds that occur in nature, mostly mono and sesqui-terpenes, tetraterpenoids, aromatics hydrocarbons and their oxidized products such as carbonyl compounds, alcohols and ester [4]. Some plants serve as a unique source of flavours, fragrances and pharmaceuticals etc, for commercial importance. One of the main and major constituent of the oil may predominantly occur in the volatile oils of many plant species. Certain plant volatile oils contents have a broad spectrum of activity against insect and mite pests, plant pathogenic and other fungi, and nematodes etc. [5].

As all are aware that the volatile oils are characteristic to that of the plant material, and as per the reported data, essential oil the oil of pepper from P. nigrum fruits consist of terpenes, l-phellandrene, caryophyllene, piperonal, dihydrocarveol, caryophellene oxide, cryptone, α-pinene, β-pinene, dl-limonene, piperidine, citronellol, phenyl acetic acid and other oxygenated compounds [6-8].The volatile oil of Piper longmun contained n-hexa, n-hepta, n-octa, n-nonadecane, n-eicosane, n-heneicosane, α-thujene, terpinolene, zingiberene, p-cymene, p-methoxy acetophenone, dihydrocarveoll, phenyl ethyl aclcohol; the presence of esters of acetic acid, caprylic caid and trace of phenols were reported [5]. Likewise the volatile oil of Zingiber officinale contained sesquiterpene hydrocarbons such as zinziberene, ar-curcumene, farnesene and smaller amounts of β-bisbolene, β-elemene, β- sesquiphillandrene, sesquiterpene alcohols such as zingiberol etc; monoterpene hydrocarbons such as camphene, α-pinene, β-pinene, cumene, myrecene, limonene, p-cymene, β-phyllanedrene; the oxygenated monoterpene hydrocarbons and associated compounds such as 2-heptanol, 2-nonanol, n-nonanal, n-decanal, methyl heprenone, 1, 8- cineol, borneol bornyl acetate, linalool, geranial and neral were reported [10-13].

In the standardization path way, the identification and verification of raw botanicals by certain set of the methods and procedures are followed. Among them comparing finger print profiles of ingredients and formulation using various chromatographic techniques are very much useful for quality control of herbal/traditional medicines. Present study aimed to evaluate the identity and quality of formulation through volatile oil content of ingredients along with finished product.

Experimental:

Collection of plant materials:

The raw botanicals namely Piper nigrum L. (fruit), Piper longum L. (fruit) and Zingiber officinale Roscoe. (rhizome) were procured from the local market, Chennai. The single drugs or plant raw materials were identified and authenticated by the Botany Department of CSMRADDI, Chennai.

Preparation of Trikatu churna (TC):

The each plant material of three ingredients was separately ground into fine powders. Equal quantities of each fine powder of Piper nigrum, Piper longum and Zingiber officinale were thoroughly mixed and then sieved through muslin cloth to get the fine powdered churna formulation as per procedure given in Ayurvedic Formulary of India, Part I, 7:14 [14].

Extraction of Volatile oil content:

Weigh 25 g each test sample of the Trikatu churna powder and powders of its three ingredients namely Piper nigrum, Piper longum, Zingiber officinalis were taken separately in 500 ml round bottom flask. Added water till the sample is completely immersed (150 to 200 ml), Clevenger′s apparatus with the double walled condenser was fixed. The hydro distillation was carried out by keeping the temperature at 80-90 ^oC in the heating mantle to continue distillation for 3 to 4 hours. The volume of the oil collected in the graduated tube of the Clevenger′s apparatus was observed to measure and collect [15].

Test Solution:

The each extracted volatile oil of the formulation Trikatu churna and its three ingredients were dissolved in n-hexane separately and made up to the 10 ml mark of 10 ml volumetric flask.

Instrumentation:

A CAMAG HPTLC system (Muttenz, Switzerland) equipped with a sample applicator TLC autosampler 4, twin trough plate development chamber, TLC Scanner 3, win CATS software version 1.4.4. and Hamilton (Reno, Nevada, USA) Syringe. HPTLC studies were carried out per reported methodology [16-18].

HPTLC method:

20 μl of the each volatile sample extract was applied on E. Merck aluminium plate pre-coated with silica gel 60 F₂₅₄of 0.2 mm thickness and the plate was developed in Toluene: Ethyl acetate (9: 1) dried the plate and kept under UV 254 nm and 366 nm and TLC profiles were documented. The TLC plate developed above, before dipping in vanillin-sulphuric acid was scanned at 254 nm using Scanner-3, Camag HPTLC instrument using Deuterium lamp. The plate was then dipped in Vanillin-sulphuric acid reagent and heated in a hot air oven at105^oC until the colour of the spots appeared and profile photo was documented under white light.

RESULTS AND DISCUSSION:

According to WHO guidelines, the quality control process of Traditional medicines and polyherbal formulations, verification and identity confirmation is a key component and it can be achieved by evaluating certain test parameters such as macro and microscopical, physicochemical and phytochemical analysis and fingerprint profiles using various chromatographic techniques such as HPTLC, HPLC, LC-MS and GC-MS etc.[19-20]. In the present study, the ingredients were identified and verified for their occurrence in the formulation with the help of volatile oil content. It is the key feature as it is the characteristic nature to that of raw botanical ingredient of the finished product and it is the first report for evaluation of quality of the formulation with the help of volatile oils profile. It is an additional way of quality assurance where the volatile oil contents are present with the raw ingredients in the course of quality control and standardization. Development of chromatographic finger print profiles using HPLTC comparative to other techniques is the easy and economic way for the verification of the presence and/or absence of the raw botanical ingredients. It is the optimal way to determine the identity and purity of ingredients with respective to their extracts.

Volatile oil content:

25 g each test sample powder was carried out the hydro distillation process and collected the volatile oil contents as shown in the (Table 1).

Table 1 Volatile oil content

S. No	Test Sample	Volatile oil content (% w/w)
1	Trikatu churna	0.33
2	Piper nigram	2.23
3	Piper logum	0.82
4	Zingiber officinale	0.26

TLC and HPTLC Fingerprint Profile:

The TLC plate was developed in Toluene: Ethyl acetate (9: 1) mobile phase, air dried, observed under visualize and documented the fingerprint profiles of UV 254 nm, 366 nm and under white light for the derivatized plate in vanillin in sulphuric acid (Figure 1).

Table 2 TLC observation and R_f values

Wave length	Track-1	Track-2	Track-3	Track-4
UV 254 nm	0.02	0.05	-	0.16
	0.09	0.10	0.08	-
	-	0.2,	-	-
	-	0.3,	-	-
	-	0.36	-	0.43
	0.63	0.61	0.58	0.59
	-	0.66	-	-
	-	0.85	0.84	-
	0.93	0.93	0.92	0.92
UV 366 nm	-	-	-	0.15
	-	-	-	0.36
	0.53	0.51	0.50	0.50

	-	0.65	-
	-	0.85	0.84
	0.93	0.93	0.92	0.91
White-light/ Visible range [after-Derivatization]	0.04	0.05	-	0.03
	0.09	0.10	0.08	-
	-	-	0.14	0.14
	0.18	0.19	0.18	0.17
	0.26	0.26	0.24	0.24
	0.30	0.30	0.28, 0.33	0.29
	0.36	0.35	0.36
	0.38	0.38
	0.42	0.41	0.39	0.39
	0.48	0.47	0.45	0.44
			0.54
	0.59	0.58	0.58	0.58
	0.66		0.62
		0.73	0.75	0.76
		0.86	0.85
	0.92	0.94	0.93	0.93

In TLC profile developed under UV 254 nm for the volatile oil of TC formulation on Track-3 (Figure 1) appeared with four prominent bands with R_f values of 0.08, 0.58, 0.84 and 0.92; It was found the similar bands with the volatile oil of Piper nigrum on Track-1. In case of volatile oil of Piper longumn on Track-2, the prominent bands observed at 0.10, 0.61, 0.85 and 0.93 R_f values are found similar with that of volatile oil of TC formulation. Similarly in case of volatile oil of the Zingiber officinale was found the bands with R_f values of 0.59 and 0.92 simailar to that of volatile oil of T.C on Track-3. In the same manner the TLC profiles of volatile oil of ingredients are compared and found similarity with the volatile oil of TC under UV-366 nm and white light of derivatized plate in vanillin in sulphuric acid (Table 2 and Figure 1). It was observed more similarity was found in TLC profile of derivatized plate when compared to TLC profiles documented under UV 254 nm and 366 nm for volatile oils of TC and its ingredients.

The plate developed above, before dipping in vanillin-sulphuric acid was scanned at 254 nm using Scanner-3 to get the volatile oil fingerprint profile of HPTLC chromatograms (Figure 2 and 3) and observed that, the R_fof values with highest peak area and next to highest peak area of the Piper nigrum (0.82 and 0.91), Piper longum (0.11 and 0.86) and Zingiber officinale (0.92 and 0.58) found similar to that of the chromatographic profile of the essential oil content of TC with R_f values of 0.84, 0.92, 0.58 and 0.08. The similarity matching of profiles, assure the presence of above said ingredients in TC formulation and its quality.

CONCLUSION:

It is the first report for the evaluation of comparative fingerprint profiles of the volatile oil content of the formulation and with its ingredients essential oil or volatile oil content. It is one of the best ways to assess the quality of the finished formulation in the course of standardization. As shown in the results part the matching similarity profile was observed in both TLC finger print and HPTLC finger prints. The study has its future scope to verify the volatile contents of ingredients and formulations with the GC and/or GC-MS profiles in qualitative and quantitative manner to furthermore assure the quality of the finished formulation.

ACKNOWLEDGEMENT:

The authors are very grateful to the Director General and Deputy Director General, Central Council for Research in Ayurvedic Sciences, New Delhi, for his encouragement and providing opportunity to conduct this study.

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Received on 25.06.2018 Modified on 20.08.2018

Research J. Pharm. and Tech 2019; 12(1): 291-296.

DOI: 10.5958/0974-360X.2019.00054.4

UV 254 nm	UV 366 nm	Derivatized in Vanillin-sulfuric acid

T1 T2 T3 T4	T1 T2 T3 T4	T1 T2 T3 T4
Traks T1,T2,T3 and T4 are 20 µl volatile oils in of Piper nigram, Piper logum, Trikatu churna and Zingiber officinale.

Piper nigrum
Piper longum
Trikatu churna (TC)
Zingiber officinale