Simultaneous detection of Rutin, Quercetin, Gallic acid, Caffeic acid, Ferulic acid, Coumarin, Mangiferin and  Catechin   in Hepatoprotective  commercial  herbal formulations by HPTLC technique

 

Ramasamy Arivukkarasu*, Aiyalu Rajasekaran, Syed haffan bin hussain, Mohammed Ajnas

KMCH College of Pharmacy, Coimbatore, Tamilnadu, India

 

 

ABSTRACT:

The aim of the study is to detect simultaneously in single mobile phase the rutin, quercetin, gallic acid, caffeic acid, ferulic acid, coumarin, mangiferin and catechin in hepatoprotective commercial herbal formulations. HPTLC method was adopted to confirm the presence of these common secondary metabolites in the tested commercial herbal formulations. Results of the study clearly revealed the presence of rutin, quercetin, caffeic acid, ferulic acid, mangiferin responsible for hepatoprotective activity.The developed HPTLC method can be employed for the routine investigations of abov revealed markers in commercial herbal formulations.

 

 

 

INTRODUCTION:

Standardization of herbal formulation in terms of raw materials, manufacturing practices and composition is important to ensure quality and  optimum level of active principles for their bio potency [1]. Identification of major and unique compounds in herbs as markers and development of analytical methodologies for  monitoring them are the key steps involved in marker based standardization. High performance thin layer chromatography (HPTLC) is a preferred analytical tool for  fingerprints and quantification of marker compounds in herbal drugs due to its simplicity, high sensitivity, accuracy and less expensive [2]. Rutin  prevented the CCl₄-induced prolongation in pentobarbital sleeping time confirming its hepatoprotectivity [3].

 

Caffeic acid and quercetin, the well-known phenolic compounds widely present in the plant kingdom, were investigated for their possible protective effects against paracetamol and CCl₄-induced hepatic damage. Paracetamol at the oral dose of 1 g/kg produced 100% mortality in mice while pretreatment of separate groups of animals with caffeic acid (6 mg/kg) and quercetin (10 mg/kg) reduced the death rate to 20% and 30%, respectively[4]. Ferulic is a phenolic compound possessing antioxidant, anticancer, and antiinflammatory activities.  They have investigated the hepatoprotective effect of Ferulic acid against carbon tetrachloride (CCl₄)-induced acute liver injury. Ferulic acid protects from CCl₄-induced acute liver injury through reduction of oxidative damage and inflammatory signaling pathways[5]. With the above background we are plan to detect and quantify the  hepatoprotective markers like rutin, quercetin ,caffeic acid, ferulic acid, and mangiferin in hepatoprotective  commercial  herbal formulations by HPTLC technique.No HPTLC method is reported in the literature for identification of phytoconstituents for the   these selected  commercial formulations and hence this paper describes detection  and quantification of rutin, quercetin, caffeic acid, ferulic acid, and mangiferin in hepatoprotective commercial  herbal formulations by HPTLC technique.

 

MATERIALS AND METHODS:

Collection of commercial herbal  formulations for HPTLC screening:

Two formulations were procured from the market. Both the formulations were tablet dosage forms namely   Liv -52  and Tefroliv forte tablets. The ingredients of two  commercial herbal formulations are provided in Table 1. Organoleptic evaluation of formulations were carried out and tabulated in Table 2.

Table 1 Ingredients present in  two selected formulations

Liv-52	Tefroliv forte
Himsra   (Capparis spinosa)	Phyllanthus niruri
Kasani  (Chichoriumintybus)	Tephrosia purpurea
Kakamachi (Solanum nigrum)	Eclipta alba
Arjuna (Terminalia arjuna)	Andrographis paniculata
Kasamarda (Cassia occidentalis)	Picrorhiza kurroa
Biranjasipha (Achilleamille folium)	Solanum nigrum
Jhavuka (Tamarix gallica)	Piper longum
	Terminalia chebula
	Ocimum sanctum

 

 

 

 

 

Table 2: Organoleptic evolution and pH of formulations

Name of the formulation	Colour	Odour	Nature of particles	Taste	pH of the 1% solution of formulation
Liv 52	Brown	Aromatic	Fine	Aromatic and mucilaginous	6.1
Tefroliv forte	Brownish green	Aromatic	Fine	Aromatic and astringent	5.8

 

Equipment: A Camag HPTLC system comprising of Linomat 5 applicator and  Camag TLC scanner and single pan balance of Shimadzu model was used, for weighing the samples.

 

Chemicals and solvents: Rutin, gallic acid caffeic acid feulic acidvitexin,and mangiferin were procured from Sigma Chemical Company Inc., USA. Solvents for extraction were purchased from Qualigens fine chemical (P) limited Mumbai. HPTLC was carried out using Merck aluminium sheet coated with silica gel GF 254  (0.2 mm).

 

Preparation of standards and extracts from the commercial herbal  Formulations:

One gram of the each formulation was taken and sonicated  with 10 ml of methanol. Filtered and the filtrate solution was used  for HPTLC analysis. Standard marker compounds were prepared using methanol to get a concentration 1 mg/1ml

 

Application of sample: The sample solutions were spotted in the form of bands of width 6 mm with a Hamilton 100 μl syringe on precoated plate 60 F254 (10 cm × 10 cm with 0.2 mm m thickness, E.Merck) using a Camag Linomat V applicator. The slit dimension was kept mm × 0.45 mm. Five μl of each sample and standard solutions were applied on to the plate.

 

Development: The chromatogram was developed in Camag glass twin -through chamber (10-10 cm) previously saturated with the  mobile phase toluene: ethyl acetate: formic acid: methanol [3:6:1.6:0.4] for 10 min (temperature 25.2°C, relative humidity 40%). The migration distance was 80 mm. TLC plates were air  dried with air dryer. Densitometric scanning was performed using Camag TLC Scanner ‑III at 254 nm and 366 nm  operated by a Wincat software[7].

 

Detection:The plate was scanned at UV 254 and 366 nm using Camag TLC Scanner-3 and Linomat V. Rf value of each compound which were separated on plate and data of peak area of each band was recorded.

 

RESULTS AND DISCUSSION:

The following different solvent compositions were tried for monitoring the elution of components in herbal formulations [6]

 

Ethyl acetate:glacial acetic acid formic acid:water (100:3:3:28)

1.     Ethyl acetate: Methanol: Water: Toluene (100: 13: 10: 13)

2.     Chloroform: Ethyl acetate : Methanol (6: 4: 0.3)

3.     Ethyl Acetate:Methanol :Water Toluene (100: 15.5: 13.5: 2)

4.     Ethyl acetate: Methanol: Water (100:15.5:13.5)

 

Among the 5 mobile phases attempted, Toluene: ethyl acetate: formic acid: methanol in the ratio of 3:6:1.6:0.4 gave better elution for all the formulations tested and hence it was used as mobile  phase for detection of constituents in herbal formulations. The optimized chamber saturation time for mobile phase was 3.0 min at room temperature (25 ± 1°C). The densitometric analysis was performed at 256 nm in reflectance mode [7].

 

The Rf values of the marker compounds were in the range of 0.09 to 0.79 (Fig 1, Fig 2 and Table 3).Liv-52  showed  Rf  values  same as that of rutin 0.09, mangiferin 0.17 and caffeic acid 0.66 .Tefroliv forte  showed Rf value equal to quercetin 0.56 and Ferulic acd 0.69 .The chromatogram of the Liv-52 and Tefroliv forte were shown in the Fig 3  to Fig 9. Catechin was absent in both the formulations. In the present paper the  ideal  method, first time we followed was simultaneously detection of standard markers rutin, quercetin, gallic acid, mangiferin and catechin present in hepatoprotective  commercial  herbal formulations by HPTLC technique.

 

 

 

 

 

Fig 1. TLC Profile methanol extracts of marketed and traditional

formulations at 254 nm.

1.Coumarin 2.Liv-52 3.Rutin gallic acid and  Quercetin 4.Caffeic acid  5.Tefroliv forte 6.ferulic acid 7. Traditional formulation  8.mangiferin 9. Traditional formulation.

 

 

 

Fig 2. TLC Profile methanol extracts of marketed and traditional

formulations at 366 nm.

1.Coumarin 2.Liv -52   3.Rutin gallic acid and  quercetin 4.caffeic acid  5.tefroliv forte6.ferulic acid 7.Traditional formulation  8.mangiferin 9.Traditional formulation

 

Fig 3 Chromatogram of  commercial Formulation Liv -52 detection of caffeic acid

 

 

Fig 4.Chromatogram of  commercial Formulation Liv -52 detection of rutin and mangiferin

 

 

 

Fig  5 Chromatogram of  Commercial Formulation  Tefroliv forte detection quercetin and ferulic  acid.

 

Fig 6 Chromatogram of standard  simultanoeus developed  rutin,quercetin and gallic acid

 

 

Fig  7 Chromatogram of standard  caffeic acid

 

Fig 8  Chromatogram of standard  mangiferin and catechin

 

 

Fig 9  Chromatogram of standard   ferulic acid

 

 

Table 3: Rf values of marker compounds and the constituents in commercial/traditional herbal formulations

Markers	Marker Rf value	Liv  -52	Tefroliv forte	Area of Marker	Area of marker in sample	Amount of marker present µg/ 5 µl	Amount of Marker mg/ g	% of marker in formulation
Rutin	0.09	0.09	-----	9747.4	624.2	0.320	0.320	0.032%
Mangiferin	0.17	0.17	-----	11543.9	3156.3	1.366	1.366	0.13%
Quercetin	0.56	-----	0.56	13436.4	4140.5	1.54	1.54	0.15%
Caffeic acid	0.66	0.66	-----	17187.3	3391.0	0.986	0.986	0.098%
Ferulic acid	0.69	-----	0.69	24157.4	17113.4	3.542	3.542	0.35%

 

 

CONCLUSION:

It can be concluded that rutin, mangiferin, quercetin and ferulic acid  were simultaneously detected in the two commercial formulations. Presence  of  rutin, mangiferin and caffeic acid in Liv-52 and  quercetin  and ferulic acid in tefroliv forte and absence of coumarin gallic acid and catechin in both the formulation. The marker compound selected for  detection was well established for hepatoprotective activity. The developed HPTLC method may be adopted for routine detection of rutin, quercetin, gallic acid, mangiferin  ferulic acid ,caffeic acid and catechin in commercial  formulations by simultaneous detection..

 

REFERENCES:

1.     Ramasamy Arivukkarasu, Aiyalu Rajasekaran . Detection of Flavonoids, Phenolic Acids and Xanthones in Commercial Herbal Formulations by HPTLC Technique. Research Journal of  Pharmacognosy and Phytochemistry. 2015,7(1):9-24.

2.     Harish Chandra Andola, Vijay Kant Purohit. High Performance Thin Layer Chromatography  (HPTLC): A Modern Analytical tool for Biological Analysis. Nature and Science 2010, 8(10): 58-615.

3.     Khalid H. Janbaz,Sheikh A. Saeed,Anwar H. Gilani. Protective effect of  rutin on paracetamol-  and CCl₄-induced hepatotoxicity in rodents. Fitoterapia  2002,73 (7) ; 557-63.

4.     Khalid H. Janbaz,, Sheikh.A. Saeed,Anwar .H Gilani. Studies on the protective effects of caffeic Acid and quercetin on chemical-induced hepatotoxicity in rodents. Phytomedicine. 2004 ,11 (5); 424-430.

5.     Hyo-Yeon Kim  Juhyun Park,   Kwan-Hoo Lee,  Dong-Ung Lee, Jong-Hwan Kwak, Yeong Shik Kim,  Sun-Mee Lee. Ferulic acid protects against carbon tetrachloride-induced liver injury in mice.Toxicology. 282(3);104-11.

6.     Nurok D, Strategies for optimizing the mobile phase in planar  chromatography Chem.Rev 1989, 9: 363-757.

7.     Bhandari P, Kumar N, Gupta AP, Singh B, Kaul VK. A rapid RP-HPTLC densitometry method for simultaneous determination of major flavonoids in important medicinal plants. Journal of Separation Science 2007, 30(13):2092-6

 

 

 

 

Received on 07.04.2017          Modified on 11.07.2017

Accepted on 20.08.2017       ©A&V Publications All right reserved