Fingerprint Analysis of Herbal Raw Materials available in Market Belongs to Combretacea And Fabaceae family by HPTLC technique using antioxidant markers

Ramasamy Arivukkarasu^*, Aiyalu Rajasekaran

KMCH College of Pharmacy, Coimbatore, Tamilnadu, India

*Corresponding Author E-mail: phytoarivu@gmail.com

ABSTRACT:

The prime aim of the study is to notice the flavonoids, phenolic acids and xanthones in five commercial herbal raw materials namely Pterocarpum marsupium Bark, Ormocarpum cochinchinense leaf, Indigofera tinctoria leaf, Terminalia arjuna bark and Terminalia chebula fruit obtained from Fabaceae and Combretacea family used in daily domestic needs to confirm the presence of common antioxidant secondary metabolites in herbal raw materials. Results of the study clearly revealed that these raw materials from Fabaceae and Combretacea family contains flavonoids, phenolic acids and xanthones. The developed simultaneous HPTLC method can be employed for the routine investigations of flavonoids, phenolic acids and xanthones in herbal raw materials. Pterocarpum marsupium Bark, and Ormocarpum cochinchinense leaf, was ascertain the presence of Rutin and reveals 0.006%,0.051% respectively. Gallic acid was only orginate in Pterocarpum marsupium Bark of about 0.113%. Quercetin was found to be 0.061%, 0.239%, 0.336%,0.066% and 0.036% respectively in all tested five extracts. Catechin was found to be 0.203% in Terminalia arjuna bark. Mangiferin was found to be 0.009%. in Indigofera tinctoria leaf.

KEYWORDS: Rutin, Gallic acid, Quercetin, Catechin, Mangiferin, HPTLC.

INTRODUCTION:

The modern scientific community has presently recognized flavonoids to be a unique class of therapeutic molecules due to their diverse therapeutic properties. 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.The primary aim of the study is to detect the flavonoids, phenolic acids and xanthones in five commercial herbal raw materials obtained from Fabaceae and Combretacea family in daily domestic needs as antioxidants, antidiabetic, antipyretic, diuretic, anti fertility, ulcer healing, anti-inflammatory agent dysentery, constipation, diarrhea, relieves morning sickness, nausea, eliminates bacteria, heals wounds, burns and Reduces stress.

Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is a flavonol, abundantly found in plants, Citrus leaves contain rutin at concentrations of 11 and 7 g/kg in orange and lime trees respectively¹ Catechin is a flavan-3-ol, a type of natural phenol and antioxidant² Catechins have been shown to demonstrate a variety of antimicrobial properties. Consumption of green tea has been shown to distribute catechin compounds and/or their metabolites throughout the body, which allows for not only the possibility of treatment of infections but also the prevention of infections³.

Gallic acid is main ingredient and responsible for pharmacological mechanisms in the pathophysiological process of the oxidative damage diseases, such as cancer, cardiovascular, degenerative and metabolic diseases.⁴

Mangiferin is a C-glycosyl compound consisting of 1,3,6,7-tetrahydroxyxanthen-9-one having a beta-D-glucosyl residue at the 6-position.Mangiferin and its derived lead molecule have proven its effectiveness as an antioxidant, analgesic, antidiabetic, antiproliferative, chemopreventive, radioprotective, cardiotonic, immunomodulatory and diuretic⁵.

Quercetin, a polyphenol derived from plants, has a wide range of biological actions including anti-carcinogenic, anti-inflammatory and antiviral activities; as well as attenuating lipid peroxidation, platelet aggregation and capillary permeability⁶ .

Vitexin (apigenin-8-C-glucoside) has recently received increased attention due to its wide range of pharmacological effects, including but not limited to anti-oxidant, anti-cancer, anti-inflammatory and neuroprotective effects⁷.

Ferulic acid is an organic compound found in the cell walls of certain plants. Rich in antioxidants such as vitamin A, vitamin C, and vitamin E. Ferulic acid is most often found in anti-aging skin creams, where is it believed to neutralize free radicals that damage and age cells ^8,9.

Ellagic acid has been marketed as a dietary supplement with a range of claimed benefits against cancer, heart disease, and other medical problems. Ellagic acid has been identified by the U.S.Food and drug Aministration as a "fake cancer 'cure'^10.

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^11,12.There is no simultaneous HPTLC method is reported in single mobile phase in the literatures for identification of seven phytoconstituents for the selected herbal raw materials from Fabaceae and Combretacea family and hence this paper describes detection of flavonoids, phenolic acids and xanthones in raw materials by HPTLC method.

MATERIALS AND METHODS:

Collection of herbal raw materials for HPTLC screening

Five herbal raw material were procured from the traditional siddha practitioner (Bala vidayambigai siddha hospital, Bommidi small town in Dharmapuri district of Tamil nadu, India ) who is using this material for various herbal formulations. The five herbal raw materials were Pterocarpum marsupium Bark, Ormocarpum cochinchinense leaf, Indigofera tinctoria leaf, Terminalia arjuna bark and Terminalia chebula fruit obtained from Fabaceae and Combretacea family. The traditional medical practitioners use this raw material for various formulations like skin care chooranam, Expectorant Chooranam, anti-histamine chooranam, antidiabetic chooranam, anti inflammatory Chooranam, Blood purifier chooranam, Blood pressure reducing chooranam and blood sugar reducing chooranam.

Equipment:

A CAMAG HPTLC system comprising of a Linomat-5 applicator and CAMAG TLC Scanner-3 and single pan balance of Shimadzu model was used, for weighing the samples.

Chemicals and solvents

Rutin, Gallic acid, Quercetin, Catechin, Vitexin, Mangiferin, Ellagic acid and Ferulic acid 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 GF₂₅₄ (0.2 mm).

Preparation of standards and extracts from the herbal raw materials

One gram of each dried powdered material 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/1 ml.

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 5 mm × 0.45 mm. Eight µl of each sample and five µl of standard solutions were applied on to the plate. The migration distance was 80 mm. TLC plates were dried with air dryer. Densitometric scanning was performed using Camag TLC Scanner-3 at 254 nm and 366 nm operated by a wincat software.

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 °C, relative humidity 40%). The development was done for 8 cm from bottom.

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 monitor the elution of components¹³ in herbal extracts:

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

· Ethyl Acetate: Methanol: Water Toluene (100:13:10:13)

· Chloroform: ethyl acetate: methanol (6:4:0.3)

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

· Ethyl acetate: methanol: water (100:15.5:13.5)

· Chloroform: ethyl acetate: formic acid (6:4:0.3)

· Toluene: ethyl acetate: formic acid: methanol (3:6:1.6:0.4)

· Ethyl acetate: methanol: water (100:13.5:10)

· Toluene: ethyl acetate (93:7)

Among the 9 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 extracts tested and hence it was used as mobile for detection of constituents in herbal extracts.

The optimized chamber saturation time for mobile phase was 10 min at room temperature (25 ± 1°C). The densitometric analysis was performed at 254 nm in reflectance mode. The Rf values of the marker compounds were in the range of 0.09 to 0.84.(Table 1) The detection and quantity of marker in herbal raw material extracts were given in Table 1. The identity of components in herbal extracts was ascertained by chromatogram (Figure 1)

Table 1 Rf values of standard markers in extracts of Pterocarpum marsupium, Ormocarpum cochinchinense ,Indigofera tinctoria, Terminalia arjuna and Terminalia chebula

Track Number	Name / Amount of Sample in µl	Rf values of compounds in extracts/Standards	Rf value of the marker in extracts	Name of marker in extracts	Area of Stan dard Marker in sample	Amount of marker present in µg/ 8 µl of extracts/ 5 µl of standards	% of marker in Extracts
T-1	Pterocarpumarsupium Bark Methanol extract / 8 µl	0.06,0.14,0.21,0.29, 0.35,0.41,0.44,0.48,0.55,0.68,0.74, 0.81and 0.84.	0.14	Rutin	277.7	0.0545	0.006%
			0.74	Gallic acid	3042.5	0.9055	0.113%
			0.84	Quercetin	1715.6	0.4953	0.061%
T-2	Ormocarpum cochinchinense leaf methanol extract /8 µl	0.07,0.13,0.18, 0.30,0.36,0.45,0.52,0.54,0.64,0.76,0.79,0.83,0.86 and 0.89	0.13	Rutin	2109.7	0.4142	0.051%
T-2	Ormocarpum cochinchinense leaf methanol extract /8 µl		0.83	Quercetin	6436.4	1.9156	0.239%
T-3	Indigofera tinctoria leaf Methanol extract /8 µl	0.01,0.21,0.28, 0.37,0.59,0.64,0.77,0.79,0.83 and 0.87	0.28	Mangiferin	429.7	0.0725	0.009%
T-3	Indigofera tinctoria leaf Methanol extract /8 µl		0.83	Quercetin	9328.0	2.693	0.336%
T-4	Terminalia arjuna bark Methanol extract /8 µl	0.07,0.36,,0.53,0.59,0.72,0.81, and 0.84.	0.72	Catechin	6571.9	1.628	0.203 %
T-4	Terminalia arjuna bark Methanol extract /8 µl	0.07,0.36,,0.53,0.59,0.72,0.81, and 0.84.	0.84	Quercetin	1854.0	0.5353	0.066%
T-5	Terminalia chebula fruit Methanol extract /8 µl	0.05,0.25, 0.31,,0.45,0.65,0.73,0.80, and 0.84.	0.84	Quercetin	1017.5	0.2937	0.036%
T-6	Rutin/ 5 µl	0.15			25462.3	5.0	100%
	Gallic acid/ 5 µl	0.74			16799.8	5.0	100%
	Quercetin / 5 µl	0.83			17317.0	5.0	100%
T-7	Catechin	0.71			20179.4	5.0	100%
T-8	Ferulic acid/ 5 µl	0.84			13964.4	5.0	100%
T-9	Vitexin/ 5 µl	0.42			9362.7	5.0	100%
T-10	Mangiferin/ 5 µl	0.26			29602.0	5.0	100%

CONCLUSION:

The findings can be concluded that flavonoids, phenolic acids and xanthones were detected in the five herbal extracts. Presence of quercetin was confirmed in all five herbal extracts. Gallic acid was attendance in Pterocarpum marsupium Bark. Mangiferin and Catechin was detected in the Indigofera tinctoria leaf and Terminalia arjuna bark respectively. Rutin was detect in Pterocarpumarsupium Bark and Ormocarpum cochinchinense leaf. The developed HPTLC method may be adopted for routine detection of flavonoids, phenolic acids and xanthones in the herbal extracts.

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Received on 06.05.2021 Modified on 11.05.2021

Asian Journal of Pharmacy and Technology. 2021; 11(2):130-134.

DOI: 10.52711/2231-5713.2021.00021