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 potency1.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 respectively 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 expensive11,12.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)

 


Figure 1  HPTLC Profile of methanol  extracts of herbal raw extracts and  markers after development  in mobile phase and visualised at 254 nm and 366 nm.

 


Pterocarpum marsupium Bark, and Ormocarpum cochinchinense leaf showed same Rf values as that of Rutin  0.14. Quercetin was detected by its Rf value 0.84 in Pterocarpum marsupium Bark, Ormocarpum cochinchinense leaf, Indigofera tinctoria leaf, Terminalia arjuna bark and Terminalia chebula fruit. Gallic acid was detected in Pterocarpum marsupium Bark with Rf value of 0.74. Mangiferin was detected in Indigofera tinctoria leaf with Rf value of 0.28.  Catechin was detected in Terminalia arjuna bark with Rf value of 0.72.

 

 


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%

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%

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 %

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.

 

REFERENCES:

1.          Pietta, PG. Flavonoids as antioxidants. Journal of Natural Products. 2000; S 63 (7): 1035–42.

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3.          Wanda C. Reygaert.   Green Tea Catechins: Their Use in Treating and Preventing Infectious Diseases BioMed Research International Volume 2018; 1-9.

4.          Jiayu Gao, Jiangxia Hu , Dongyi Hu , and Xiao Yang  A Role of Gallic Acid in Oxidative Damage Diseases: A Comprehensive Review  Natural Product Communications  2019;1–9.

5.          Jyotshna Puja Khare and Karuna Shanker. Mangiferin: A review of sources and interventions for biological activities 2016; 42(5):504–514.

6.          Yao Li et al Quercetin, Inflammation and Immunity Nutrients 2016; 8: 167.

7.          Miao He et al. A review on the Pharmacological effects of vitexin and isovitexin.  Fitoterapia   2016; 115: 74-85.

8.          Zhao, Zhaohui; Moghadasian and Mohammed H."Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: A review". Food Chemistry 2008; 109 (4): 691–702. 

9.          Kumar, Naresh Pruthi and Vikas. "Potential applications of ferulic acid from natural sources". Biotechnology Reports.  2014; 4: 86–93.

10.        187 Fake Cancer 'Cures' Consumers Should Avoid". U.S. Food and Drug Administration Archived from the original   2018

11.        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): 58-61.

12.        Meier B, Spriano D. Modern HPTLC--a perfect tool for quality control of herbals and their preparations. J AOAC Int.    2010; 93(5):1399-409.

13.        Arivukkarasu R, and Rajasekaran A. Detection of flavonoids, phenolic acids and xanthones in commercial herbal formulations by HPTLC technique. Research Journal of Pharmacognosy and Phytochemistry 2015;76 (1):19-24.

 

 

 

 

 

Received on 06.05.2021            Modified on 11.05.2021           

Accepted on 13.05.2021      ©Asian Pharma Press All Right Reserved

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

DOI: 10.52711/2231-5713.2021.00021