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 three commercial herbal raw materials obtained from Rutaceae family in daily domestic needs as antioxidants, antidiabetic, antipyretic, ulcer healing, diuretic, anti-fertility and anti-inflammatory agent and dysentery, constipation and 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 7g/kg in orange and lime trees respectively² Catechin is a flavan-3-ol, a type of natural phenol and antioxidant³ 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 infections⁴.

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^5,6. 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 diseases⁷. 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'⁸. 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 permeability⁹. 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 effects¹⁰.

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 diuretic¹¹. 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^12,13. There is no simultaneous HPTLC method is reported in single mobile phase in the literatures for identification of eight phytoconstituents for the selected herbal raw materials from rutaceae 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:

Three 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 Three herbal raw materials were Aegle marmelos, Citrus aurantifolia and Murraya koenigii. 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, Catechin, Quercetin, Gallic acid, Ellagic acid, Ferulic acid, Vitexin 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 GF₂₅₄ (0.2 mm).

Preparation of standards and extracts from the herbal raw materials:

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

Application of sample:

The sample solutions were spotted in the form of bands of width 6mm with a Hamilton 100μl syringe on precoated plate 60 F254 (10cm × 10cm with 0.2mm m thickness, E. Merck) using a Camag Linomat V applicator. The slit dimension was kept 6mm × 0.45mm. Eight µl of each sample and five µl of standard solutions were applied on to the plate. The migration distance was 80mm. TLC plates were dried with air dryer. Densitometric scanning was performed using Camag TLC Scanner-3 at 254nm and 366nm operated by a wincat software.

Development:

The chromatogram was developed in CAMAG glass twin-through chamber (10-10cm) 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 366nm 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 components¹⁴ 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 0.79. (Table 1) Aegle marmelos, Citrus aurantifolia and Murraya koenigii showed same Rf values as that of Rutin 0.15. Xanthone was detected in the Murraya koenigii with Rf value of 0.23. Rf value of standard marker Ellagic acid coincided with Rf value 0.68 of Aegle marmelos and Citrus aurantifolia. A Common flavonoid vitexin was identified by its Rf value 0.41 in Citrus avantifolia. Catechin was detected in Murraya koenigii matching the Rf Value 0.64. Quercetin was detected by its Rf value 0.77 in Aegle marmelos, Citrus aurantifolia and Murraya koenigii. The detection and quantity of marker inherbal raw material extracts were giving in Table 1 The identity of components in herbal extracts was ascertained by chromatogram Fig. 1 and 2 and overlaying their absorption spectra with standards (Fig. 3 and 4)

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.

1. Aegle marmelos leaf methanol ext 2. Citrus aurantifolia root methanol ext 3. Murraya koenigii leaf ext 4. Quercetin, Rutin, Gallic acid 5. Catechin 6. Ellagic acid 7. Ferulic acid 8. Vitexin

9. Mangiferin

Table 1: Rf values of standard markers in extracts of Aegle marmelos, Citrus aurantifolia and Murraya koenigii

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	Aegle marmelos Leaf Methanol extract/8 µl	0.06,0.18,0.33,0.47,0.55,0.62,0.70,0.75,0.79 and 0.90.	0.18	Rutin	20755.5	5.80	0.072%
			0.70	Elagic acid	543.9	0.38	0.004%
			0.79	Quercetin	6272.2	2.76	0.034%
T-2	Citrus aurantifolia root methanol extract /8 µl	0.06,0.10,0.17, 0.29,0.39,0.51, 0.70,0.78, and 0.86	0.17	Rutin	1709.2	0.478	0.005%
			0.39	Vitexin	2391.1	1.52	0.019%
			0.70	Elagic acid	540.2	0.37	0.004%
			0.78	Quercetin	6231.4	4.96	0.062%
T-3	Murraya koenigii leaf ext /8 µl	0.11,0.17,0.19, 0.22,0.34,0.37,0.55,0.64,0.71,0.77 and 0.88	0.17	Rutin	760.1	0.2126	0.002%
			0.22	Mangiferi	918.2	0.1827	0.002%
			0.64	Catechin	78.7	0.120	0.001%
			0.77	Quercetin	3830.6	1.689	0.021%
T-4	Quercetin / 5 µl	0.77			11333.3	5.0	100%
T-4	Gallic acid/ 5 µl	0.65			14268.5	5.0	100%
T-4	Rutin/ 5 µl	0.15			17875.5	5.0	100%
T-5	Catechin/ 5 µl	0.64			3277.4	5.0	100%
T-6	Elagic acid/ 5 µl	0.68			7222.5	5.0	100%
T-7	Ferulic acid/ 5 µl	0.78			11436.3	5.0	100%
T-8	Vitexin/ 5 µl	0.41			7814.2	5.0	100%
T-9	Mangiferin/ 5 µl	0.23			25119.8	5.0	100%

Figure 2: Chromatogram of Raw materials extracts and Standard markers


Figure 3: Overlay of Ferulic acid in *Citrus aurantifolia* and *Murraya koenigii*	Figure 4: Overlay of Rutin in *Aegle Marmelos*

CONCLUSION:

The findings can be concluded that flavonoids, phenolic acids and xanthones were detected in the three herbal extracts. Presence of rutin was confirmed in all three herbal extracts. Ellagic acid was attendance in Aegle marmelos and Citrus aurantifolia. Mangiferin and Catechin was detected in the Murraya koenigii. Vitexin was well-known in Citrus avantifolia. Quercetin was detecting in Aegle marmelos, Citrus aurantifolia and Murraya koenigii. The developed HPTLC method may be adopted for routine detection of flavonoids, phenolic acids and xanthones in the herbal extracts.

REFERENCES:

1. Bauer R et al. Pharmaceutical quality, standardization and normalization of phytopharmaceuticals (in German). Z Phytother. 1994; 15: 82–91.

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

3. Krewson CF, and Naghski J. Some physical properties of rutin. Journal of the American Pharmaceutical Association. 1952; 41 (11): 582–7.

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

5. 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.

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

7. 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.

8. 187 Fake Cancer 'Cures' Consumers Should Avoid". U.S. Food and Drug Administration Archived from the original 2018.

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

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

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

12. 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.

13. 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.

14. 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 11.01.2021 Revised on 05.02.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):151-155.

DOI: 10.52711/2231-5675.2021.00026