VALIDATED SPECTROSCOPIC AND CHROMATOGRAPHIC TECHNIQUES FOR QUALITY CONTROL ANALYSIS OF APIGENIN IN HERBAL MEDICINES

VALIDATED SPECTROSCOPIC AND CHROMATOGRAPHIC TECHNIQUES FOR QUALITY CONTROL ANALYSIS OF APIGENIN IN HERBAL MEDICINES

Shweta B. Chougule, Arati B. Jivaje, V. S. Mannur and Shailendra S. Suryawanshi *

Department of Pharmaceutical Chemistry, KLE College of Pharmacy Belagavi, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India.

ABSTRACT: The herbal medicines have been largely used since form several thousands of years in all developing and developed countries in the primary health care of community and society. Flavonoids are the class of polyphenolic secondary metabolites, which are mainly found in plants. Apigenin is a flavonoid which shows several pharmacological activities and used effectively for the management and treatment of several diseases and disorders. Many herbal medicines and their preparation containing Apigenin are available in market and hence Quality Control and standardization of Apigenin is important and essential in ayurvedic industries. The scientific research articles published on analytical development and validation of Apigenin and its estimation in its extract, isolated form and other herbal or polyherbal formulations were reviewed. We have mainly focused on chromatographic and spectroscopic techniques for qualitative and quantitative analysis of Apigenin and presented in this review work. We conclude here that the spectroscopic and chromatographic methods play significant role in the quality control and standardization of Apigenin in herbal medicines.

Keywords: Apigenin, Flavonoid, Validation, Quality Control, Herbal Medicines

INTRODUCTION: Herbal medicinal plants have been recognised for millennia as a rich source of therapeutic agent for prevention of diseases and disorders and are highly respected all over the world. Nature has bestowed upon our land an abundance of herbal medicines in the form of plants. Among ancient civilisation, India has been known to be rich storehouse of herbal medicinal plants, treatment with herbal medicinal plants is considered as safe because there is no or least side effects.

More than three- quarters of the world’s population depends mainly on herbal medicinal plants for their health care needs. Plant-based medications are becoming more popular in both developed and developing countries as people realise that natural products have fewer side effects, non-toxic and readily available ¹. With the increasing use of herbal goods, a wide range of phytomedines, herbal medicine and their formulation are widely available in the market all around the world.

Increase in demand of herbal medicine has faith that all natural products are safe and effective. However, there are still some concerns are associated with herbal medicine concerning its pharmacognosy, standardization and Quality control of drugs and it has become a public health concern ². The herbal drug used in developing countries of the world lacks official quality control standards, there is no way to ensure that the quality of different batches is consistent. Quality control includes safety of herbal drugs, efficacy and toxicity in herbals and their interactions these quality control parameters need to be tested before the finished product is manufactured, marketed, and ingested by patients. Standardization of herbal formulation is required for the evaluation of high-quality medications. There are variety of tools available that can be used to both qualitative and quantitative measures of herbal medicine. To isolate, identify the phytoconstituents responsible for the medicinal potential of herbs/plants, sophisticated analytical techniques are continually being developed und upgraded. Mainly the spectroscopic and chromatographic methods play major role in quality control and standardization of herbal formulation ³.

Flavonoids are the class of polyphenolic secondary metabolites. They are the important class of natural products, widely observed in flowers, plants, tea, grains, bark, stem, roots, vegetables, fruits and wine. Flavonoids, being the main active ingredient, provide a variety of health-promoting pharmacological effects such as anti-allergic, anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic effects. Apigenin is a flavonoid that has been used as a traditional herbal medicine because of its anti-oxidant, anti-inflammatory, antibacterial and antiviral properties. Apigenin is also known for its anticancer properties, which can be used to treat a variety of cancers. It has great demand due to its safety and effectiveness ⁴.

Apigenin is a flavonoid, widely found in a variety of fruits, vegetables and plants. Vegetables such as parsley, oranges, onions, maize, tea, rice, wheat sprouts chamomile, wheat sprouts. Parsley and peppers are two most common sources of flavones containing Apigenin. Apigenin is a polyphenolic metabolite, chemically known as 4ˈ,5,7, -trihydroxy flavone ⁵.

MATERIALS AND METHODS: Numerous herbal medicines and their preparation containing Apigenin are available in market. Various scientific research articles published in quality control analysis and standardization of Apigenin in its extract, isolated from or any other herbal or polyherbal formulation is being reviewed in the present article, we have mostly focused on the chromatographic and spectroscopic methods. The details of methodological analysis and interpretation of results were presented as below:

Quality Control Analysis of Apigenin in Herbal Medicines by Spectroscopic and Chromato-graphic Techniques: Numerous research articles, data, and reports are published by scientist on spectroscopic and chromatographic method for quality control and standardization of herbal medicine were presented below and reported as follows:

Spectroscopic Techniques: Arun P. et. al., have reported validation of UV- method for quantification of Apigenin- hydrogenated Soy phosphatidylcholine complex. The UV spectra run in the range of 200-400nm and the maximum wavelength of Apigenin was observed at 335nm and 268nm.

They have used methanol as solvent system. Calibration curve was constructed using concentration range of 2-12µg/ ml at 335nm and 2-14µg/ml at 268nm. The reported method was not economical because they have used high percentage organic phase ⁶. Rathode P. et. al., have reported validation of UV- method for quantification of Apigenin in leaves extract of Parsley. In this method maximum wavelength of Apigenin was observed at 208nm. They have used methanol as solvent system in ratio of 50:50 % v/v. Calibration curve was constructed using concentration range of 0.5 to 12µg/ml ⁷.

Shetti P. et. al., have reported optimization of a validated UV-spectrophotometric methodology for assessment of Apigenin in bulk powder. In this method maximum absorbance wavelength of Apigenin was observed at 267nm. They have used methanol as solvent. The detector response for apigenin was linear in the sure concentration range of 2µg/ml-10µg/ml with relation coefficient of 0.9995. This reported method was found to be specific, selective linear, precise, rugged, reproducible for estimation of apigenin with %RSD values less than 2 ⁸. The detailed summery of spectroscopic standardization of Apigenin is presented in Table 1.

TABLE 1: SPECTROSCOPIC METHODS FOR ANALYSIS OF APIGENIN IN HERBAL MEDICINES

Chromatographic Techniques: Rajgopal S. et. al.,have reported reverse phase high- performance liquid chromatography (RP-HPLC) technique for concurrent determination of Apigenin and Luteolin in ethanol extract of Clerodendrum Serratum leaves. Method using C18G column (250mm×4.6mm i.d, 5µm)  and methanol- acetonitrile- acetic acid- orthophosphoric acid- water (40:20:0.05:0.05:40) as solvent system at flow rate of 0.6mL min^ˉ1  and detection wavelength at 352nm. LOD was observed 0.05µg/mL for Luteolin along with 0.6µg/ml for Apigenin. LOQ was observed 0.15µ/ml for Luteolin along with 1.8µg/ml for Apigenin ⁹. Rajshekaran A. et. al., have reported simultaneous quantification of Luteolin and Apigenin in method leaf extract of Bacopa monnieri Linn by HPLC. The separation is achieved on C₁₈ column (5µm, 200mm×4.6mm, id) and potassium dihydrogen phosphate buffer (20mM, pH 3-5 modified with ortho phosphoric acid, v/v) and acetonitrile as solvent system with flow rate of 1ml/min and detection wavelength achieved at 348nm.

LOD was established 0.03µg/ml for Luteolin and 0.04µg/ml for Apigenin. LOQ was established 0.91µg/ml for Luteolin along with 0.13µg/ml for Apigenin ¹⁰. Che Zain MS. et. al., have reported ultra-high performance liquid chromatography- ultraviolet/ photodiode array(UHPLC-UV/ PDA) methodfor simultaneous estimation of Luteolin and Apigenin derivatives from leaf extracts of Elaeis guineensis. method is carried out using BEH C₁₈ column with 1.7µm particle size, 100mm length and 2-1 mm internal diameter for separation and the elution of compound was assisted with two mobile phase water 0.1% formic acid and 0.063% ammonium formate as solvent A and 0.1% formic acid and acetonitrile as solvent B, at flow rate of 0.40mL/min ¹¹. Subramanian G. et. al., have reported RP-HPLC technique for concurrent quantification of Luteolin and Apigenin from Achillea millefolium linn. Method using Hibar Lichrospher C₈ column with ultraviolet detector for separation at a wavelength of 269nm and methanol (20:80 v/v) with 0.5% trifluoracetic acid as solvent systemat flow rate of 1.0ml/min ¹². Gudzenko A. et. al., have reported simultaneous determination of Luteolin and Apigenin in Achillea Millefolium L. herb RP-HPLC method. Method using waters x-terra C₁₈ column (250×4.6nm i.d, 5µm particle size) for separation. Mobile phase consists of two different solution A & B, both solutions consist of acetonitrile and acetic acid in the ratio of 90:10 for solution A and 10:90 for solution B at flow rate of 1mL/min and detection wavelength at 350nm. The LOD was found to be 0.02µg/ml for Luteolin and 0.02 for Apigenin. The LOQ was observed at 0.05µg/ml for Luteolin along with 0.02µg/ml for Apigenin ¹³.

Guzelmeric E. et. al., have reported high performance thin layer chromatography (HPTLC)technique for Apigenin 7-O-glucoside in chamomile flowers and its application for fingerprint discrimination of chamomile-like materials. Method using silica gel 60NH2F254s HPLC plate for separation and formic acid-ethyl acetate- acetic acid-water (1.5:30:1.5:3, v/v/v/v) as solvent system ¹⁴. Sharafi A. et. al., have reported HPLC technique for quantification of Apigenin from dried powder of Cosmos Bipinnatua, Apium Graveolens and Petroselinum Crispum. Separation is achieved by using C₈stationary phase (250mm*4.6mm, particle size 5µm; Perfectsill, MZ-Analysentechnik, Germany) provided with a guard column of the same packing. Water and acetonitrile (55:45v/v) as mobile phase with flow rate of 1ml/min at 340nm wavelength the eluted peaks were detected by UV detector. The LOD was established at 0.005µg/ml. LOQ was established at 0.01µg/ml ¹⁵. Gomathy S. et. al., have reported HPLC technique for the concurrent quantification of Luteolin and Apigenin in commercial preparation. Method using HibarLichrospher C₈ (150×4.6mm i.d., 5µ) for separation. methanol (20:80 %, v/v) with0.5% trifluoracetic acid  as solvent system with flow rate of 1ml/min at 269 wavelenght. The developed technique was established linear above the concentration ranging from 10 to 110ng/ml with >0.99 R2. LOD was established 5.0 for Apigenin with 7.0ng/ml for Luteolin. The LOQ was established at 10ng/ml for both Luteolin and Apigenin ¹⁶.

Panchal H. et. al., have reported validated HPTLC technique for concurrent quantification of Luteolinand Apigenin in Achillea millefolium. As a stationary phase, method uses a silica gel 60 F254 HPTLC plates. Ethyl acetate: formic acid: toluene (4:0.3:6 v/v/v) as solvent system. The determination was accomplished at 254nm using densitometric absorbance-reflection mode for Apigenin and luteolin. The calibration curve was found to be linear between 200-1200ng/spot for Apigenin and 500-3000ng/spot for Luteolin. The LOD was found to be 71.06ng/spot for Apigenin and 74.05ng/spot for Luteolin. LOQ was found to be 230.33ng/spot for Apigenin and 243.72ng/spot for Luteolin ¹⁷.

Gtowacki R. et. al., have reported micellar electrokinetic chromatography fordetermination of total Apigenin in herbs. Method using a buffer solution consisting of 30mmol/L sodium borate (pH 10.2), 10% acetonitrile, and 10 mmol/L sodium dodecyl sulfate for separation. The method was validated and calibrated for total Apigenin in the range of 1.0-100µmol/L (R² = 0.994). The LOD was found to be 0.48µmol/L and LOQ was found to be 0.92µmol/L¹⁸. Gomathy S. et. al., have reported RP-HPLC technique for the concurrent estimation of Luteolin and Apigenin from Achillea millefolium linn. The estimation is carried out using HibarLichrospher C₈ column (150mm×4.6mm i.d., particle size 5µ) and methanol (20:80 v/v) with 0.5% trifluoroacetic acidas solvent system at a flow rate of 1.0ml/min and detection wavelength of 269nm. The standard curve was constructed using concentration range of 1-5µg/ml with linear correlation coefficient of R² = 0.99 ¹⁹.

Attarde DL et. al., have reported HPLC technique concurrent quantification of Luteolin and Apigenin in ethyl acetate extract of Premna integrifolia Land inselected marketed herbal formulation of dashmula. Method using Pre- coated Silica Gel G 60F₂₅₄ HPTLC Plates (0.2mm Thick, Size-20×10cm) as stationary phase and ethyl acetate: toulene: formic acid (4:6:0.15) as mobile phase. Retention factor was 0.39 and 0.29 for standard Apigenin and Luteolin at 347nm iso absorptive wavelength ²⁰. Satpathy S. et. al., have reported HPTLC method for simultaneous estimation of Apigenin and Luteolin in Hygrophilia spinosa T anders.

Method using HPTLC plates for separation and toluene-ethyl acetate-formic acid (6.0:4.0:1.0 v/v) as solvent system and observation was achieved at 349nm. The calibration curve range between 80-560ng/band (R² =0.997) for Apigenin and 40-280ng/band (R² =0.998) for Luteolin. The LOD was found to be 6.25 for Apigenin and 2.36 for Luteolin. LOQ was found to be 18.95ng for Apigenin and 7.55ng for Luteolin ²¹. Patel NG. et. al., have reported HPTLC method for estimation of Luteolin and Apigenin in Premna mucronate Roxb., Verbenaceae. Method using precoated silica gel 60 F₂₅₄ HPTLC aluminium plates (10×10cm, 0.2mm thick) as stationary phase and ethyl acetate: formic acid: toluene (4:0.3:6) as mobile phase and detection was achieved at 366 nm.

Calibration curve was found to be linear between 200 and 1000ng/band for Luteolin and 50 and 250ng/band for Apigenin. The LOD was found to be 42.6ng/band for Luteolin and 7.97ng/band for Apigenin. The LOQ was found to be 129.08ng/band for Luteolin with24.155ng/band for Apigenin ²². Shambhu N. et. al., have reported HPTLC method for simultaneous estimation of Luteolin, Apigenin from Cardiospermum halicacabum Linn. and Hydnocarpus pentadra (BUCH. -HAM.) OKEN. Method using silica gel 60F₂₅₄ TLC plates as stationary phase and methanol: formic acid: chloroform (1.0:2.0:8.0) as mobile phase. The LOD was found to be 0.01µg/band for both Luteolin and Apigenin. The LOQ was found to be 0.02µg/band for Luteolin and 0.03µg/band for Apigenin ²³. Elgin G. et. al., have reported HPLC method for the determination of antioxidant flavonoids of some Hypericum L. Species. Method using C₁₈ (ODS) analytical column for separation and methanol and 0.01M orthophosphoric acid (pH 7) (50:50, v/v) as mobile phase. Rutin, Quercitin-3’-glucoside (isoquercitrin), Luteolin-4’glucoside, Quercitin-4’glucoside, Quercitin, Naringenin, Luteolin and Apigenin were chosen as the model compounds for the study ²⁴. Koneru S. et. al., have reported newly developed analytical method for standardization of Apigenin by RP-HPLC method. Method using Merk C₁₈ analytical column (5µm, 250mm× 4.6mm, I.D) and acetonitrile and phosphate buffer at acidic pH (70:30 v/v) as solvent system and observation was achieved at 268nm. Calibration curve was found to be linear between range of 10-50µg/ml with correlation coefficient of 0.9996. Retention time was found to be 3.53min. The LOD was found to be 3.3 /s and LOQ was found to be 10 /s ²⁵.

Kim J. et. al., have reported HPLC-UV based quantitative analytical method for Apigenin-7-O-glucoside in Matricaria chamomilla extract. Method using Capcell Pak MGII C₁₈ (250×4.6mm, 5um) for separation and 20% acetonitrile in aqueous 0.05% (v/v) trifluoroacetic acid as solvent system at a flow rate of 1.0 mL/min and observation was achieved at 330nm. The LOD was established at 0.57mg/ml and LOQ was established at 1.73mg/ml ²⁶. Watak S. et. al., have reported column chromatography method for isolation of Apigenin from Trigonella foenum Graecum seed extract on lab scale. Method using butanol: ethylacetate (1:1) as mobile phase and Silica Gel is used for column packing. The compound isolated from column was evaluated by performing HPTLC, UV, IR and HPLC with standard Apigenin ²⁷. Parikh N. H. et. al., have reported HPTLC method for estimation of apigenin in Ocimum basilium L. Seed (takhmaria). Method using precoated silica gel G60, HPTLC plate for separation and toluene: formic acid: acetone (5:1:4 v/v) as mobile phase and densitometric estimation was done at 340nm. Calibration curve was achieved in the range of 100-600ng and method was found to be linear 0.995 ²⁸.

Khuluk R F et. al., have reported high performance liquid chromatography- diode array detection (HPLC-DAD) method for estimation of flavonoids in Sonchus arvensis. The estimation was carried out by using reverse phase C₁₈ column and methanol and 0.2% aqueous formic acid as solvent system with flow rate at 1 mL/min. The LOD was observed at 0.006-0.015µg/mL and LOQ was observed at 0.020-0.052µg/mL. The method for the estimation of eight flavonoids that are apigenin, kaempferol, orientin, rutin, hyperoside, myricetin, luteolin and quercetin ²⁹. The detailed summery of chromatographic standardization of Apigenin is Presented in Table 2.

TABLE 2: CHROMATOGRAPHIC ANALYSIS OF APIGENIN IN HERBAL MEDICINES

CONCLUSION: Quality control analysis is major and essential step in the manufacturing of herbal formulations as the quality of product affects the safety and efficacy of medicinal products. The quality control parameters need to be tested before the finished product is manufactured, marketed, and ingested by patients. Flavonoids are the class of polyphenolic secondary metabolites, which are derived from plants. Apigenin is a flavonoid that has a variety of medicinal properties. Many herbal medicines containing Apigenin are available in market and hence quality analysis is essential. The spectroscopic and chromatographic methods play major role in the quality control and standardization of Apigenin in its extract, isolated from or any other herbal or polyherbal formulation.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Chougule SB, Jivaje AB, Mannur VS and Suryawanshi SS: Validated spectroscopic and chromatographic techniques for quality control analysis of Apigenin in herbal medicines. Int J Pharm Sci & Res 2022; 13(12): 4857-64. doi: 10.13040/IJPSR.0975-8232.13(12).4857-64.

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