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ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 4  |  Page : 169-177  

Quality evaluation with reference to clitoriacetal and in vitro antioxidant activities of Clitoria macrophylla root


1 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
2 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok; Department of Pharmacognosy, College of Pharmacy, Rangsit University, Pathum Thani, Thailand

Date of Web Publication1-Oct-2019

Correspondence Address:
Dr. Nijsiri Ruangrungsi
Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok 10330
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.JAPTR_67_19

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  Abstract 

Clitoria macrophylla Wall. (syn. Clitoria hanceana Hemsl.), is commonly known in Thai as Nontai-yak, An-chan-pa, or Ueang-chan-pa, which belongs to Leguminosae family. According to traditional Thai medicine, the root has been used for the treatment of skin diseased as well as for pest control in horticulture and animal husbandry. The aim of this study is to investigate for the pharmacognostic specification, the clitoriacetal content, and in vitro antioxidant capacities of C. macrophylla roots from 12 different sources throughout Thailand. Clitoriacetal content was quantitatively analyzed by thin-layer chromatography (TLC) - densitometry with winCATS software and TLC image analysis with ImageJ software. Antioxidant activities were evaluated by 2, 2-diphenyl-1-picrylhydrazyl radical scavenging, ferric reducing antioxidant power assay, β-carotene bleaching assay, total phenolic, and total flavonoid contents. The pharmacognostic specification of C. macrophylla roots in Thailand was established. The loss on drying, total ash, acid-insoluble ash, and water contents should be not more than 6.40%, 12.29%, 8.89%, and 8.16% of dry weight, whereas ethanol and water-soluble extractive values should be not less than 4.95% and 14.72% of dry weight, respectively. Furthermore, the clitoriacetal content of C. macrophylla roots determined by TLC-densitometry and TLC image analysis was found to be 2.20 ± 1.31 and 2.22 ± 1.16 g/100 g of dry roots, respectively. The clitoriacetal contents of both methods were not significantly different using paired t-test. Moreover, the ethanolic extract of C. macrophylla roots showed its antioxidant potential compared to the standard butylated hydroxyl toluene and quercetin.

Keywords: Antioxidant activity, Clitoria macrophylla, clitoriacetal, pharmacognostic specification, thin-layer chromatography


How to cite this article:
Pitakpawasutthi Y, Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. Quality evaluation with reference to clitoriacetal and in vitro antioxidant activities of Clitoria macrophylla root. J Adv Pharm Technol Res 2019;10:169-77

How to cite this URL:
Pitakpawasutthi Y, Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. Quality evaluation with reference to clitoriacetal and in vitro antioxidant activities of Clitoria macrophylla root. J Adv Pharm Technol Res [serial online] 2019 [cited 2020 Dec 3];10:169-77. Available from: https://www.japtr.org/text.asp?2019/10/4/169/268458




  Introduction Top


Clitoria macrophylla Wall. (syn. Clitoria hanceana Hemsl.), is commonly known in Thai as Nontai-yak, An-chan-pa, or Ueang-chan-pa, which belongs to Leguminosae family. It is widely found in tropical countries and South East Asia.[1] According to traditional Thai medicine, the root has been used for the treatment of skin diseased as well as for pest control in horticulture and animal husbandry.[2] The previous research reported that tuber juice of C. macrophylla is sprayed on vegetable to kill green flies in Thailand, and the root juice is used to kill worms in the back of buffaloes.[3] From the phytochemical investigation, C. macrophylla has been reported to contain rotenoid compounds such as clitoriacetal, 6-deoxyclitoriacetal, and stemonacetal. In addition, it has been informed that clitoriacetal (C19H18O9) is the major compound which demonstrates remarkable antipyretic and anti-inflammatory activities.[2],[4]

Nowadays, the roots of Stemonaceae (Stemona tuberosa Lour., Stemona collinsae Craibr., Stemona burkillii Prain.) and Leguminosae (C. macrophylla Wall.) crude drugs are sold under the same name of “Nontai-yak” in the market of Thailand. However, it is very important to choose the right herbs to heal health conditions, according to pharmacognostical study. Therefore, the pharmacognostic specification is primarily important tool for identification, authentication, and standardization of herbal medicines. This research was attempted to investigate the standardization parameters of C. macrophylla root in Thailand, to determine the clitoriacetal content using thin-layer chromatography (TLC)-densitometry compared to TLC image analysis and to evaluate its antioxidant capacities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, β-carotene bleaching assay, total phenolic, and total flavonoid contents for evidence-based efficacy of this crude drug.


  Materials and Methods Top


Plant materials

C. macrophylla roots were purchased from 12 Thai traditional drug stores throughout Thailand and authenticated by Assoc. Prof. Dr. Nijsiri Ruangrungsi. The voucher specimens were deposited at College of Public Health Sciences, Chulalongkorn University, Thailand.

Plant extraction

After removal of any foreign matters, the dried roots were pulverized into powders and exhaustively extracted with 95% ethanol by Soxhlet apparatus. The extract was filtered and evaporated to dryness in vacuo. The extract yield was recorded.

Isolation of standard clitoriacetal

Dried powdered roots (500 g) were macerated with 95% ethanol for 2-month period and filtered. The combined filtrates were evaporated in vacuo, and the residue was chromatographed on a silica gel column using chloroform: ethanol (19: 1) as eluent. The fractions were collected and monitored by TLC. The homogeneous fractions were combined and evaporated to afford a pale yellow solid. After recrystallization from a mixture of chloroform and ethanol, pale yellow powder of clitoriacetal was obtained.[2] Then, this compound was examined using Fourier Transform Nuclear Magnetic Resonance (NMR) Spectrometer 500 MHz (Liquid) (NMR 500 MHz, model: Avance III HD) at Scientific and Technological Research Equipment Centre, Chulalongkorn University, Thailand.

Determination of pharmacognostic specification

The pharmacognostic parameters including macroscopic and microscopic characters, determination of loss on drying, total ash, acid-insoluble ash, water content, ethanol, and water extractive values were examined according to standard methods of the World Health Organization.[5] All samples were analyzed in triplicate. The results were represented by grand mean ± pooled standard deviation. For the determination of TLC fingerprint, the ethanolic extract solution was applied onto the TLC plate (silica gel 60 GF254). The plate was developed in a mixture of chloroform: Ethanol (19: 1) and then examined under ultraviolet (UV) light (254, 365 nm), and detected by spraying with anisaldehyde reagent.

Determination of clitoriacetal contents by thin-layer chromatography-densitometry and thin-layer chromatography image analysis

The extract was dissolved with 95% ethanol to get the concentration of 3–6 mg/ml. The standard clitoriacetal was dissolved in 95% ethanol and diluted to obtain the series of standard solution range from 0.2 to 1.2 mg/ml.

For TLC-densitometry, 3 μ of 12 ethanol extracts and standard solutions were applied on the same TLC plate. The TLC plate was developed in a TLC chamber with a mobile phase consisted of toluene: Ethyl acetate: Formic acid (7.5: 2.5: 0.5), then the plate was removed and allowed to dry at room temperature. The developed TLC plate was scanned with CAMAG TLC Scanner 4 (CAMAG, Switzerland) under the wavelength of maximum absorbance (298 nm) and expressed as a chromatographic peak by winCATS software (Camag, Switzerland).

For TLC image analysis, the developed TLC plate was photographed under short wave UV light (254 nm) using a digital camera and saved as a TIFF file. Peak area of each band was quantitated using ImageJ software. The content of clitoriacetal was determined by comparing the peak area to the calibration curve obtained from the same TLC plate.

Method validation

According to the International Conference on Harmonisation guidelines, the method validation including calibration range, specificity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness was performed.[6]

Antioxidant activities

The antioxidant activities including DPPH radical scavenging assay, FRAP assay, β-carotene bleaching assay, total phenolic and total flavonoid contents of the extract, and standard clitoriacetal were evaluated according to the previous researches using quercetin and butylated hydroxyl toluene (BHT) as a positive control.[7],[8],[9]


  Results and Discussion Top


Pharmacognostic specification of Clitoria macrophylla root

The herbal medicines need to provide the quality control evidence which indicate the quality evaluation of plant materials and make them more reliable. Hence, macroscopic and microscopic examination is the first process to establish the identity and purity and thereby ensure quality of a particular sample.[5] The dried roots and the drawing of C. macrophylla were shown in [Figure 1] and [Figure 2]. The anatomical characterization of C. macrophylla root demonstrated periderm, cortical fiber, xylem vessel, xylem parenchyma, xylem ray with starch granules, xylem fiber [Figure 2]. The histological evaluation of C. macrophylla root showed cork in surface view, xylem parenchyma in longitudinal view, fragment of fibers, fragment of spiral vessel, starch granules, sclerenchymatous sclereid, parenchyma in longitudinal view, parenchyma in sectional view, and fragment of reticulate vessel [Figure 3]. The pharmacognostic parameters of C. macrophylla root were shown in [Table 1]. The loss on drying, total ash, acid-insoluble ash, and water contents should be not more than 6.40, 12.29, 8.89, and 8.16 g/100 g of dry weight, whereas ethanol, and water-soluble extractive values should be not less than 4.95 and 14.72 g/100 g of dry weight, respectively. Furthermore, TLC fingerprint of ethanolic extract of C. macrophylla root demonstrated the pattern of phytochemical characteristic constituents which revealed good separation of bands on the TLC plate [Figure 4]. Thus, this TLC fingerprint could be used as a reference standard for further identification of C. macrophylla root.
Figure 1: The twig of Clitoria macrophylla with flowers and pods

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Figure 2: (a) Dried Clitoria macrophylla root crude drug, (b) the anatomical character of Clitoria macrophylla root transverse section; (1) periderm, (2) cortical fiber, (3) xylem vessel, (4) xylem parenchyma, (5) xylem ray with starch granules, (6) xylem fiber

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Figure 3: Histological character of Clitoria macrophylla root powder; (1) cork in surface view, (2) xylem parenchyma in longitudinal view, (3) fragment of fibers, (4) fragment of spiral vessel, (5) starch granules, (6) sclerenchymatous sclereid, (7) parenchyma in longitudinal view, (8) parenchyma in sectional view, (9) fragment of reticulate vessel

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Table 1: The pharmacognostic parameters of Clitoria macrophylla root

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Figure 4: Thin-layer chromatography fingerprint of ethanolic extract of Clitoria macrophylla root

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Clitoriacetal content in Clitoria macrophylla root

Subsequent crystallization of a related fraction from the mixture of chloroform: ethanol (19: 1) afforded clitoriacetal compound, whose identity was examined by 1 H NMR and 13 C NMR in comparison with previous studies. The proton magnetic resonance spectrum revealed the presence of three methoxyl groups, four aromatic protons, a hydrogen-bonded hydroxyl group, and two alcoholic hydroxyl groups.[4] The 1 H NMR spectrum,13 C NMR spectrum, and structure of clitoriacetal were shown in [Figure 5].
Figure 5: (a) The 1H nuclear magnetic resonance spectrum, (b) 13C nuclear magnetic resonance spectrum, and (c) structure of clitoriacetal

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The percentage yield of ethanolic extracts of C. macrophylla root by Soxhlet extraction was 11.85 ± 4.71 g/100 g by dry weight. The quantitative analysis of clitoriacetal in the extracts was performed by TLC-densitometry and TLC image analysis. The TLC plate developed with toluene: ethyl acetate: formic acid (7.5: 2.5: 0.5) and photographed under UV 254 nm is shown in [Figure 6]. Densitometry is the quantitative and qualitative measurement of absorbed visible, UV light, or emitted fluorescence on excitation with UV light.[10] In addition, ImageJ (Department of Health and Human Services, National Institutes of Health (NIH), United State) is a free software which can quantitate and calculate pixel intensity in digital image of TLC spot and transform to chromatographic peak.[11] The clitoriacetal content of C. macrophylla roots determined by TLC-densitometry and TLC image analysis was found to be 2.20 ± 1.31 and 2.22 ± 1.16 g/100 g of dry roots, respectively. The clitoriacetal contents of both methods were not significantly different (P > 0.05) using paired t-test. Eventually, it can be concluded that TLC image analysis is inexpensive and convenient technique and is demonstrated to be used as an alternative method for quantitative analysis of clitoriacetal content in C. macrophylla roots.
Figure 6: The thin-layer chromatography plate photographed under ultraviolet 254 nm; standard clitoriacetal (track 1–6), Clitoria macrophylla root extracts from 12 different sources (track 7–18)

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Method validation

The method validity of TLC-densitometry and TLC image analysis was summarized in [Table 2]. The specificity was confirmed by comparing light absorption spectrum of the peak at apex among standard clitoriacetal and all samples which representing peak identity and comparing the absorption spectrum of the sample peak at up-slope, apex, and down-slope which representing peak purity [Figure 7]. The maximum UV spectrum of clitoriacetal could be detected at 298 nm. The calibration curve of standard clitoriacetal was polynomial relationships with good correlation coefficients (R2 > 0.99) ranged from 0.6 to 3.6 μg/spot [Figure 8]. The recovery was determined to evaluate the accuracy by spiking known three concentrations of standard clitoriacetal in a sample and found to be within acceptable limits (97.87%–118.24%). The repeatability and the intermediate precision were determined on the same day and in 3 different days. The repeatability and the intermediate precision were expressed as percentage relative standard deviation (RSD) in all cases and found to be <10% RSD for both methods. The detection limit and quantitation limit were calculated based on the standard deviation of the regression line and the slope of the calibration curve. In this study, the LOD and LOQ values displayed sufficient sensitivity of both methods. The robustness was performed by adjusting the mobile phase ratio which demonstrated the values of 1.87% RSD for TLC-densitometry and 1.48% RSD for TLC image analysis. The results demonstrated that there were no differences in the peak area of clitoriacetal in both methods. As the results of method validation, TLC-densitometry and TLC image analysis were efficient and reliable to quantitate the clitoriacetal contents in C. macrophylla root.
Table 2: Method validation of clitoriacetal in Clitoria macrophylla root by thin-layer chromatography-densitometry and thin-layer chromatography image analysis

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Figure 7: (a) The absorbance spectra of clitoriacetal in Clitoria macrophylla root extracts from 12 different sources and standard clitoriacetal representing peak identity, (b) the absorption spectra of the sample peak at up-slope, apex, and down-slope representing peak purity

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Figure 8: The calibration curve of clitoriacetal by (a) thin-layer chromatography-densitometry and (b) thin-layer chromatography image analysis

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Antioxidant activities

The antioxidant activities of ethanolic extract of C. macrophylla root, standard clitoriacetal, and positive control (quercetin and BHT) were summarized in [Table 3].
Table 3: The antioxidant activities of Clitoria macrophylla root extract, standard clitoriacetal, and positive control

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2,2-diphenyl-1-picrylhydrazyl radical scavenging activity

The color of DPPH radical turns from purple to yellow when receives hydrogen atom from antioxidant compounds.[12] The results demonstrated that the ethanolic extract of C. macrophylla root was able to decolorized DPPH radical with IC50 of 465.92 μg/ml compared to quercetin, BHT and standard clitoriacetal with IC50 of 3.34, 28.78, and 4279.29 μg/ml, respectively [Figure 9]. The previous study also reported DPPH radical scavenging activity of quercetin and BHT which showed nearly IC50 values of 4.84 and 24.82 μg/ml, respectively.[13]
Figure 9: Percent 2,2-diphenyl-1-picrylhydrazyl inhibition of Clitoria d root extract, standard clitoriacetal, and positive control

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Ferric reducing antioxidant power

FRAP represents the ability of an antioxidant compound to reduce Fe 3+-tripyridyltriazine (TPTZ) into Fe 2+-TPTZ that gives the blue color at the maximum absorbance of 593 nm.[14] The ethanolic extract of C. macrophylla root and standard clitoriacetal demonstrated the reducing power ability with FRAP values of 0.18 and 0.02 mM Fe (II)/mg, compared to BHT and quercetin which showed FRAP values of 0.93 and 2.49 mM Fe (II)/mg, respectively.

Beta-carotene bleaching inhibitory activity

The inhibition of β-carotene bleaching is used for the antioxidant ability to inhibit lipid peroxidation. When β-carotene exposed to radicals or oxidizing species, the compound loses their color because their double bond is interrupted by oxidation.[15] In this study, C. macrophylla ethanolic extract at 1 mg/ml showed 49.56% antioxidant activity compared to 13.16%, 90.70%, and 93.44% of clitoriacetal, quercetin, and BHT, respectively. The antioxidant activities of these tested samples demonstrated the dose-response relationship [Figure 10]. The results of β-carotene bleaching inhibition of quercetin and BHT in this study were in accordance with the previous study which showed the antioxidant activity of 87.18% and 90.88% at a concentration of 1 mg/ml, respectively.[8]
Figure 10: The antioxidant activity of Clitoria macrophylla extract, clitoriacetal compared to BHT and quercetin by β-carotene bleaching assay

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Total phenolic content

Folin–Ciocalteu assay is widely used to measure the total concentration of phenolic hydroxyl groups in the plant extract, and gallic acid is often used as a reference substance to set up a calibration curve.[16] The ethanolic extract of C. macrophylla root was evaluated for the quantification of total phenolic content from the calibration curve of gallic acid, as shown in [Figure 11]. The total phenolic content of the ethanolic extract of C. macrophylla root was 229.00 ± 1.49 mg GAE/g extract.
Figure 11: (a) Gallic acid calibration curve for total phenolic quantification and (b) quercetin calibration curve for total flavonoid quantification

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Total flavonoid content

The aluminum chloride colorimetric assay is widely used to evaluate the flavonoid content in plant materials. The carbonyl and hydroxyl groups present in flavonoids can interact with metal complexes. Particularly, flavones and flavonols tend to form a stable complex with AlCl3 which can be quantified by spectrophotometry.[9],[17]

The total flavonoid content of the ethanolic extract of C. macrophylla was found to be 23.53 ± 2.75 mg QE/g extract regarding to the calibration curve of quercetin [Figure 11].


  Conclusion Top


The pharmacognostic specification of C. macrophylla roots in Thailand was established. Clitoriacetal was isolated and purified by column chromatography and used as a reference compound. TLC-densitometry as well as TLC image analysis were developed, validated, and performed for quantification of clitoriacetal in this crude drug. Moreover, the antioxidant potential of the ethanolic extract of C. macrophylla roots was demonstrated.

Acknowledgments

The authors wish to thank College of Public Health Sciences, Chulalongkorn University, and all staff members for necessary assistance and instrument supports.

Financial support and sponsorship

This study was financially supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University.

Conflicts of interest

There are no conflicts of interest.



 
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Brand WW, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebenson Wiss Technol 1995;28:25-30.  Back to cited text no. 12
    
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Chaowuttikul C, Palanuvej C, Ruangrungsi N. Pharmacognostic specification, chlorogenic acid content, and in vitro antioxidant activities of Lonicera japonica flowering bud. Pharmacognosy Res 2017;9:128-32.  Back to cited text no. 13
    
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Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal Biochem 1996;239:70-6.  Back to cited text no. 14
    
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Mueller L, Boehm V. Antioxidant activity of β-carotene compounds in different in vitro assays. Molecules 2011;16:1055-69.  Back to cited text no. 15
    
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Blainski A, Lopes GC, de Mello JC. Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from Limonium brasiliense L. Molecules 2013;18:6852-65.  Back to cited text no. 16
    
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Mabry T, Markham KR, Thomas MB. The Systematic Identification of Flavonoids. New York: Springer Science and Business Media; 2012.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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