Journal of Advanced Pharmaceutical Technology & Research

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 12  |  Issue : 1  |  Page : 8--13

In vitro cytotoxic, genotoxic, and antityrosinase activities of Clitoria macrophylla root


Yamon Pitakpawasutthi1, Maneewan Suwatronnakorn2, Somchai Issaravanich2, Chanida Palanuvej2, Nijsiri Ruangrungsi3,  
1 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok; Department of Thai Traditional Medicine, Faculty of Health and Sport Science, Thaksin University, Phatthalung, Thailand
2 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
3 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

Correspondence Address:
Dr. Nijsiri Ruangrungsi
Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok 10330
Thailand

Abstract

Clitoria macrophylla Wall. (Leguminosae), locally known as Non-tai-yak or An-chan-pa, commonly distributed in tropical nations and Southeast Asia. Regarding traditional Thai medical system, C. macrophylla roots carry out a potential in dermatology. Its roots are also used as insecticide in agriculture and animal farming. Moreover, clitoriacetal is the major component that can be detected in C. macrophylla root. This research aimed to assess the efficacy of C. macrophylla root extract and clitoriacetal for its anticancer and antityrosinase activities as well as to assess in vitro safety potential for its cytotoxic and genotoxic effects. C. macrophylla root and clitoriacetal were tested by brine shrimp lethality, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, comet assay, and antityrosinase activity. C. macrophylla root, clitoriacetal, and rotenone demonstrated the toxicity against brine shrimp nauplii with LC50 of 332.15, 136.54, and 0.15 μg/mL, respectively. C. macrophylla root and clitoriacetal showed cytotoxic potential against breast ductal carcinoma (BT-474), liver hepatoblastoma (Hep-G2), and colon adenocarcinoma (SW-620). At 100 μg/mL, the percent DNA damage of C. macrophylla root and clitoriacetal was 37.84% and 36.01%, respectively. C. macrophylla root and clitoriacetal were able to inhibit the tyrosinase enzyme with IC50 of 12.27 and 7.30 mg/mL, respectively, which less effective than glutathione (positive control). The present study revealed the in vitro biological activities of C. macrophylla root and its clitoriacetal constituent which proposed the scientific evidences in efficacy and safety evaluation including in vitro cytotoxicity, DNA damage as well as antityrosinase activities.



How to cite this article:
Pitakpawasutthi Y, Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. In vitro cytotoxic, genotoxic, and antityrosinase activities of Clitoria macrophylla root.J Adv Pharm Technol Res 2021;12:8-13


How to cite this URL:
Pitakpawasutthi Y, Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. In vitro cytotoxic, genotoxic, and antityrosinase activities of Clitoria macrophylla root. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 May 10 ];12:8-13
Available from: https://www.japtr.org/text.asp?2021/12/1/8/306568


Full Text



 Introduction



Clitoria macrophylla Wall. (Leguminosae), locally named Non-tai-yak or An-chan-pa, commonly distributed in tropical nations and Southeast Asia.[1] Regarding traditional Thai medical system, C. macrophylla roots carry out a potential in dermatology. Its roots are also used as insecticide in agriculture and animal farming.[2] Tuber juice and root juice of C. macrophylla were reported to get rid of green flies on vegetable and clear up worms on the buffalo's back.[3] For the phytochemical investigation of C. macrophylla root, the main compositions of rotenoid were isolated and identified such as clitoriacetal, 6-deoxyclitoriacetal, and stemonacetal. Especially, clitoriacetal (C19H18O9) is the major component that can be detected in the root part which demonstrates antipyretic, anti-inflammatory, and antioxidant activities [Figure 1].[2],[4],[5]{Figure 1}

Most herbal medicine still needs to be studied scientifically such as standardization, biological activities, and efficacy of each plant material to become important concerns for both authorities and the public. Moreover, the biological experimental assessments have been used as standard safety studies together with the efficacy tests. The herbal medicines and natural products are mostly comprised complex compounds. It is important to investigate the biological studies to get scientific information before clinical trials. Thus, this research aimed to assess the efficacy of C. macrophylla root ethanolic extract and standard clitoriacetal for its anticancer and antityrosinase activities as well as to assess in vitro safety potential for its cytotoxic and genotoxic effects by brine shrimp lethality and comet assays.

 Materials and Methods



Plant materials

C. macrophylla roots were obtained from traditional Thai drug store in Thailand and authenticated by the expert (Ruangrungsi N). The voucher specimens were prepared and kept at College of Public Health Sciences, Chulalongkorn University.

Plant extraction

C. macrophylla roots were cleaned, dried, and ground into powder before exhaustive extraction with 95% ethanol through Soxhlet apparatus. The yields after filtration and evaporation under vacuum were kept for bioactivity tests.

Isolation of clitoriacetal

Dried powders of C. macrophylla roots (500 g) were successively undergone 95% ethanol maceration for 60 days and filtered. The combined filtrates were collected and evaporated to obtain a crude extract. The extract was subjected to column chromatography packed with silica gel 60G and eluted with the solvent system, chloroform-ethanol (19:1). Each fraction (10 ml) was collected and observed by thin-layer chromatography. The homogenous fractions were evaporated to give a pale yellow solid that was further purified by recrystallization. Clitoriacetal was obtained as a pale yellow powder and confirmed by NMR (Bruker Avance III™ HD 500 MHz).[2],[5]

Brine shrimp nauplii lethality assay

According to Meyer et al., 1982,[6] artificial sea water at the concentration of 36.66% (w/v) was prepared and aerated for 24 h in brine shrimp hatching box under illumination. Artemia salina cysts were added and incubated at room temperature. Ten brine shrimps were transferred after 48 h using pasture pipette to individual vial containing 5 ml saline water. Various concentration of C. macrophylla root ethanolic extract, citoriacetal, and positive control (rotenone) in methanol were pipetted into a small filter paper and left until methanol was dried. Then, the prepared filter paper was placed into each vial containing the brine shrimp. Each concentration was performed in five replicates. The percent death of nauplii at 6, 12, 18, and 24 h was counted, recorded, and calculated for the LC50.

Cell viability assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the modified method as described by Masmann.[7] Five human cancer and one human normal cell lines including BT-474 (breast ductal carcinoma), CHAGO-K1 (undifferentiated lung carcinoma), SW-620 (colon adenocarcinoma), KATO-3 (gastric carcinoma), Hep-G2 (liver hepatoblastoma), and Wi-38 (Lung fibroblast) were studied. Tested cell lines were incubated in tissue culture flask in RpMI-1640 supplemented with 5% (v/v) fetal calf serum at 37°C in 5% CO2 for 3 days. Two hundred microliters of these cells were transferred to 96 well culture plates (about 1 × 104 cells/well) and incubated for 24 h at 37°C, 5% CO2, and 100% relative humidity condition.

The sample solution (2 μl) was dispensed into the appropriate wells. This analysis was performed in four replicates. The plates were further incubated for 72 h then added with 10 μl of MTT solution. After 4 h incubation, the supernatant medium was removed, dimethyl sulfoxide (DMSO) (150 μl) was added to solubilize the formazan crystal in the cells, mixed for 2–3 min on an orbital shaker. The absorbance was measured at 540 nm. The IC50 was determined from % cell survival as follows:

[INLINE:1]

DNA damage

Lymphocytes isolation

Fresh blood specimen from healthy donor was aseptically collected in sterile tube containing heparin. Six milliliters of diluted fresh blood was layered over 3 mL of Ficoll-Histopaque 1077 in a conical centrifuge tube, then centrifuge at 1800 rpm, 4°C for 30 min. The lymphocyte cells were 3-time rinsed in phosphate buffer saline (PBS, pH 7.4) and added with 10 ml of incomplete RPMI-1640 medium to discharge the buffer. Each step was centrifuge at 1600 rpm, 4°C for 10 min. Then, complete RPMI-1640 medium was added to get the lymphocyte suspension about 4 × 105 cells/mL using hemocytometer. Four hundred microliters portions were aliquoted into microcentrifuge tube and kept at −80°C.

Comet assay

According to Singh et al.,[8] each lymphocyte suspension was rinsed in PBS, pH 7.4 for 3 times, and added incomplete RPMI-1640 medium to obtain 4 ml as suspension. C. macrophylla root ethanolic extract and citoriacetal at concentration of 50 and 100 μg/mL were dissolved in 2% DMSO. Hydrogen peroxide and PBS (pH 7.4) were used as positive and negative controls, respectively. One hundred microliters of lymphocyte suspension were added into microcentrifuge tube that containing 100 μl of sample and incubated at 37°C for 1 h, centrifuged at 3000 rpm, 4°C for 5 min, and the supernatant was discarded.

The slides and coverslips were cleaned with ethanol and air dried before used. The slide was coated with 1% normal agarose which melt in PBS (pH 7.4) as the 1st layer and placed in low humidity before use to ensure the agarose adhesion. The treated samples were mixed with 1% low melting agarose which melt with PBS (pH 7.4) as ratio 1:1 at 37°C and spread onto the precoated slide, placed the coverslip over the second layer, and kept on ice until agarose gel solidified. After agarose gel has harden, the coverslip was slid off and spread with 0.5% low melting agarose which melt with PBS (pH 7.4) as the third layer, cover with coverslip, and kept in a cool temperature until agarose forming harden. Then, the freshly lysis solution was prepared by mixing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) with 10% DMSO. 1% Triton X-100 was added just before use. The coverslip was slid off, and the slide was immersed into a cool freshly lysis solution at 4°C for 1 h. After that, the slides were placed in horizontal gel electrophoresis chamber. The electrophoresis solution was 200 mM EDTA and 10 N NaOH, pH 13. For electrophoresis process, the slide was placed horizontally and washed three times with the neutralization buffer containing 0.4 M Tris buffer (pH 7.5) for 5 min.

Each slide was stained with 20 μg/mL ethidium bromide for 5 min, washed with water, and covered with coverslip, kept in a cool temperature. The migrated DNA (comet) was observed under fluorescent microscope (Axio Imager A2; Carl Zeiss, Germany) with the magnification of ×400. The degrees of damage were categorized into four classes of visual scoring depended on the size and intensity of the comet tail. Each comet was authorized a value of 0–3 conforming to its class; Class 0 interpreting comets with no or barely detectable tails (undamaged cells) and Class 1–3 interpreting increasing relative tail intensities [Figure 2].[9] DNA damage was calculated as equation below.{Figure 2}

[INLINE:2]

Antityrosinase activity

Antityrosinase activity was evaluated by dopachrome method in 96-well microplate.[10],[11] The C. macrophylla root extract, citoriacetal, and positive control (L-glutathione) were dissolved in 1 ml of DMSO (50% DMSO in water) and diluted to different concentrations. Each sample (40 μl) was mixed with 80 μl of 0.1 M sodium phosphate (pH 6.8) and 40 μl of L-DOPA substrate solution (19.7 mg in 0.1 M sodium phosphate, pH 6.8). Mushroom tyrosinase solution (31 U/mL) 40 μl was added in the reaction. The dopachrome was measured at 475 nm. The tyrosinase inhibitory activity was determined as equation below and IC50 was calculated.

[INLINE:3]

 Results and Discussion



Brine shrimp lethality activity

The toxicity investigation on brine shrimp nauplii has been reported by Meyer et al., 1982.[6] Brine shrimp lethality assay is always set for toxicological activity test of the natural products because it is a sensitive indicator species. It is rapid, reliable, nonexpensive, and convenient preliminary test for plant extracts which correlates reasonably well with cytotoxic properties. In several studies, brine shrimp lethality assay has been an authentic assay to estimate toxicity of the compounds or the extracts.[12],[13],[14]

The results of brine shrimp lethality activity of C. macrophylla root ethanolic extract, clitoriacetal, and positive control (rotenone) were evaluated and expressed as LC50 values: LC50 values <500 μg/mL (toxic), ≥500 ≤1000 μg/mL (weak toxicity), and >1000 μg/mL (nontoxicity).[15] It was found that C. macrophylla root ethanolic extract, clitoriacetal, and rotenone (positive control) were toxic against brine shrimp nauplii with LC50 of 332.15, 136.54, and 0.15 μg/mL, respectively [Figure 3]. On the contrary, clitoriacetal that was one of rotenoids isolated from the roots of Clitoria fairchildiana. Howard[16] was reported as weak toxic with the LC50 of 515.3 μg/mL.[17] The methanolic extract of the root of C. fairchildiana was found to be more toxic than C. macrophylla root ethanolic extract due to the LC50 of 158.0 μg/mL.[17] Brine shrimp lethality assays on Clitoria ternatea L. leaf extracts by two studies showed different LC50 as well. Kamilla et al. studied the exposure of 24 h aged nauplii to the methanolic extract of C. ternatea leaf and LC50 were found to be 1.46 and 0.49 mg/mL for 24 and 48 h of incubation periods, respectively.[18] Das and Chatterjee studied the effect of C. ternatea leaf extracted with 50% aqueous ethanol on 48 h aged nauplii. The LC50 after 24 h incubation period was 3.25 mg/mL.[19] Brine shrimp cytotoxic potency of C. macrophylla and clitoriacetal in this study was determined using 48 h aged nauplii and 24 h exposure period.{Figure 3}

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability activity

The MTT assay has been used successfully to quantitate cell survival and proliferation in macrophage-mediated cytotoxicity. This assay is used depending on the ability of succinate dehydrogenase in mitochondria of viable cells capable to transform the MTT tetrazolium salt into intracellular purple formazan that can be solubilized by DMSO and quantified by spectrophotometry.

C. macrophylla root ethanolic extract, clitoriacetal, and doxorubicin (positive control) were evaluated for cytotoxic activity against five human cancer cell lines and one normal cell line. The results were shown in [Table 1]. Cytotoxicity cutoff criteria of crude extract and pure compound are IC50 <20 μg/mL and <4 μg/mL, respectively.[20] As the result, C. macrophylla ethanolic extract showed more cytotoxic potential against breast ductal carcinoma (BT-474), liver hepatoblastoma (Hep-G2), and colon adenocarcinoma (SW-620) with IC50 of 6.6, 5.8, and 12.1 μg/mL, respectively. Similarly, clitoriacetal showed cytotoxic potential with IC50 of 4.1, 1.4, and 3.0 μg/mL, respectively. Furthermore, clitoriacetal was the most potent anticancer compound against HEP-G2 cells which showed IC50 less than positive control. Another rotenoid, 6-deoxyclitoriacetal isolated from C. macrophylla roots demonstrated strong cytotoxic activity against cultured P-388 lymphocytic leukemia cells and marginal active against multidrug-resistant KB-V1 oral cavity cancer cells. Vinblastin enhanced the cytotoxicity against KB-V1. The mechanism might involve P-glycoprotein affinity with rotenoids.[2] Sangthong et al. studied the cytotoxic activity of 6-deoxyclitoriacetal and its derivatives against various cancer cell lines. The results showed promising cytotoxic property that might occur through inhibition of topoisomerase IIα and DNA intercalation.[21]{Table 1}

DNA damage (comet assay)

Comet assay is a rapid standard procedure to observe DNA damage in eukaryotic cells depended on the detection of denatured DNA fragments migrating out of the cell nucleus during electrophoresis.[22] The percent of DNA damage was shown in [Figure 4]. Hydrogen peroxide was used as positive control and PBS (pH 7.4) was used as negative control. At 100 μg/mL, the percent DNA damage of C. macrophylla root ethanolic extract and clitoriacetal was 37.84% and 36.01%, respectively. C. macrophylla root ethanolic extract and clitoriacetal showed DNA damage potential with a dose-dependent relationship between the intensity of DNA damage and concentration of the sample. The aromatic methoxylation was reported as one of toxophores of the rotenoid molecule that contributed to the oxygen radical production, leading to DNA damage.[23],[24],[25]{Figure 4}

Antityrosinase activity

Tyrosinase is the enzyme in melanosomes of melanocyte, which involves in melanin biosynthesis. Antitirosinase activities were examined using dopachrome method.[10],[11] L-dopa was used as a substrate of tyrosinase in vitro. Dopaquinone product chemically changed to color substance, dopachrome. As the result, C. macrophylla root ethanolic extract and clitoriacetal were able to inhibit the tyrosinase enzyme with IC50 of 12.27 and 7.30 mg/mL, respectively, which less effective than positive control (glutathione) with IC50 of 0.01 mg/mL [Figure 5]. Chen et al. revealed that Clitoria ternatea Lindl. (Butterfly pea) flower extract increased whitening effect of the mask preparation.[26] Ruksounjik and Khunkitti studied the tyrosinase inhibition of C. ternatea flower ethanolic extract. It appeared that 0.2 mg/mL of the extract and glutathione could inhibit 22.04 ± 2.42% and 95.72 ± 2.00% of tyrosinase activity, respectively.[27] The marginal activity on tyrosinase inhibition of C. macrophylla root and a rotenoid, clitoriacetal was revealed in this study.{Figure 5}

 Conclusion



The in vitro biological activities of C. macrophylla root ethanolic extract were demonstrated with reference to its rotenoid, clitoriacetal. Cytotoxicity against A. salina nauplii as well as various cancer cell lines was revealed. DNA damage effect was studied using comet assay. Tyrosinase inhibitory property was also reported.

Financial support and sponsorship

This study was financially supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. The authors wish to thank College of Public Health Sciences, Chulalongkorn University and all staff members for necessary assistance and instrument supports.

Conflicts of interest

There are no conflicts of interest.

References

1Ruangrungsi N, Tunsaringkarn T, Palanuvej C, Rungsiyothin A, Issaravanich S, Vipanngeun N. Pharmacognostic Specification of Thai Crude Drugs. Bangkok: Institute of Thai Traditional Medicine, Department for Development of Thai Traditional and Alternative Medicine; 2007. p. 51-7.
2Lin LJ, Ruangrungsi N, Cordell GA, Shieh HL, You M, Pezzuto JM. 6-deoxyclitoriacetal from Clitoria macrophylla. Phytochemistry 1992;31:4329-31.
3Fantz PR. Ethnobotany of Clitoria (Leguminosae). Econ Bot 1991;45:511-20.
4Taguchi H, Kanchanapee P, Amatayakul T. The constituents of Clitoria macrophylla Wall. Cat., a Thai medicinal plant. The structure of a new rotenoid, clitoriacetal. Chem Pharm Bull (Tokyo) 1977;25:1026-30.
5Pitakpawasutthi 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.
6Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 1982;45:31-4.
7Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
8Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184-91.
9Cortés-Gutiérrez EI, Hernández-Garza F, García-Pérez JO, Dávila-Rodríguez MI, Aguado-Barrera ME, Cerda-Flores RM. Evaluation of DNA single and double strand breaks in women with cervical neoplasia based on alkaline and neutral comet assay techniques. J Biomed Biotechnol 2012;2012:385245.
10Masuda T, Yamashita D, Takeda Y, Yonemori S. Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Biosci Biotechnol Biochem 2005;69:197-201.
11Chan EW, Lim YY, Wong LF, Lianto FS, Wong SK, Lim KK, et al. Antioxidant and tyrosinase inhibition properties of leaves and rhizomes of ginger species. Food Chem 2008;109:477-83.
12Montanher AB, Pizzolatti MG, Brighente IM. An application of the brine shrimp bioassay for general screening of Brazilian medicinal plants. Acta Farm Bonaerense 2002;21:175-8.
13Michael AS, Thompson CG, Abramovitz M. Artemia salina as a test organism for bioassay. Science 1956;123:464.
14Caldwell GS, Bentley MG, Olive PJ. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. Toxicon 2003;42:301-6.
15Bastos ML, Lima MR, Conserva LM, Andrade VS, Rocha EM, Lemos RP. Studies on the antimicrobial activity and brine shrimp toxicity of Zeyheria tuberculosa (Vell.) Bur. (Bignoniaceae) extracts and their main constituents. Ann Clin Microbiol Antimicrob 2009;8:16.
16Silva BP, Bernardo RR, Parente JP. Rotenoids from roots of Clitoria fairchildiana. Phytochemistry 1998;49:1787-9.
17Santos RA, David JM, David JP. Detection and quantification of rotenoids from Clitoria fairchildiana and its lipids profile. Nat Prod Commun 2016;11:631-2.
18Kamilla L, Ramanathan S, Sasidharan S, Mansor SM. Toxicity evaluation of methanol extract of Clitoria ternatea L. leaf. Malays J Med Health Sci 2012;8:33-9.
19Das N, Chatterjee P. Evaluation of brine shrimp cytotoxicity of 50% aqueous thanolic leaf extract of Clitoria ternatea L. Asian J Pharm Clin Res 2013;7:15-7.
20Geran RI, Greenberg NH, Macdonald MM, Schumacher AM, Abbott BJ. Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep 1972;3:1-102.
21Sangthong S, Krusong K, Ngamrojanavanich N, Vilaivan T, Puthong S, Chandchawan S, et al. Synthesis of rotenoid derivatives with cytotoxic and topoisomerase II inhibitory activities. Bioorg Med Chem Lett 2011;21:4813-8.
22Liao W, McNutt MA, Zhu WG. The comet assay: A sensitive method for detecting DNA damage in individual cells. Methods 2009;48:46-53.
23Crombie L, Josephs JL, Cayley J, Larkin J, Weston JB. The rotenoid core structure: Modifications to define the requirements of the toxophore. Bioorg Med Chem Lett 1992;2:13-6.
24Ji BC, Yu CC, Yang ST, Hsia TC, Yang JS, Lai KC, et al. Induction of DNA damage by deguelin is mediated through reducing DNA repair genes in human non-small cell lung cancer NCI-H460 cells. Oncol Rep 2012;27:959-64.
25Aviello G, Canadanovic-Brunet JM, Milic N, Capasso R, Fattorusso E, Taglialatela-Scafati O, et al. Potent antioxidant and geno-protective effects of boeravinone G, a rotenoid isolated from Boerhaavia diffusa. PLoS One 2011;6:e19628.
26Chen LH, Chen IC, Chen PY, Huang PH. Application of butterfly pea flower extract in mask development. Sci Pharm 2018;86:1-9.
27Ruksounjik O, Khunkitti W. The comparative study in bioactivities of Rang Jeud, Butterfly pea and red grape peel. Isan J Pharm Sci 2016;12:61-9.