Evaluation of cytotoxicity potential of Garcinia cambogia (Gaertn.) Desr. male flower extract using Allium cepa model

Research Articles

2023 | Vol: 8(5) | Issue: 5(September-October) | https://doi.org/10.31024/apj.2023.8.5.2

Evaluation of cytotoxicity potential of Garcinia cambogia (Gaertn.) Desr. male flower extract using Allium cepa model

Shilpa K.J.^*, Krithi Puthukulathil Gopi, Aswathi Muzhapravan, Kavya Prabhakaran Kinattumkara

Department of Botany, Jnana Kaveri P.G. Centre Chikka Aluvara, Mangalore University, Kodagu, India

^*Address for Corresponding Author

Shilpa K. J.

Department of Botany, Jnana Kaveri P.G. Centre Chikka Aluvara, Mangalore University, Kodagu, India

Abstract

The cytotoxic and genotoxic effects of various solvent extracts of Garcinia cambogia male flower was carried out using Allium cepa model. The solvents such as distilled water, ammonia, ethyl acetate and hexane were used for extraction. The percent mitotic index of all extracts decreased with increase in extract concentrations. A dose dependent decrease in root length with an increase in the extract concentration was also observed. The results revealed aberrations such as chromosome bridge, nuclear lesions, vagrant chromosomes, c- metaphase, abnormal separation and disturbed metaphase. Hence it can be concluded from the results that the plant extracts are cytotoxic and genotoxic in nature.

Keywords: Aberration, Allium cepa, cytotoxicity, Garcinia cambogia, mitotic index, vagrant

Introduction

The medicinal plants have profound therapeutic advantages but have been found to be potentially toxic, mutagenic and carcinogenic. The short - or long - term use raises concern about their toxic effects (Soetan and Aiyelaagbe, 2009). Hence the toxicological effects of herbal extracts need to be tested (Ping et al., 2012). Allium cepa test is an in vivo model used to analyze chromosomal alterations. This genetic model evaluates the disturbances in mitotic cycle (Lubini et al., 2008).

Garcinia cambogia (Gaertn.) Desr. Malabar tamarind or Kodampuli belongs to family Guttiferae (Clusiaceae) (Bohra and Waman, 2019)_.The tree is commonly found in semi - evergreen to evergreen forests. Pharmacological activities such as anti-obesity, antimicrobial, anticancer, anti - alzheimer, anti-inflammatory, hepatoprotective and anti - HIV activities are reported in G. cambogia (Rauf et al., 2012). Several compounds from different parts of G. cambogia such as Xanthones, Benzophenones, Organic Acids were isolated (Santo et al., 2020). The sub - compounds of xanthones are Garbogiol (roots), Rheedia xanthone A (peel), Oxy- guttiferone I, Oxy-guttiferone k, Oxy-guttiferone k2, Oxy-guttiferone m (Fruits). In case of Benzophenones, the sub - compounds are garcinol and isogarcinol (peel), Guttiferone I, Guttiferone n, Guttiferone j, Guttiferone k, Topoisomerase II inhibitor, Guttiferone m (Fruits). In case of Organic Acids, the sub - compounds are heterocyclic amines Tartaric acid, Citric acid, Malic acid, Garcinialactone (Fruits). There are no reports on the cytotoxicity studies of G. cambogia. The work has been undertaken to study the cytotoxic effects of extract of male flowers of G. cambogia using Allium cepa assay.

Materials and Methods

Study area

The district of Wayanad is situated on the eastern portion of Kerala (Figure 1). This district is a part of Western Ghats. The district lies between north latitude 11º 27’ and 11 º 58’35"and the east longitudes 75º 47’50" and 76º 26’ 35". On the north the district is bounded by Kodagu district of Karnataka state, on the east by Mysore district of Karnataka state and Nilagiri district of Tamilnadu state, on the south by Ernad taluk of Malappuram district and Kozhikode taluk of Kozhikode district, on the west by Vadakara and Quilandy taluk of Kozhikode district and Thalassery taluk of Kannur district. The total area of the district is 2131 sq.km. i.e., 5.48 % of the total geographical area of the state (Prasad et al., 2014).

Figure 1. Location map of study area (Image source: John et al., 2020)

Collection of Sample

The male flowers were collected from Irukanni region, Wayanad, Kerala (Figure 2a and b). The sample was collected during the month of March 2022. Approximately 250 grams of fresh male flowers were collected. Information about the plant was collected from local people.

Figure 2. G. cambogia male (a) flowers; (b) Twig with male flowers

Preparation of extracts

Fresh male flowers were collected, washed with distilled water and air dried. The sample was ground into fine powder using mixer grinder. The powdered material was extracted with distilled water, ammonia, ethyl acetate and hexane for 72 hours at room temperature. The samples were then filtered using filter paper (Whatman No. 1). The filtrates were evaporated to obtain different extracts.

Allium cepa assay

Allium cepa assay was carried out following the method of Owalarafe et al., 2020, with slight modification: Onion bulbs were obtained commercially. The outer scales were removed and the roots were scraped leaving the ring of the primordial root intact. The onion bulbs were treated with five concentrations of each extract, viz: 20, 40, 60, 80, 100 mg/ml. Five onion bulbs were utilized for each concentration for each extract. Tap water was taken as control, and five onion bulbs were kept in tap water (control). The base of each of the bulb was suspended in the extract inside 100 mL beakers for 72 h. At the end of exposure period, the length of the roots of five onion bulbs with the best growth at each concentration was measured (in cm) with a ruler. Average length for each concentration and the control was obtained. The effect of each sample on the morphology of growing root cells was examined. Root tips 1-3 cm long were cut and placed in a watch glass, the root tips were hydrolyzed in 1 N HCl at 60^°C for 10 min and stained with acetocarmine for 20 min. The tip was then squashed on glass slide with 45 % acetic acid to determine the mitotic index and the presence of chromosomal aberrations by viewing slides under the light microscope. Percentage mitotic index was calculated as:

Statistical analysis

The results from the studies were represented as Mean ± SEM. The data were analyzed by one- way analysis of variance (ANOVA), P value less than 0.05 was considered as statistically significant. Microsoft Excel 2010 was used for statistical analysis.

Results

A decrease in the percentage mitotic index was observed as the concentration of each of solvent extract increased. Water extract showed % mitotic index 60.90 % (2 mg/ml) and 47.02 % (10 mg/ml), Ammonia extract 41.37 % (2 mg/ml) and 47.30 % (10 mg/ml), Hexane extract 35.41 % (2 mg/ml) and 76.48 % (10 mg/ml), Chloroform extract 83.28 % (2 mg/ml) and 87.81 % (10 mg/ml) (Table 1). In the water extract a significant increase was observed between control to 6 mg/ml, while a significant decrease was observed from 6 mg/ml – 10 mg/ml. In ammonia extract a significant increase was observed between control and 2 mg/ml, and from 4mg/ml – 10 mg/ml. A similar trend was observed in the hexane extract. In chloroform extract significant increase was observed between control and 2 mg/ml and then a significant decrease from 2 mg/ml – 10 mg/ml (Table 1). The effect of different concentrations on root length of G. cambogia flower extracts when compared with the control is represented in Table 2. A decrease in percentage inhibition with an increase in concentration of extract was observed. The extracts showed a decline in meristematic growth relative to the control as the concentrations of the extracts increased. The most significant decrease in growth was observed in hexane and chloroform extract. The chromosomal aberrations observed in the A. cepa assay with different solvent extracts of G. cambogia flowers are presented in photomicrographs in Figure 3. The result revealed aberrations such as chromosome bridge, nuclear lesions, vagrant chromosomes, c-metaphase, abnormal separation and disturbed metaphase.

Table 1. Effect of different solvent extracts of Garcinia gummi - gutta on cell division in A. cepa

Aqueous extract

Ammonia extract

Hexane extract

Chloroform extract

Concentration (mg/ml)

of cell

Mitotic index

Total aberrations

No cells

Mitotic index

Total aberrations

No cells

Mitotic

index

Total aberrations

No of

cells

Mitotic index

Total aberrations

Control

638

20.66±

0.01^aa

0. 00 ± 0.0^aa

638

20.66±

0.01^aa

0.00±0^aa

638

20.6 ±0.01^aa

0.00±0.0^aa

634

20.66 ±0.015^aa

0.00±0.0^aa

434

5.76 ± 0.01^ba

10.56 ± 0.002^ba

598

7.04 ± 0.02^bb

6.03± 0.01^bb

494

9.7±0.03^bc

10.55±0.03^ba

434

4.83 ± 0.01^bd

7.15 ± 0.015^g

418

3.83±

0.015^ca

10.25±

0.02^ba

556

6.85 ±0.02^cb

5.56± 0.03^cb

441

8.86 ±

0.02^cc

9.26 ± 0.02^cc

402

4.45 ±

0.015^bd

6.96 ± 0.015^cd

402

4.45±

0.015^da

6.94 ± 0.02^ca

528

5.46±0.02^db

4.94± 0.02^db

366

8.46 ±

0.02^cc

6.25 ± 0.02^da

372

4.33 ±

0.03^ba

5.93 ± 0.02^cb

388

4.36±

0.015^da

6.95 ± 0.02^ca

501

6.15±0.02^cb

4.19± 0.01^db

355

4.23 ±

0.01^da

5.93 ± 0.02^ec

349

3.13 ±

0.02^cc

4.29 ± 0.02^eb

349

3.44±

0.015^ca

5.46 ± 0.02^da

482

4.94±0.02^eb

3.74± 0.02^eb

327

3.66 ±

0.02^ea

5.53 ± 0.03^ea

301

3.95 ±

0.02^ca

3.96 ± 0.015^fb

Note: Results are expressed as mean ± standard deviation. First superscript represent comparison along the column and second superscript represent comparison along the row. All test values with different superscripts across the rows and column are significantly different

Table 2. Inhibitory effect of different solvent extracts of G. gummi - gutta flowers on A. cepa meristematic growth

Concentration (mg/L)

WE (cm)

AE (cm)

CE (cm)

HE (cm)

Control

3.53 ± 0.02^a

(0 %)

3.53 ± 0.02^a

(0 %)

3.53 ± 0.02^a

(0 %)

3.53 ± 0.02^a

(0 %)

1.38 ± 0.01^b

(60.90 %)

2.07 ± 0.02^b

(41.37%)

0.59 ± 0.02^c

(83.28%)

2.28 ± 0.01^b

(35.41%)

1.1 ± 0.15^b

(68.83 %)

2.06 ± 0.02^b

(41.64%)

0.86 ± 0.01^c

(75.63%)

0.66 ± 0.02^c

(81.30%)

2.07 ± 0.02^b

(41.35%)

2.75 ± 0.02^b

(22.09%)

1.49 ± 0.03^b

(7.79%)

0.39 ± 0.01^c

(88.95%)

1.37 ± 0.02^b

(61.18%)

1.16 ± 0.02^b

(67.13%)

0.72 ± 0.02^c

(79.60%)

0.35 ± 0.01^c

(90.08%)

1.87 ± 0.02^b

(47.02 %)

1.86 ± 0.02^b

(47.30%)

0.43 ± 0.02^c

(87.81%)

0.83 ± 0.02^c

(76.48%)

Note: value in bracket are percentage decrease in root length of Allium cepa.; All test values with different superscripts across the rows are significantly different. p<0.001

Figure 3. Different chromosomal aberrations observed in Allium cepa(a) Chromosome Bridge; (b) Double nuclear Lesions; (c) Vagrant Chromosome; (d) C – Metaphase; (e) Abnormal separation; (f) Disturbed metaphase

Discussion

In this study, toxic effect of G. cambogia male flower extracts was evaluated by analysing percent mitotic index (%), inhibition of root tip growth and occurrence of chromosomal aberration. Mitotic index is characterized by the total number of dividing cells in cell cycle. It is a parameter that evaluates cytotoxicity of the sample under consideration (Fernandes et al., 2007). Reduction in the mitotic index with an increase in the extract concentration is an indication of delay in cell division, alteration of cell cycle and cell death (Rank and Nielsen, 1997). Significant differences (p<0.001) between control and treated group were observed in mitotic index. Our results are in agreement with previous findings (Ping et al., 2012 and Owalarafe et al., 2020). A dose dependent decrease in root length might be due to adverse effect on cell division such as disturbances in cell cycle or chromatin dysfunction. The appearance of stunted growth in roots indicated growth retardation and cytotoxicity (Yildiz et al., 2009). Our results are in accord with the findings of (Celik and Aslanturk, 2010; Irfan et al., 2012; Akwu et al., 2019) where the root growth is inhibited in a dose dependent manner. Chromosomal aberrations are characterized either by change in total chromosome number or in structure of chromosome in response to chemical treatment. Chromosomal aberrations such as bridges, lagging, stickiness, vagrant, binucleated, nuclear lesions, giant cell, c – mitosis, delayed anaphase, laggard, abnormal anaphase, abnormal metaphase, chromosome breaks, ring chromosome, have been reported in chemicals, pesticides, fertilisers and extract treated A. cepa treated roots (Soodan et al., 2014; Sabeen et al., 2020). The aberrations observed were of both clastogenic and physiological. Chromosome bridges occur during the translocation of unequal chromatid exchange and cause structural chromosome mutation. A chromosome moves ahead from its chromosomal group leading to unequal separation of number of chromosomes in the daughter cells in vagrant (Khanna and Sharma, 2013). C–metaphase occurs due to reduction in stainability in certain regions of chromosomes which results from inhibition of non – histone protein synthesis (Ronne, 1977). C–Metaphase has been reported by (Abderrahman, 1998; Vicentini et al., 2001; Obute et al., 2016). The chromosomal aberrations decreased as the mitotic index decreased with increased extract concentrations. This could be due to mitogenic agents that can overcome intracellular braking mechanisms (Owalarafe et al., 2020).

Conclusion

It can be concluded from the results that the solvent extracts of Garcinia cambogia male flowers have potential cytotoxic and genotoxic effect. Hence care must be taken while using these extracts in pharmacology.

Ethical Approval: Not applicable

Consent to Participate: Not applicable

Consent to publish: We voluntarily agree to publish our research work

Funding: The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Competing Interests: The authors declare no conflict of interest.

Financial interests: The authors have no relevant financial or non - financial interests to disclose

Availability of data and materials: Not applicable

Author contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Krithi Puthukulathil Gopi], [Kavya Kinattumkara Prabhakaran], [Aswathi Muzhapravan], [Shilpa KJ]. The first draft of the manuscript was written by [Shilpa K J] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

References

Abderrahman SM. 1998. Cytogenetic effects of Peganum harmala extracts on maize root tips. Cytologia 63:283 - 291.

Akwu NA, Naidoo Y, Singh M. 2019. Cytogenotoxic and biological evaluation of the aqueous extracts of Grewia lasiocarpa: An Allium cepa. South African Journal of Botany125: 371 - 380.

Bohra P, Waman AA. 2019. Morphological and Biochemical Studies in Garcinia gummi- gutta (L.). Roxb. Erwerbs - Obstbau 61:217 - 223. https://doi.org/10.1007/s10341 – 019-00419-3

Celik TA, Aslanturk OS. 2010. Evaluation of cytotoxicity and genotoxicity of Inula viscosa leaf extracts with Allium test. Journal of Biomedicine and Biotechnology https://doi.org/10.1155/2010/189252.

Fernandes TCC, Mazzero MA, Marin – Morales. 2007. Mechanism of micronuclei formation in polyploidizated cells of Allium cepa exposed to trifluralin herbicide. Pesticide Biochemistry and Physiology 88: 252 - 259. https://doi.org/ 10.1016/j.pestbp.2006.12.003

Irfan IA, Umut G, Semih Y, Yasar DM. 2012. Cytotoxicity of Aloe vera gel extracts on Allium cepa root tip cells. Turkish Journal of Botany 36(3):263 - 268. https://doi.org/10.3906/bot-1102-5.

John J, Bindu G, Srimuruganandam B, Wadhwa A, Rajan P. 2020. Land use/land cover and land surface temperature analysis in Wayanad district, India, using satellite imagery. Annals of GIS 26(4): 343 – 360. https://doi.org/10.1080/19475683.2020.1733662

Khanna N, Sharma S. 2013. Allium cepa root chromosomal aberration assay: A review. Indian Journal of Pharmaceutical and Biological Research 1(3):105 - 119. https://doi.org/10.30750/ijbr.1.3.15.

Lubini G, Fachinetto JM, Laughinghouse HD, Paranhos JT, Ferreira da Silva AC, Tedesco SB. 2008. Extracts affecting mitotic division in root – tip meristematic cell. Biologia 63(5): 647 - 651. https://doi.org/10.2478/s11756-008-0108-x

Obute G, Chimezie E, Nwagwu UA. 2016. Assessing the genotoxic effects of aqueous leaf extract of Neem (Azadirachta indica A. Juss), cosmetics and Alomo Bitters (Alcoholic Drink) using Allium test. Biotechnology Journal International 16 (3): 1-13. https://doi.org/10.9734/BJI/2016/29042

Owalarafe TL, Salawu K, Ihegboro GO, Ononamadu CJ, Alhassan AJ, Wudil AM. 2020. Investigation of cytotoxicity potential of different extracts of Ziziphus mauritiana (Lam) leaf Allium cepa model. Toxicology Reports 7: 816 - 821. https://doi.org/10.1016/j.toxrep.2020.06.010

Ping KY, Darah I, Yusuf UK, Yeng C, Sasidharan S. 2012. Genotoxicity of Euphorbia hirta. An Allium cepa assay. Molecules 17:7782 – 7791. https://doi.org/10:3390/molecules17077782

Prasad DAG, Raghavendra MP, Shyma TB. 2014. Antimicrobial activity of Tribal Medicines collected from Wayanad District Kerala. World Journal of Pharmaceutical Research 3:2476 - 2492.

Rauf AA, Khan S, Rasool Z, Ali S, Saleem M. 2012. In vitro antifungal activity of three selected Pakistani medicinal plants. Journal of medicinal plant research 1(2):41 - 43. https://doi.org/ 10.5829/idosi.mejmpr.2011.1.2.1110

Rank J, Nielsen MH. 1997. Allium cepa anaphase - telophase root tip chromosome aberration assay on N – methyl – N – nitrosourea, maleic hydrazide, sodium azide and ethyl methylsulfonate. Genetic Toxicology and Environmental Mutagenenesis 390:121 - 127. https://doi.org/ 10.1016/S0165 – 1218(97)00008-6.

Ronne M. 1977 Invitro induction of G – bands with cycloheximide. Cytologia 63: 283 -291. https://doi.org/10.1111/j.1601-5223.1977.tb01217.x

Sabeen M, Mahmood Q, Bhatti ZA, Farisulla, Irshad M, Billal M, et al .2020. Allium cepa assay based comparative study of selected vegetables and the chromosomal aberrations due to heavy metal accumulation. Saudi Journal of Biological Science 27(5): 1368-1374. https://doi.org/ 10.1016/j.sjbs.2019.12.011

Santo, Santana, Junior, Araujo, Bogo, Frietas, et al. 2020. Medicinal Potential of Garcinia Species and their compounds. Molecules 25(19): 4513. https://doi.org/10.3390/molecules25194513

Soetan KO, Aiyelaagbe OO. 2009. The need for bioactivity - safety evaluation and conservation of medicinal plants – A review. Journal of Medicinal Plant Research 3(5): 324 -328.

Soodan RK, Katnora JK, Nagpal A. 2014. Allium cepa root chromosomal aberration assay: An efficient test system for evaluating genotoxicity of agricultural soil. International Journal of Scientific Research 3(8):245 -250.

Vicentini VEP, Camparoto ML, Teixeira RO, Mantovani MS. 2001.Medicinal herb tea effects on vegetal and test systems. Acta Scientific 23(2):593 - 598.

Yildiz M, Cigerci IH, Konuk M, Fidan AF, Terzi H. 2009.Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays. Chemosphere 75: 934-938. https://doi.org/10.1016/j.chemosphere.2009.01.023.

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Research Articles

2023 | Vol: 8(5) | Issue: 5(September-October) | https://doi.org/10.31024/apj.2023.8.5.2

Evaluation of cytotoxicity potential of Garcinia cambogia (Gaertn.) Desr. male flower extract using Allium cepa model

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