IN-VITRO STUDIES OF EXTRACTS OF PLUMBAGO ZEYLANICA IN CERVIX CARCINOMA CELLS

IN-VITRO STUDIES OF EXTRACTS OF PLUMBAGO ZEYLANICA IN CERVIX CARCINOMA CELLS

V. Rama Devi, K. Ganesh Kadiyala and Amrutha V. Audipudi *

Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

ABSTRACT: The present study evaluated the antimicrobial and cytotoxic activities of chloroform, hexane methanol, and petroleum ether extracts of Plumbago zeylanica (PEC, PEH, PEM, and PEP). Antimicrobial activities of the extracts were evaluated against gram-positive bacteria (Staphylococcus aureus-ATCC 6538 and Bacillus cereus-NCIM 2155) and gram-negative bacteria (E. coli-ATCC 8739) along with cytotoxicity activity against cervix carcinoma cell lines (HeLa and SiHa cell lines). Out of the four extracts tested, PEM and PEC extracts showed significantly high value of zone of inhibition against tested bacteria. PEM showed zone of inhibition of 10 mm, 13mm and 8 mm in ATCC 6538, NCIM 2155 and ATCC 8739, respectively. On the other hand, chloroform extract showed a zone of inhibition of 8mm, 10mm and 7 mm in ATCC 6538, NCIM 2155 and ATCC 8739, respectively. The four extracts PEC, PEH, PEM, and PEP showed a significant cytotoxicity activity with IC₅₀ concentration at 238.93ug/ml, 694.44ug/ml, 580.40ug/ml and 593.04ug/ml against the HeLa Cell line and 142.71ug/ml, 547.16ug/ml, 448.04ug/ml and 424.27ug/ml respectively against the SiHa Cell line after the treatment of 24 h. Based on the results of the anti-microbial and anticancer analysis, it was identified that extracts of P. zeylanica may show anticancer activity and also suggest that the PEC has satisfactory cytotoxic potential properties against both cervix carcinoma cell lines based on the dosage of the drug after the incubation period of 24 h. Therefore our results strongly support the antimicrobial and cytotoxicity activities of P. zeylanica provide a scientific basis to develop novel drug formulations from this traditional medicinal plant.

Keywords: Plumbago zeylanica, Cervix carcinoma cell line, Cytotoxicity, Gram-positive, Gram-negative, HeLa cell line, Si Ha cell line

INTRODUCTION: One of the richest sources of phytochemicals is the roots of Plumbago zeylanica ¹. P. zeylanica, a wide-ranging curative herb, is available all over Africa and Asia. It is a long-winded, lasting herb belonging to the Plumbaginaceae family (Plumbago L. genus).

Enormous literature is available that explains the medicinal uses of P. zeylanica ². It is used as a remedy for skin infections and intestinal worm’s viz. scabies, hookworms, leprosy, dermatitis, acne, sores, ringworm, and ulcers ³.

The ancient systems of medicine in different places have been using P. zeylanica for a variety of treatments. The powdered root and the bark are used to treat syphilis, gonorrhea, tuberculosis, swellings, rheumatic pain, and wound healing ⁴. The decoction obtained from roots with boiled milk is swallowed to treat inflammation in the mouth, chest and throat.

A paste of the root in vinegar, water, and milk shows a significant effect against influenza and black water fever, although root extracts are taken orally to treat shortness of breath ⁵. In Ayurveda, it was mentioned that the extracts of P. zeylanica plant have anticancer ^{6, 7}, antitumor, anti-inflammatory, antioxidant, anti-mycobacterial, and antimicrobial activities ^8-15.

The constituents in the roots of the plant are credited with potential properties towards therapeutic uses, including hepatoprotective, anti-atherogenic, neuroprotective, cardiotonic, and stimulating the central nervous system ¹⁶. Different extracts from P. zeylanica have various vital activities like acetone extract of P. zeylanica, which shows effects on chromosomal aberrations produced by ethinyl estradiol in human lympho-cytes culture ¹⁷.

In view of the vast applications of P. zeylanica to treat various diseases as a medicine, the present investigation aimed to analyze anti-microbial activity against gram-positive (Staphylococcus aureus (ATCC 6538), Bacillus cereus (NCIM 2155)) and gram-negative (E. coli (ATCC 8739)), and anticancer activities in cervix carcinoma cell lines (HeLa and SiHa cell lines) of chloroform, hexane methanol, and petroleum ether extracts (PEC, PEH, PEM, and PEP) of P. zeylanica.

MATERIALS AND METHODS:

Chemicals and Cell Lines: Gram-positive bacteria (Staphylococcus aureus (ATCC 6538), bacillus cereus (NCIM 2155)), gram-negative bacteria (E.coli (ATCC 8739)), Cell lines - HeLa-Human Cervix Adenocarcinoma Cell line (NCCS, Pune), SiHa-Human Cervix Squamous Carcinoma Cell line (NCCS, Pune), Cell culture medium: DMEM- High Glucose - (#AL111, Himedia), Adjustable multichannel pipettes and a pipettor (Benchtop, USA) , Fetal Bovine Serum (#RM10432, Himedia), MTT Reagent (5 mg/ml) (# 4060 Himedia) , DMSO (#PHR1309, Sigma), Camptothecin (#C9911, Sigma), D-PBS (#TL1006, Himedia), 96-well plate for culturing the cells (From Corning,USA), T25 flask (# 12556009, Biolite - Thermo), 50 ml centrifuge tubes (# 546043 TORSON), 10 ml serological pipettes (TORSON), 10 to 1000 ul tips (TORSON). Chloroform, Methanol, Pet Ether, and Hexane solvents were purchased from Sigma-Aldrich Co., USA.

Equipment: Centrifuge (Remi: R-8 °C), Pipettes: 2-10μl, 10-100μl, and 100-1000μl, Inverted micro-scope (Biolink), 37°C incubators with a humidified atmosphere of 5% CO₂ (Healforce, China).

Anti-microbial Analysis: Paper discs impregnated with specific antibiotics or the test substances are placed on the surface of the Muller Hinton agar medium inoculated with the target organisms, which is recommended for the diffusion of antimicrobial agents as described in NCCLS approved standard. The plates are incubated, and the zones of inhibition around each disc are measured. Muller Hinton Agar plates were prepared, and the test microorganisms were inoculated by the spread plate method. Filter paper discs approximately 6mm in diameter were soaked with 15µl of the plant extract and placed in the previously prepared agar plates. Each disc was pressed down to ensure complete contact with the agar surface and distributed evenly so that they are no closer than 24 mm from each other, center to center. The agar plates were then incubated at 37ºC. After 16 to 18 h of incubation, each plate was examined. The resulting zones of inhibition were uniformly circular with a confluent lawn of growth. The diameters of the zones of complete inhibition were measured, including the diameter of the disc where the chloramphenicol was used as control.

Cytotoxicity Studies:

MTT Assay: Briefly, cells were seeded at 1 × 105 cells/mL in 96 well microtiter plates in Minimum Essential Medium with fetal bovine serum. The cells were incubated overnight for attachment. Drug concentrations in serial three-fold dilutions were added in triplicates and incubated for 48h at 5% CO₂ at 37°C (see list of drugs and corresponding cell line used in Table 1). Thereafter, the cells were treated with 3-[4,5-dimethylthiazol- 2- yl]- 2, 5- diphenyltratrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO). Four hours later, all of the medium, including MTT solution (5 mg/mL) was aspirated from the wells. The remaining formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a 96 well microplate reader (SynergyTM HT, Bio-Tek Instruments, Inc.). The cytotoxicity index was determined using the untreated cells as a negative control. The percent[1]age of cytotoxicity was calculated using the background-corrected absorbance follows (3, 4): IC₅₀ determination The IC₅₀ was extrapolated from the dose-response graph. The drug concen-tration that reduced the viability of cells by 50% (IC₅₀) was determined by plotting triplicate data points over a concentration range and calculating values using regression analysis of PRISM program. MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on the reduction of the yellow-colored water-soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570 nm ^20-22.

TABLE 1: DETAILS OF PLANT EXTRACTS

The IC₅₀ value was determined by using a linear regression equation i.e.

Y = Mx +C.

Here, Y = 50. M and C values were derived from the viability graph ^23-25.

Concentrations Used for the Study: In this study, four test compounds were used to check the cytotoxicity in two different carcinoma cell lines, namely, HeLa and SiHa Table 1. The used concentrations of the compound to treat the cells are tabulated in Table 2.

TABLE 2: DETAILS OF DRUG TREATMENT TO RESPECTIVE CELL LINES USED FOR THE STUDY

RESULTS AND DISCUSSION:

Anti-microbial Analysis: Antimicrobial activity of four different plant extracts (PEC, PEH, PEM, and PEP) against Staphylococcus aureus (ATCC 6538), Bacillus cereus (NCIM 2155), and E. coli (ATCC 8739) were analyzed by means of the zone of inhibition and compared with the standard antibiotic (streptomycin). The plant extracts showed the inhibition of the growth of both gram-positive and negative bacteria Fig. 1.

FIG. 1: ANTIMICROBIAL ANALYSIS OF FOUR DIFFERENT EXTRACTS OF P. ZEYLANICA (PEH, PEM, PEC AND PEP AT A CONCENTRATION OF (40ug/100ul) AGAINST THE GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA

Out of the four extracts tested, methanol and chloroform solvent extracts showed a good zone of inhibition against the gram-positive and gram-negative bacteria Fig. 1 and also revealed that secondary metabolites resolved in methanol and chloroform extracts of P. zeylanica are showing significant antibiotic efficiency on par with standard antibiotic streptomycin.

Cytotoxicity Studies: The direct microscopic observations of drug-treated images of HeLa and SiHa Cell lines by Inverted biological microscope after incubation of 24 h were present in plate 1 and 2. Percentage of cell viability of HeLa and SiHa Cell lines at different concentrations of PEC, PEH, PEM, and PEP showed variation in their cytotoxicity efficacy Fig 3. Statistical analysis of cell cytotoxicity study by ELISA reader suggest that against HeLa Cells, the test Compounds viz., PEC, PEH, PEM, and PEP, showing significant cytotoxicity with the IC₅₀ concentrations at 238.93 µg/ml, 694.44µg/ml, 580.40µg/ml and 593.04 µg/ml respectively. And 142.71µg/ml, 547.16 µg/ml, 448.04µg/ml and 424.27µg/ml respectively in SiHa cell line after the treatment of 24 h of incubation at 37 °C temperature Fig. 2.

FIG. 2: % OF CELL VIABILITY OF HeLa AND SiHa CELL LINES TREATED WITH DIFFERENT CONCENTRATIONS OF PEP, PEM, PEH AND PEC AFTER 24 h Of THE INCUBATION PERIOD

Earlier studies reported the antibacterial activities of P. zeylanica against many human and agricultural pathogens ¹⁸ and cytotoxic activities in HGE-17 cell lines ¹⁹. It was found that the cytotoxic activity of methanol extracts from different parts of five Arthemisia species was evaluated by Gordanian ²⁶. A. absinthium was found to have a greater cytotoxic effect on MCF-7 cells with an IC₅₀ value of 221.5 µg/mL ²⁷. The highest cytotoxicity of Consolida orienta-lis L. extract against HeLa cell was found in 5 and 2.5 mg/ml (500 µg/ml and 250 ug/ml), and it was found that the percentage of growth inhibition is increasing with increasing the concentration of test compounds, and IC₅₀ value was 1.6 mg/ml (1600 µg/ml) ²⁸. Our results also revealed that chemical constituents are solvent specific and showed variation in their activity against two different cancer cell lines (Hela and SiHa) causing cervical cancer and also strongly suggest that the chloroform extracts (PEC) have satisfactory cyto-toxic potential properties against the both cervix carcinoma cell lines based on the dosage of the extracted sample after the incubation period of 24 h. Therefore, the chloroform extracts of Plumbago zeylanica are useful to treat the cervix cancers caused by HeLa and SiHa cell lines effectively.

CONCLUSION: Although the secondary meta-bolites of P. zeylanica are known for their drug properties, their bioactive potential solvent-specific activity. Chloroform extract (PEC) has satisfactory cytotoxic potential properties against both cervix carcinoma cell lines and antimicrobial activities against both gram-positive (Staphylococcus aureus (ATCC 6538) and Bacillus cereus (NCIM 2155)) and gram-negative (E. coli (ATCC 8739)) clinical pathogens and provide a scientific basis to develop novel drug formulations from this traditional medicinal plant.

FIG. 3: IC₅₀ CONCENTRATIONS OF THE TEST COMPOUNDS AGAINST THE DRUG TREATED HeLa AND SiHa CELL LINES AFTER THE INCUBATION PERIOD OF 24 h

PLATE 1: CYTOTOXICITY STUDIES OF THE PLANT EXTRACTS (PEC, PEH, PEM, AND PEP) IN THE HELA CELL LINE AT DIFFERENT CONCENTRATIONS AFTER INCUBATION FOR 24 h

PLATE 2: CYTOTOXICITY STUDIES OF THE PLANT EXTRACTS (PEC, PEH, PEM, AND PEP) IN THE SiHa CELL LINE AT DIFFERENT CONCENTRATIONS AFTER INCUBATION FOR 24 h

ACKNOWLEDGEMENT: Authors are thankful to the Department of Botany and Microbiology (DST-first and UGC-SAP-Phase II), Acharya Nagarjuna University, Guntur, A.P 522510, India and Lavin laboratories, Hyderabad, Telangana-500 044, India.

CONFLICTS OF INTEREST: No conflict of interest.

REFERENCES:

1. Rajakrishnan R, Lekshmi R, Benil PB, Thomas J, AlFarhan AH, Rakesh V and Khalaf S: Phytochemical evaluation of roots of Plumbago zeylanica and assessment of its potential as a nephroprotective agent. Saudi J Biol Sci 2017; 24(4): 760-76.
2. Mukul C: A review on Morphology, Phytochemistry and Pharmacological activities of medicinal herb Plumbago zeylanica Journal of Pharmacognosy and Phytochemistry 2014; 3(2): 95-118.
3. Datta S, Sarangi B and Mishra RN: In-vitro antioxidant activities of Plumbago zeylanica and Plumbago rosea Linn: a comparative study. International Journal of Pharmacognosy and Phytochemical Research 2014; 6(3): 531-35.
4. Chitrak (Plumbago zeylanica): Properties, Benefits & Dosage. 2019. https://www.planetayurveda.com .
5. Teshome K, Gebre-Mariam T, Asres K, Perry F and Engidawork E: Toxicity studies on dermal application of plant extract of Plumbago zeylanica used in Ethiopian traditional medicine, J Ethnopharmacology 2008; 117: 236-48.
6. Hema Mani M and Jayachitra A: Anticancer activity of ethanolic extract of Plumbago zeylanica against Dalton’s ascitic lymphoma in mice. International Journal of Applied Engineering Research 2019; 14(7): 1715-21.
7. Kapare HS, Metkar SR and Shirolkar SV: Anticancer potential of Plumbago zeylanica and its isolated constituent plumbagin: a review. Int J Pharm Sci & Res 2020; 11(10): 4859-65.
8. Yang SJ, Chang SC, Wen HC, Chen CY, Liao JF and Chang CH: Plumbagin activates ERK1/2 and Akt via superoxide, Src and PI3-kinase in 3T3-L1 Cells, Eur J Pharmacol 2010; 638: 21-28.
9. Aleem M: Anti-Inflammatory and anti-microbial potential of Plumbago zeylanica a review. JDDT 2020; 10(5-s): 229-35.
10. Subramaniyan V and V. Paramasivam: Potential anti-inflammatory activity of Plumbago zeylanica. Asian Journal of Pharmaceutical and Clinical Research 2017; 10: 372-5.
11. Sivakumar V, Prakash R, Murali MR, Devaraj H and Devaraj SN: In-vivo micronucleus assay and GST activity in assessing genotoxicity of plumbagin in Swiss albino mice, Drug Chem Toxicol 2005; 28: 499-507.
12. Singh MK, Pandey A, Sawarkar H, Gupta A, Gidwani B, Dhongade H and Tripathi DK: Methanolic Extract of Plumbago zeylanica - a remarkable antibacterial agent against many human and agricultural pathogens. Journal of Pharmacopuncture 2017; 20(1): 18-22.
13. Mossa JS, El-Feraly FS and Muhammad I: Antimycobacterial constituents from Juniperus procera, Ferula communis and Plumbago zeylanica and their in vitro synergistic activity with isonicotinic acid hydrazide, Phytother Res 2004; 18: 934-37.
14. Shukla B, Saxena, S and Usmani S: Phytochemistry and pharmacological studies of Plumbago zeylanica L: a medicinal plant review. Clin Phytosci 2020; 7(34). DOI

https://doi.org/10.1186/s40816-021-00271-7.

15. Hariram NS, Bapurao PU and Won PS: HPTLC analysis, antioxidant, anti-inflammatory and xanthine oxidase inhibitory activity of Plumbago zeylanica L, Chiang Mai J. Sci 2015; 42(4): 886-95.
16. Jain P, Sharma HP and Singh P: Antifungal, antioxidant and phytochemical analysis of Plumbago zeylanica Vegetos 2020; 33(2): 247-57.
17. Bopaiah CP and Pradhan N: Central nervous system stimulatory action from the root extract of Plumbago zeylanica in rats. Phytother Res 2001; 15: 153-56.
18. Siddique YH, Ara G, Faisal M and Afzal M: Protective role of Plumbago zeylanica extract against the toxic effects of ethinylestradiol in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg9 and cultured human peripheral blood lymphocytes, Alternative Medicine Studies 2011; 1: 726-29.
19. Mukesh KS, Ajit P, Hemant S, Anshita G, Bina G, Hemant D and Dulal KT: Methanolic extract of Plumbago zeylanica - a remarkable antibacterial agent against many human and agricultural pathogens. J Pharmacopuncture 2017; 20(1): 18-22.
20. Ann SP, Aneesul I and Yuvaraj P: Anti-helicobacter pylori and cytotoxic activity of detoxified root of Plumbago auriculata, Plumbago indica and Plumbago zeylanica. The Journal of Phytopharmacology 2013; 2(3): 4-8.
21. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH and Boyd MR: Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay, Cancer Res 1988; 48(3): 589-601.
22. Mosmann TJ: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, Immunol. Methods 1983; 65(1-2): 55-63.
23. Ganesh KK, Anupama D, Jyoti T, Anupriya A, Hemanth BS, Krishna Ch, Meganathan T and Anil KM: Metal Based Imaging Probes of DO3A-Act-Met for LAT1 Mediated Methionine Specific Tumors : Synthesis and Preclinical Evaluation, Pharm Res 2015; 32: 955-67.
24. Gerlier D and Thomasset N: Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 1986; 94 (1-2): 57-63.
25. Alley MC, Scudiere DA, Monks A, Czerwinski M, Shoemaker R and Boyd MR: Validation of an automated microculturetetrazolium assay (MTA) to assess growth and drug sensitivity of human tumor cell lines, Proc. Am. Assoc Cancer Res 1986; 27: 389-91.
26. Mosmann T: Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays, J. Immunol. Methods 1983; 65(1-2): 55-63.
27. Gordanian B, Behbahani M, Carapetian J and Fazilati M: In-vitro evaluation of cytotoxic activity of flower, leaf, stem and root extracts of five Artemisia species. Res Pharm Sci 2014; 9: 91-96.
28. Deniz U. Hatice G, Fatma G and Ramazan M: Cytotoxic Activities of Certain Medicinal Plants on different Cancer Cell Lines. Turk J Pharm Sci 2017; 14(3): 222-30.
29. Farkhondeh N, Abbas AD, Bahman E, Vahid M and Sepideh M: Cytotoxic Properties of Some Medicinal Plant Extracts from Mazandaran, Iran. Iranian Red Crescent Medical Journal 2013; 15(11): e8871.
    

    ---

    How to cite this article:

    Devi LVR, Kadiyala KG and Audipudi AV: In-vitro studies of extracts of Plumbago zeylanica in cervix carcinoma cells. Int J Pharm Sci & Res 2021; 12(12): 6668-74. doi: 10.13040/IJPSR.0975-8232.12(12).6668-74.

    ---

    All © 2021 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.