Research Article

Cytotoxic, Antibacterial, and Leishmanicidal Activities of Paullinia pinnata (Linn.) Leaves

Background

Plants arenow being explored for use in the treatment and management of clinical diseases (1-4). Paullinia pinnata is a woody or sub-woody climber of the family Sapindaceae. It originates from tropical America and is now common in the savanna zones of tropical Africa and Madagascar (5). The common names are “bread and cheese plant” and “sweet gum” (5,6).

Phytochemical screening of the leaves has shown the presence of cardiac glycosides, saponins, alkaloids, anthraquinones, flavonoids, and tannins (6-10). These secondary metabolites have been proposed and shown to be responsible for the observed effects in various investigations where the leaves of P. pinnata have shown antimalarial (11,12), antioxidant (12-15), antidiarrhoeal (16), hematological (17), anti-typhoid (18), wound healing (19,20), phytotoxic (21), analgesic, and anti-inflammatory activities (8,22,23).

These investigations have served to support or validate some of the traditional applications of different preparations of the leaf of P. pinnata in the treatment and management of diverse diseases and ailments. However, its application in the amelioration of other health challenges still needs to be explored.

In the light of this, the aim of this study was to investigate the antileishmanial, antibacterial, and cytotoxic effects of P. pinnata leaves using various in-vitro bioassays, which may possibly lead to further investigations. The results of this study would further contribute to the existing knowledge on P. pinnata and provide observations which can be further explored.

Materials and Methods

Sample Collection and Extraction

Fresh leaves of P. pinnata were collected from the Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria. They were authenticated and given the specimen voucher number FHI 106555 at the same institute. The leaves were shade-dried at room temperature and the dried leaves were milled and extracted using absolute methanol for 6 hours in a Soxhlet extractor. The extract was concentrated with a rotary evaporator (Heidolph HB, Germany) and a vacuum oven (Gallenhamp, England) at a temperature of 40^oC. A 14% yield of the extract was realized that was stored refrigerated.

In Vitro Assays

Brine Shrimp Toxicity Assay

The modified method of Kivçak et al (24) was employed. First, 20 mg of P. pinnata was dissolved in 2 mL of methanol. Various volumes of the solution (5, 50, and 500 µL) were prepared and transferred into three separate vials to make final concentrations of 10, 100 and 1000 µg/mL, respectively, and the solvent was evaporated overnight. This was done in triplicates. Then, 50 mg of the eggs of Artemia salina (brine shrimp) kept at 4^oC was sprinkled into a hatching tray half-filled with filtered brine solution. For the eggs to hatch and develop into nauplii (larvae), the solution was incubated at 37^oC for 2 days. With the help of a Pasteur pipette, 10 nauplii were deposited into each of the 9 vials and the volume was made up to 5 mL with sea water. To attract the nauplii, the vials were illuminated by a lamp and incubated at 22-27^oC for 24 hours. For the negative and positive controls, 2 sets of 3 vials were prepared for the solvent and etoposide (7.4625 µg/mL, a cytotoxic drug), respectively. The lethal concentration 50 of the nauplii (LC₅₀) within 24 hours was determined.

Antibacterial Bioassay

Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri,and Staphylococcus aureus were the bacteria strains used for the bioassay. Overnight cultures containing 10⁸ colony forming unit/mL of microorganisms were used and diluted with sterile distilled water to obtain turbidity equivalent to a 0.5 McFarland turbidity standard. The agar well diffusion method was employed (24,25). On day 1, the nutrient broth was inoculated with single colony of bacterial culture and incubated for 24 hours at 37^oC. On day 2, 100 µL of fresh bacteria was inoculated into soft agar that was melted and cooled to 40^oC before shaking the mixture, which was then added to the plate containing the nutrient agar. For even distribution, the plate was rotated and allowed to solidify. A 6 mm diameter sterile cork borer was employed to make wells in the plate and appropriate codes were assigned to them. Subsequently, 100 µL of the stock solution of the methanol leaf extract of P. pinnata (3 mg of extract dissolved in 1 mL of dimethyl sulfoxide (DMSO) (i.e. the concentration of 3 mg/mL) was placed in respective agar well plate according to bacterial culture. Wells supplemented with DMSO and imipenem (standard drug) served as negative and positive controls, respectively. The zone of inhibition was documented after incubating the plates at 37^oC for 24 hours.

Leishmanicidal Assay

The method of Saeed et al (26) was applied in this study. Leishmania major (DESTO) promastigotes were cultured at 22-25^oC in RPMI- 1640 (Sigma) supplemented with 10% fetal bovine serum (FBS) which had been inactivated by heat at 56^oC for 30 minutes. Under this condition, the promastigote culture in the logarithmic phase of growth was washed three times with physiological saline after centrifugation at 2000 rpm for 10 minutes. Subsequently, to have a final concentration of 10⁶cells/mL, fresh culture medium was used to dilute the parasites. In a 96-well microtiter plate, 180 µL of the medium was placed in the first row and 100 µL of the same medium was placed in the other well. Serial dilution of the extract was carried out after adding 20 µL of the extract to the medium and 100 µL of parasite culture was added into all the wells. One row contained DMSO and served as control. Another row contained Amphotericin B and Pentamidine which were the standard drugs. The numbers of the parasites which survived were counted microscopically with the aid of the Neubauer chamber (Marienfeld, Germany) after incubating the plate at 21-22^oC for 72 hours. The mean of the three different experiments was used as the final result. A Windows-based EZ-Fit Enzyme Kinetics Software version 5.03 (Perrella Scientific Inc.) was used to calculate the 50% inhibitory concentrations (IC₅₀).

Results

The in vitro brine shrimp lethality bioassay of the methanol leaf extract of P. pinnata showed that the standard drug (etoposide) had 50% lethality at 7.4625 µg/mL while the extract was not toxic at any of the three concentrations (Table 1).

In Table 2, the antibacterial bioassay shows that the zone of inhibition of the methanol leaf extract of P. pinnata for E. coli and B. subtilis were 9 mm and 12 mm, respectively, while it was zero for S. flexneri, S. aureus, P. aeruginosa, and S. typhi. The zone of inhibition of the standard drug, imipenem, ranged from 24 to 33 mm for various bacteria species.

Table 3 reveals that the methanol leaf extract of P. pinnata showed no inhibitory activity on L. major at the concentration of 100 µg/mL while the standard drugs (amphotericin B and pentamidine) had 50% inhibition of L. major at 0.48 ± 0.02 µg/mL and 2.53 ± 0.01 µg/mL, respectively.

Discussion

In this study, it was discovered that the methanol extract of P. pinnata leaves does not possess cytotoxic and leishmanicidal activities. Its effect against bacterial pathogens was insignificant. This is a preliminary study from which further studies can be developed.

Brine shrimp lethality bioassay is a simple, rapid, low-cost but highly efficient and sensitive laboratory assay which is considered a useful tool for the detection of antitumor compounds (27), nano-structures (28), pesticidal compounds (29), fungal toxins (30), plant extract toxicity (31-33), heavy metals (34), cyanobacteria toxins (29), anti-Trypanosoma cruzi (35), and cytotoxicity of dental materials (36). The assay is based on the ability to kill laboratory cultured A. salina nauplii. Moreover, Hamidi et al(37) have shown that crude plant extracts give more accurate results than pure compounds. Additionally, isolated pure compounds seem to lose specific bioactivity, especially with brine shrimp. The LD₅₀ values below 249 µg/mL are regarded as highly toxic, 250-499 µg/mL as median toxicity, and 500-1000 µg/mL as light toxicity, and values above 1000 µg/mL are regarded as non-toxic (38). Since the methanol leaf extract of Paullinia pinnata did not show any sign of toxicity against A. salina at 10, 100, and 1000 µg/mL, it implies that the extract may have no anti-tumor, pesticidal, anti-Trypanosoma cruzi or anti-fungal properties. This observation is inconsistent with the findings of the study conducted by de Dieu Tamokou et al (15), which showed that P. pinnata leaves had anti-cancer properties. This may be because these investigators employed various specific cancer cell lines, suggesting that cancer cell lines are more sensitive in assessing cytotoxicity.

Bacteria are prokaryotes that are usually single-celled organisms, most of which have cell walls that contain peptidoglycan. They are abundant in air, water, and soil, and are major inhabitants of the skin, mouth, and intestines (39). Some of them are pathogenic and are therefore responsible for various bacterial diseases. The agar well diffusion method has been shown to be more sensitive than the disc diffusion method (40). Moreover, it is used to test the antimicrobial activity of plants or microbial extracts (41). The methanol leaf extract of P. pinnata showed no significant activity against E. coli and B. subtilis, and had no activity against S. flexneri, S. aureus, P. aeruginosa, and S. typhi. This supports the findings of de Dieu Tamokou et al who investigated the leaf extract of P. pinnata for activity against Salmonella typhimurium, Listeria monocytogens, E. coli, and S. aureus but reported no activity (15). Our findings also corroborate the observations of Ajayi et al(42) who investigated essential oil extracted from P. pinnata but did not report any activity against Klebsiella pneumonia, Bacillus megaterium, B. subtilis, Proteus mirabilis, P. aeruginosa, and E. coli. However, Ikhane et al (6) demonstrated that the methanol leaf extract had activity against P. aeruginosa and S. aureus at high concentrations. It can be suggested from the results of this study that fractionation of the methanol extract of the leaves may improve its activity against B. subtilis which had an inhibition zone of 12 mm.

Leishmaniasis, a tropical and sub-tropical disease, is endemic in 98 countries and the most common in Europe, Northern Africa, the Middle East, Asia, and parts of South America (43,44). It is mainly caused by a genus of parasitic protozoa known as Leishmania, which is transmitted by the bite of infected female phlebotomine sand flies. There are three forms of leishmaniasis: visceral (which is the most serious form of the disease), cutaneous (the most common form), and mucocutaneous (45). leishmaniasisaffects either the skin or the internal organs. Leishmania major was used in this study because it is a species of protozoa in the genus which is responsible for the disease condition known as zoonotic cutaneous leishmaniasis (46). The methanol leaf extract of P. pinnata showed no activity against L. major; therefore, it does not possess antileishmanial activity and may not be recommended for the management of the disease.

Conclusion

Based on the findings of this study, it can be concluded that the leaves of Paullinia pinnata (Linn.) possess insignificant activity against bacterial pathogens; however, the activity against Bacillus subtilis may be explored. Additionally, the leaves had no activity against Leishmania and may not be cytotoxic; therefore, they may not be of considerable benefit when used for these purposes.

Acknowledgements

The Third World Organization for Women in Science (TWOWS), now known as Organization for Women in Science for the Developing World (OWSD), is appreciated for granting the award of a Postgraduate Training Sandwich Fellowship to Oluwatoyin A. Adeyemo-Salami with which these experiments were conducted at the Hussein Ebrahim Jamal (H.E.J.) Research Institute of Chemistry, Karachi, Pakistan, is also highly acknowledged.

Conflict of Interest Disclosures

The authors declare no conflict of interests.

Ethical Issues

This is not applicable because they are all in-vitro assays.

Funding

Third World Organization for Women in Science (TWOWS), now known as Organization for Women in Science for the Developing World (OWSD), financially supported this study.

References

1. Hasan SS, Ahmed SI, Bukhari NI, Loon WC. Use of complementary and alternative medicine among patients with chronic diseases at outpatient clinics. Complement Ther Clin Pract 2009; 15(3):152-7. doi: 10.1016/j.ctcp.2009.02.003 [Crossref] [ Google Scholar]
2. Salomonsen LJ, Skovgaard L, la Cour S, Nyborg L, Launsø L, Fønnebø V. Use of complementary and alternative medicine at Norwegian and Danish hospitals. BMC Complement Altern Med 2011; 11:4. doi: 10.1186/1472-6882-11-4 [Crossref] [ Google Scholar]
3. Eddouks M, Chattopadhyay D, De Feo V, Cho WC. Medicinal plants in the prevention and treatment of chronic diseases 2013. Evid Based Complement Alternat Med 2014; 2014:180981. doi: 10.1155/2014/180981 [Crossref] [ Google Scholar]
4. Yuan H, Ma Q, Ye L, Piao G. The traditional medicine and modern medicine from natural products. Molecules 2016; 21(5):559. doi: 10.3390/molecules21050559 [Crossref] [ Google Scholar]
5. Burkill HM. The Useful Plants of West Tropical Africa. Vol 5. Kew: Royal Gardens; 2000.
6. Ikhane D, Banwo K, Omotade O, Sanni A. Phytochemical and antimicrobial activities of methanolic extract of Paullinia pinnata leaves on some selected bacterial pathogens. J Herbs Spices Med Plants 2015; 21(1):59-74. doi: 10.1080/10496475.2014.906015 [Crossref] [ Google Scholar]
7. Abourashed EA, Toyang NJ, Choinski J Jr, Khan IA. Two new flavone glycosides from Paullinia pinnata. J Nat Prod 1999; 62(8):1179-81. doi: 10.1021/np990063z [Crossref] [ Google Scholar]
8. Ior LD, Uguru MO, Olotu PN, Ohemu TL, Ukpe A. Evaluation of analgesic and anti-inflammatory activities and ahytochemical screening of the leaves extract of Paullinia pinnata (Sapindaceae). J Chem Pharm Res 2011; 3(4):351-6. [ Google Scholar]
9. Adeyemo-Salami OA, Makinde JM. Acute and sub-acute toxicity studies of the methanol extract of the leaves of Paullinia pinnata (Linn) in Wistar albino mice and rats. Afr J Med Med Sci 2013; 42(1):81-90. [ Google Scholar]
10. Imade FN, Nosakhare NG, Mensah JK. Phytochemical and antibacterial properties of the leaf, stem and root of Paullinia pinnata Linn. Niger Ann Nat Sci 2015; 15(1):79-84. [ Google Scholar]
11. Maje IM, Anuka JA, Hussaini IM, Katsayal UA, Yaro AH, Magaji MG. Evaluation of the anti- malarial activity of the ethanolic leaves extract of Paullinia pinnata Linn (Sapindaceae). Niger J Pharm Sci 2007; 6(2):67-72. [ Google Scholar]
12. Adeyemo-Salami OA, Ademowo OG, Farombi EO. Antioxidant and antiplasmodial activities of methanol leaf extract of Paullinia pinnata. J Herbs Spices Med Plants 2020; 26(3):315-28. doi: 10.1080/10496475.2020.1740905 [Crossref] [ Google Scholar]
13. Zamble A, Carpentier M, Kandoussi A, Sahpaz S, Petrault O, Ouk T. Paullinia pinnata extracts rich in polyphenols promote vascular relaxation via endothelium-dependent mechanisms. J Cardiovasc Pharmacol 2006; 47(4):599-608. doi: 10.1097/01.fjc.0000211734.53798.1d [Crossref] [ Google Scholar]
14. Jimoh FO, Sofidiya MO, Afolayan AJ. Antioxidant properties of the methanol extracts from the leaves of Paullinia pinnata. J Med Food 2007; 10(4):707-11. doi: 10.1089/jmf.2006.253 [Crossref] [ Google Scholar]
15. de Dieu Tamokou J, Chouna JR, Fischer-Fodor E, Chereches G, Barbos O, Damian G. Anticancer and antimicrobial activities of some antioxidant-rich Cameroonian medicinal plants. PLoS One 2013; 8(2):e55880. doi: 10.1371/journal.pone.0055880 [Crossref] [ Google Scholar]
16. Nyegue MA, Afagnigni AD, Ndam YN, Djova SV, Fonkoua MC, Etoa FX. Toxicity and activity of ethanolic leaf extract of Paullinia pinnata Linn (Sapindaceae) in Shigella flexneri-induced diarrhea in Wistar rats. J Evid Based Integr Med 2020; 25. doi: 10.1177/2515690x19900883 [Crossref]
17. Adeyemo-Salami OA, Ewuola EO. Hematological effects of repeated graded doses of the methanol extract of Paullinia pinnata (Linn) leaves in Wistar albino rats. Pharmacognosy Res 2015; 7(Suppl 1):S34-8. doi: 10.4103/0974-8490.150522 [Crossref] [ Google Scholar]
18. Lunga PK, Tamokou Jde D, Fodouop SP, Kuiate JR, Tchoumboue J, Gatsing D. Antityphoid and radical scavenging properties of the methanol extracts and compounds from the aerial part of Paullinia pinnata. Springerplus 2014; 3:302. doi: 10.1186/2193-1801-3-302 [Crossref] [ Google Scholar]
19. Annan K, Houghton PJ, Govindarajan R. In vitro and in vivo wound healing actions of Paullinia pinnata L. Planta Med 2007; 73:463. doi: 10.1055/s-2007-987243 [Crossref] [ Google Scholar]
20. Annan K, Govindarajan R, Kisseih E. Wound healing and cytoprotective actions of Paullinia pinnata L. Pharmacogn J 2010; 2(10):345-50. doi: 10.1016/s0975-3575(10)80107-5 [Crossref] [ Google Scholar]
21. Salami OA, Fafunso MA. Phytotoxic activity of the methanol leaves extract of Paullinia pinnata (Linn). Niger J Nat Prod Med 2016; 20:1-3. doi: 10.4314/njnpm.v20i1.1 [Crossref] [ Google Scholar]
22. Aiyelero M, Salawu K, Yaro AH, Enape OJ, Usman S. Phytochemical, analgesic and anti-inflammatory analysis of the ethylacetate fraction of Paullinia pinnata leaf L (Sapindaceae). J Pharm Res Dev Pract 2018; 2(1):34-40. [ Google Scholar]
23. Tseuguem PP, Nguelefack TB, Piégang BN, Mbankou Ngassam S. Aqueous and methanol extracts of Paullinia pinnata (Sapindaceae) improve monosodium urate-induced gouty arthritis in rat: analgesic, anti-inflammatory, and antioxidant effects. Evid Based Complement Alternat Med 2019; 2019:5946291. doi: 10.1155/2019/5946291 [Crossref] [ Google Scholar]
24. Kivçak B, Mert T, Öztürk HT. Antimicrobial and cytotoxic activities of Ceratonia siliqua L extracts. Turk J Biol 2002; 26(4):197-200. [ Google Scholar]
25. Das K, Tiwari RK, Shrivastava DK. Techniques for evaluation of medicinal plant products as antimicrobial agents: current methods and future trends. J Med Plants Res 2010; 4(2):104-11. doi: 10.5897/jmpr09.030 [Crossref] [ Google Scholar]
26. Saeed M, Khan H, Khan MA, Simjee SU, Muhammad N, Khan SA. Phytotoxic, insecticidal and leishmanicidal activities of aerial parts of Polygonatum verticillatum. Afr J Biotechnol 2010; 9(8):1241-4. doi: 10.5897/ajb09.1448 [Crossref] [ Google Scholar]
27. Anderson JE, Goetz CM, McLaughlin JL, Suffness M. A blind comparison of simple bench-top bioassays and human tumour cell cytotoxicities as antitumor prescreens. Phytochem Anal 1991; 2(3):107-11. doi: 10.1002/pca.2800020303 [Crossref] [ Google Scholar]
28. Maurer-Jones MA, Love SA, Meierhofer S, Marquis BJ, Liu Z, Haynes CL. Toxicity of nanoparticles to brine shrimp: an introduction to nanotoxicity and interdisciplinary science. J Chem Educ 2013; 90(4):475-8. doi: 10.1021/ed3005424 [Crossref] [ Google Scholar]
29. Jaki B, Orjala J, Bürgi HR, Sticher O. Biological screening of cyanobacteria for antimicrobial and molluscicidal activity, brine shrimp lethality, and cytotoxicity. Pharm Biol 1999; 37(2):138-43. doi: 10.1076/phbi.37.2.138.6092 [Crossref] [ Google Scholar]
30. Harwig J, Scott PM. Brine shrimp (Artemia salina L) larvae as a screening system for fungal toxins. Appl Microbiol 1971; 21(6):1011-6. doi: 10.1128/am.21.6.1011-1016.1971 [Crossref] [ Google Scholar]
31. McLaughlin JL, Chang CJ, Smith DL. . Bench-top bioassays for the discovery of bioactive natural products: an update 1991.
32. Syahmi AR, Vijayarathna S, Sasidharan S, Latha LY, Kwan YP, Lau YL. Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq, (oil palm leaf) methanol extract. Molecules 2010; 15(11):8111-21. doi: 10.3390/molecules15118111 [Crossref] [ Google Scholar]
33. Kibiti CM, Afolayan AJ. Antifungal activity and brine shrimp toxicity assessment of Bulbine abyssinica used in the folk medicine in the Eastern Cape province, South Africa. Bangladesh J Pharmacol 2016; 11(2):469-77. doi: 10.3329/bjp.v11i2.24405 [Crossref] [ Google Scholar]
34. Martı́nez M, Del Ramo J, Torreblanca A, Dı́az-Mayans J. Effect of cadmium exposure on zinc levels in the brine shrimp Artemia parthenogenetica. Aquaculture 1999; 172(3-4):315-25. doi: 10.1016/s0044-8486(98)00431-1 [Crossref] [ Google Scholar]
35. Zani CL, Chaves PP, Queiroz R, De Oliveira AB, Cardoso JE, Anjos AM. Brine shrimp lethality assay as a prescreening system for anti-Trypanosoma cruzi activity. Phytomedicine 1995; 2(1):47-50. doi: 10.1016/s0944-7113(11)80048-6 [Crossref] [ Google Scholar]
36. Pelka M, Danzl C, Distler W, Petschelt A. A new screening test for toxicity testing of dental materials. J Dent 2000; 28(5):341-5. doi: 10.1016/s0300-5712(00)00007-5 [Crossref] [ Google Scholar]
37. Hamidi MR, Jovanova B, Panovska TK. Toxicоlogical evaluation of the plant products using brine shrimp (Artemia salina L) model. Maced Pharm Bull 2014; 60(1):9-18. doi: 10.33320/maced.pharm.bull.2014.60.01.002 [Crossref] [ Google Scholar]
38. Bussmann RW, Malca G, Glenn A, Sharon D, Nilsen B, Parris B. Toxicity of medicinal plants used in traditional medicine in Northern Peru. J Ethnopharmacol 2011; 137(1):121-40. doi: 10.1016/j.jep.2011.04.071 [Crossref] [ Google Scholar]
39. Willey JM, Sherwood LM, Woolverton CJ. Prescott’s Principles of Microbiology. New York: McGraw Hill; 2009.
40. Valgas C, Machado de Souza S, Smânia EF, Smânia A Jr. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol 2007; 38(2):369-80. doi: 10.1590/s1517-83822007000200034 [Crossref] [ Google Scholar]
41. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal 2016; 6(2):71-9. doi: 10.1016/j.jpha.2015.11.005 [Crossref] [ Google Scholar]
42. Ajayi IA, Jonathan SG, Adewuyi A, Oderinde RA. Antimicrobial screening of the essential oil of some herbal plants from Western Nigeria. World Appl Sci J 2008; 3(1):79-81. [ Google Scholar]
43. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012; 7(5):e35671. doi: 10.1371/journal.pone.0035671 [Crossref] [ Google Scholar]
44. Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R. Leishmaniasis: a review. F1000Res 2017; 6:750. doi: 10.12688/f1000research.11120.1 [Crossref] [ Google Scholar]
45. World Health Organization (WHO). Leishmaniasis. Geneva: WHO; 2020. https://www.who.int/news-room/fact-sheets/detail/leishmaniasis.
46. Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major. N Engl J Med 2002; 346(12):891-5. doi: 10.1056/NEJMoa011882 [Crossref] [ Google Scholar]