N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 BMC Complementary https://doi.org/10.1186/s12906-020-03195-w Medicine and Therapies RESEARCH ARTICLE Open Access Ethanolic extract of Nymphaea lotus L. (Nymphaeaceae) leaves exhibits in vitro antioxidant, in vivo anti-inflammatory and cytotoxic activities on Jurkat and MCF-7 cancer cell lines Benoit Banga N’guessan1* , Adwoa Dede Asiamah1, Nana Kwame Arthur1, Samuel Frimpong-Manso2, Patrick Amoateng1, Seth Kwabena Amponsah1, Kennedy Edem Kukuia1, Joseph Adusei Sarkodie3, Kwabena Frimpong-Manso Opuni2, Isaac Julius Asiedu-Gyekye1 and Regina Appiah-Opong4 Abstract Background: Nymphaea lotus L. (N. lotus) is an aquatic plant with anecdotal reports suggesting its use in the traditional management of cancer. However, there is a paucity of data on the antioxidant, anti-inflammatory and cytotoxic properties of N. lotus in relation to its phytochemical and elemental contents. This study aimed at determining the antioxidant, anti-inflammatory and cytotoxic properties of the hydro-ethanolic extract of N. lotus leaves (NLE), and its phenolic, flavonoid and elemental constituents. Methods: The antioxidant property of NLE was determined using total phenolic and flavonoid, DPPH radical scavenging, lipid peroxidation and reducing power assays. The anti-inflammatory activity of NLE (100–250-500 mg/ kg), diclofenac and hydrocortisone (positive controls) were determined by paw oedema and skin prick tests in Sprague Dawley rats. Also, the erythrocyte sedimentation rate (ESR) was determined by Westergren method. The macro/micro-elements content was determined by the XRF method. The cytotoxic property of NLE was determined by the MTT assay, on two cancer cell lines (MCF-7 and Jurkat) and compared to a normal cell line (Chang liver). Inhibitory concentrations were determined as IC50 values (±SEM). (Continued on next page) * Correspondence: bbnguessan@ug.edu.gh; benoitnguessan@yahoo.com 1Department of Pharmacology and Toxicology, School of Pharmacy, College of Health Sciences, University of Ghana, P.O. Box LG 43, Legon, Accra, Ghana Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 2 of 13 (Continued from previous page) Results: The extract had appreciable levels of phenolic and flavonoids compounds and was two-fold more potent in scavenging DPPH radicals than Butylated hydroxytoluene (BHT). However, NLE was three- and six-fold less potent than ascorbic acid and BHT, respectively, in reducing Fe3+ to Fe2+. The extract was six-fold more potent than gallic acid in inhibiting lipid peroxidation. The extract caused a dose-dependent decrease in rat paw oedema sizes, comparable to diclofenac, and a significant decrease in wheel diameters and ESR. The elemental analysis revealed relevant concentrations of Mg2+, P2+, S2+, K2+, Mn+, Fe+, Cu+, Zn+ and Cd+. The extract exhibited cytotoxic activity on both MCF-7 (IC50 = 155.00 μg/ml) and Jurkat (IC50 = 87.29 μg/ml), with higher selectivity for Jurkat cell line. Interestingly, the extract showed low cytotoxicity to the normal Chang liver cell line (IC50 = 204.20 μg/ml). Conclusion: N. lotus leaves extract exhibited high antioxidant, anti-inflammatory and cancer-cell-specific cytotoxic properties. These aforementioned activities could be attributed to its phenolic, flavonoid and elemental constituents. Keywords: Nymphaea lotus, Cytotoxicity, Micro/macro-elements, MCF7, Jurkat Background receptor (PR) and/or estrogen receptor (ER) are consid- Non-communicable diseases (NCDs) such as cardiomy- ered hormone receptor-positive breast cancers, while in opathies, chronic respiratory diseases, diabetes, and can- triple-negative breast cancer (TNBC), tumours do not cer are among the commonest causes of mortality and express any of these receptors. Just as in the rest of the morbidity worldwide. In sub-Saharan-Africa, NCDs rep- world, invasive ductal carcinoma is the commonest resent a significant cause of death and disability. NCDs histological type of breast cancer in Africa [12, 13]. are predicted to surpass infectious diseases as the most Acute T-cell leukemia (ALT) exhibits a very invasive important cause of morbidity and mortality by 2030 [1– pattern of progression associated with a reduced overall 3]. Oxidative stress (OS) and inflammation are primary survival due to factors such as chemoresistance and im- molecular mechanisms that play critical roles in the on- munosuppression [14, 15]. Due to high mortality associ- set and development of NCDs [4, 5]. Understanding the ated with the aforementioned cancers, there is the need role of OS and inflammation in NCDs has paved way for for agents with high efficacy (and few adverse effects) to the discovery of novel compounds with high antioxidant help in managing these conditions. and anti-inflammatory potential, that can be used in the During the past 18 years, cancer treatment strategies management of NCDs and cancer in particular. have markedly shifted from the use of cellularly-targeted Cancer is reported to be the second leading cause of therapies to the elaboration of molecularly-targeted death worldwide, with an estimated 9.6 million deaths in drugs. However, using a single molecularly-targeted 2018 [6, 7]. This estimation has been projected to double agent would often not yield good clinical response. by the next coming decade despite therapeutic advances Therefore, the use of combination therapy targeting made in recent years and the improvement in the man- multiple molecular sites could be of benefit in managing agement of cancer patients [7, 8]. It is noteworthy, that, cancers [16]. Medicinal plants used to manage cancers nearly 70% of these cancer-related deaths happen in traditionally have been shown to possess different prop- low- and middle-income countries (https://www.who. erties such as antioxidant, anti-inflammatory, cytotoxic, int/news-room/fact-sheets/detail/cancer). According to immunostimulant, anti-bleeding, providing minerals, vi- the New Global Cancer Data [6], Africa has the highest tamins, enzymes, and micronutrients to the body, etc. proportion of cancer deaths compared with its inci- [17, 18]. Therefore, the use of medicinal plant (that are dence, and this is likely due to the high occurrence of combinations of active phytochemicals, micro and macro cancer types related to poor prognosis along with re- elements) with established and promising antioxidant, stricted access to timely diagnosis and treatment [9]. Ex- anti-inflammatory and cytotoxic potentials could repre- amples of common and/or neglected cancers are breast sent an alternative to conventional cancer chemotherapy cancer and leukaemia. in Africa [19]. Breast cancer is a common invasive cancer found in Although questioned by some authors [20], it is gener- females. Data suggests that prevalence of breast cancer ally reported in the literature that up to 80% of the is on the increase among Ghanaian women, unlike in population in Africa depend on traditional medicines for North American women [10, 11]. Currently, a surrogate their primary healthcare needs [21]. In Ghana, there classification of five subtypes of breast cancer (based on have been efforts to integrate herbal medicines into the histological and molecular characteristics) is being used conventional health care system [22]. The interest for in clinical practice. Tumours expressing progesterone herbal products is also on the rise even in developed N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 3 of 13 countries such as the United States of America, where was placed (voucher number PH/2015/001). Plant ex- approximately 20% of people use herbal products for traction was performed as previously described [37], various health conditions [23]. However, many phy- with slight modification. Briefly, the fresh leaves of N. totherapies remain poorly studied. lotus were separated from the other parts of the plant, Nymphaea lotus L. (N. lotus), from the family Nym- dried at room temperature for 2 weeks and then ground phaeaceae (water lily family), is a perennial aquatic flow- into a powder. The powder was then dried again in an ering plant. It is native to Egypt and grows in various incubator at 45 °C for 36 h. A mass of 157.5 g of the parts of Central Africa, West Africa, and Madagascar powdered plant was weighed and dissolved in 20:80 v=v [24, 25]. N. lotus is known to possess numerous ethno- of water and ethanol. Dissolution was in the ratio 1:10 (1 medicinal properties and often used in the treatment of part of powder to 10 parts of solvent). The mixture was rheumatic pains, tumours and cancers [26–30]. Reports macerated with intermittent stirring for 24 h and filtered. of phytochemical analysis showed that the major bio- Filtrates were pulled together and concentrated using a active metabolites in Nymphaea species are flavonoids rotary evaporator (Rotavapor, Switzerland) at 40 °C. The and phenolic compounds, and that N. lotus contains very resulting concentrate was freeze-dried. The powder ob- special macrocyclic flavonoids [31, 32]. Based on earlier tained (referred to as NLE) was kept in an air-tight con- studies, one can say that N. lotus would be a good tainer and stored at room temperature. source of new isolates with high antioxidant, anti- inflammatory and cytotoxic potentials. Phytochemical screening Secondary metabolites found in plants that have been Tests for the presence of phytochemicals (triterpenoids, shown to be pharmacologically active include flavonoids, saponins, flavonoids, tannins, phenolic compounds and alkaloids, tannins, glycosides and sugars, among others alkaloids) contained in the NLE extract were conducted [33]. Interaction between secondary metabolites and mi- according to protocols previously reported [38]. cro and macro elements in plants are known to either decrease or enhance bioavailability of these secondary Total flavonoid content assay metabolites [34]. Conversely, toxic heavy metals that are The total flavonoid content of NLE extract was deter- environmental pollutant from the soil, water or air can mined as previously described [39]. Briefly, a mass of be absorbed by medicinal plants at high concentrations, 100 μg of different concentration of NLE extract was and this could partly explain reported toxic effects of placed in a 96 well plate, 2% aluminium chloride was some plants [35]. The World Health Organization there- added to each well and incubated at room temperature. fore recommends systemic determination of heavy metal Absorbances were then read at 420 nm. Total flavonoid content and maximum permissible levels of some heavy was evaluated as quercetin equivalent (mg/ml). metals (cadmium, arsenic, and lead) in raw consumable plant materials [36]. The aim of the current study was to determine the in- Total phenolic assay (modified Folin-Ciocalteau method) vitro antioxidant potential, in-vivo anti-inflammatory ac- The total phenol content of NLE extract was determined tivity and the cytotoxic property of the hydro-ethanolic as previously described [39]. Briefly, aliquots (10 μl) of extract of the leaves of N. lotus on Jurkat (leukaemia) NLE extract and gallic acid of different concentrations and MCF7 (breast cancer) cell lines, and Chang liver were placed in Eppendorf tubes. Distilled water (0.79 ml) (normal) cell line. Also, this study sought to determine and Folin-cicocalteau reagent (~ 50 μl) were subse- and relate the total phenol, total flavonoid and the quently added and mixed totally. The mixture was after- macro/micro-elemental constituents of the leaves of N. wards incubated at room temperature for 8 min. After lotus to its pharmacological activities. incubation, Na2CO3 solution (150 μl) was added, mixed and again incubated at room temperature for 2 h. Ali- Methods quots were put in well-plates, and absorbance read at Plant collection and extraction 750 nm. Triplicates concentrations of NLE and gallic The plant, N. lotus, was collected, following the local le- acid prepared were used. gislation, in August 2015 from the wild in Koumassi (Latitude: 5° 16′ 60.00“ N; Longitude: -3° 58’ 59.99” W), Macro and micro-elements screening: energy dispersive a suburb of Abidjan (Cote d΄Ivoire). No permission was x-ray fluorescence spectroscopic (ED-XRF) measurement required to collect these plants. The plant was identified The screening of macro and micro-elements found in N. by Mr. Digbeu Luc Gouda (a herbalist) and authenti- lotus was determined as previously described [40]. cated by Dr. Cindy Kitcher at the Department of Briefly, the powder of N. lotus leaves was kept at 60 °C Pharmacognosy and Herbal Medicine (School of Phar- overnight in an oven before pelletisation and subsequent macy, University of Ghana), where a voucher specimen measurement. Triplicate measured samples (4 g/sample) N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 4 of 13 and 0.9 g of a binder (Fluxana H Elektronic BM-0002-1, added and centrifuged at 300 g for 10 min. Two hundred Licowax C micro powder PM-Hoechstwax, Germany) (200) μL of the supernatant were pipetted into a tube, were homogenized using the RETSCH Mixer Mill and 200 μL of distilled water and 40 μL of ferric chloride (MM301, Germany) and pressed with a manual hy- were added. The blank was a solution with all reagents draulic press (SPECAC, UK) to obtain pellets of 32 mm except the NLE. The resulting solution was incubated in diameter and 3mm thickness. These were used for for 30 min and pipetted into well plates, after which ab- subsequent XRF measurements. The detection limit for sorbance was read at 700 nm. Ascorbic acid and BHT light elements (Si, Al, Mg and Na) was in the range of were used as positive controls. 25–50 ppm. For heavy metals, 1–5 ppm was the limit of detection. Calibration was factory done using inter- Anti-inflammatory assays national rock standards. Simultaneous measurement and Animals care and safety analysis of the elemental contents of the samples of N. This research was reviewed and approved by the Ethical lotus leaves were performed using a SPECTRO X-Lab and Protocol Review Committee of the College of Health 2000 spectrometer. Sciences, University of Ghana (Protocol Identification Number: CHS-Et/M.6-P1.5/2017–2018) and was con- Antioxidant assays ducted in accordance with the internationally accepted 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) assay principles for laboratory animal use and care as found in The DPPH assay was performed as previously described the US guidelines (NIH publication #85–23, revised in [39]. Briefly, different concentrations of NLE extract 1985). were made by threefold serial dilutions starting from an Sixty pathogen-free male Sprague-Dawley rats (Hsd: initial concentration of 20 mg/ml. The test samples SD strain), aged 2 to 3months and weighing 150–200 g, (100 μL) were added in triplicates to 100 μL of DPPH in were purchased from the Center for Plant Medicine Re- well plates and gently shaken. The plate was covered search (CPMR), Mampong, Eastern Region, Ghana. Ani- with a foil and incubated in the dark, at room mals were housed in stainless steel cages of 2 cubic feet temperature, for 20 min. Absorbances were then read at (61 cm × 31 cm × 31 cm) with softwood shavings as bed- 517 nm. The amount of sample required to react with ding. They were maintained under standard laboratory one half of the DPPH was expressed as the relative conditions (temperature ~ 25 °C, relative humidity 60– amount of the positive control, butylated hydroxytoluene 70%, and 12 h light-dark cycle), fed with standard pellet (BHT), that reacted. Antioxidant activity of samples was diet (AGRIMAT, Kumasi, Ghana) and allowed access to expressed as DPPH radical scavenging by the formula: water ad libitum. Animals were acclimatised under these ¼ Sample absorbance conditions for 7 days before the experiment. To prevent%DPPH radical scavenging x 100 Control absorbance contamination, animals’ feeding, and water troughs were washed frequently. Lipid peroxidation assay The lipid peroxidation assay was performed as previ- Paw edema test ously described [41]. Briefly, 100 μL of different concen- The paw edema test was performed as previously de- trations of NLE extract, obtained by twofold dilutions, scribed [43]. Briefly, rats were randomly divided into 6 were added to 1 ml of egg lecithin [3 mg/ml] in phos- groups of 5 each. Oedema was induced by sub-plantar phate buffer. 10 μL of FeCl3 [400 mM] and 10 μL of L- injection of 100 μL of 1% freshly prepared solution of ascorbic acid [200 mM] were then added to induce lipid carrageenan into the right-hind paw of each rat of all peroxidation. After incubation for 1 h, at 37 °C, 1 ml of groups. Group 1 served as negative control and was ad- 15% trichloroacetic acid (TCA) and 1ml of 0.375% tert- ministered distilled water; groups 2, 3 and 4 were treated butyl alcohol (TBA) in 20% acetic acid were added to with NLE at 100, 250 and 500mg/kg of body weight stop the reaction. The mixture was boiled for 15 min, (bw) respectively, 3 days prior to the induction of inflam- allowed to cool and centrifuged at 300 g. Absorbance of mation. Groups 5 and 6 were given diclofenac (2 mg/kg the supernatant was measured at 532 nm. bw) orally and hydrocortisone succinate (4 mg/kg bw) intraperitoneally 30 min before carrageenan injection. Reducing power assay Paw thickness was measured just before the carrageenan The reducing power assay was performed as previously injection (initial) and then at 1, 2, 3, 4, and 24 h after described [42]. Briefly, 200 μL of NLE extract were carrageenan injection. Increase in paw thickness was added and mixed to 200 μL of potassium ferricyanide. measured as the difference in paw thickness at the initial The mixture was incubated at 50 °C for 20 min, and a time and paw thickness at respective hours using a volume of 200 μL of trichloroacetic acid (TCA) was then plethysmometer. N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 5 of 13 Skin prick test and erythrocyte sedimentation rate (ESR) added to the cells and incubated for 48 h. For the treat- The skin prick test was performed, as previously de- ment of suspension cells (Jurkat), 1 × 104cells/well were scribed [44]. Briefly, rats were randomly divided into 6 seeded and varying concentrations of the extract (62.5 to groups of 5 each and sensitized (group 2, 3, 4, 5 and 6) 1000 μg/ml) and curcumin (0 to 36.84 μM) were added or not (group 1) by intraperitoneal and subcutaneously in the wells. The plates were incubated at 37 °C in a hu- injections of albumin (OVA) emulsified in aluminum hy- midified atmosphere containing 5% CO2. droxide on days 1 and 7. Group 1 (non-sensitized con- trol) and group 2 (OVA-sensitized control) received MTT assay distilled water orally. In contrast, groups 3, 4 and 5 were Cytotoxic effects of NLE and curcumin on cell lines pre-treated with daily oral administration of NLE ex- were determined using an MTT assay as previously de- tracts (100, 250 and 500 mg/kg bw) and group 6 with in- scribed [47]. Briefly, for adherent cells, after 48 h of traperitoneal administration of hydrocortisone succinate treatment incubation, 15 μl of MTT [5 mg/ml] in (4 mg/kg bw) over 8 days, from day 1. On day 9, two phosphate-buffered saline (PBS) were added and incu- separate areas on the skin of all rats were shaved using a bated at 37 °C for 4 h. MTT was flicked off and the blade and the prick test was carried out on all rats by formed formazan crystals solubilised in 100 μl of DMSO. subcutaneous injection of histamine. The time for the Absorbance was measured at 570 nm, using a microplate appearance of the swelling was noted and the wheal di- spectrophotometer (Tecan Infinite M200 Pro plate ameters were recorded for all rats at 1 h and 2 h after reader, Austria). For suspension cells (Jurkat), after 48-h histamine injection. The average skin oedema was calcu- treatment, the medium was discarded and 20 μl/well of lated for each group and expressed as a percentage of MTT solution [5 mg/ml] added. The plate was incubated the OVA-sensitized control. Percentage of skin oedema for 3 h at 37 °C. Finally, 20 μl of isopropanol was added relative to that of OVA-sensitized control for each group to each well and the plate read using a spectrophotom- was calculated according to the formula: eter at a wavelength of 590 nm. The percentage of cell inhibition was determined using the formula: ¼ oedema in a particular treated group%Skin oedema x 100 oedema in sensitized control group  Mean absorbance of treated cells %Cell growth inhibition ¼ 100 − x 100 Mean absorbance of untreated cells Rats were then sacrificed by cervical dislocation and the whole blood was collected by cardiac puncture into The experiment was done in triplicate. A graph of citrate tubes under sterile conditions. The erythrocyte mean percentage viability against concentration was sedimentation rate was determined as previously de- plotted, using Graph pad prism 5 and the IC50s of cur- scribed [45, 46]. Briefly, the Westergren pipette was gen- cumin and the extract (where applicable) were tly inserted into the citrate tube, and by capillary action, determined. the anticoagulated blood rose to the 0 mm mark to the top of the pipette. The pipette was placed into a stand Statistical analysis and fixed in a vertical position for 1 h. The sedimenta- Percentage inhibition against the log of concentration tion rate was then measured in mm/h by reading from was plotted for each sample tested, and 50% inhibitory the calibrations on the pipette. concentration (IC50) interpolated from the curves. IC50 values were determined using Graph Pad Prism software In-vitro cytotoxic activity of NLE version 5.0 for Windows (GraphPad Software, San Cell culture and treatment Diego, CA, USA) and expressed as IC50 mean value (± Cell culture and treatment were performed as previously SEM). Graphs were plotted using Sigma Plot for Win- described [47]. Jurkat, MCF-7 and Chang liver cell lines, dows Version 11.0 (Systat Software Inc., Germany). A provided by the Noguchi Memorial Institute for Medical duplication method was used before the actual analysis Research, University of Ghana, were cultured in EMEM of the micro and macro-elements. The method employs containing 10% FBS and 1% penicillin/streptomycin and three-axial geometry, thereby reducing background noise all cells were maintained at 37 °C, 100% relative humid- by radiation polarisation. ity, 5% CO2, 95% air, and culture media changed twice a week. The cells were subcultured when they reached Results 80% confluence. For the treatment of adherent cells Phytochemical, total flavonoid and total phenolic (MCF-7and Chang Liver cells), well plates at a density of contents of NLE 1 × 104 cells/well were seeded with 100 μl of cell suspen- The phytochemical screening of NLE revealed the pres- sion and incubated for 24 h. The extract concentrations ence of triterpenoids, saponins, flavonoids, tannins and ranged from 62.5 to 1000 μg/ml and curcumin (positive phenolic compounds and the absence of alkaloids. The control) from concentrations of 0 to 36.84 μM were then total flavonoid content of NLE was expressed as N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 6 of 13 quercetin equivalent and extrapolated from the standard Table 1 Content and limit of detection (ppm) of micro and curve of quercetin (Fig. 1.1-A). Concentrations of 0.31 macro-elements in Nymphaea lotus leaves and 0.63 mg/ml of NLE contained 1.8 and 2.2 g of total Elements Mean ± 3σ (ppm) Limit of detection LOD (ppm) flavonoid per 100 g of quercetin equivalent, respectively Mg 11,433 ± 0.43 1100 (Fig. 1.1-B). In the other hand, the total phenolic content Al 7733 ± 0.01 95 of NLE, expressed as gallic acid equivalent, was extrapo- Si 38,403 ± 0.036 160 lated from the standard curve of gallic acid (Fig. 1.2-A). At concentrations of 2.5; 5.0 and 10.0 mg/ml of NLE, the P 2959 ± 97 10 total phenolic contents determined were 10.5; 11.8 and S 3225 ± 68 13 10.6 g /100 g of gallic acid equivalent, respectively K 21,500 ± 0.021 16 (Fig. 1.2-B). Ca 13,800 ± 0.009 17 Mn 414 ± 28 5 Micro and macro-elements content of NLE Fe 5859 ± 87 12 A total of nine 9 macro-elements and 11 micro- elements were identified and quantified in NLE samples Cu 6 ± 3 2 (Table 1). The macro-elements of mean values between Zn 33 ± 3 1 414 and 38,403 ppm identified were magnesium (Mg), Rb 28 ± 1 1 aluminum (Al), silicon (Si), phosphorus (P), sulphur (S), Sr 19 ± 1 1 potassium (K), calcium (Ca), manganese (Mn), and iron Y 2 ± 1 1 (Fe). While the micro-elements of mean values between Zr 9 ± 2 1 2 and 40 ppm were copper (Cu), zinc (Zn), rubidium (Rb), strontium (Sr), yttrium (Y), zirconium (Zr), nio- Nb 3 ± 2 1 bium (Nb), silver (Ag), cadmium (Cd) and thorium (Th). Ag 40 ± 8 2 The content of the elements quantified in N. lotus leaves Cd 16 ± 9 2 decreases in the order: Si > K > Ca >Mg > Al > Fe > S > Pb – 2 P >Mn > Ag > Zn > Rb > Sr > Cd > Th > Zr > Cu > Nb > Y. Th 15 ± 5 2 Macro and micro-elements content were measured on pellets of N. lotus leave Antioxidant activities of NLE samples (32 mm diameter × 3mm thickness) using energy dispersive x-ray The extract and BHT exhibited a concentration- fluorescence (ED-XRF) spectroscopy (SPECTRO X-Lab 2000 spectrometer). Measurements were done in triplicate and data presented are expressed in dependent scavenging activity of DPPH (Fig. 2a). The ppm and are mean value ±3σ. The limit of detection for light elements (Si, Al, concentration of NLE required to inhibit 50% of free Mg, and Na) was in the range of 25–50 ppm and 1–5 ppm for heavy metals radicals (IC50) was 0.0976 ± 0.25 mg/ml compared to 0.2188 ± 0.02 mg/ml obtained for BHT (positive control). lipid peroxidation (Fig. 2b). The IC50 values deter- The difference in IC50 values between the NLE and BHT mined for NLE and gallic acid were 0.2367 ± 0.006 was statistically significant (p < 0.001) and NLE was and 1.418 ± 0.01 mg/ml, respectively. The difference in found to be about two-fold more potent than BHT, in IC50 values between the NLE and gallic acid was sta- scavenging DPPH radicals. Also, both NLE and gallic tistically significant (p < 0.001) and NLE was about acid caused a concentration-dependent inhibition of six-fold more potent in inhibiting lipid peroxidation Fig. 1 Total flavonoids and total phenolic contents of the hydro-ethanolic extract of Nymphaea lotus (NLE) leaves. Panel 1.1: Total flavonoid content of NLE (2.5mg/ml – 10mg/ml) expressed as quercetin equivalent (QE). Panel 1.2: Total phenolic content of various concentrations of NLE (2.5mg/ml- 10 mg/ml) expressed as gallic acid equivalent (GAE). Each point represents the mean value ± standard error on the mean (SEM), (n = 3) N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 7 of 13 Fig. 2 Antioxidant activities of the hydro-ethanolic extract of Nymphaea lotus (NLE). a: 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) radical scavenging activity, with each point representing mean value ± standard error on the mean (SEM), (n = 3). b: Inhibition of lipid peroxidation by NLE and gallic acid, with each point representing mean ± SEM (n = 3). c: Reducing power of NLE, Ascorbic acid and butylated hydroxytoluene (BHT), with each point representing mean ± SEM (n = 3) than gallic acid. The extract, ascorbic acid and BHT NLE and the standard drug on the wheal diameter were showed a concentration-dependent reducing power time- and dose-dependent and were statistically signifi- (Fig. 2c). IC50 determined were 0.447 ± 0.02; 0.151 ± cant at NLE 250 and 500mg/kg (p < 0.05 and p < 0.03 and 0.07 ± 0.04 mg/ml, for NLE, ascorbic acid 0.0001). and BHT, respectively. There were significant differ- ences (p < 0.001) between the IC50 values of NLE, Erythrocyte sedimentation rate (ESR) ascorbic acid and BHT. However, NLE was about 3 Control rats sensitised by ovalbumin showed a signifi- and 6-fold less potent than ascorbic acid and BHT, cantly increased ESR (p < 0.01) when compared to the respectively, in reducing Fe3+ to Fe2+. non-sensitized rats. Ovalbumin-sensitized rats treated with increasing doses of NLE exhibited a dose- Anti-inflammatory activities of NLE dependent decrease of the ESR. However, this NLE- Rat paw oedema reduction induced decrease of ESR was significant at 250 and 500 Sub-plantar injection of carrageenan in the paw induced mg/kg (p < 0.05 and p < 0.01 respectively). Also, ESR was oedema (in all rats) which size increased with time till 4 significantly decreased (p < 0.01) in rats treated with h and then spontaneously decreased till 24 h after injec- hydrocortisone when compared the control sensitised tion (Fig. 3). However, rats pre-treated with NLE or rats (see Fig. 5). standard drugs (diclofenac and hydrocortisone) exhibited significantly reduced paw oedema sizes at all points Effect of NLE on MCF-7, Jurkat and Chang liver cell lines measured in time when compared to the negative con- proliferation trols. These effects of NLE and standard drugs on the The effect of NLE on MCF-7, Jurkat and Chang liver cell oedema size were time- and dose-dependent and were lines proliferation is presented in Fig. 6. The extract and statistically significant among groups (p < 0.05 and p < curcumin (positive control) exhibited concentration- 0.0001 respectively). dependent inhibition of MCF-7 with IC50 values of 5.180 and 155 μg/ml for curcumin and NLE, respect- Skin prick test ively. The difference between the IC50 values of NLE Intradermal injection of histamine induced the forma- and curcumin were statistically significant (p < 0.001). tion of a wheal on the skin of all rats (Fig. 4). An hour Both NLE and curcumin inhibited the growth of Jur- after histamine injection, only rats pre-treated with kat cells in a concentration-dependent fashion with hydrocortisone presented a significantly reduced wheal IC50 values of 2.056 and 87.29 μg/ml for curcumin diameter, when compared to the negative control group and NLE respectively and the difference was found to (p < 0.05). However, 2 h after histamine injection, rats be statistically significant (p < 0.001). The extract and pre-treated with NLE or the standard drug (hydrocorti- curcumin exhibited a concentration-dependent inhib- sone) exhibited a significantly reduced wheal diameter, ition on Chang liver cell growth with IC50 values of as compared to the negative control. These effects of 204.2 and 2.568 μg/ml for NLE and curcumin N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 8 of 13 Fig. 3 Anti-inflammatory activities of the hydro-ethanolic extract of Nymphaea lotus leaves (NLE, 100, 250 and 500mg/kg, p.o.), diclofenac (2 mg/ kg) and hydrocortisone (4 mg/kg) on the paw oedema size in normal SD rats. Chart a shows the time course effects over a 24-h period; chart b shows the total percentage paw oedema’s area under the curve (AUC) over the 24-h period. Data are mean value ± standard error on the mean (SEM), (n = 5). *p < 0.05, ***p < 0.001 when compared to the negative control group, using one-way ANOVA followed by a Dunnett’s Multiple Comparison Test respectively and this difference was statistically signifi- The phytochemical screening of the hydro-ethanol ex- cant (p < 0.001). tract of N. lotus leaves (NLE) revealed the presence of triterpenoids, saponins, flavonoids, tannins and phenolic Discussion compounds and the absence of alkaloids. Also, the deter- There is a growing body of evidence that suggests that mination of the total phenol and total flavonoids con- oxidative stress and inflammation are two primary tents of NLE extract revealed appreciable amounts of mechanisms leading to the initiation and development these two secondary metabolites. The presence of sap- of cancers. This study sought to contribute to the man- onin, triterpenoids and considerable amounts of flavo- agement of cancer by investigating the antioxidant, anti- noids and phenolic compounds in our extract could inflammatory and cytotoxic activities of N. lotus (a medi- explain the antioxidant, anti-inflammatory and cytotoxic cinal plant traditionally used to manage cancer patients) activities of NLE, as similarly reported by previous stud- in relation to its phytochemical and elemental ies on aqueous and acetone extracts of N. lotus [48–50]. constituents. Likewise, these findings are consistent with a study Fig. 4 Anti-inflammatory activities of the hydro-ethanolic extract of Nymphaea lotus leaves (NLE, 100, 250 and 500mg/kg, p.o.) and hydrocortisone (4 mg/kg) on wheal diameter in ovalbumin (OVA)-sensitized SD rats, 1 and 2 h after intradermal injection of histamine. Data are mean value ± standard error on the mean (SEM), (n = 5). **p < 0.01, ***p < 0.001 when compared to the negative control group, using one-way ANOVA followed by a Dunnett’s Multiple Comparison Test N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 9 of 13 showing the presence of vitamins A, C and E in N. lotus leaves [51]. Previous reports showed the presence of al- kaloids in the chloroform, ethyl acetate, n-butanol, acet- one and aqueous extracts of N. lotus leaves, in addition to the presence of phenolic compounds (flavonoids, cou- marins and tannins), and sterols [50, 52]. The absence of alkaloids in our extract (NLE) could be due to solvent system used for the extraction or the geographical loca- tion of plants collected. Our choice for the hydro- ethanolic system was because we wanted to mimic, as much as possible, the traditional preparation of N. lotus. The elemental content of NLE sample was measured by energy dispersive x-ray fluorescence spectroscopy (ED-XRF). It is a fast, reliable and powerful analytical tool for the determination of the elemental composition of diverse materials (particularly for high energy ele- ments) and does not produce waste and requires no Fig. 5 Anti-inflammatory activities of the hydro-ethanolic extract of chemical reagents [53]. A total of nine 9 macro and 11 Nymphaea lotus leaves (NLE, 100, 250 and 500mg/kg, p.o.) and hydrocortisone (4 mg/kg) on the rate of sedimentation of micro-elements were identified and quantified in the erythrocyte (ESR) from blood samples of ovalbumin (OVA)-sensitized NLE sample (Table 1). The content of various elements SD rats. Data are mean value ± standard error on the mean (SEM), analyzed in N. lotus leaves decreases in the order: Si > (n = 5). *p < 0.05, **p < 0.01 when compared to the negative control K > Ca >Mg > Al > Fe > S > P >Mn > Ag > Zn > Rb > Sr > group, using one-way ANOVA followed by a Dunnett’s Multiple Cd > Th > Zr > Cu > Nb > Y. Macro-elements such as Comparison Test magnesium (Mg), phosphorus (P), sulphur (S) and Fig. 6 Cytotoxic activities of the hydro-ethanolic extract of Nymphaea lotus leaves (NLE) and curcumin on cell growth of breast cancer (MCF-7) cell line (a), leukaemia (Jurkat) cell line (b) and Chang liver cells (c). Each point represents the mean value ± standard error on the mean (SEM), (n = 3) N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 10 of 13 micro-elements such as manganese (Mn), copper (Cu) study, the hydro-alcohol and chloroform extracts of Nyc- and zinc (Zn) present in NLE sample have been reported tantes arbour were found to be as potent as ascorbic to be relevant elements as anti-cancer adjuvants [54, 55]. acid in reducing power property [65]. These elements are known to play important roles in The anti-inflammatory activity of NLE extract was cellular metabolism, as co-factors in numerous enzym- measured using carrageenan-induced rat paw oedema atic activities, or as important components of structural assay, skin prick test and erythrocyte sedimentation rate proteins such as hormones involved in cancer therapy assay. Carrageenan-induced rat paw oedema model is an [56]. Copper and zinc have been reported to be involved appropriate test commonly used in screening of anti- in the elimination of free radicals through cascading en- inflammatory agents. The biphasic acute inflammation zyme systems [57]. Thus, the presence of these elements induced by carrageenan is characterized by the release of in NLE could support the traditional use of N. lotus as pro-inflammatory mediators such as kinins, histamine an anticancer medicinal plant and also explain the anti- and serotonin, in the first phase [66], followed by the re- oxidant, anti-inflammatory and cytotoxic activities of lease of prostaglandins-like substances, the main media- NLE reported in this study. Moreover, cadmium (Cd), a tors causing the acute inflammation [67]. This later toxic heavy metal, was detected in the NLE sample. Lead phase is responsive to clinically useful anti-inflammatory was below the detectable limit. These findings are almost agents [68]. The extract significantly decreased paw similar to results reported [51] using a flame photometer oedema sizes in a dose-dependent manner, thus, might and an atomic absorption spectrophotometer (AAS). be containing some anti-inflammatory compounds caus- This finding proved that results obtained using ED-XRF ing the inhibition of prostaglandin-induced inflamma- or AAS methods are not significantly different [58]. tory pathway. Subcutaneous injection of histamine (or However, it is important to note that acute or chronic other allergens) causes mast cells to degranulate and re- exposure of an organism to these toxic elements (which lease histamine and other mediators, following the are usually contaminants from the environment) present cross-linkage of specific IgE bound to their surface re- in medicinal plants could result in the development ceptors. This leads to a triple response characterized by of unwanted toxicopathies [59]. Therefore, the pres- a capillary dilatation (causing a red spot), an arteriolar ence of these toxic elements (but not N. lotus itself) dilatation mediated by axon reflex leading to a flare could explain the toxic effects of the hydro-ethanolic (causing the redness in the surrounding area) and an ex- and hydro-methanolic extracts of N. lotus reported in udation of fluid from venules and capillaries (causing the the literature. These toxic effects were characterised formation of a wheal), which wheal can be quantitated by an induction of a chromosomal aberration in rat by measuring its diameter [44]. Thus, the skin prick test lymphocytes, an increase of aberrant sperm cells and was used to measure the anti-inflammatory effect of somatic genotoxicity [49, 60]. NLE on the immunoglobulin E (IgE) response to ovalbu- The antioxidant activity of NLE extract was deter- min (OVA) and on the vasodilatory activity of a pre- mined using DPPH radical scavenging assay, lipid perox- formed mediator, histamine, in sensitized rats. The idation inhibition assay and reducing power assay. The extract significantly decreased wheal diameters in a DPPH radical scavenging assay revealed that NLE was dose-dependent manner, thus, might be containing anti- about two times more potent than BHT in scavenging inflammatory compounds that could also block mast DPPH. This result shows that NLE has a strong antioxi- cells degranulation and the release histamine (or other dant potential, and that could be attributed to its phen- mediators). Erythrocyte sedimentation rate assay mea- olic, flavonoid and elemental contents. A correlation sures the distance that erythrocytes have sedimented between phenolic compounds, micro-element content (under the influence of gravity) after 1 h in a vertical col- and antioxidant activity has been reported in the litera- umn of anticoagulated blood. The main factor influen- ture [61–63]. In the current study, NLE was also found cing and directly correlating with the sedimentation rate to be about six-fold more potent in inhibiting lipid per- is the amount of fibrinogen in the blood. The extract oxidation than gallic acid. The extract’s ability to inhibit significantly decreased the ESR in a dose-dependent lipid peroxidation could be attributed to the presence of manner, thus, might be containing anti-inflammatory lipid-soluble antioxidant compounds, hence, could do- compounds that could block the synthesis of fibrinogen. nate protons to stabilize lipid radicals or exquisitely Altogether, these results showed a consistent anti- scavenges lipid peroxyl radicals, thereby, terminating inflammatory activity of NLE extract. These findings are subsequent chain propagation reactions [64]. In a redu- consistent with the traditional use of N. lotus as anti- cing power assay, the oxidative form of iron (Fe3+) in inflammatory medicine [69] to treat arthritis and rheum- ferric chloride is converted to ferrous (Fe2+) by antioxi- atic pains [70]. dant compounds. The extract was not as potent as as- Invasive ductal carcinoma is the commonest histo- corbic acid or BHT in reducing Fe3+ to Fe2+. In a similar logical type of breast cancer in Africa and acute T-cell N’guessan et al. BMC Complementary Medicine and Therapies (2021) 21:22 Page 11 of 13 leukemia exhibits a very aggressive course of progression concentration 50; MTT: 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium with poor prognosis associated with shorter overall sur- bromide; NCDs: Non-communicable diseases; NLE: Nymphaea lotus ethanolic extract; OS: Oxidative stress; SEM: Standard error on the mean; SD: Sprague- vival. Therefore, there is a need to discover new effective Dawley (rats); WHO: World health organisation agents against these two types of cancer. MCF-7 is a hu- man adenocarcinoma breast cancer cell line with estro- Acknowledgements gen, progesterone and glucocorticoid receptors, while The authors are grateful to the Non-Governmental Organisation for the Pro-motion of Scientific Research in African Traditional Medicine (NGO “PRORES- Jurkat cell line was derived from human acute T-cell MAT”) and to the late Mr. Kafongo Kignelman Ouattara (Abidjan, Cote leukaemia. These two cell lines are excellent models for d’Ivoire) for introducing Dr. Benoit Banga N’guessan to the herbalist. Also, we screening anticancer (cytotoxic) agents. The cytotoxic wish to thank the herbalist Mr. Digbeu Luc Gouda (Abidjan, Cote d’Ivoire) foraccepting to collaborate with us and for helping in the collection of the activity of NLE extract was determined using an MTT plant material. We are also grateful to all laboratory technicians at the School assay. Both NLE and curcumin (positive control) exhib- of Pharmacy and Noguchi Memorial Institute for Medical Research (NMIMR), ited cytotoxic activities on MCF-7 and Jurkat cell lines, University of Ghana, for their help and technical support. however, NLE was not as potent as curcumin. This dif- Authors’ contributions ference could be explained by the fact that curcumin is a BBN conceived the study. BBN, PA, SFM and RAO designed the pure isolated compound and NLE is a crude extract con- methodology. ADA, NKA performed the experimental work under the supervision of BBN and RAO. BBN, ADA, NKA and SFM conducted the taining several active compounds which could have an- collection of data. BBN, ADA, NKA, SFM, SKA, KEK, JAS, KFMO, IJAG and RAO tagonistic effects on each other. The cytotoxic activity of performed the data analysis. BBN, ADA, NKA, SFM, SKA, KEK, JAS, KFMO, IJAG NLE revealed some cell-specificity for Jurkat cell line, as and RAO were involved in the writing of the manuscript. All authors read and approved the final manuscript. shown by the IC50 values of NLE for Jurkat and MCF-7. Interestingly, NLE showed less cytotoxic activity on Funding Chang liver cells (normal cell line) when compared to The authors have no support or funding to report. the cancer cell lines (Jurkat and MCF-7). In the opposite, Availability of data and materials curcumin rather showed a potent cytotoxic activity on The data used to support the findings of this study are available from the Chang liver cell line. These results corroborate similar corresponding author upon request. antiproliferative studies on the cytotoxic activities of Ethics approval and consent to participate Nymphea lotus (whole plant), Nymphea alba and Nym- This research was reviewed and approved by the Ethical and Protocol phaea mexicana [27, 71] and could be attributed to the Review Committee of the College of Health Sciences, University of Ghana phytochemical and elemental contents of N. lotus leaves (Protocol Identification Number: CHS-Et/M.6-P1.5/2017–2018) and was con-ducted in accordance with the internationally accepted principles for labora- and therefore, could justify its traditional use as cancer tory animal use and care as found in the US guidelines (NIH publication treatment. Further bioactivity-guided fractionation stud- #85–23, revised in 1985). ies would be necessary to isolate the active compounds Consent for publication responsible for the aforementioned activities. Not applicable. Conclusion Competing interestsDr. Patrick Amoateng declares that he is a member of the editorial board Our results showed that the hydro-ethanolic extract of (Associate Editor) of this journal. All other authors declare that they have no N. lotus leaves possesses high antioxidant potential, anti- competing interests. inflammatory property and cytotoxic activity on MCF-7 Author details and Jurkat cancer cell lines. These properties could be 1Department of Pharmacology and Toxicology, School of Pharmacy, College attributed to the presence of appreciable amounts of of Health Sciences, University of Ghana, P.O. Box LG 43, Legon, Accra, Ghana. 2 total phenol and flavonoids, as well as to the presence of Department of Pharmaceutical Chemistry, School of Pharmacy, College ofHealth Sciences, University of Ghana, P.O. Box LG 43, Legon, Accra, Ghana. macro/micro-elements such as magnesium, phosphorus, 3Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, sulphur, manganese, copper and zinc. However, the College of Health Sciences, University of Ghana, P.O. Box LG 43, Legon, 4 presence of heavy metals such as cadmium detected in Accra, Ghana. Department of Clinical Pathology, Noguchi Memorial Institutefor Medical Research (NMIMR), College of Health Sciences, University of N. lotus leaves could explain the toxic effects of the Ghana, Accra, Ghana. hydro-ethanolic extract of N. lotus previously reported in the literature. Altogether, our findings could explain Received: 13 March 2020 Accepted: 17 December 2020 the use of N. lotus leaves in the traditional management of cancer patients and justify the use of this plant as a References source for the discovery of new anticancer agents with 1. Holmes MD, Dalal S, Volmink J, Adebamowo CA, Njelekela M, Fawzi WW, Willett WC, Adami H-O. 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