Metabolite Characterization, Antioxidant, Anti-proliferative And Enzyme Inhibitory Activities Of Lophira Lanceolata Tiegh. Ex Keay Extracts

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Kouadio Ibrahime Sinan a,1 , Lara Safti´c Martinovi´c b,1 , Zeljka ˇ Perˇsuri´c b , Sandra Kraljevi´c Paveli´c b , Petra Grbˇci´c b , Dario Matulja b , Ouattara Katinan Etienne c , Mohamad Fawzi Mahomoodally d,e , Devina Lobine e , Tapan Behl f , Gokhan Zengin a, *

ABSTRACT

Lophira lanceolata Tiegh. ex Keay, a native tropical plant to the west and central Africa, is a multifunctional plant used as timber and also exploited in traditional medicine in Africa. The present study presents the pharmacological properties of Lophira lanceolata leaf and stems bark extracts prepared from different extraction procedures (infusion, homogenizer-assisted extraction, maceration, soxhlet). The detailed phytochemical composition, in vitro antioxidant and antiproliferative assays, as well as the key enzyme (cholinesterases, tyrosinase, α-amylase and α-glucosidase) inhibitory potentials of the extracts were evaluated. Chemical profiling confirmed the presence of lanceolatins and lophirones in all analyzed plant parts. Higher levels of total phenolics (156.42 mg gallic acid equivalent (GAE)/g for maceration-water), phenolic acids (165.84 mg caffeic acid equivalent (CE)/g for maceration-water) and flavanols (101.51 mg catechin equivalent (CAE)/g for soxhlet-methanol (MeOH)) were observed in the stem bark extracts. Antioxidant assays showed remarkable free radical scavenging and reducing power activities for all extracts, among which stem bark showed the highest potential. Stem bark extracts (IC50 values: 57.55–93.10 μg/ml for colorectal adenocarcinoma (HT29) and 78.18–89.58 μg/ml for metastatic breast cancer cell line (MCF7)) also showed better antiproliferative effect than leaf (424.14–790.27 μg/ml for HT29 and 374.46–943.09 μg/ml for MCF7) on carcinoma cells while they induced proliferation in normal human cell lines (normal skin fibroblasts (HFF)). In the enzyme inhibitory assays, the methanol extracts of both plant parts were confirmed as effective against AChE, BChE (only stem barks extracts) and α-glucosidase enzymes. Regarding anti-tyrosinase effects, the methanol leaf extracts (122.21–131.17 mg kojic acid equivalent (KAE)/g) and all stem bark extracts (94.58–153.21 mg KAE/g) exhibited inhibitory effects. Findings amassed herein tend to validate the traditional uses of L. lanceolata and advocate for the development of phyto-medicaments based on its extracts.

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1. Introduction

African traditional medicine is one of the oldest and the most diverse of all healthcare systems (Mothibe and Sibanda, 2019). For centuries, the population of Africa has exploited the unique and rich plant biodiversity for their healthcare needs. African plant biodiversity, coupled with the deeply rooted African ethnobotanical knowledge, has yielded several novel biologically active compounds that are now components of the modern drugs; notable examples include the anti-tumor agents' vinblastine and vincristine derived from Catharanthus roseus (Madagascar periwinkle), yohimbine from Pausinystalia Yohimbe and physostigmine from calabar bean, the seeds of Physostigma venosum (Khalid, 2012). Therefore, with the high interest in plant-derived medicaments, Africa remains a promising resource for the recovery of new chemical entities that can be exploited for the further development of new drugs (Nafiu et al., 2017).

Lophira lanceolata Tiegh. ex Keay (family Ochnaceae), commonly known as ironwood or red oak, is a multipurpose tree, widely distributed in the woody savannahs of tropical Africa. It is reputed for its medicinal attributes in West African traditional medicine (Dicko et al., 2017; Etuk and Muhammad, 2010; Mapongmetsem, 2007). The plant is a small to medium-sized tree that can grow up to 12 m in height, with leaves that are alternate, elongated, and 11–45 cm × 2–9 cm in size, and clustered at end of short or twisted branches. The stem bark of the plant is corky grey, rough, and breaks into flakes while the inner bark is yellow to brownish red. The plant flowers from October to January and the fruit is conical and fairly woody, 0.1 cm in diameter with one elongated seed. Fruiting occurs between February to April (Etuk and Muhammad, 2010; L´eandre et al., 2013; Mapongmetsem, 2007).

The seeds of L. lanceolata are mainly consumed by the West African people, where it is predominantly used as an edible oil called ‘menu oil’. Meni oil is used for medicinal purposes or in cosmetics; it is traditionally used to alleviate dermatosis, toothache and muscular tiredness and is employed in soap making. Additionally, this oil can be incorporated in porridge or used as a tonic to feed children (Mapongmetsem, 2007). In Nigeria, infused young twigs are used for treating fever, respiratory problems and dysentery (Ali et al., 2011). The root powder can be mixed with flour and consumed to cure constipation, while its concoction can be used to cure chronic wounds (Onyeto, 2014). Other ethnomedicinal uses of the L. lanceolata plant include curation of abdominal pain, diarrhea, rheumatism, cardiovascular diseases, and pulmonary diseases (Ali et al., 2011; L´eandre et al., 2013; Mapongmetsem, 2007).

The diverse medicinal attributes of L. lanceolata have attracted the attention of many researchers and thus, this species has been the focus of several phytochemical and pharmacological studies. Phytochemical investigations revealed the presence of abundant flavonoids, alkaloids, glycosides, tannins, and terpenes (Audu et al., 2007; Garba and Yaro, 2015; Igboeli et al., 2015). Researchers have evidenced that L. lanceolata possesses anti-malarial (Lopatriello et al., 2019), anti-bacterial (Awachie and Ugwu, 1997), anti-diarrhoeal, anti-plasmodial (Igboeli et al., 2015), anti-anthelmintic (Ndjonka et al., 2014; Samje et al., 2014), antioxidant (Onyeto, 2014; Oussou et al., 2016), antihyperglycemic (Camille et al., 2017), anti-convulsion (Garba and Yaro, 2015) and anti-tuberculosis (Nkot et al., 2018) effects. Additionally, several studies have been performed in order to assess the safety of L. lanceolata usage (Igboeli et al., 2015; Nkot et al., 2018; Onyeto, 2014).

Although significant effort has been put into the investigation of L. lanceolata active constituents and their biological activities, to our best knowledge, none of them have made detailed phytochemical screening and attempted to evaluate the inhibitory effects against key enzymes that are involved in the pathogenesis of chronic diseases. In this direction, the present study was designed to investigate the pharmacological attributes and chemical contents of L. lanceolata leaf and stem bark extracts obtained using different extraction methods. Biological investigations were comprised of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) cytotoxicity assay, antioxidant and enzyme inhibitory (cholinesterases, tyrosinase, α-amylase and α-glucosidase) assessments. Direct injection quadrupole time-of-flight (Q-TOF) and phytochemical screening assays were performed in order to provide insights into the phytochemical profiles of the different extracts from L. lanceolata.

2. Material and methods

2.1. Plant material and preparation of extracts

L. lanceolata plant was collected in Cote ˆ d’Ivoire (Gbˆekˆe region, Prikro village) and this plant was confirmed by one co-author (Dr. Ouattara Katinan Etienne). The voucher specimen was deposited in the Department of Biology, Science Faculty, Selcuk University (KIS-19− 1110). Selected plant stem barks and leaves were dried at room temperature and powdered by the use of a laboratory mill. For extracts preparation, infusion, Soxhlet (SOX), maceration (MAC), and homogenizer-assisted extraction (HAE) were performed. Methanol and water were used as solvents in the extraction methods. The details for extractions were given in supplemental material. Methanol was removed by using a rotary evaporator, while infusion was lyophilized. All dried extracts were protected in a refrigerator (+ 4 ◦C) until use.

2.2. Phytochemical profiling

2.2.1. Phytochemical screening

The phytochemical qualitative screening was performed in order to determine the presence of secondary bioactive metabolites by specific tests: phenolic compounds (ferric chloride test), terpenoids (Salkowski test), alkaloids (Mayer’s test), glycosides (Keller-Killiani test), steroids (Libermann-Burchard’s test) and saponins (froth test) according to Edeoga et al. with minor modifications (Edeoga et al., 2005). All details were given in supplemental materials. The total phenolic, phenolic acid, flavanol and flavonoid contents of the extracts were measured and detailed methods were described in our previous paper (Vladimir-Kneˇzevi´c et al., 2011; Zengin and Aktumsek, 2014). Total phenolic content was determined by using the colorimetric Folin-Ciocalteu method and absorbances were recorded at 765 nm. Total flavonoid content was measured with AlCl3 methods and absorbances were read at 415 nm. Total flavanol content was detected by using DMACA (p-dimethylaminocinnamaldehyde) and absorbances were read at 640 nm. Total phenolic acid content was determined by the Arnow reagent and the absorbances were recorded at 490 nm. Standards, namely gallic acid (GAE) for phenolics, caffeic acid (CE) for phenolic acid, catechin (CAE) for flavanol and rutin (RE) for flavonoids, were used to explain the results. The standards were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The experimental details are given in supplemental materials.

2.2.2. Direct injection quadrupole time-of-flight (Q-TOF) analysis

Plant extracts were dissolved in methanol to a concentration of 7.5 mg/mL, sonicated for 10 min at room temperature (Sonorex digitec, Bandelin, Germany) and afterward diluted 10 times. For phytochemical profiling, an Agilent 1290 series UPLC equipped with a degasser, binary pump, autosampler and column oven coupled to an Agilent 6550 iFunnel quadrupole time-of-flight mass spectrometer equipped with dual AJS ESI source (Agilent Technologies) was used. The direct injection Quadrupole Time-Of-Flight (Q-TOF) method was already published in our previous work (Safti´c et al., 2019). Identification of the phenolic compounds was done using Mass Hunter PCDL Manager (Version B.04.00), MassBank (MassBank, 2019), mzCloud (mzCloud, 2019) and MassBank of North America (MoNA) (MassBank of North America, 2020).

2.3. Assays for biological activity

To detect antioxidant properties, we used several chemical assays including different mechanisms namely, radical scavenging, reducing power, and metal chelating. Trolox (TE) and ethylenediaminetetraacetic acid (EDTA) were used as standard antioxidant compounds Obtained results were expressed as equivalents of these compounds (Bursal et al., 2019; Eruygur et al., 2019; Grochowski et al., 2017; Topal et al., 2016). To detect inhibitory effects on enzymes, we used colorimetric enzyme inhibition assays and these assays included tyrosinase, α-glucosidase, α-amylase, and cholinesterases. Some standard inhibitiors (galatamine (GALAE), kojic (KAE) acid and acarbose (ACAE)) were used as positive controls. The experimental details are given in supplemental materials.

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2.4. Cell culturing and evaluation of antiproliferative and cytotoxic effects

Human tumor cell lines: colorectal adenocarcinoma (HT29) and metastatic breast cancer cell line (MCF7) as well as normal skin fibroblasts (HFF) obtained from ATCC (American Type Culture Collection), were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ mL penicillin and 100 μg/mL streptomycin (Capricorn Scientific, Germany) with 5% CO2 at 37 ◦C in a humidified atmosphere. Samples were prepared in cell growth medium in 10 mg/mL stock concentration and further dissolved in the ultrasonic bath for 30 min prior to the experiment. For the experiment, carcinoma cell lines and normal human cell lines were seeded in 96-well microtiter plates at a density of 5000 cells per well. Test compounds were then added in five, 10-fold dilutions (0.0001–1 mg/ml) that were freshly prepared on the day of testing in growth medium and incubated for a further 72 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, USA) assay was used to calculate the cell growth rate. Percentage of growth was calculated by transforming the experimentally determined absorbance values using the formulas proposed by the National Institutes of Health (NIH) and IC50 values were calculated. Experimental measurements were performed in tetraplicates in two individual biological experiments.

2.5. Statistical analysis

The outcomes of all the analyses were expressed as the mean ± standard deviations. First, the statistical analysis was done to calculate the difference between samples using one-way ANOVA followed by Tukey post hoc test. Then, data were submitted to exploratory principal component multivariate analysis (PCA) to identify the discriminative factors. Clustered image map was done to characterize the clusters obtained from PCA. All procedures were done under R software v. 3.6.2.

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3. Results and discussion

3.1. Phytochemical composition

Numerous biologically active compounds have been documented and isolated from plants due to their specific and strong pharmacological properties (Bursal and Gülçin, 2011; Bursal et al., 2013; Gülçin et al., 2011; Kose ¨ et al., 2015). In particular, phenolic compounds have been found to possess several medicinal properties such as antimicrobial, antioxidants, anti-inflammation, anti-atherosclerosis, anti-aging, cardioprotective, hepatoprotective and anti-cancer (Abbas et al., 2017; Han et al., 2007; Quideau et al., 2011). In this study, the leaf and stem bark of L. lanceolata were extracted using different extraction methods; infusion, maceration (MAC), Soxhlet (SOX) and homogenizer assisted extractions (HAE) using water and methanol as solvents. Initially, preliminary phytochemical screening of L. lanceolata extracts was performed, and the results revealed the presence of phenolic compounds, steroids and saponins in each tested sample (Table 1). The stem bark extracts also contained terpenoids and glycosides that were not detected in all leaf extracts. However, only methanol leaf extracts were positive for the presence of alkaloids.

A deeper insight into the possible correlation between pharmacological activities and biologically compounds present in the extracts was done by assessing the phytochemical composition of L. lanceolata leaf and stem bark extracts for the total phenolic content (TPC), total flavonoid content (TFC), total phenolic acid (TPAC), and total flavonol content (TFlvC) and the results are presented in Table 2. For the leaf extracts, the HAE-MeOH (92.13 ± 1.52 mg GAE/g) extract, followed by the SOX (81.07 ± 1.64 mg GAE/g) extract were found to have the highest TPC, while the extract obtained using the Soxhlet method (116.58 ± 2.78 mg RE/g), followed by the HAE-MeOH (113.72 ± 3.00 mg RE/g) displayed the highest TFC. The TPaC varied from 13.42 ± 0.72–48.61 ± 3.58 mg CE/g and the TFlvC varied from 0.77 ± 0.08–17.77 ± 0.40 mg CAE/g, with highest content observed for HAE-MeOH extract.

The stem bark extracts showed notable TPC, with MAC-water extract, followed by SOX extract displaying the highest content (Table 2). The TFC varied from 1.65 ± 0.40 (MAC-Water) and 33.73 ± 1.35 mg RE/g (MAC-MeOH). A significant amount of TPaC was observed in all the stem bark extracts in the following order: MAC-Water > infusion > SOX > HAE-MeOH > MAC-MeOH >HAE-Water. For the TFlvC, the SOX-MeOH (101.51 ± 2.56 mg CAE/g), HAE-MeOH (92.12 ± 1.03 mg CAE/ g) and MAC-MeOH (88.33 ± 0.69 mg CAE/g) have shown noteworthy TFlvC as compared to other studied stem bark extracts. Overall, the stem bark extracts were found to possess a higher level of phenolic compounds.

Clearly, obtained results indicated that the amounts of total bioactive compounds depended on the used extraction methods and solvents. Generally, the homogenizer-assisted extraction method could be considered as the best technique for preparing extracts from L. lanceolata. According to our findings, several authors reported that the homogenizer-assisted technique was the most effective technique with short extraction times (Dall’Acqua et al., 2020; Rocchetti et al., 2019; Sinan et al., 2020; Zheleva-Dimitrova et al., 2020). In addition, methanol was more active than water in the extraction techniques used. This fact was also supported by several researchers who noted methanol as the best extraction solvent (Barbouchi et al., 2020; Sarikurkcu et al., 2020). As far as our literature survey could ascertain, we observed few studies on total bioactive compounds of L. lanceolata extracts. Among the studies, the total phenolic level of ethanol extract of leaves of L. lanceolata was found to be 414.07 mg GAE/g extract by Kalmob´e et al. (2017). The phenolic level was higher than our findings. This difference could be explained by environmental and climatic differences. In addition, in recent times, spectrophotometric methods for total bioactive compounds have had several doubts. For example, the Folin-Ciocalteu reagent could react with peptides or other chemicals as well as phenolics (Sanchez-Rangel ´ et al., 2013). Thus, the assay could not reflect accurate levels of phenolics in the tested extracts. Thus, the phytochemical studies should be included at least one chromatographic method for qualification and quantification of bioactive compounds.

Thereafter, the individual phytochemicals in the extracts were detected using direct infusion QTOF analysis (Table 3). Based on the experimental data, 23 compounds were putatively identified with the majority of them being flavonoids and bioflavonoids. Orientin derivatives, isovitexin, saporanin, scoparin (a flavone), vitexin and its derivatives were identified only in the leaf extracts. Lanceolatin A was detected only in MAC-water leaf extracts and, infusion and MAC-MeOH stem bark extracts, while the biflavonoid Lanceolatin A was presented only in SOX-MeOH stem bark extract and in all leaf extracts (except the HAE-MeOH). The presence of lanceolatin B was observed in all extracts of both plant parts except in the infused and HAE-MeOH leaf extracts. The bioflavonoid lanceolatin B was detected in all extracts except in the macerated stem bark extract. All the extracts, except HAE-MeOH and HAE-Water leaf extracts contained lanceolatin C. Obtained data also revealed that the extracts were rich in lophirones. Lophirones A, D, E, I, J and L were detected in most of the extracts of both plant parts. Our results are in the line with the literature where the occurrence of biflavanoids lophirones in L. lanceolata was previously reported by Lopatriello et al. (Lopatriello et al., 2019). Biflavanoids lophirones are biogenetically produced from the dimerization of chalcones with different regiochemistry (Ghogomu et al., 1987; Lopatriello et al., 2019; Tih et al., 1989, 1994). Presence of flavonoids such as orientin and vitexin derivatives only in the leaf extracts correlated with a higher level of total flavonoid content.

3.2. Antioxidant property

In the present study, the antioxidant potential of the different extracts of L. lanceolata leaf and stem bark using phosphomolybdenum, free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), and ferrous-ion chelating assays were evaluated. Based on the experimental findings (Table 3), all studied extracts have shown remarkable free radical scavenging and reducing power. For the leaf extracts, the highest free scavenging activity was observed in the HAEMeOH (DPPH: 93.82 ± 0.23 and ABTS: 132.25 ± 0.09 mg TE/g), followed by infused (for DPPH: 93.81 ± 0.43 mg TE/g) and SOX-MeOH (for ABTS: 132.09 ± 0.07 mg TE/g) extracts. The HAE-MeOH extract has displayed the highest cupric and ferric reducing power, with values of 287.73 ± 21.98 and 231.21 ± 0.57 mg TE/g, respectively.

Among the stem bark extracts, the highest free radicals quenching activity was displayed by the SOX-MeOH (DPPH: 195.18 ± 0.55 and 265.04 ± 0.99 mg TE/g), followed by HAE-MeOH (DPPH: 194.83 ± 0.21and 264.43 ± 0.13 mg TE/g) extracts. Notable reducing capacity was displayed by all the tested stem bark extracts, with the aqueous extracts obtained using maceration (CUPRAC: 853.99 ± 13.27 and FRAP: 853.99 ± 13.27 mg TE/g) and homogenizer assisted (CUPRAC: 683.57 ± 11.09 and FRAP: 648.34 ± 3.86 mg TE/g) methods being superior sources of reducing agents in both CUPRAC and FRAP assay. Modest total antioxidant capacity was displayed by the tested leaf and stem bark extracts. With respect to metal chelating properties, the activity varied between 6.76 ± 1.76 (HAE-Water) and 22.68 ± 0.74 (HAE-MeOH) mg EDTAE/g and 2.09 ± 0.45 (MAC-MeOH) and 6.15 ± 1.12 mg EDTAE/g for the leaf and stem bark extracts, respectively.

As far as the literature could justify, there are few studies regarding the antioxidant properties of L. lanceolata (Onyeto, 2014; Oussou et al., 2016). Clearly, obtained antioxidant results are in line with the total phenolic content observed from the extracts. This fact indicated that phenolic compounds could be considered as main contributors to the observed antioxidant properties. This fact also was obtained by Pearson’s correlation analysis and the results are given in Fig. 1. In accordance with our findings, several researchers reported a positive correlation between total phenolic content and antioxidant properties (Chu et al., 2020; Ribeiro et al., 2020; Thummajitsakul et al., 2020). In addition to correlation analysis, the presence of flavonoid (lanceolatin derivatives) and bioflavonoids (lophirone derivatives) in the tested extracts could be attributed to the antioxidant properties. Similarly, Ajiboye et al. (2014) reported that lophirone B and C exhibited significant antioxidant properties in DPPH and reduced power assays.

3.2.1. Enzyme inhibitory properties

The enzyme inhibitory properties of L. lanceolata leaf and stem bark extracts toward cholinesterases (AChE and BChE), α-amylase, α-glucosidase, and tyrosinase were investigated, and the experimental data are illustrated in Table 4. Among leaf extracts, only the methanol extracts (HAE-MeOH: 4.40 ± 0.10, SOX-MeOH: 4.22 ± 0.21 and MAC-MeOH: 4.01 ± 0.30 mg GALAE/g) were potent inhibitors of AChE, while none of the extracts were active against BChE. All stem bark extracts have shown inhibitory activity against AChE with values ranging from 1.93 ± 0.19 (infusion) to 5.18 ± 0.03 (HAE-MeOH) mg GALAE/g, while only methanol extracts (HAE-MeOH: 7.87 ± 1.68, SOX-MeOH: 8.66 ± 1.75 and MAC- MeOH: 7.31 ± 1.33 mg GALAE/g) were potent against BChE. Notable anti-tyrosinase effects were displayed for the methanol leaf extracts, with the highest activity recorded for extract obtained using the Soxhlet extraction method (131.17 ± 1.96 mg KAE/g). No inhibitory activity against tyrosinase was displayed by the HAE-Water and MAC-Water extracts. All stem bark extracts showed significant inhibitory potency against tyrosinase in the following order: MAC-MeOH > SOX-MeOH > HAE-MeOH > Infusion > MAC -Water > HAE-Water. With regard to anti-diabetic effects, all tested leaf and stem bark extracts showed modest activity against α-amylase enzyme and only methanol extracts of both plant parts were effective at inhibiting α-glucosidase, with values ranging from 2.77 ± 0.01–2.86 ± 0.16 mmol ACAE/g for leaf extracts and 2.74 ± 0.01–2.76 ± 0.01 mmol ACAE/g for stem bark extracts.

To the best of my knowledge, the purported enzyme inhibitory results are the first report for L. lanceolata. At this point, results from this study could provide a valuable baseline date for future investigations. As can be seen in Fig. 1, we observed a significant correlation (R > 0.5) between total phenolics and enzyme inhibitory assay (except for glucosidase). In addition, high correlation coefficient values were found between total flavanol and the enzyme inhibition assays. These findings indicated that the biological activities of the tested extracts were strongly linked to their chemical profiles. Our findings are inconsistent with previous papers conducted by several researchers, who reported a linear relationship between total bioactive compounds and enzyme inhibitory effects (Ali Reza et al., 2018; Kaewnarin et al., 2016; Llorent-Martínez et al., 2020; Muddathir et al., 2017; Wu et al., 2020). Taken together, L. lanceolata could be considered as a potential source of natural enzyme inhibitors (Table 5).

3.3. Evaluation of antiproliferative and cytotoxic activities

Antiproliferative and cytotoxic activities of leaf and stem bark L. lanceolata extracts were evaluated on two carcinoma cell lines, HT29 and MCF7, and on a non-transformed human fibroblast cell line (HFF-1). Infusion and maceration-water leaf extracts showed modest anti-proliferative activity and were the most effective on all tested cell lines (Table 6). Other extracts showed similar modest activity only on carcinoma cell lines while inducing the proliferation of fibroblasts (Figure S1). The most potent antiproliferative effects were noticed in all stem bark extracts, showing the same, non-selective antiproliferative effect on carcinoma cell lines, regardless of the extraction method used (Table 6). Interestingly, this more pronounced antiproliferative effect was accompanied by a strong proliferative activity observed on the nontransformed fibroblast cell line (Figure S2). Both leaf and stem bark extracts were not cytotoxic on tested cell lines. These results indicate a potential for further development of antiproliferative agents with low systemic cytotoxicity based on the L. lanceolata plant that might be interesting in the area of cancer drug development.

In an earlier study conducted by Ajiboye et al. (2014), the cytotoxic effects of L. alata and lophirones B and C were investigated on Ehrlich ascites carcinoma cells, whereby significant anticancer properties were recorded. The authors also reported that the presence of lophirones B and C were strongly linked to the cytotoxic effect of the tested extract. In the light of this information, the bioflavonoids (lophirone derivatives) might be responsible for the observed anticancer abilities of L. lanceolata in the presented paper.

3.4. Multivariate analysis

Univariate statistical analysis confirmed differences in the phytochemical content, antioxidant activities and in vitro enzyme inhibitory activities of L. lanceolata samples. More precisely, these differences were observed between both studied plant parts, as well as between the different technique-solvent used for extraction. It is common knowledge that the distribution of molecules in plant change from one plant part to another, thus affecting biological activities. In addition, several studies highlighted the impact of technique-solvent used for the extraction on the presence and final concentrations of phytochemical compounds of interest. Based upon this information, it was important to estimate the level of impact of both factors on the analyzed biological activities. In this regard, exploratory multivariate analysis i.e. principal component analysis appeared to be appropriate for that purpose.

PCA reduced the descriptors into two main PCs enclosing 90.1 % of the variability (Fig. 2A). PC1, the most prominent component with 66.7 % of explained variability, was positively associated with antioxidant (phosphomolybdenum (PPBD), 2,2′ -azino-bis(3-ethylbenzothiazoline-6- sulphonic acid (ABTS), cupric reducing antioxidant (CUPRAC), 2,2- diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP)) and α-amylase assays. PC2 was found to be positively bound to α-glucosidase and ferrous chelating ability assays (Fig. 2B). Two main discriminative clusters representing each studied plant part were observed along PC1. In addition, samples of each plant part were separated along PC2, in relation to the solvent used in the extraction process (Fig. 3A).

Stem bark extracts were characterized by the strongest antioxidant properties, whereas leaf extracts had the higher ferrous chelating ability (Fig. 3B). Additionally, only a few stem bark extracts were found to exert an inhibitory effect on butyrylcholinesterase (BChE) enzyme activity. Secondary metabolite biosynthesis in plants is a process influenced by several factors, some of which are related to the plant itself and some influenced by the environment. For the factors associated with the plant itself, it has been shown that the distribution and concentration of phytoconstituents in plants vary greatly depending on their ontogenetic development and plant part (Feduraev et al., 2019).

Additionally, the influence of the extraction solvent on the biological activities of L. lanceolata has been emphasized. This effect is more noticeable on the leaves (Fig. 4B). Besides, our results showed that methanol was a more adequate solvent for the extraction of bioactive constituents as it provided qualitatively and quantitatively richer extracts. This finding can be attributed to the higher solubility of phytochemical compounds of L. lanceolata in methanol, suggesting their lipophilic nature. Taken together, these findings recognized L. lanceolata stem bark as the richest plant part in phytonutrients and methanol as the suitable solvent for its extraction.

4. Conclusion

This study reports the phytochemical characterization of L. lanceolata leaf and stem extracts and evaluation of the antioxidant, enzyme inhibitory and antiproliferative/cytotoxic effects. Lanceolatins and lophirones were found in all analyzed plant parts, whereas several apigenin and luteolin glucosides were found only in leaf extracts. Overall, the extract also showed interesting antioxidant activities whereby the stem bark extracts showed the best antiproliferative effect towards carcinoma cell lines, thereby advocating for further studies to demonstrate its potential in the management of cancer. The inhibition of key clinical enzymes also showed the potential of the extracts in the management of Alzheimer’s, hyperpigmentation, and diabetes mellitus. These findings evidenced that L. lanceolata is a promising source of bioactive constituents with an envisaged potential application in skin tone formulations. However, further studies (efficacy, toxicity, bioavailability, etc.) on L. lanceolata should be performed with the aim to develop novel antioxidants and enzyme inhibitors with improved properties over synthetic ones.

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