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Analytical Letters
ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: https://www.tandfonline.com/loi/lanl20
Exploring Chemical Profiles and Bioactivities
of Harungana madagascariensis Lam. ex Poir.
Leaves and Stem Bark Extracts: A New Source of
Procyanidins
Eulogio J. Llorent-Martinez, Alba Ruiz-Riaguas, Kouadio Ibrahime Sinan,
Kouadio Bene, Maria Luisa Fernández-de Cordova, Carene Picot-Allain, Fawzi
Mahomoodally, Hammad Saleem & Gokhan Zengin
To cite this article: Eulogio J. Llorent-Martinez, Alba Ruiz-Riaguas, Kouadio Ibrahime Sinan, Kouadio Bene, Maria Luisa Fernández-de Cordova, Carene Picot-Allain, Fawzi Mahomoodally, Hammad Saleem & Gokhan Zengin (2020) Exploring Chemical Profiles and Bioactivities of
Harungana�madagascariensis Lam. ex Poir. Leaves and Stem Bark Extracts: A New Source of
Procyanidins, Analytical Letters, 53:3, 399-412, DOI: 10.1080/00032719.2019.1653903
To link to this article: https://doi.org/10.1080/00032719.2019.1653903
Published online: 19 Aug 2019. Submit your article to this journal
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NATURAL PRODUCT ANALYSIS
Exploring Chemical Profiles and Bioactivities of Harungana
madagascariensis Lam. ex Poir. Leaves and Stem Bark
Extracts: A New Source of Procyanidins
Eulogio J. Llorent-Martineza, Alba Ruiz-Riaguasa, Kouadio Ibrahime Sinanb, Kouadio Benec, Maria Luisa Fernandez-de Cordovaa, Carene Picot-Allaind, Fawzi Mahomoodallyd, Hammad Saleeme,f, and Gokhan Zenginb
a
Faculty of Experimental Sciences, Department of Physical and Analytical Chemistry, University of Jaen, Jaen, Spain;bScience Faculty, Department of Biology, Selcuk Universtiy, Konya, Turkey;cUnite de Formation et de Recherche Sciences de la Nature, Laboratoire de Botanique et Phytotherapie,
Universite Nangui Abrogoua, Abidjan, C^ote d’Ivoire;dFaculty of Science, Department of Health Sciences, University of Mauritius, Reduit, Mauritius;eSchool of Pharmacy, Monash University, Selangor Darul Ehsan, Malaysia;fInstitute of Pharmaceutical Sciences, University of Veterinary & Animal Sciences, Lahore, Pakistan
ABSTRACT
This study attempts to valorize the multiple pharmacological proper-ties of Harungana madagascariensis Lam. ex Poir., also known as dragon’s blood tree, with wide applications in African traditional medicine. The antioxidant and inhibitory activity of H. madagascar-iensis leaves and stem bark extracts (ethyl acetate, aqueous extracts, and methanol) against enzymes related to diabetes (a-glucosidase, a-amylase), epidermal hyperpigmentation problems (tyrosinase), and Alzheimer’s disease (acetyl and butyryl cholinesterase) were eval-uated. The phytochemical profiles of the extracts were studied by high-performance liquid chromatography with diode array detection and mass spectrometry (HPLC-DAD-MS), observing the presence of procyanidins and flavonoids, particularly in the leaves’ extracts. The radical scavenging and reducing power of H. madagascariensis leaves’ extracts were greater than the stem bark extracts. The metha-nol extracts of leaves (4.61 mg galantamine equivalent (GALAE)/g extract) and stem bark (4.68 mg galantamine (GALAE)/g extract) of H. madagascariensis inhibited acetyl cholinesterase. Methanol extracts (153.55 and 147.07 mg kojic acid equivalent (KAE)/g extract, for leaves and stem bark extracts, respectively) of H. madagascariensis showed high tyrosinase inhibition. Correlation and principal compo-nent analysis (PCA) were also performed. The observed pharmaco-logical effects of H. madagascariensis support that this plant may be a promising candidate for the development of novel pharmaco-phores for the treatment of diabetes, epidermal hyperpigmentation problems, Alzheimer’s disease, and other oxidative-stress-related complications. ARTICLE HISTORY Received 27 June 2019 Accepted 6 August 2019 KEYWORDS Alzheimer’s disease; diabetes; high-performance liquid chromatography with diode array and mass spectrometry detection (HPLC-DAD-MS); phenolic; principal component analysis (PCA)
CONTACT Gokhan Zengin gokhanzengin@selcuk.edu.tr Department of Biology, Faculty of Science, Selcuk University, Campus/Konya, Konya, 42050 Turkey
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ß 2019 Taylor & Francis Group, LLC
2020, VOL. 53, NO. 3, 399–412
Introduction
In the recent decades, natural products and their applications are gaining great interest because synthetics present serious concerns. From this point, plants or their metabolites are considered as main sources of natural products and, for this reason, many studies have been performed on plants in the scientific platform (Oyenihi and Smith2019). For example, in 2015, artemisinin has been isolated from Artemisia annua for treating mal-aria and this work was awarded the Nobel Prize (Su and Miller 2015). This case is regarded as a starting point for further studies on plants, especially uninvestigated plants, and the number of phytochemical studies are increasing day by day.
Most of these studies have focused on plant secondary metabolites, including phen-olic compounds, terpenoids, and alkaloids. It is documented that secondary metabolites provide significant health benefits such as antioxidant, antimicrobial, anticancer, or anti-inflammatory agents (Roleira et al. 2015; Ahangarpour, Sayahi, and Sayahi 2019; Zhang, Virgous, and Si 2019). As an example, procyanidins are polyphenolic com-pounds of which plants, grape seeds and berries are their main sources (Cos et al.
2004). Procyanidins are active antioxidants and thus they exhibit significant radical scavenging, anticancer, and antitumour activities (Zhang et al. 2016). In this sense, the discovery of new procyanidins sources may provide significant effects on nutraceutical and pharmaceutical applications.
Harungana madagascariensis Lam. ex Poir. belongs to the Hypericaceae family and is native to Madagascar and tropical African regions (Tankeo et al. 2016). In African med-ical lore, the leaves and stem bark of H. madagascariensis are used to treat skin diseases, malaria, and aneamia (Kengni et al. 2016). H. madagascariensis forms part of the Jubi formulation, used to restore hemoglobin level and pack cell volume (Iwalewa et al.
2008). In the Cameroonian pharmacopeia, H. madagascariensis leaves and seed oil are used to treat typhoid fever and malaria (Ndjakou Lenta, Vonthron-Senecheau, et al. 2007). Ethnobotanical evidence mentioned the use of H. madagascariensis stem bark for the management of nephrosis and gastrointestinal complications (Iwalewa et al. 2008). H. madagascariensis has been traditionally used to treat angina, wounds, diarrhea, dys-entery, syphilis, gonorrhea, asthma, and tuberculosis (Tankeo et al. 2016). A decoction prepared from H. madagascariensis stem bark is administered to manage diabetes, amebiasis, and cough (Mpiana et al. 2008).
Numerous phytochemicals have been identified from different parts of H. madagas-cariensis. Bazouanthrone, harunganol A, feruginin A, harunganin, betulinic acid friede-lan-3-one, and harunganol B were isolated from the root bark of H. madagascariensis (Ndjakou Lenta, Ngouela, et al. 2007). Harunmadagascarins A and B, harunganol B, methyl 3-formyl-2,4-dihydroxy-6-methyl benzoate, harungin anthrone, friedelin, betu-linic acid, and lupeol were identified from the stem bark (Kouam et al. 2005). Madagascin, kaempferol-3-O-b-D-glucopyranoside, and ferruginin A were also identified
from the bark of the plant (Tankeo et al. 2016).
Astilbin, a flavanone, has been characterized and isolated from H. madagascariensis leaves (Moulari et al.2006). Glycosides, tannins, saponins, and alkaloids were also iden-tified in H. madagascariensis leaves (Okoli et al. 2002). Previous pharmacological studies support the anti-oxidant, antimalarial, antifungal, anticancer, antityphoid, antibacterial, antiprotozoal, anti-amoebic, and antisickling activities of H. madagascariensis (Tona
et al. 1998; Ndjakou Lenta, Ngouela, et al. 2007; Mpiana et al.2008; Iwalewa et al.2009; Biapa et al. 2013; Kengni et al. 2016; Tankeo et al. 2016; Lemma et al.2017; Ochwang’i
et al. 2018).
In an endeavor to valorize H. madagascariensis, we assessed the in vitro inhibitory activities of the leaves and stem bark extracts against enzymes associated with Alzheimer’s disease (cholinesterases), diabetes (a-glucosidase and a-amylase), and epi-dermal hyperpigmentation (tyrosinase) problems, as well as different antioxidant mecha-nisms (free radical scavenging, reducing power, metal chelating, and total antioxidant). Various solvents (ethyl acetate, water, and methanol) were used to characterize their effects on the biological properties. The chemical characterization of the methanol extracts of H. madagascariensis tissue was performed by high-performance liquid chro-matography – electrospray ionization – tandem mass spectrometry (HPLC-ESI-MSn). To provide more details on the analysis of the plant tissues and solvents, multivariate analysis was performed.
Materials and methods
Plant material and preparation of extracts
Sampling of the plant species was done in the Gontougo region (Kokomian) of Ivory Coast in the year 2018. Botanical authentication of the plant was done by the botanist Dr. Bene Kouadio (Universite Nangui Abrogoua, Ivory Coast). The stem bark and leaves were dried at room temperature in the shade for approximately 10 days. These materials were then powdered using a laboratory mill.
Methanolic and ethyl acetate were selected as extraction solvents in the maceration techniques (5 g plant sample was mixed with 100 ml of each solvents for 24 h.). Next the extracts were filtered and evaporated in vacuo at 40C. Water extract was prepared as a traditional infusion (5 g plant sample was treated with 100 ml of boiling water for 20 min). The infusion was filtered and then dried by a lyophilizer. All extracts were stored at þ4C protected from the light until analysis.
Profile of bioactive compounds
By referring to our previous paper (Uysal et al. 2017), the flavonoids (TFC) and total phenolic (TPC) contents were determined on the basis of the AlCl3 and the standard
Folin-Ciocalteu assays, respectively. The results are expressed as equivalent of rutin (mg RE/g) for TFC and gallic acid equivalent (mg GAE/g) for TPC.
Determination of enzyme inhibitory and antioxidant effects
The in vitro enzyme inhibitors effects of extracts on the four enzymesa-amylase, a-glu-cosidase, cholinesterases, and tyrosinase were evaluated as previously reported (Uysal et al. 2017). The enzyme inhibitor actions of samples were assessed as equivalents of kojic acid (KAE) for tyrosinase, galantamine for acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE), and acarbose fora-amylase and a-glucosidase.
Regarding the antioxidant capacity of the samples, different experiments as ferrous ion chelating, phosphomolybdenum, and radicals scavenging tests (FRAP, ABTS, CUPRAC, and DPPH) were spectrophotometrically screened. The findings were given as standard compounds equivalents (mg EDTAE/g and mmol TE/g). The assay methods were provided in our earlier work (Uysal et al.2017).
HPLC-ESI-MSn
To characterize the chemical profiles of methanolic extracts, an Agilent Series 1100 with a G1315B diode array detector was used as HPLC system. The separation of the com-pounds was achieved with a Luna Omega Polar C18 analytical column of 150 3.0 mm
and 5mm particle size (Phenomenex). A Polar C18 Security Guard cartridge
(Phenomenex) with dimensions of 4 3.0 mm was also used. The HPLC system was connected to an ion trap mass spectrometer (Esquire 6000, Bruker Daltonics) with an electrospray interface operating in the negative mode. The chromatographic conditions are detailed in our previous paper (Llorent-Martınez et al. 2018).
Data evaluation
One-way ANOVA followed by Tukey’s multiple range was done to investigate signifi-cant differences (p< 0.05) between the tested samples. Pearson’s correlation coefficients were calculated and a correlation map was generated to pinpoint the link between the studied biological activities and phenolic classes. Unsupervised principal component analysis (PCA) and hierarchical cluster analysis for both biological activities and sam-ples, using“ward” as linkage rule and the Euclidean similarity measure, were conducted. The variability of the tested extracts was observed and obtained clusters were character-ized after checking by the multivariate analysis of variance (MANOVA). The statistical procedures were achieved by R software v. 3.5.1.
Results and discussion
Phytochemical characterization
The total phenolic and flavonoid contents of each extract of H. madagascariensis are summarized in Table 1. The highest total content of phenolic compounds (195.72 mg GAE/g) was detected in the aqueous extract of H. madagascariensis leaves and the high-est flavonoid content (40.61 mg RE/g) was observed in the methanol extract of the same
Table 1. Total phenolic and flavonoid contents of H. madagascariensis.
Plant tissue Solvents Total phenolic content (mg GAE/g) Total flavonoid content (mg RE/g)
Leaves Ethyl acetate 93.49 ± 1.59e 1.51 ± 0.09de
Methanol 185.00 ± 2.57b 40.61 ± 1.20a
Aqueous 195.72 ± 2.25a 26.32 ± 0.66b
Stem bark Ethyl acetate 66.64 ± 4.50f 0.47 ± 0.02e
Methanol 153.85 ± 2.12c 2.82 ± 0.19c
Aqueous 134.24 ± 3.71d 2.30 ± 0.26cd
Values are the mean ± standard deviation of three parallel measurements. GAE: gallic acid equivalent; RE: rutin equiva-lent. The superscript letters indicate the differences between the tested extracts (p< 0.05).
plant part. It is worth mentioning that the occurrence of phenolics and flavonoids was greater in leaves compared to stem bark. This finding is in line with previously pub-lished material (Thabti et al. 2014; Iqbal, Salim, and Lim 2015; Dutta and Ray 2018). The total content of phenolic compounds (153.85 mg GAE/g) of the stem bark methanol extract of H. madagascariensis reported in the current study was greater compared to the value (132.24 ± 0.61 mg GAE/g) reported by (Antia, Ita, and Udo 2015). In fact, environmental conditions, such as soil composition, temperature, rainfall, and sunlight exposure, determine the occurrence of phenolics in plants (Borges et al.2013).
In addition to spectrophotometric determinations, the detailed phenolic composition of the methanol extracts, which contained higher levels of TPC and TFC than other extracts, was established by HPLC-ESI-MSn. Figure 1 shows the base peak chromato-grams of the leaves and stems bark extracts of H. madagascariensis while mass fragmen-tation of compounds is shown inTable 2.
As presented in Table 2, leaves’ extract of H. madagascariensis contained high
amounts of procyanidin dimers and trimers, formed by condensation of catechin and epicatechin. Seven dimers and three trimers were observed in leaves’ extracts, whereas five dimers and five trimers were identified in bark stem extracts (Table 2). Procyanidin dimers, all of them B-type, were identified based on the deprotonated molecular ion at m/z 577 and fragment ions at m/z 451, 425, 407, and 289 (Ruiz et al. 2005). An analyt-ical standard was also analyzed, observing the same fragmentation pattern. Trimers were characterized by their [M H] ion at m/z 865 and their fragmentation pattern, in agreement with bibliographic data (Ruiz et al.2005).
Flavonoids were also identified in the leaves and stem bark extracts of H. madagas-cariensis. Compounds 6 and 10 were observed in both leaves’ and stem bark extracts; they corresponded to catechin and epicatechin, respectively. They presented the same fragmentation patterns but different retention times (compound 6 at 8.8 min and
Figure 1. Base peak chromatograms of the extracts of H. madagarascariensis. The peaks are identified inTable 2.
Table 2. Characterization of the extracts of H. madagascariensis. Peak number Retention time (min) [M H] m/z m/z (% base peak) Assigned identification Leaves Stem bark 1 1.7 341 MS2[341]: 179 (100), 161 (7), 143 (56), 131 (20), 119 (8), 113 (17 Disaccharide 2 2.2 577 MS2[577]: 451 (17), 425 (100), 407 (54), 289 (25) Procyanidin dimer 3 2.2 865 MS2[865]: 739 (49), 695 (100), 577 (51), 575 (50), 425 (20), 407 (42), 287 (34) Procyanidin trimer 4 6.8 577 MS2[577]: 451 (30), 425 (100), 407 (69), 289 (30), 287 (10) Procyanidin dimer 5 8.0 577 MS2[577]: 451 (22), 425 (100), 407 (74), 289 (34), 287 (16) Procyanidin dimer 6 8.8 289 MS2[289]: 245 (100), 205 (33), 203 (22), 179 (19), 161 (9) MS3[289!245]: 227 (35), 203 (100), 188 (54), 161 (28) Catechin 7 9.3 865 MS2[865]: 739 (53), 695 (100), 577 (61), 425 (23), 407 (40), 287 (30) Procyanidin trimer 8 9.8 431 MS2[431]: 179 (100), 143 (9) MS3 [431!179]: 161 (85), 143 (73)119 (100) Hexose derivative 9 10.7 577 MS2[577]: 425 (100), 407 (72), 289 (20), 287 (15) Procyanidin dimer 10 12.1 289 MS2[289]: 245 (100), 205 (40), 203 (22), 179 (18), Epicatechin 11 12.4 449 MS2[449]: 269 (100), 225 (34) Apigenin derivative 12 12.7 577 MS2[577]: 425 (100), 407 (36), 289 (20), 287 (18) Procyanidin dimer 13 14.2 865 MS2[865]: 739 (61), 695 (100), 577 (78), 425 (18), 407 (57), 287 (44) Procyanidin trimer 14 15.2 577 MS2[577]: 425 (100), 407 (50), 289 (27), 287 (42) Procyanidin dimer 15 16.1 865 MS2[865]: 739 (55), 695 (43), 577 (100), 425 (27), 407 (47), 287 (31) Procyanidin trimer 16 18.3 463 MS2[463]: 301 (100), 151 (18) MS3 [463!301]: 179 (100), 151 (66) Quercetin-O-hexoside 17 19.6 609 MS2[609]: 301 (100) MS3[609!301]: 271 (35), 179 (75), 151 (100) Rutin 18 20.1 577 MS2[577]: 425 (100), 407 (65), 289 (16), 287 (28) Procyanidin dimer 19 20.4 865 MS2[865]: 739 (88), 695 (100), 577 (63), 425 (42), 407 (38) Procyanidin trimer 20 21.4 449 MS2[449]: 303 (82), 285 (100), 151 (27) MS3[449 !303]: 285 (100), 177 (8), 125 (23) Taxifolin-O-deoxyhexoside 21 21.6 519 MS2[519]: 357 (100) MS3[519!357]: 151 (100), 136 (24) Pinoresinol-O-hexoside 22 22.4 433 MS2[433]: 301 (100) MS3[433 !301]: 271 (44), 179 (18), 151 (100) Quercetin-O-pentoside 23 23.2 505 MS2[505]: 307 (100) Unknown 24 23.6 449 MS2[449]: 303 (100), 285 (82), 151 (30) MS3[449 !303]: 285 (100), 177 (9), 125 (12) Taxifolin-O-deoxyhexoside 25 24.5 447 MS2[447]: 301 (100) MS3[447!301]: 271 (36), 179 (54), 151 (100) Quercetin-O-deoxyhexoside 26 28.5 567 MS2[567]: 273 (100), 258 (25) MS3 [567!273]: 258 (100) Unknown 27 28.7 431 MS2[431]: 285 (100) MS3[431!285]: 257 (39), 255 (100), 229 (19) Kaempferol-O-deoxyhexoside 28 35.8 301 MS2[301]: 179 (67), 151 (100) Quercetin 29 36.6 563 MS2[563]: 269 (100) MS3[563!269]: 225 (100) Apigenin-O-pentoside-hexoside
compound 10 at 12.1 min), so they were unequivocally identified by the use of analytical standards.Table 3 shows that the leaves extract of H. madagascariensis contained higher concentrations of catechin and epicatechin compared to the stem bark extract.
Two derivatives of the trihydroxyflavone apigenin, namely compounds 11 and 29, were characterized in the stem bark extracts. Both compounds exhibited the 269!225 fragmentation, typical of apigenin aglycone. In contrast, compound 28 was identified to be quercetin. Four derivatives of quercetin were also identified in the studied extracts, all of them showing the aglycone at m/z 301 (fragment ions at m/z 179 and 151). According to the neutral losses observed, compounds 16, 22, and 25 were characterized as quercetin-O-hexoside, quercetin-O-pentoside, and quercetin-O-deoxyhexoside, respectively. Compound17 was identified to be rutin or quercetin-3-rutinoside.
Compounds 20 and 24, identified in the leaves’ extracts only, showing [M H] at m/z 449, exhibited the neutral loss of 146 Da (deoxyhexoside) to yield the aglycone at m/z 303. The aglycone was characterized as the flavanol taxifolin, due to the fragment ions at m/z 285, 177, and 125 (Hashim et al. 2013). Compound 27, present in leaves’ extracts, suffered the neutral loss of 146 Da to yield kaempferol and was thus character-ized to be kaempferol-O-deoxyhexoside.
Compound 1 was characterized to be a disaccharide, whereas compound 8 as a hex-ose derivative. In both cases, the fragment ions at m/z 179, 161, 143, and 119 were observed, all typical of hexoside moieties (Brudzynski and Miotto 2011). Compound21, identified in the stem bark extracts only, exhibited deprotonated molecular ion at m/z 519 and, after the neutral loss of 162 Da yielded the lignan pinoresinol at m/z 357. Hence, this compound was identified to be pinoresinol-O-hexoside (Ye, Yan, and Guo2005).
Quantification of the main biomolecules
The main constituents pinpointed in the extracts were semi-quantified by comparison with analytical standards and results are given inTable 3. It was observed that the con-centrations of phenolics in leaves’ extracts were greater compared to the extracts of stem bark; this finding is in agreement with spectrophotometric determinations. Leaf extracts were richer in flavonoids (54 mg/g DE) compared to procyanidins (16.3 mg/g DE). The stem bark extracts contained a higher concentration of procyanidins (8.1 mg/g DE) compared to flavonoids (5.7 mg/g DE).
The most abundant biomolecules in leaves’ extracts were catechin, epicatechin, taxifolin-O-deoxyhexoside isomers, and quercetin-taxifolin-O-deoxyhexoside. These five compounds accounted for approximately 76% of TIPC and are suspected to be accountable for the pro-nounced bioactivity of the tested extracts. In the stem bark extracts, epicatechin was the most abundant compound (34% of TIPC), followed by procyanidin dimers and trimers. The TIPC of leaves’ extracts (70 mg/g DE) was higher than stem bark extracts (13.8 mg/g DE).
Antioxidant effects
The results of the antioxidant activity of the different extracts of the stem bark and leaves of H. madagascariensis are summarized in Table 4. The methanol aqueous and
methanol extracts of H. madagascariensis stem bark and leaves showed higher radical scavenging properties against DPPH and ABTS compared to the extract of ethyl acetate. Likewise, the reducing potential of the tested extracts, determined using the CUPRAC and FRAP assays, revealed the higher reducing capacities of the aqueous and methanol extracts. It was noted that the radical scavenging and reducing power of the leaves extracts were greater than the stem bark extracts. This observation may be correlated to the amounts of phenolics and flavonoids identified in the different plant parts.
The total antioxidant ability estimations of the tested extracts revealed that aqueous and methanol extracts of leaves were more active. Regarding metal chelating activity, the ethyl acetate extracts (65.63 and 55.43 mg EDTAE/g, for the stem bark and leaves extracts, respectively) displayed higher chelating activity compared to the methanol and aqueous extracts. In accordance with our study, the antioxidant activity of H. madagas-cariensis has been previously described (Biapa et al. 2008; Antia, Ita, and Udo 2015). Tannins, saponins, terpenoids, alkaloids, flavonoids, cardiac glycosides, and phenols pre-sent in H. madagascariensis are known to possess potent antioxidant capacities and may be responsible for the observed antioxidant activities (Moronkola et al. 2018). Besides, harunmadagascarins A and B, harungin anthrone, and harunganol B isolated from the stem bark of H. madagascariensis have been reported to exhibit significant antioxidant activity (Kouam et al. 2005).
Catechin, epicatechin, and quercetin, identified in the polyphenolic pool of H. mada-gascariensis extracts, are widely acknowledged to be antioxidants (Allgrove and Davison
2014; Zanwar et al. 2014; Lesjak et al. 2018) and may be responsible for the observed antioxidant activities. It is worth indicating that the TPC values of the studied extracts of H. madagascariensis were positively correlated to the antioxidant results (Figure 2a), highlighting that as the TPC increased, the antioxidant capacity also increased.
Table 3. Characterization of the main compounds found in the extracts of H. madagascariensis.
Number Identification Leaves Stem bark
Procyanidins 4 Dimer 4.1 ± 0.1 0.76 ± 0.04 5 Dimer 1.42 ± 0.08 – 7 Trimer 1.05 ± 0.07 0.30 ± 0.01 9 Dimer 3.54 ± 0.2 3.0 ± 0.1 12 Dimer 0.49 ± 0.03 – 13 Trimer 3.2 ± 0.1 1.70 ± 0.04 14 Dimer 2.0 ± 0.1 1.38 ± 0.03 15 Trimer – 0.53 ± 0.02 18 Dimer 0.45 ± 0.02 0.42 ± 0.01 Total 16.3 ± 0.3 8.1 ± 0.1 Flavonoids 6 Catechin 12.9 ± 0.6 1.01 ± 0.04 10 Epicatechin 11.0 ± 0.5 4.7 ± 0.2 20 Taxifolin-O-deoxyhexoside 14.5 ± 0.5 – 24 Taxifolin-O-deoxyhexoside 1.01 ± 0.05 – 25 Quercetin-O-deoxyhexoside 14.2 ± 0.5 – 27 Kaempferol-O-deoxyhexoside 0.23 ± 0.01 – 28 Quercetin 0.18 ± 0.01 – Total 54 ± 1 5.7 ± 0.2
Total individual phenolic content 70 ± 1 13.8 ± 0.2
Sum of all the quantified compounds.
Inhibitory effects on key enzymes
The methanolic, ethyl acetate, and aqueous extracts of the stem bark and leaves of H. madagascariensis were screened for their cholinesterase (butyryl and acetyl cholinester-ase), tyrosinase, a-amylase, and a-glucosidase inhibitory activities. These enzymes have been targeted for the front-line management of epidermal hyperpigmentation problems, Alzheimer’s disease and diabetes. In general, the aqueous extracts were the least active against the studied enzymes, except against tyrosinase. The inhibition of cholinesterase enzymes, namely acetyl cholinesterase and butyryl cholinesterase, offers new therapeutic possibilities for the treatment of Alzheimer’s disease. The role of the different cholin-esterase enzymes in the pathogenesis of Alzheimer’s disease highlights the need for therapeutic agents having the potential to modulate the activity of both enzymes.
Table 4. Antioxidant properties of H. madagascariensis.
Plant part Solvents
DPPH (mmol TE/g) ABTS (mmol TE/g) CUPRAC (mmol TE/g) FRAP (mmol TE/g) Phosphomolybdenum (mmol TE/g) Metal chelating ability (mg EDTAE/g) Leaves Ethyl acetate 0.34 ± 0.01d 0.85 ± 0.02d 1.11 ± 0.15d 0.52 ± 0.06d 3.12 ± 0.10e 55.43 ± 5.66b
Methanol 1.89 ± 0.01a 3.38 ± 0.04b 3.92 ± 0.16b 2.28 ± 0.12b 4.20 ± 0.06b 28.57 ± 1.47d Aqueous 1.69 ± 0.08b 4.20 ± 0.03a 4.97 ± 0.03a 3.06 ± 0.09a 4.92 ± 0.11a 24.28 ± 0.86d Stem bark Ethyl acetate 0.33 ± 0.01d 0.65 ± 0.01e 0.97 ± 0.03d 0.33 ± 0.02e 3.18 ± 0.45de 65.63 ± 3.34a Methanol 1.87 ± 0.02a 2.86 ± 0.19c 3.41 ± 0.13c 2.03 ± 0.12c 3.85 ± 0.06bc 43.45 ± 2.24c Aqueous 1.12 ± 0.09c 2.74 ± 0.09c 3.36 ± 0.09c 2.04 ± 0.06c 3.54 ± 0.18cd 18.44 ± 2.40e Values expressed are means ± standard deviation of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA
equivalent. The superscript letters indicate the differences between the tested extracts (p< 0.05).
Figure 2. Statistical evaluations of H. madagarascariensis: (a) relationship between the total bioactive compounds and biological activities (Pearson correlation coefficient (R), p< 0.05); (b) explained vari-ance of each of the principal components; (c) distribution of biological activities on the correlation cir-cle based on PCA; (d) and (e) scatter plot of PCA and heat map obtained from the matrix of biological activities for the extracts. Definitions: TPC: total phenolic content; TFC: total flavonoid con-tent; PPBD: phosphomolybdenum assay; MCA: metal chelating assay; TYR: tyrosinase inhibition.
Indeed, substantially increased activity of butyryl cholinesterase and acetyl cholinesterase has been associated with the increased hydrolysis of acetylcholine, a neurotransmitter (Zengin et al.2018).
The results presented in Table 5 clearly show that the methanolic stem barks and leaves extracts inhibited acetyl cholinesterase. However, H. madagascariensis stem bark methanol extract (5.01 mg GALAE/g) showed the highest butyryl cholinesterase inhibi-tory activity. Taxifolin, identified in H. madagascariensis extracts, has been reported to exert an inhibitory action on acetyl cholinesterase (Gocer et al. 2016). The methanolic extract of Rhizophora mucronata leaves, rich in catechin, a flavan-3-ol also identified in H. madagascariensis, showed potent inhibitory action against acetyl and butyryl cholin-esterase (Suganthy and Devi 2016). Methanol extracts (153.55 and 147.07 mg KAE/g extract, for leaves and stem bark extracts, respectively) of H. madagascariensis showed the highest tyrosinase inhibition.
Lupeol, previously identified from H. madagascariensis stem bark, has been reported to possess significant activity against tyrosinase (Azizuddin, Khan, and Choudhary
2011). Astilbin (IC50 value 1mm), identified from the leaves of H. madagascariensis, has
been reported to possess tyrosinase inhibitory activity (Hanamura, Uchida, and Aoki
2008). The characterization of phytochemicals in the H. madagascariensis extracts studied here, reveals the presence of a kaempferol derivative (m/z 563), catechin (m/z 289), and rutin (m/z 609). Interestingly, detailed studies have shown these compounds inhibited tyrosinase activity (Kim et al.2004; Chang2009; Si et al.2012).
The inhibitory activity of the methanol, aqueous and ethyl acetate extracts of H. madagascariensis leaves and stem bark on enzymes targeted for the treatment of dia-betes was investigated. In general, the inhibitory activity of the tested extracts against a-amylase was relatively low (0.32 to 0.93 mmol ACAE/g extract). In contrast, the extracts showed higher inhibitory action against a-glucosidase (1.16 to 1.26 mmol ACAE/g extract), with the aqueous extracts being the most active.
Interestingly, two anthracenones, namely, madagascenone A and B, isolated from H. madagascariensis bark showed a-glucosidase inhibition with IC50 values lower than the
standard, acarbose (Onajobi et al. 2016). Furthermore, alloxan-induced diabetic rats treated with the aqueous extract of H. madagascariensis leaves (500 mg/kg body weight for 7 days) showed a reduced blood glucose level (Nimenibo-Uadia and Nwachukwu
2017). The findings gathered from these pharmacological studies support the traditional use of H. madagascariensis for the management of diabetes.
Table 5. Enzyme inhibitory activity of H. madagascariensis.
Plant tissue Solvents
AChE inhibition (mg GALAE/g) BChE inhibition (mg GALAE/g) Tyrosinase inhibition (mg KAE/g) Amylase inhibition (mmol ACAE/g) Glucosidase inhibition (mmol ACAE/g) Leaves Ethyl acetate 3.80 ± 0.09b 3.27 ± 0.16c 86.15 ± 2.16f 0.73 ± 0.03b 1.16 ± 0.06b
Methanol 4.61 ± 0.06a 4.51 ± 0.19b 153.55 ± 0.90a 0.93 ± 0.01a 1.17 ± 0.02b Aqueous 2.88 ± 0.09c 2.03 ± 0.16d 123.68 ± 1.36d 0.32 ± 0.03d 1.26 ± 0.02a
Stem bark Ethyl acetate 3.73 ± 0.09b 4.44 ± 0.19b 134.88 ± 1.40c 0.75 ± 0.03b Inactive
Methanol 4.68 ± 0.08a 5.01 ± 0.11a 147.07 ± 0.81b 0.91 ± 0.01a 1.18 ± 0.01b Aqueous 2.97 ± 0.08c 2.04 ± 0.13d 116.58 ± 1.86e 0.40 ± 0.04c 1.26 ± 0.01a Values expressed are the mean ± standard deviation of three parallel measurements. GALAE: galantamine equivalent;
KAE: kojic acid equivalent; ACAE: acarbose equivalent. The superscript letters indicate the differences between the tested extracts (p< 0.05).
Statistical evaluation
The obtained results from biological activity assays were evaluated by multivariate statis-tical analysis and the outcomes are shown in Figure 2. Apparently, a highly significant positive correlation was noted between total phenolic components and reducing power (FRAP and CUPRAC), free radical scavenging assays (DPPH and ABTS), as well as total antioxidant assays (PPBD). However, this component demonstrated a significantly posi-tive correlation with glucosidase inhibition abilities (Figure 2a). Two principal compo-nents expressing respectively 65.1% and 34.9% of total variances were obtained by the PCA (Figure 2b).
The PCA scatter plot and heat map analysis clustered the samples into three groups according to similarities in their biological activities (Figure 2d,e). These results were confirmed by the MANOVA, which showed the weakest p-value for all of the per-formed statistical tests (Table 6). This observation demonstrated that the studied bio-logical activities taken collectively discriminate the samples in three distinct groups dependent on the types of solvent used.
The first cluster was represented by methanol extracts of both studied tissues and the second cluster by water extracts. The samples obtained from the ethyl acetate were included in the third cluster. Figure 2c–e shows that the third cluster was characterized by a high metal chelating activity while the members of the first cluster showed overall good properties against all studied biological activities except metal chelating ability, including the DPPH, tyrosinase, BChE, amylase, and AChE assays.
Conclusion
In this study, the biological properties and chemical composition of H. madagascariensis leaves’ and stem bark extracts were evaluated. The methanol and water extracts exhib-ited significant antioxidant properties and high total phenolic and flavonoid contents. In addition, the methanol extracts were very active against cholinesterases, tyrosinase, and a-amylase. Flavonoids and procyanidins were the main components in the metha-nolic extracts. The results obtained from this study clearly demonstrated the choice of solvent is one of the most important steps in the phytochemical studies of H. madagascariensis.
A strong correlation was observed between the total bioactive compounds and the antioxidant properties. To the best of our knowledge, the present paper is the first regarding biological properties and chemical characterization of H. madagascariensis extracts. At this point, this investigation provides useful baseline data for further investi-gation, especially with green extraction techniques including pressurized liquid, micro-wave-assisted or ultrasound-assisted extraction, geared toward the development of novel pharmacophores and functional applications.
Table 6. Evaluation of the differences between the three clusters using multivariate analysis of vari-ance (MANOVA).
Pillai’s trace Wilks’ lambda Hotelling’s trace Roy’s largest root
Value 1.98 0.0001 895.89 808.08
Acknowledgements
The technical and human support provided by CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucıa, FEDER) is gratefully acknowledged.
ORCID
Gokhan Zengin http://orcid.org/0000-0002-5165-6013
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![Table 2. Characterization of the extracts of H. madagascariensis. Peak number Retention time (min) [M H] m/z m/z (% base peak) Assigned identification Leaves Stembark 1 1.7 341 MS 2 [341]: 179 (100), 161 (7), 143 (56), 131 (20), 119 (8), 113 (17 Disacch](https://thumb-eu.123doks.com/thumbv2/9libnet/4971794.100631/7.756.90.657.92.931/characterization-extracts-madagascariensis-retention-assigned-identification-stembark-disacch.webp)
