PMIO 2017; 4(03): e108-e113
DOI: 10.1055/s-0043-122397
Letters
Georg Thieme Verlag KG Stuttgart · New York

Constituents of Acacia nilotica (L.) Delile with Novel Kinase Inhibitory Activity

Augustine A. Ahmadu1, *, Abdulkarim Agunu2, Thi-Ngoc-Dung Nguyen3, Blandine Baratte3, Béatrice Foll-Josselin3, Sandrine Ruchaud3, Benoît Serive3, *, Stéphane Bach3, *
  • 1Department of Pharmaceutical and Medicinal Chemistry, Kaduna State University, Kaduna, Nigeria
  • 2Department of Pharmacognosy and Drug development, University of Ilorin, Ilorin, Nigeria
  • 3Sorbonne Universités, UPMC Univ. Paris 06, CNRS USR3151, Protein Phosphorylation and Human Disease Laboratory, KISSf Screening Facility, Station Biologique, Roscoff, France
Further Information

Correspondence

Dr. Augustine Ahmadu
Department of Pharmaceutical and Medicinal Chemistry
Faculty of Pharmaceutical Sciences
Kaduna State University
Tafawa Balewa Way
800241 Kaduna
Nigeria   
Phone: +23/480/370 335 05   

Publication History

received 07 July 2017
revised 25 September 2017

accepted 21 October 2017

Publication Date:
05 December 2017 (online)

 

Abstract

Acacia nilotica (L.) Delile belongs to the genus Acacia, which includes about 1400 species in subtropical and tropical Africa including Nigeria, Senegal, Egypt, and Mozambique as well as Asia from India to Burma. This plant is traditionally used to treat several pathologies such as mouth, ear, and bone cancer. Moreover, it possesses many other biological activities (antidiarrheal, anti-inflammatory, antimicrobial, and antifungal). We report here the extraction, purification, and identification of two known compounds [ethylgallate and (+)-catechin] from the bark of the tree that were further tested for their inhibitory activities against a panel of disease-related protein kinases. Both compounds were active, and (+)-catechin showed the best activity by inhibiting nine out of fourteen protein kinases with an IC50 value in the µg/mL range. This compound gave the highest activity against CLK1 with an IC50 of 2.1 µg/mL. The ethyl acetate extract and its components, such as catechins and other polyphenols, which also had protein kinase inhibitory activity, can be exploited in the research for anticancer agents.


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The genus Acacia includes some 1400 species of trees and shrubs widespread throughout warm and semiarid regions of the world including Nigeria. Acacia nilotica (L.) Delile belongs to the subgenus Acacia [1] [2]. It grows naturally in tropical Africa including Nigeria, Senegal, Egypt, and Mozambique as well as Asia from India to Burma, where it was probably introduced. A. nilotica is a shrubby tree, 5–20 m high and is widely used in traditional medicine in Africa ([Fig. 1a]). In the traditional Hausa ethnomedicine of Northern Nigeria, the leaves and bark are used to treat diarrhea and inflammation [3]. While naringenin, and several galloyl and catechin derivatives have been isolated from the bark [4] [5], flavonol glycosides from the seeds [6], antimicrobial, antifungal, and anti-inflammatory properties have been reported previously [7] [8] [9] [10]. Studies regarding the Fabaceae family (i.e., those of A. nilotica) have shown that 106 of their phytochemicals possess activities related to cancer treatment. Amongst them, A. nilotica compounds were described for anticancer activity and for cancer preventive activity [11].

Zoom Image
Fig. 1 a Pictures of A. nilotica branches (Zaria, Nigeria). b Chemical structures of the purified compounds.

Previously, we have isolated two new peltogynoids from the stem bark of this plant [12]. In the present study, as part of our continuing chemical exploration from the species A. nilotica, we report here the isolation of two phenolic compounds. For the first time, ethyl gallate and (+)-catechin were tested against a panel of 14 protein kinases for their potential inhibitory activity.

Bark samples of A. nilotica were collected in Zaria, Nigeria, in July 2015, as described previously [12]. After an ethanolic extraction, chloroform, ethyl acetate, and n-butanol fractions were produced and two major compounds from the ethyl acetate fraction were purified (compounds 1 and 2). Compound 1 is a pale white solid corresponding to a known compound ethyl gallate (MW=198.17) according to various types of spectroscopy analyses. The chemical structure of the purified compound 1 is depicted in [Fig. 1b] .

Compound 2 is a pale yellow powder. It was validated and confirmed as the known compound (+)-catechin (MW=290.26) according to the spectroscopy analysis. The chemical structure of the purified compound 2 is depicted in [Fig. 1b] .

UV, NMR, and MS of these compounds were consistent with that reported in the literature for ethyl gallate [13] [14] and (+)-catechin [15] [16].

A primary screening of the extracts and isolated compounds against 14 disease-related protein kinases was undertaken. [Table 1] and [Fig. 2a] show the results of the primary screening of the extracts and compounds against a panel of 14 protein kinases. The result indicates the activity that remains in the tube after treating the mentioned kinases with 50 µg/mL of the extracts and compounds compared to the control assay treated with DMSO. The results revealed that the chloroform extract showed the worst inhibition against the tested kinases (e.g., 50 µg/mL of the chloroform extract inhibits only 10% of the total CDK5 kinase activity). In contrast, the ethylacetate extract is highly active against the Haspin kinase: 50 µg/mL of the extract inhibits 99% of the maximal kinase activity. A similar trend was observed for the n-butanol soluble fraction and compounds 1 and 2. The ethyl acetate and n-butanol fractions and the isolated compounds 1 and 2 were tested over a wide range of concentrations (from 0.016 to 50 µg/mL) and the IC50 values were determined from the dose-response curves. [Table 2] reports the inhibitory activity of the fractions and isolated compounds against the mentioned kinases. The result revealed that the ethylacetate fraction is the most active against the Haspin kinase (IC50 of 1.2 µg/mL) followed by Aurora B kinase (IC50 of 2.9 µg/mL). This extract is more active than the n-butanol. Chloroform extract was not tested since the primary screening did not show any activity against the kinase panel at 50 µg/mL. Inhibitory activity of the two compounds showed that (+)-catechin (2) was the most active protein kinase inhibitor as it inhibits the activity of nine out of the fourteen protein kinases. As shown here, (+)-catechin is inactive against HsCDK5/p25, PfGSK3, HsRIPK3, SscCK1, and LmCK1 (IC50 >172 µM). Note that the IC50 value of compound 2 against MmCLK1, the best target, was estimated at 2.1 µg/mL for the natural product purified from Acacia and 2.5 µg/mL for the molecule commercially available ([Fig. 2b]). The kinase panel tested in this study represents only 2% of the human kinome (total of 518 protein kinases). Since plant flavonoids have been shown to modulate the activities of some enzyme systems, e.g., those involved in cell surface signal transduction, immune function and transformation, tumor growth, and metastasis [17] [18], some other targets might also be affected.

Zoom Image
Fig. 2 Effect of purified compounds 1 and 2 on the catalytic activity of a selected set of mammalian and parasitic kinases. a Kinase activities in the presence of 50 µg/mL of tested compound are expressed in % of maximal activity, i.e., measured in the absence of inhibitor. ATP concentration used in the kinase assays was 15 µM (mean±range; n=2). Hs, Homo sapiens; Ssc, Sus scrofa; Rn, Rattus norvegicus; Mm, Mus musculus; Pf, Plasmodium falciparum; Lm, Leishmania major; Ld, Leishmania donovani. b Dose-dependent effect of compound 2 and commercial (+)-catechin on mouse CLK1. Recombinant GST-CLK1 was assayed in the presence of increasing concentrations of the two batches of (+)-catechin. Kinase activities are expressed in % of maximal activity, i.e., measured in the absence of inhibitor (mean±SD; n=3).

Table 1 Primary screening of fractions from A. nilotica and purified compounds against 14 disease-related protein kinases.

Extract or compound

CDK2/CyclinA

CDK5/p25

CDK9/CyclinT

Mm CLK1

Ssc GSK-3

Pf GSK-3

Rn Dyrk1A

Pim1

Haspin

RIPK3

Lm CK1

Ld TLK

Ssc CK1

AuroraB

Chloroform

87

90

67

44

71

74

81

61

45

114

75

65

73

52

Ethyl acetate

37

25

11

10

15

11

12

7

1

23

20

50

9

-2

n-Butanol

59

60

15

18

11

9

17

20

0

43

29

73

23

10

Compound 1

51

47

55

35

35

68

55

52

42

93

79

58

64

48

Compound 2

29

36

16

10

20

62

11

23

6

132

53

27

40

8

The table reports the results of the primary screening performed using 50 µg/mL of the mentioned extract or purified compound. Data are expressed in % of maximal activity, i.e., measured in the absence of inhibitor. ATP concentration used in the kinase assays was 15 µM (values are means; n=2). Kinases are from human (Hs, Homo sapiens) origin unless specified: Ssc, Sus Scrofa; Rn, Rattus norvegicus; Mm, Mus musculus; Pf, Plasmodium falciparum; Lm, Leishmania major; Ld, Leishmania donovani.

Table 2 Effect of fractions and purified compounds on kinase inhibitory activity of A. nilotica (IC50).

Extract or compound

Cdk2 /CyclinA

Cdk5 /p25

Cdk9 /CyclinT

Mm CLK1

Ssc GSK3

Pf GSK3

Rn Dyrk1A

Pim1

AuroraB

Haspin

RIPK3

Ssc CK1

Lm CK1

Ld TLK

Ethyl acetate

>50

31.0

8.0

6.5

11.0

4.5

10.0

3.9

2.9

1.2

27.0

4.0

12.0

>50

n-Butanol

>50

>50

11.0

15.0

8.1

6.0

19.0

15.0

3.0

2.2

>50

27.0

8.5

>50

Compound 1

>50

>50

>50

27.0

30.0

>50

>50

35.0

>50

45.0

>50

>50

>50

>50

Compound 2

14.0

>50

11.0

2.1

11.0

>50

11.0

7.0

21.0

18.0

>50

>50

>50

32.0

Values are reported in µg/mL for both extracts and purified compounds (n=3; independent experiments). ATP concentration used in the kinase assays was 15 µM. Kinases are from human origin unless specified: Ssc, Sus scrofa; Rn, Rattus norvegicus; Mm, Mus musculus; Pf, Plasmodium falciparum; Lm, Leishmania major; Ld, Leishmania donovani.

Regarding ethyl gallate, although the literature reported numerous activities as an anti-inflammatory and antioxidant compound against various cancer cell lines and mycobacteria [19] [20] [21] [22], no strong significant kinase inhibition was reported in this study. Compound 1 affected only four kinases with IC50s under 50 µg/mL (<252 µM).

(+)-Catechin (2) is naturally present in green tea. Green tea has been used daily since before 3000 BC in China for its medicinal effects. These last decades, it has been demonstrated that catechins represent the molecular family, which hold the activity. Furthermore, the presence of polyphenol derivatives with reported proapoptotic activity [23] could prevent cancer [24]. Such molecules are not only present in green tea but also in many other land plants like Acacia. Their role is not fully understood but plants can acquire an ecological/survival benefit (e.g., space colonization, protection against grazing, activity against pathogens) to synthesize such compounds [25]. This phenomenon of so-called allelopathy is a source to identify new inhibitors of human therapeutic targets. As an example, it has been shown that the inhibitory effect of flavonoids in the growth of malignant cells could be a consequence of their interference with the protein kinase activities involved in the regulation of cellular proliferation and apoptosis [26]. Studies have shown that some polyphenols (e.g., catechins that are flavanols) possess suppressive effects in human cancer [27]. In diabetic rats, they may delay the loss of functional beta-cell mass and delay the progression of diabetes by preventing oxidative stress and beta-cell apoptosis [28]. Moreover, (+)-catechin has been known as an effective antioxidant by scavenging hydroxyl radicals, delaying the consumption of other antioxidants such as α-tocopherol and β-carotene, and inhibiting lipid peroxidation. This molecule has a hepatoprotective effect against CCl4-induced acute liver injury as well, and has displayed anti-hyperglycemic activity in STZ-diabetic rats. Thus, (+)-catechin might be further explored as a lead in the discovery of anticancer agents.

Materials and Methods

Plant collection and extraction

Dried pulverized bark (400 g), collected in Zaria (Nigeria), was extracted with 70% ethanol to exhaustion at room temperature. Removal of the solvent at a reduced temperature afforded a brownish crude extract (25 g). A portion of this extract (20 g) was resuspended in distilled water (100 mL) and partitioned successively to exhaustion with 2 × 500 mL each of chloroform, ethyl acetate, and n-butanol. Removal of the organic solvents afforded 1.25 g of chloroform, 3.2 g of ethyl acetate, and 5.2 g of n-butanol, respectively. Commercial (+)-catechin was obtained from Sigma-Aldrich (99% purity, reference product #43412).


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The authors wish to acknowledge the assistance of Dr. Zulfiqar Ali of the National Center for Natural Products Research, University of Mississippi, Oxford, MS, USA, for MS of isolated compounds and Dr. Nikolas Fokialakis of the Department of Pharmacognosy and Natural Products Chemistry, University of Athens, Greece, for the LC-MS analysis. The authors also thank Cancéropôle Grand-Ouest (axis: Marine Natural Products in Cancer Treatment), GIS IBiSA (Infrastructures en Biologie Santé et Agronomie, France), and Biogenouest (Western France Life Science and Environment Core Facility Network) for supporting the KISSf screening facilty (Roscoff, France). T.-N.-D. Nguyen is supported by the government of Vietnam (recipient of a “Project 911” PhD fellowship) and the French Embassy in Vietnam (Campus France). B. Serive is supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (grant #622735). The S. Bach project is supported by the ANR/Investissements d’Avenir program via the OCEANOMICs project (grant #ANR -11-BTBR-0008) and INCa (NECROTRAIL Program).

* These authors share senior authorship.


Supporting Information


Correspondence

Dr. Augustine Ahmadu
Department of Pharmaceutical and Medicinal Chemistry
Faculty of Pharmaceutical Sciences
Kaduna State University
Tafawa Balewa Way
800241 Kaduna
Nigeria   
Phone: +23/480/370 335 05   


Zoom Image
Fig. 1 a Pictures of A. nilotica branches (Zaria, Nigeria). b Chemical structures of the purified compounds.
Zoom Image
Fig. 2 Effect of purified compounds 1 and 2 on the catalytic activity of a selected set of mammalian and parasitic kinases. a Kinase activities in the presence of 50 µg/mL of tested compound are expressed in % of maximal activity, i.e., measured in the absence of inhibitor. ATP concentration used in the kinase assays was 15 µM (mean±range; n=2). Hs, Homo sapiens; Ssc, Sus scrofa; Rn, Rattus norvegicus; Mm, Mus musculus; Pf, Plasmodium falciparum; Lm, Leishmania major; Ld, Leishmania donovani. b Dose-dependent effect of compound 2 and commercial (+)-catechin on mouse CLK1. Recombinant GST-CLK1 was assayed in the presence of increasing concentrations of the two batches of (+)-catechin. Kinase activities are expressed in % of maximal activity, i.e., measured in the absence of inhibitor (mean±SD; n=3).