Abstract
In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC50 = 260 ± 0.112 µg/ml) and antiplasmodial (IC50 = 8.5 ± 1.6 µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31 µg/ml ≥ IC50 ≥ 5.6 µg/ml; antiplasmodial: 14 µg/ml ≥ IC50 ≥ 2 µg/ml) with very minimal toxicity.
Introduction
Diabetes mellitus (DM) is a chronic disease that arises when the pancreas does not produce enough insulin or when the body cannot effectively use it, resulting in hyperglycemia. Type II DM, or non-insulin-dependent diabetes, results from ineffective use of insulin as a result of excess body weight, physical inactivity and genetic susceptibilityCitation1. Treatment of diabetes involves lowering blood glucose through different mechanisms, including insulin secretion, glucose absorption and metabolism. One of the approaches to control diabetes is to block carbohydrate digestion and absorption. Alpha-glucosidase, which is involved in the cleavage of glucose from disaccharides and oligosaccharides, is the most important enzyme among those participating in the carbohydrate digestion process. Inhibitors of this enzyme are now receiving much more attention towards the development of new anti-diabetic drugs. Malaria remains one of the most threatening diseases in the developing world today. The widespread resistance to commonly used antimalarial drugs has necessitated the search for new antimalarial drugs.
Medicinal plants, an integral part of South African traditional medicine, continue to play a major role in the public health of most developing countries. They remain a reliable source of therapeutics for several illnesses that threaten public health. In South Africa, Buddleja saligna Willd. (Loganiaceae), locally known as iGqeba-elimhlope by the Zulu, is a well-known medicinal plant used locally to treat different ailments. It is a small- to medium-sized evergreen tree (10 m) growing in warm moist areas but usually 4–5 m at high elevationCitation2. It is used as a purgative; to treat coughs, colds, diabetes, tuberculosis, thrush and sores, anasarca and chest painsCitation3,Citation4.
The biological activities reported for B. saligna include antiplasmodial, acetylcholinesterase and alpha-glucosidase inhibitoryCitation2, anti-mycobacterialCitation5, antibacterial and antioxidantCitation6, antimutagenicCitation7 activities. In our previous studies, we reported the interesting antiplasmodial and alpha-glucosidase inhibitory activities exhibited by the hexane fraction of B. salignaCitation2. We report here the alpha-glucosidase inhibitory activity and antiplasmodial properties of isolated terpenoids from the hexane fraction.
Materials and methods
Plant collection
Leaves of B. saligna were collected in May 2013 from the Botanical Garden of the University of KwaZulu-Natal, Pietermaritzburg, South Africa. The plant was appropriately identified by the Curator, and a voucher specimen (Chukwujekwu #7 NU) was deposited in the Herbarium of the University of KwaZulu-Natal, Pietermaritzburg. Plant material was dried at 50 °C, powdered and stored in paper containers at ambient temperature for less than 24 h prior to extraction.
Extraction and bioassay-guided fractionation
The oven-dried powdered leaves (583 g) were extracted with 80% methanol with sonication for 1 h and then soaked overnight. The extracts were filtered through a Büchner funnel using Whatman No. 1 filter paper, and the solvent was evaporated under reduced pressure at 30 °C. Liquid–liquid partitioning was done as described by Chukwujekwu et alCitation8. Gravity-aided column chromatography (Merck 9385, Darstadt, Germany; 123 g, 2.5 cm × 73 cm) of the hexane fraction (11 g), using hexane:ethyl acetate (1:0–0:1) and ethyl acetate:MeOH (1:0–0:1) step gradients, produced 19 fractions. These fractions were pooled by TLC profile into 8 fractions and subjected to antiplasmodial bioassay, in which fraction 3 (392 mg) was found to be the most active (IC50 = 3.12 µg/ml). Repeated purification of fraction 3 by column chromatography and subsequently preparative TLC (Merck glass plates, 20 × 20 cm, silica gel F254, 0.25 cm thickness), using hexane:ethyl acetate (3:1) as the solvent system, afforded three active pure compounds: (1) (11 mg); (2) (19 mg) and (3) (16 mg) ().
Figure 1. Structures of isolated terpenoids (1, Betulonic acid; 2, Betulone; 3, Spinasterol) from B. saligna leaves.
Alpha-glucosidase inhibitory activity
Alpha-glucosidase inhibitory activity was determined as previously described by Rengasamy et alCitation9. Alpha-glucosidase from Saccharomyces cervisiae (EC 3.2.1.20), p-nitrophenyl-α-d-glucopyranoside and acarbose were obtained from Sigma-Aldrich (WGK, Germany). The control experiment contained the same reaction mixture, but the sample solution was replaced with the same volume of phosphate buffer. Acarbose dissolved in dimethyl sulphoxide (DMSO), was used as a positive control. The determinations were carried out in triplicate. The percentage inhibition (%) was calculated using the following equation:
(1)
where Acontrol is the absorbance of the control and Asample is the absorbance of the sample. The IC50, which is the concentration of the sample required to inhibit 50% of the enzyme was determined for each sample using GraphPad Prism 5.0 (La Jolla, CA).
In-vitro antiplasmodial assay
Samples were tested for antiplasmodial activity in triplicate against chloroquine-sensitive (CQS) strain of Plasmodium falciparum (NF54) as described by Chukwujekwu et alCitation10. The reference drugs used were chloroquine diphosphate (CQ) (Sigma) and artesunate (Sigma, WGK, Germany).
Cytotoxicity assay
Samples were tested for in vitro cytotoxicity against a mammalian cell-line, Chinese Hamster Ovarian (CHO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)-assay. The assay was performed as previously described by Chukwujekwu et alCitation10.
Identification of compounds
Full structural elucidation of found compounds was made using NMR and MS spectroscopy. NMR spectra were measured in CDCl3 on Bruker AVANCE 600 (1H at 600.1 MHz and 13C at 150.9 MHz) NMR spectrometer (Billerica, MA). Chemical shifts (in ppm, δ scale) were referenced to the residual solvent signals (7.26 for 1H and 77.0 for 13C). Coupling constants (J) are given in Hz. The complete assignment of 1H and 13C signals was performed by an analysis of the correlated homonuclear H,H-COSY, H,H-ROESY and heteronuclear H,C-HSQC and HMBC spectra. Mass spectra were collected on an LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA) using ESI or APCI ionization.
3-oxolup-20(29)-en-28-oic acid (betulonic acid; 1)
13C NMR (150.9 MHz, CDCl3): 14.61 (CH3-27); 15.81 (CH3-26); 15.93 (CH3-25); 19.35 (CH3-30); 19.61 (CH2-6); 20.98 (CH3-24); 21.36 (CH2-11); 25.48 (CH2-12); 26.62 (CH3-23); 29.67 (CH2-15); 30.54 (CH2-21); 32.09 (CH2-16); 33.59 (CH2-7); 34.10 (CH2-2); 36.90 (C-10); 37.02 (CH2-22); 38.50 (CH-13); 39.59 (CH2-1); 40.62 (C-8); 42.47 (C-14); 46.88 (CH-19); 47.31 (C-4); 49.18 (CH-18); 49.83 (CH-9); 54.92 (CH-5); 56.37 (C-17); 109.75 (CH2-29); 150.30 (C-20); 182.03 (C-28); 218.18 (C-3).
1H NMR (600.1 MHz, CDCl3): 0.92 (br.s, 3H, H-25); 0.97 (s, 3H, H-26); 0.99 (br.s, 3H, H-27); 1.01 (s, 3H, H-24); 1.07 (s, 3H, H-23); 1.05 (m, 1H, H-12 a); 1.21 (br.dt, 1H, Jgem = 13.5 Hz, J15a,16a = J15a,16b = 3.3 Hz, H-15a); 1.27–1.35 (m, 2H, H-11a, 5); 1.34–1.54 (m, 10H, H-9, 7, 1a, 16a, 21a, 6, 11b, 22a); 1.53 (m, 1H, H-15b); 1.63 (t, 1H, J18,19 = J18,13 = 11.4 Hz, H-18); 1.69 (dd, 3H, J30,29a = 1.4 Hz, J30,29b = 0.7 Hz, H-30); 1.72 (m, 1H, H-12b); 1.89 (ddd, 1H, Jgem = 13.3 Hz, J1b,2b = 7.6 Hz, J1b,2a = 4.4 Hz, H-1b); 1.94 – 2.03 (m, 2H, H-21b, 22b); 2.22 (ddd, 1H, J13,12a = 12.9 Hz, J13,18 = 11.6 Hz, J13,12b = 3.7 Hz, H-13); 2.28 (br.dt, 1H, Jgem = 13.0 Hz, J16b,15 = 3.4 Hz, H-16b); 2.40 (ddd, Jgem = 15.7 Hz, J2a,1a = 7.6 Hz, J2a,1b = 4.4 Hz, H-2a); 2.49 (ddd, 1H, Jgem = 15.7 Hz, J2b,1b = 9.8 Hz, J2b,1a = 7.6 Hz, H-2b); 3.01 (td, 1H, J19,18 = J19,21 = 10.9 Hz, J19,21 = 5.0 Hz, H-19); 4.61 (dq, 1H, Jgem = 2.3 Hz, J29a,30 = 1.4 Hz, H-29a); 4.74 (bd, 1H, Jgem = 2.3 Hz, H-29b). HRMS (ESI) for C30H46O3 [M+H] calc: 455.35197; found: 455.35183.
Lup-20(29)-en-28-ol-3-one (betulone; 2)
13C NMR (150.9 MHz, CDCl3): 14.68 (CH3-27); 15.79 (CH3-26); 15.95 (CH3-25); 19.10 (CH3-30); 19.66 (CH2-6); 21.03 (CH3-24); 21.36 (CH2-11); 25.22 (CH2-12); 26.63 (CH3-23); 27.03 (CH2-15); 29.12 (CH2-16); 29.74 (CH2-21); 33.52 (CH2-7); 33.96 (CH2-22); 34.13 (CH2-2); 36.87 (C-10); 37.43 (CH-13); 39.60 (CH2-1); 40.89 (C-8); 42.78 (C-14); 47.34 (C-4); 47.77 (CH-19); 47.77 (C-17); 48.69 (CH-18); 49.75 (CH-9); 54.92 (CH-5); 60.55 (CH2-28); 109.76 (CH2-29); 150.38 (C-20); 218.07 (C-3).
1H NMR (600.1 MHz, CDCl3): 0.93 (d, 3H, J25,LR = 0.9 Hz, H-25); 0.99 (d, 3H, J27,LR = 1.0 Hz, H-27); 1.03 (s, 3H, H-24); 1.06 (s, 3H, H-26); 1.07 (s, 3H, H-23); 0.96–1.13 (m, 3H, H-12a, 15a, 22a); 1.12–1.35 (m, 4H, H-5, 11a, 16a, 21a); 1.35–1.54 (m, 7H, H-9, 7, 1a, 6, 11b); 1.60 (t, 1H, J18,19 = J18,13 = 11.7 Hz, H-18); 1.69 (dd, 3H, J30,29a = 1.4 Hz, J30,29b = 0.8 Hz, H-30); 1.63–1.75 (m, 3H, H-12b, 13, 15b); 1.80–2.04 (m, 4H, H-1b, 16b, 21b, 22b); 2.39 (br.td, 1H, J19,18 = J19,21 = 11.1 Hz, J19,21 = 6.0 Hz, H-19); 2.40 (ddd, Jgem = 15.7 Hz, J2a,1a = 7.6 Hz, J2a,1b = 4.4 Hz, H-2a); 2.49 (ddd, 1H, Jgem = 15.7 Hz, J2b,1b = 9.8 Hz, J2b,1a = 7.6 Hz, H-2b); 3.35 (br.d, 1H, Jgem = 10.7 Hz, H-28a); 3.80 (br.d, 1H, Jgem = 10.7 Hz, H-28b); 4.59 (br.dq, 1H, Jgem = 2.4 Hz, J29a,30 = 1.4 Hz, H-29a); 4.69 (br.d, 1H, Jgem = 2.4 Hz, H-29b). HRMS(ESI) for C30H48O2 [M+Na] calc: 463.35465; found: 463.35466.
(3β,5α,22E)-Stigmasta-7,22-dien-3-ol (α-spinasterol; 3)
13C NMR (150.9 MHz, CDCl3): 12.05 (CH3-18); 12.24 (CH3-29); 13.04 (CH3-19); 18.99 (CH3-27); 21.08 (CH3-26); 21.37 (CH3-21); 21.55 (CH2-11); 23.01 (CH2-15); 25.39 (CH2-28); 28.50 (CH2-16); 29.64 (CH2-6); 31.48 (CH2-2); 31.87 (CH-25); 34.22 (C-10); 37.14 (CH2-1); 37.99 (CH2-4); 39.46 (CH2-12); 40.27 (CH-5); 40.81 (CH-20); 43.29 (C-13); 49.45 (CH-9); 51.25 (CH-24); 55.13 (CH-14); 55.91 (CH-17); 71.07 (CH-3); 117.46 (CH-7); 129.45 (CH-23); 138.16 (CH-22); 139.56 (C-8).
1H NMR (600.1 MHz, CDCl3): 0.55 (s, 3H, H-18); 0.80 (d, 3H, J27,25 = 6.5 Hz, H-27); 0.80 (br.s, 3H, H-19); 0.81 (t, 3H, J29,28 = 7.4 Hz, H-29); 0.85 (d, 3H, J26,25 = 6.5 Hz, H-26); 1.03 (d, 3H, J21,20 = 6.7 Hz, H-21); 1.08 (m, 1H, H-1a); 1.12–1.33 (m, 6H, H-4a, 6a, 12a, 16a, 17, 28a); 1.33–1.61 (m, 9H, H-2a, 5, 11, 15, 24, 25, 28b); 1.61–1.86 (m, 7H, H-1b, 2b, 4b, 6b, 9, 14, 16b); 2.00 (ddd, 1H, Jgem = 12.7 Hz, J12,11 = 4.4 Hz, J12,11 = 2.8 Hz, H-11); 2.03 (m, 1H, H-20); 3.60 (tt, 1H, J3,4 = J3,2 = 11.1 Hz, J3,4 = J3,2 = 4.6 Hz, H-3); 5.03 (ddd, J23,22 = 15.2 Hz, J23,24 = 8.9 Hz, J23,LR = 0.7 Hz, H-23); 5.16 (m, 1H, H-7); 5.16 (dd, 1H, J22,23 = 15.2 Hz, J22,20 = 8.8 Hz, H-22). HRMS (APCI) for C29H48O [M+H] calc: 413.37834; found: 413.37724.
Results and discussion
Three known compounds () were isolated from the hexane fraction. Compound 1 (betulonic acid; a derivative of betulinic acid), compound 2 (betulone; derivative of betulin) and compound 3 (α-spinasterol). With an exception of compound 3Citation11,Citation12, this is the first report of isolation of the compounds from the genus Buddleja.
The alpha-glucosidase inhibitory properties of the isolated compounds are presented in . There is an improvement in both the inhibitory activities and selective index of the compounds over the hexane fraction. All the compounds showed higher activity than acarbose, the drug commercially available to treat type II diabetes. Among the compounds tested, compound 2 was the most potent inhibitor against yeast alpha-glucosidase with an IC50 value 5.96 ± 0.563 μg/ml. Besides compound 2, compound 3 also showed a good alpha-glucosidase inhibitory activity followed by compound 1. Similar lupan-type triterpenes have been reported to exhibit alpha-glucosidase inhibitory activityCitation13. In a different anti-diabetic assay model, betulinic acid was found to increase glucose uptake and enhanced glycogen synthesisCitation14. It is proposed that a steroid nucleus is important for a high hypoglycemic effect and possibly conditioning the high degree of lipophilicityCitation15 and they exhibit anti-diabetic properties with relatively low toxicityCitation16,Citation17. According to Li et al.Citation18, the mechanism of action of terpenoids and steroids in in-vivo studies might occur through stimulation of pancreatic islets leading to an increase in insulin-induced glucose uptake.
Table 1. Alpha-glucosidase inhibitory properties of isolated terpenoids from B. saligna leaf extract.
presents the antiplasmodial and cytotoxicity activities of the isolated compounds as well as their selective index (SI). Compound 1 was the most promising compound as it displayed the highest antiplasmodial activity with little cytotoxicity. This was followed closely by compound 2 and lastly compound 3. It is interesting to note the enhancement in the antiplasmodial activity of the compounds, especially compounds 1 and 2, and the decrease in toxicity of the compounds compared to the hexane fraction. The high toxicity of the hexane fraction may be attributed to the interaction of different constituents of the hexane fraction as it is a complex mixture or the elimination of certain toxic constituents of the hexane fraction during the process of isolation of the compounds. The antiplasmodial activity of the isolated compounds, except compound 3, has been reported. The antiplasmodial and cytotoxicity results of compound 1 displayed similar results with that reported in the literatureCitation19. Although compound 3 displayed weak antiplasmodial activity, to the best of our knowledge, it is the first report of its antiplasmodial property. Compound 2 has been reported to possess antiplasmodial activity against a multidrug resistant strain of Plasmodium falciparumCitation20.
Table 2. In-vitro antiplasmodial activity of isolated terpenoids from B. saligna leaf extract against chloroquine-sensitive (CQS) strain of P. falciparum (NF54) and cytotoxicity evaluation using Chinese Hamster Ovarian (CHO) cell line.
Although these are known compounds, the study has revealed a new source of the compounds. To the best of our knowledge, this is the first report of antiplasmodial activity of compound 3 and alpha-glucosidase inhibitory activity of the three compounds. However, further research in line with structure-activity studies of the compounds, particularly compounds 1 and 2 might lead to the synthesis of a more active compound. The establishment of anti-diabetic activity, especially for compound 2, requires further research. The mechanism of action of this compound needs to be unravelled as it might contribute towards developing an anti-diabetic drug of terpenoid origin.
Declaration of interest
The authors report no declarations of interest.
JCC and KRRR thank the University of KwaZulu-Natal and National Research Foundation for financial assistance in the form of post-doctoral fellowships.
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