Alepidea amatymbica, an herbaceous plant with a broad ethnomedicinal application among the native of Eastern and Southern Africa. The isolation of diterpenoids from A. amatymbica and evaluation of their biological activities, based on the ethnomedicinal information, was the primary focus of this investigation. Five bioassay guided isolated diterpenoids: ent-13-hydroxy-16-kauren-19-oic acid (1), 16-hydroxy-kaur-6-en-19-oic acid (2), 14- acetoxy ent- kaur-16-en-19-oic acid (3), 14-oxokaur-16-en-19-oic acid (4), and 14-acetoxo-12-oxokaur-16-en-19-oic acid (5) were screened in vitro for their anti-inflammatory, cytotoxicity, and antimicrobial. The diterpenoids were isolated and purified using open column chromatography, PTLC, and characterised with FTIR, NMR, and HRMS EI. The diterpenoids were not cytotoxicity on the normal cell but showed a significant effect of cancer cell lines. 14-acetoxo-12-oxokaur-16-en-19-oic acid showed a high inhibitory effect on lipoxygenase with an EC₅₀ of 19.10±3.15µg/ml compared to standard indomethacin with EC₅₀ of 17.22±5.48µg/ml. Among the diterpenes isolated and tested, 14-oxokaur-16-en-19-oic acid and 14-acetoxo-12-oxokaur-16-en-19-oic acid showed significant antibiotic activities against bacteria (MIC 125µg/ml) compared to gentamycin. Consequently, the antibiotic activity is structurally linked to the positions of acetate and oxo groups at C-14 and C-12 which enhances the activity of the diterpenoids. The in-vitro biological activities confirmed that the diterpenoids are sources of treatment and management of inflammation-related diseases, justifying its traditional applications.

Keywords: a. amatymbica, diterpenoids, bioautographic assay, cytotoxicity, lipoxygenase

EC₅₀, half maximal effective concentration; MIC, minimum inhibitory concentration; TLC, thin-layer chromatography; PTLC, preparative thin layer chromatography; FTIR, fourier transform infrared spectroscopy; HRMS E, high-resolution mass spectrometer electron ionization; 1H NMR; Proton Nuclear Magnetic Resonance; ¹³C NMR; Carbon Nuclear Magnetic Resonance COSY, Homonuclear Correlation Spectroscopy; HSQC, heteronuclear single quantum coherence or heteronuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; ATCC: American type culture collection

Alepidea amatymbica Eckl. & Zeyh is associated with the genus Alepidea a member of the Apiaceae family and known as ikhathazo by the Zulu and larger tinsel flower by the British. It is one out of the twenty-eight species of commonly used medicinal plants,¹ endemic to lowlands of Eastern and Southern Africa.^2,3It is a herbaceous plant arising from a single branched rhizome with dark green leaves, which is used in the treatment of conditions such as malaria, diarrhoea, flu, chest complaints, asthma and rheumatism.⁴ Extract of this species is reported to be highly regarded as active against HIV.⁵

The developing world has been on the receiving end of the direct impact of infectious diseases on the human health, as a consequence of the unavailability of vaccines or limited chemotherapy. The recent upsurge in the trend of infectious diseases in the world with greater impact in the Sub-Sahara Africa has led to the continuous search of reliable alternative medicine.⁶ The anti-inflammation property of A. Amatymbica was evaluated to determine its potential for preventing the initiation and development of diseases such as cancer^7,8 The report on the ethnobotanical survey of medicinal plants in the south eastern, Karoo, South Africa, documented that over 80% of South Africa population depend on herbal medicine and this dependence on traditional medicine is due to the high cost of western medication. Reviews of A. amatymbica literature has revealed various pharmacological activities which include anti-Inflammatory, antibacterial, antifungal, antihelminthic, antimalarial, antihypertensive and diuretic activities. Also, the claimed ethnomedicinal efficacy of the plant has been linked to some secondary metabolites present.⁹ However, all these metabolites have not been tested individually. Some diterpenoids with promising activities have been isolated over the past decade from A. amatymbica such as ent-kaur-16-en-19-oic acid, 15𝛽-Acetoxy-(-)-kaur-16-en-19-oic acid and 16𝛼-Methoxy-ent-kaur-en-19-oic acid.¹⁰

The encouragement of WHO on the use of traditional medicine for the treatment and prevention of diseases and its broad ethnomedicinal application¹¹ have made the primary focus of this study the isolation and characterisation of diterpenoids from A. amatymbica. Consequentially, to evaluate the in vitro inhibitory activity against 15-soybean lipoxygenase (15-LOX) enzyme, antibiotic, and cytotoxicity in normal Vero and cancerous bovine dermis cells.

Chemicals

All reagents and chemicals used in this study are from Sigma-Aldrich Chemicals Co., St Louis, MO, USA and are of analytical grade.

Collection and Authentication of amatymbica root

The selection of A. amatymbica was based on the ethnobotanical survey (oral interviews) with Mabandla village traditional healers of Umzimkhulu Local Municipality, Kwa-Zulu Natal, South Africa. The collection and identification of A. amatymbica roots were done by the head of traditional healers from the village (Mr S.P Dlamini). Authentication of the of A. amatymbica root was carried out by the South Africa National Biodiversity Institute, Pretoria, and voucher specimen number: 2116-0 was deposited at Pretoria National Botanical Garden.

Maceration and Liquid-liquid extraction

Exactly, 2000 g of A. amatymbica roots were air dried, pulverised using Lasec Polymix PX-MFC 90D grinder and macerated with acetone in a weight-solvent ratio of 1:10 ml for 6 h.¹² The extractants were recovered by use of a BUCHI Rotavapor R-210 with bath B-491 coupled to a vacuum pump V-210, and the resultant extract was air dried at ambient temperature in the fume hood under an air stream. The dried extract was re-dissolved in 70% acetone and sequentially partitioned with hexane, dichloromethane, ethyl acetate, acetone, methane, and water (freeze-dried at -55°C for 72 h). The crude acetone extract and partitions were stored at 4°C for further biological assays, fractionation, and isolation. Fractionation and Isolation of Diterpenoids from the root of A. Amatymbica.

Ethyl acetate partition (25 g) was pre-absorbed on Si-gel to produce a slurry, then freeze-dried at -55 ⁰C and Si-gel (625 g) was loaded on a 640 mm by 60 mm chromatographic column. Subsequently, eluted with different polarity of acetone-ethyl acetate solvent system. The fractions were collected from the column, purified by PTLC to give five compounds (1) (60 mg), (2) (62.1 mg), (3) (52 mg), (4) (108 mg), and (5) (62 mg) from F_8-19 (100 mg), F_75-87 (87 mg), F_52-64 (80 mg), F_20-28 (310 mg), and F_35-45 (74 mg), respectively.

All the compounds were identified by the comparison with the data in the literature and the extensive analysis of IR, 1D and 2D NMR involving COSY, HSQC, and HMBC spectra. Figure 1 shows the molecular structure of the compounds.

Figure 1 Chemical structures of diterpenoids isolated from A. Amatymbica.

Analytical data

Diterpenoids 1-5 were isolated in this investigation from an ethyl acetate fraction of the A. amatymbica root, and derivative of diterpenoids 1, 3-4 had previously been isolated.^13-16

1. Ent-13-hydroxy-16-kauren-19-oic acid (1): a colourless crystalline solid. Melting point: 215-216⁰C. (KBr)/cm^-1 3331 (v, O-H), 1600, 1475 (v, C=C), 3100 (v, =C-H), 1250, 1050 (v, C-O), 1710 (v, C=O). M/z (HRMS EI) 318.2195; C₂₀H₃₀O₃ requires [M⁺] 319.5882. - (300 MHz, (CD₃)₂CO) 2.24 (d J2.7,2H),1.20(m, 2H),1.75( m, 2H),1.75( m, 1H), 1.92(m, 2H), 1.20-1.75 (m, 2H), 2.61 (m, 2H), 1.87(m, 2H), 1.64-1.66 (m 2H),1.31(m, 2H), 2.95 (s, 2H), 2.89 (s, 2H), 4.78 ( s, 2H) 4.72 (s,2H), 1.20 (s, 3H),1.13(s,3H) (Supporting File 1 for full experimental data). δ_c (300 MHz, (CD₃)₂CO) 41.3 (C-1), 19.0(C-2), 39.8(C-3), 43.3(C-4), 55.5(C-5), 126.5(C-6), 133(C-7), 39.4(C-8), 61.3(C-9), 38.6(C-10), 34.0(C-11), 21.9(C-12), 49.9(C-13), 37.9(C-14), 58.6(C-15), 82.5(C-16), 28.5(C-17), 25.3(C-18), 178.3(C-19), 15.3(C-20) (Supporting File 2 for full experimental data).
2. 16-hydroxy-kaur-6-en-19-oic acid (2): a white crystalline. Melting point: 230-232 ⁰C. (KBr)/cm^-1 3450 (v, O–H), 1642, 1470 (v, C=C), 3080 (v, =C-H), 1240 (v, C-O), 1715 (v, C=O). M/z (HRMS EI) 318.2195; C₂₀H₃₀O₃ requires [M⁺] 318.2216. - (300 MHz, (CD₃)₂CO) 5.94 (t, J 7.2, 1H),5.55 (dd, J 2.7 1H), 2.19 (d, J 2.4), 2.16, (s, 2H), 1.87 (m, 2H), 1.80 (d, J 1.5, 2H), 1.79 (m, 2H), 1.75 (m, 2H), 1.44 (m, 1H), 1.39 (m, 2H), 1.38-1.53 (m, 2H), 1.25 (s, 3H), 1.20 (s, 3H), 1.15 (s, 1H), 0.88, (s, 3H) (Supporting File 3 for full experimental data). δ_c (300 MHz, (CD₃)₂CO) 40.4(C-1), 19.1(C-2), 37.8(C-3), 49.4(C-4), 49.3(C-5), 32.2(C-6), 34.3(C-7), 43.9(C-8), 42.5(C-9), 43.3(C-10), 21.9(C-11), 28.9(C-12), 76.4(C-13), 36.1(C-14), 43.8(C-15), 155.8(C-16), 102.5(C-17), 28.6(C-18), 178.6(C-19), 17.3(C-20) (Supporting File 4 for full experimental data).
3. 14-acetoxy ent- kaur-16-en-19-oic acid (3): is a white crystalline solid. Melting point: 219-220 ⁰C. (KBr)/cm^-1 2971 (v, O–H), 1664, 1470 (v, C=C), 3016 (v, =C-H), 1188(v, C-O), 1739 (v, C=O). M/z (HRMS EI) 376.2250; C₂₂H₃₂O₅ requires [M⁺] 376.2725. - (300 MHz, CDCl₃) 5.09(t, J 2.7,1H), 4.73 (d 2H), 2.68(d, 1H), 2.56(m, 2H), 2.18 (m, 2H), 1.98(m, 2H), 1.98 (s, 3H), 1.90 (m, 2H),1.88 (m, 2H), 1.63 (m, 2H), 1.33(t, 1H), 1.28 (s, 3H), 1.24 (m,2H), 1.15 (t, 1H), 1.01(m, 2H), 0.94 (s, 3H) (Supporting File 5 for full experimental data). δ_c (300 MHz, CDCl₃) 39.2 (C-1), 19.0 (C-2), 41.1 (C-3), 38.6 (C-4), 56.9 (C-5), 21.7 (C-6), 37.7 (C-7), 43.8 (C-8), 60.9 (C-9), 43.1 (C-10), 40.2 (C-11), 47.8 (C-12), 42.2 (C-13), 69.4 (C-14), 39.5 (C-15), 155.1 (C 16), 103.2 (C-17), 29.0 (C-18), 183.7 (C-19), 15.4 (C-20),170.1 (C-21), 21.6 (C-22) (Supporting File 6 for full experimental data).
4. 14-oxokaur-16-en-19-oic acid (4): is a brown waxy liquid. M/z (HRMS EI) 332.1988; C₂₀H₂₈O₄ requires [M⁺] 332.1445. (KBr)/cm^-1 2971 (v, O–H), 1651 (v, C=C), 3008 (v, =C-H), 1296 (v, C-O), 1721 (v, C=O). - (300 MHz, CDCl₃) 5.0 (s,2H) ,4.90 (s,2H), 2.77 (s, 1H), 2.20-2.40 (m, 2H), 2.15-2.35 (m,2H), 1.75 (d, J 3.6 ,2H), 1.66 (m, 2H), 1.61 (m, 2H), 1.57-1.75 (m, 2H), 1.50 (m,2H), 1.40-1.70 (m, 2H), 1.40-1.60 (m, 2H), 1.39 (s, 3H), 1.12 (s, 3H) (Supporting File 7 for full experimental data). δ_c (300 MHz, CDCl₃) 43.5(C-1), 19.6(C-2), 36.7(C-3), 48.3(C-4), 55.6(C-5), 35.9(C-6), 41.8(C-7), 56.9(C-8), 84.9(C-9), 22.0(C-10), 36.5(C-11), 32.8(C-12), 42.1(C-13), 212.9(C-14), 33.6 (C-15), 152.0 (C-16), 106.9(C-17), 22.0(C-18), 180.4(C-19), 18.0(C-20) (Supporting File 8 for full experimental data).
5. 14-acetoxo-12-oxokaur-16-en-19-oic acid (5): is a brown waxy solid. M/z (HRMS EI) 390.2037; C₂₂H₃₀O₆ requires [M⁺] 390.2024. (KBr)/cm^-1 3120, (v, O–H), 1664, (v, C=C), 3010 (v, =C-H), 1379, 1296 (v, C-O), 1712, 1730, 1755 (v, C=O) 3120, 1470 (v, O–H). - (300 MHz, CDCl₃) 5.08 (s,2H), 4.99(s,2H), 4.92 (d, J 3.0, 1H), 2.89 (s 1H), 2.37(m, 2H), 2.19-2.52(s,1H), 2.19 (s, 3H), 2.13-2.52 (m, 2H), 1.83 (m, J 2.7, 2H), 1.83 (d, J 2.7, 2H), 1.71 (m, 2H), 1.55 (s, 3H),1.51 (m, 2H), 1.48-2.19 (m, 2H), 1.22 (s, 3H) (Supporting File 9 for full experimental data). δ_c (300 MHz, CDCl₃) 41.9(C-1), 33.5(C-2), 32.7(C-3), 30.9(C-4), 48.6(C-5), 33.7(C-6), 33.0(C-7), 57.0(C-8), 85.5(C-9), 52.5(C-10), 36.0 (C-11), 213.2(C-12), 42.2(C-13), 73.0(C-14), 43.6(C-15), 152.0(C-16), 107.0(C-17), 21.9(C-18), 178.2(C-19), 15.5(C-20), 170.2(C-21), 21.2(C-22) (Supporting File 10 for full experimental data).

Supporting File 1 ¹H NMR Spectrum for Compound 1.

Supporting File 2 ¹³C NMR Spectrum for Compound 1.

Supporting File 3 ¹H NMR spectrum (300 MHz) for Compound 2.

Supporting File 4 ¹C NMR spectrum (300 MHz) for Compound 2.

Supporting File 5 ¹H NMR spectrum for Compound 3.

Supporting File 6 ¹C NMR spectrum (300 MHz) for Compound 3.

Supporting File 7 ¹H NMR spectrum (300 MHz) for Compound 4.

Supporting File 8 ¹³C NMR spectrum for Compound 4.

Supporting File 9 ¹H NMR spectrum (300 MHz) for Compound 5.

Supporting File 10 ¹³C NMR spectrum (300 MHz) for Compound 5.

Thin Layer Chromatography

Compounds 1-5 were developed on a TLC silica gel 60 F₂₅₄ Al-sheets using ethyl acetate in acetone as the mobile phase.¹⁷ After development, the chromatograms were subjected to direct TLC bioautographic assay.

Direct TLC bioautographic assay

The air dried chromatograms were sprayed in an esco class II biological safety cabinet (Esco Technologies) with the inoculum suspension of Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Escherichia coli (ATCC 25922) cells. The sprayed chromatograms were incubated at 37 ºC for 24h with 100% relative humidity in the dark. Solution of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (2mg/ml) were sprayed on the bioautograms¹⁸ and are re-incubated at 37 ºC for 3 h.¹⁹ Bacteria growth inhibition is confirmed by white bands on a purple background, indicating the reduction of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride to formazan. The zone of inhibition of compounds with antibacterial activity was measured.^19-21

Minimum Inhibitory Concentrations (MIC)

Minimum inhibitory concentrations of the compounds 1-5 were determined using 96-well microtitre plate against S. aureus, P. aeruginosa, E. faecalis and E. coli. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride was used as a growth indicator.²² In brief, 100 μL distilled water was dispensed into each well using a multichannel micropipette. After that, 100 μL of compound (10 mg/ml) was added to the first well of the column and serially diluted to obtain a working solution of concentrations 5.000 -0.078 mg/ml. Gentamycin and DMSO were used as positive and negative controls.

Lipoxygenase inhibition assay

Inhibitory activity of the compounds against 15-soybean lipoxygenase (15-LOX) was evaluated as described by Malterud & Rydland²³ in a borate buffer (0.2M, pH 9.00). Absorbance was read at 234 nm using UV-vis spectrophotometer for 5 min at an interval of 30 sec after addition of 15-LOX, using linoleic acid (134 μM) as substrate. The enzyme solution was kept on the ice, and controls (1.6% DMSO) were measured at intervals throughout the experiment to ensure that the activity was constant. All the reactions were performed in triplicates. The percentage of inhibition was calculated as:

$\text{\% Inhibition}=\frac{{\text{A}}_{\text{c}}-{\text{ A}}_{\text{tc}}}{{\text{A}}_{\text{c}}}\text{ X }100\text{\%}$

Where

A_c = absorbance of control

A_tc = absorbance of the test compound

Cytotoxicity assay

Dermal mesenchymal stem cells line (DMSC) and monkey Vero cells (ATCC® CCL-81™), harvested and centrifuged at 200 RPM for 5 min. The cells line were maintained in Minimal Essential Medium (MEM) to 1×10⁶ cells/ml/well, supplemented with gentamicin (0.1%) (Virbac) and foetal calf serum (5%).²⁴ A cell suspension (200 μl) was pipetted into well 2 to 11 of the sterile 96-well microtitre plate; the MEM was aspirated from the cells, and replaced with 200 μl of test compounds. At 37°C the cells were cultured at a humidity of 5% CO₂ incubator for 24 h. After the incubation period, MTT (20 µl, 5 mg/mL) was added to each well and incubated for another 4 h until purple precipitates were visible under a microscope. The concentration of MTT was measured immediately at 570 and 630 nm (reference) using a Plate reader. The blank was made up of the medium and MTT with no cells in wells in column 1. The concentration of the compounds resulting in a 50% reduction of absorbance compared to untreated cells is reported as the LC50.

Characterization of ent-13-hydroxy-16-kauren-19-oic acid (1)

¹³C-NMR reveals an exocyclic double bond resonances at δ 155.8 (C-16) and at 102.5 (C-17), a carbonyl resonance at 178.6 (C-19), an oxymethine carbon resonance at δ 73.4 (C-13) which is typical of a kaurenoic acid.^{15,16 1}H NMR showed the presence of two methyl groups with proton resonance at δ 1.20(s, 3H, H-20), and δ1.13(s, 3H, H-18) ppm. The DEPT-135 pulse sequence produces ten methene (CH₂) carbons with a total of 20 carbon atoms.The COSY spectrum indicates that proton δ 4.72 (s, H-17) coupled with the protons δ 1.64-1.66 (m, H-12).The two singlet protons δ 2.89 and 2.95 ppm (2H,s, H-14) also exhibited coupling with δ 1.64-1.66(2H,m, H-12) and also with δ 2.24 (d, J 2.7, H-15). The COSY spectrum also displays a correlation between δ 2.61 ppm (m, 2H, H-9) with δ 1.20 (s, 3H, H-20) and protons δ 2.24 (d, J 2.7, H-1). The position of the -OH group was as a result of the HMBC correlation of δ 1.31(m, 2H, H-11) and δ 1.92(m, 2H, H-12) with the quaternary carbon C-13. Compound 1, therefore, was classified as a 13-hydroxy-kaur-16-en-19-oic acid.

Characterization of 16-hydroxy-kaur-6-en-19-oic acid (2)

¹³C-NMR showed olefinic carbons resonances at δ 126.4 (C-6) and at 133.0 ppm (C-7), a carbonyl carbon resonance at 178.3ppm(C-19), an oxymethine carbon resonance at δ 82.5 ppm (C-16) which are the characteristics of a kaurenoic acid.¹⁵ DEPT 135 pulse sequence produces seven methene (CH₂) carbons with a total of 20 carbon atoms. ¹H NMR showed the presence of three methyl groups with proton resonances at δ 0.88 (s, 3H, H-20), δ1.25ppm (s, 3H, H-18), and δ1.93ppm (s, 3H, H-17). The proton on δ 5.55ppm, (dd, J 2.7, H-6), and δ 5.94ppm, (t, J 7.2, H-7), are deshielded by the π bond between the two carbons. The position of the OH group at C-16 was as a result of coupling displayed by the HMQC spectrum with the methyl protons on H-17. The double bond placement on C-6 is justified by the HMQC spectrum that revealed a ³J proton correlation between the H-7 proton δ 5.94 (t, J 7.2, 1H) with C 14 protons δ 2.16 ppm, (s, 2H) , hence the double bond is between C-6 (δ 126.5 ppm) and C 7 (δ 133.0 ppm). HMBC spectrum also showed coupling of proton H-18 (δ 0.88 ppm) ³J correlation with the tertiary carbon C-19 (δ 178.3 ppm). Similar the derivatives; 15α-hydroxy-ent-kaur-16-en-19oic acid and ent-12β-hydroxykaur-16-en-19-oic acid have been reported.^25,26 Compound 2 was therefore named as 16- hydroxy-kaur-6- en-19-oic-acid.

Characterization of 14- acetoxy ent- kaur-16-en-19-oic acid (3)

¹H NMR displayed the presence of three methyl groups with protons resonating at δ 1.23 (s, H-18) ppm and δ 0.94 (s, H-20) and the relatively deshielded δ 1.98 ppm (s, H-22) because it’s in the same chemical environment as the carbonyl group at C-21. ¹³C-NMR shows exocyclic alkene carbon resonances at δ 155.1 ppm (C-16) and 103.2 ppm (C-17), a carbonyl carbon resonance at 170.1 ppm (C-21), an oxymethine carbon resonance at δ 69.4 ppm (C-14). The DEPT-135 pulse sequence produces nine methene (CH₂) carbons with a total of 22 carbon atoms. There is a direct correlation between C-17 and the H-17 and also C-14 and H-14 on the HSQC spectrum. The COSY spectrum indicates that proton δ 4.73, s (C-17) is coupled with the protons δ 1.98 (s, 3H) (C-22). The position of the O-COCH₃ group at C-14 was as a result of the H-14³J coupling with the C-17 (δ 103.2). Also, the H-13 (δ 2.68) displaying a ¹J coupling with the C-17 (δ 103.2), as confirmed by the HMBC spectrum. In comparison Somova et al.²⁶ who isolated 11α-acetoxy ent- kaur-16-en-19-oic acid, an isomer of compound (3) which only differ by the position of the acetoxy group. Compound 3 was therefore classified as a 14-acetoxy ent-kaur-16-en-19-oic acid.

Characterization of 14-oxokaur-16-en-19-oic acid (4)

¹³C-NMR reveals olefin carbons resonating at δ 106.9 (C-17) and δ 152.0 (C-16), a carbonyl carbon resonance at δ 180.4 (C-19), a resonance typical of an oxymethine carbon at δ 84.9 (C-9). ¹H NMR displayed the presence of two methyl groups with proton resonance at δ 1.39 (s, H-20) and δ 1.20 (s, H-18) and exocyclic proton resonances δ 4.90 -5.0 (s, H-17). The DEPT-135 pulse sequence produces nine methene (CH₂) carbons with a total of 20 carbon atoms. The HSQC reveals that the carbon at 106.9(C-17) is directly attached to the two protons at H-17(5.0 and 4.90 ppm), which agrees with its DEPT 135 methylene peak classification. Also, the 41.8 (C-7) is in direct correlation with the methylene protons at H-7. The position of the keto group on C-14 was based on the COSY spectrum which established a correlation between H-17 protons coupling with H-12, H-13, and H-15 protons. The -OH group position on C-9 was determined based on its correlation with H-20 protons. A summary of all the ¹J, ²J, and ²J coupling is presented in Table 1. HMBC spectrum also displayed a correlation of H-18 (δ 1.12) proton, coupling with C-2, (δ 36.5), and C-3 (δ 36.7) placing the C-20 methyl group on quaternary C-10. Compound 4 was therefore classified as 14-oxokaur-16-en-19-oic acid, a functional group isomer of 15β-acetoxy-(-)-kaur-16-en-19-oic acid synthesised by acetylation of 15β-Hydroxy-(-)-kaur-16-en-19-oic acid.^27,28 Compound 4 is the first isolation of an oxo derivative of 15β-Hydroxy-(-)-kaur-16-en-19-oic acid from A. amatymbica.

Characterization of 14-acetoxo-12-oxokaur-16-en-19-oic acid (5)

¹³C-NMR showed an exocyclic double bond resonating at δ 152.0 ppm (C-16) and 107.0 ppm (C-17), three carbonyl resonances at δ 178.2 (C-19), δ 170.2 (C-12) and δ 213.2 (C-12), typical oxymethine carbons resonance at δ 85.5 (C-9) and δ 73.5 (C-19). ¹H NMR - (400 MHz, CDCl₃): showed the presence of three methyl groups with proton resonances at δ 2.19 (s, H-22), 1.22 (s, H-21) and 1.55 (s, H-18). These signals agree with the report of Somova et al.²⁶ & Langat et al.¹⁵ on kaurenoid isolation and characterisation. The DEPT-135 pulse sequence produces eight methene (CH₂) carbons with a total of 22 carbon atoms. The HSQC reveals that the carbon at 106.9(C-17) is directly attached to the to the two protons at H-17(5.0 and 4.90 ppm), which agrees with its DEPT 135 methylene peak classification. Also, the 41.8 (C-7) is in direct correlation with the methylene protons at H-7. The position of the keto group on C-12 was based on the COSY spectrum which established a correlation between H-17 protons coupling with H-13, and H-14 protons. The -OH group position on C-9 was determined based on the H-9 correlation with H-22 protons. A summary of all the ¹J, ²J, and ²J coupling is presented in Table 1. HMBC spectrum also displayed a correlation of H-21 (δ 2.19, s, 3H) proton, Coupling with C-20 (δ 36.5) placing the C-21 methyl group on quaternary C-20. The diterpenoid contains both the acetate group on C-14, carboxylic acid group at C-4 and a keto group on C-12. The name proposed for compound (5) is 14-acetoxo-12-oxokaur-16-en-19-oic acid.

TLC Antibacterial Bioautography Assay of pure compounds 1-5

Compounds 1-5 were screened on the selected organism to determine the susceptibility and MIC.^29,30 The sensitivity test by the direct TLC bioautographic assay showed clear zones of inhibition at a concentration of 10 mg/ml, with differing percentages of 19.26 % S. aureus, P. aeruginosa, E. faecalis, and E. coli. P. aeruginosa and E. faecalis were the most sensitive organism to all the compounds; this is in agreement with the antimicrobial report of kaurenoid acid and its epimer; xylopic.³¹ 14-acetoxo-12-oxokaur-16-en-19-oic acid (5) is the most potent against S. aureus, E, faecalis, and E.coli with a MIC 125 µg/ml while 14- acetoxy ent- kaur-16-en-19-oic acid (3) has a MIC of 125 µg/ml against P. aeruginosa (Figure 2). The structure-activity relationship of compound 3 and 5 explains the potency; the C-14 acetate enhances the antibiotic activity of the diterpenoids and the further substitution by the oxo group at position C-12. Liu et al.³² reported that acetylation increases the activity and ability of the molecule to cross the selectively permeable membrane. Also, acetylation helps a given drug reach the target site more quickly, making the effect of the medication's more intense and increasing the effectiveness of a given dose.³³

Figure 2 Antibiotic activities expressed as MIC (μg/ml) for compounds 1-5.

The lower MIC of compounds 3 and 5 establishes their possible application as a complementary antibiotic, and the presence of these bioactive diterpenoids confirms the antibacterial activity of A. amatymbica reported in the literature³⁴ and entho-medicinally.³⁵

Inhibition of Lipoxygenase Activity of Compound 1-5

Literature has shown that cell metabolism and apoptosis are regulated by signalling pathways of lipoxygenase.³⁶ Excessive release of lipoxygenase metabolites has been implicated in proliferative cancer cell, stroke, arthritis, asthma, cancer, inflammation, and many other diseases conditions.^37-42 Hence, lipoxygenase is an essential enzyme for designing selective and safe inhibitors suitable for medical application. The highest lipoxygenase inhibition at a concentration of 100μg/ml was displayed by 14-acetoxo-12-oxokaur-16-en-19-oic acid (5) followed by 14-acetoxy ent-kaur-16-en-19-oic acid (3) with 16-hydroxy-kaur-6-en-19-oic acid (2) demonstrating the least inhibitory activity when compared to the positive control of indomethacin (Figure 3).

Figure 3 % Soyabean derived 15-LOX- Inhibition by compounds 1-5.

Our results showed that only Compound 5 exhibited a significant inhibition (EC₅₀ of 19.10 ± 3.15 µg/ml), Compound 3 (EC₅₀ of 25.98 ± 1.12 µg/ml), and compound 4 (EC₅₀ of 42.76 ± 3.22 µg/ml) displayed moderate LOX activity. Compound 2 and 1 had an effective concentration of 81.18 ± 7.50 µg/ml and 60.37 ± 5.64 µg/ml respectively, consequently, exhibited moderate activity.

Previous reports on the petroleum ether and dichloromethane extracts of A. amatymbica root have been reported to demonstrate COX-1 and COX-2 inhibition.⁴³ This observed activity is attributed to the presence of 14- acetoxy ent- kaur-16-en-19-oic acid (3) and 14-acetoxo-12-oxokaur-16-en-19-oic acid (5) due to their significant EC₅₀. These findings should, therefore, be cautiously applied to the anti-inflammatory activity in humans because the mechanism of human-derived 15 LOX differs slightly from LOX, soybean lipoxygenase.

Cytotoxic Effects of the Compounds 1-5

Aqueous extracts of A. amatymbica root screened against cancerous cells did not show toxicity at all test concentration as reported in literature.⁴⁴ The root has found application as leads for the development therapeutic drug in clinical medicine, but the cytotoxicity of isolated compounds need to be established.⁴⁵ The cytotoxicity results showed potent cytotoxic effects on bovine dermis cells with compounds 1-5 (Figure 4) with an IC₅₀ less or equal to the stringent value noted for cancerous bovine dermis which is in agreement with NCI criteria.⁴⁶ The American National Cancer Institute (NCI) guidelines limit the activity of a pure compound at 50% inhibition (IC₅₀) of a proliferation of less than 30 µg/mL after the exposure time of 72 hours.⁴⁷

Figure 4 Cytotoxic effects of diterpenoids 1-5 against DMSC and Vero cell lines.

These data are also of interest as it suggests that the compounds are more toxic to cancer cells than on normal cells. The investigation provides evidence for the cytotoxicity of A. amatymbica towards bovine dermis which may be due to existing diterpenoids as reported in the previous preliminary study.^44,48

In this study, we isolated five diterpenic acids; 13-hydroxy-16-kauren-19-oic acid (1), 16-hydroxy-kaur-6-en-19-oic acid (2), 14-acetoxy ent-kaur-16-en-19-oic acid (3), 14-oxokaur-16-en-19-oic acid (4), and 14-acetoxo-12-oxokaur-16-en-19-oic acid (5). The bioautographic assay showed that all the diterpenoids were potential antimicrobial diterpenoid with P. aeruginosa and E. faecalis been very sensitive to all test diterpenoids. 14-acetoxo-12-oxokaur-16-en-19-oic acid is the most potent against S. aureus, E. faecalis, and E.coli at MIC 125 µg/ml, with a relatively high lipoxygenase inhibition activity when compared to standard indomethacin. These isolated diterpenoids did not show toxicity to normal but toxic to tumour cell lines. Thus, we conclude that A. amatymbica contain components such as diterpenic acids, which are responsible for contributing to anti-inflammatory effects and other biological activities. The acetylated diterpenic acids have potent biological activities and no toxicity of the diterpenoid from A. amatymbica root. This isolated diterpenoids can be synthesised and acetylated to confirm the in vivo activities.

We appreciate University of Pretoria Phytomedicine Programme, the Chemistry Department, University of Botswana, and the Vaal University of Technology, for providing the facilities for the study.

The author declares no conflict of interest.

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