Rubiaceae is the largest family in the Magnoliopsida class, encompassing around 550 genera and 9,000 species being used in several ethnomedicinal practices [
The
Two collections were performed, the first one, at the “A. Ducke” Forest Reserve, 26 km from Manaus, was carried out on December 5th, 2008, and a voucher specimen (222383) was deposited at the Herbarium of the Botanical Research Coordination of the National Research Institute of Amazonia (INPA). The second one at the Natural Heritage Private Reserve, locally known as “Cachoeira da Onça,” in “Presidente Figueiredo” County, AM, was carried out on May 18th, 2011. A voucher specimen (222501) was deposited at the same Herbarium.
Plant material (leaves and branches) was dried in an oven at 50°C and powdered. Each plant part was extracted three times separately, first with dichloromethane (DCM) followed by methanol (MeOH), in a sonic bath for 20 minutes. After filtration, DCM and MeOH extracts were concentrated under reduced pressure.
The extracts were analyzed following the methodology described by Matos [
Dichloromethane extract from leaves (1st collection) (9 g) was submitted to a chromatographic column (CC) fractionation on silica gel (332 g), eluted with gradients of hexane/ethyl acetate and ethyl acetate/methanol, yielding 99 fractions with 50 mL each. Fraction 25–40 (900 mg) was fractionated on silica gel (90 g) CC and eluted with hexane/ethyl acetate and ethyl acetate/methanol gradients, yielding 42 fractions with 20 mL each. Fraction 25–40.6 (130 mg) was fractionated on silica gel (17 g) CC and eluted with hexane/ethyl acetate and ethyl acetate/methanol gradients, yielding 19 fractions with 10 mL each. Afterwards, fraction 25–40.6.4 (4 mg) was submitted to high-performance liquid chromatography (HPLC) analysis. HPLC was performed with a Shimadzu system SCL-10AVP, processing software programs CLASS VP, dual LC-6AD pumps, 10AF autosampler, SPD-M20 diode-array detector, cyanopropyl column (
Schematic representation of
All fractions were evaluated by TLC analysis, eluted with appropriated systems, and revealed under UV light exposure (
The NMR data was obtained at 295 K on a Bruker AVANCE 400 NMR spectrometer operating at 9.4 Tesla, observing 1H and 13C at 400 and 100 MHz, respectively. The spectrometer was equipped with a 5 mm multinuclear direct detection probe, with z-gradient. One-bond (HSQC) and long-range (HMBC) 1H-13C NMR correlation experiments were optimized for coupling constants
Resazurin microtiter assay (REMA) was used to evaluate the antimycobacterial activity. This method uses resazurin as an oxidoreduction indicator to evaluate the bacterial viability and contamination, in addition to analyzing the antimicrobial activity [
The extracts activity was evaluated against three
Samples were first evaluated in 96-well microplates at a 200
The extracts presenting an antimycobacterial activity at the 200
Following the incubation period, 30
All
Following crude extracts chemical and biological analysis, the dichloromethane extract from leaves (1st collection) was chosen to be fractionated, since it showed to be the most active against the three
Fraction 25–40.6 1H-NMR data showed the presence of several signals in the shielded region between
HPLC fractionation of this mixture was performed in order to isolate them, and yielded two fractions,
The substance
1H and 13C NMR chemical shifts (
Position | Oleanolic acid | Ursolic acid | ||||
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|
|
|
|
|
1 | 38.5 | 1.63 (m) | 39.0 | 38.6 | 1.72 (m) | 39.2 |
2 | 28.1 | 1.60 (m) | 28.1 | 28.2 | 1.60 (m) | 28.2 |
3 | 79.1 | 3.23 (dd; |
78.2 | 78.7 | 3.22 (dd; |
78.2 |
4 | 38.8 | — | 39.4 | 38.5 | — | 39.6 |
5 | 55.3 | 0.74 (m) | 55.9 | 55.2 | 1.34 (m) | 55.9 |
6 | 18.8 | 1.54 (m) | 18.8 | 18.3 | 1.60 (m) | 18.8 |
7 | 32.7 | 1.49 (m) | 33.4 | 32.9 | 1.72 (m) | 33.7 |
8 | 39.3 | — | 39.8 | 39.5 | — | 40.1 |
9 | 47.6 | 1.54 (m) | 48.2 | 47.3 | 1.60 (m) | 48.1 |
10 | 37.0 | — | 37.4 | 37.0 | — | 37.5 |
11 | 23.8 | 0.94 (m) | 23.8 | 23.7 | 1.91 (m) | 23.7 |
12 | 122.8 | 5.31 (dd; |
122.6 | 125.9 | 5.27 (dd; |
125.7 |
13 | 143.5 | — | 144.8 | 137.9 | — | 139.3 |
14 | 41.5 | — | 42.2 | 42.0 | — | 42.6 |
15 | 27.7 | 1.60 (m) | 28.4 | 28.1 | 1.60 (m) | 28.8 |
16 | 23.7 | 0.94 (m) | 23.8 | 25.0 | 1.34 (m) | 25.0 |
17 | 46.7 | — | 46.7 | 48.1 | — | 48.1 |
18 | 42.1 | 2.82 (m) | 42.1 | 53.8 | 2.2 (m) | 53.6 |
19 | 46.0 | 2.87 (m) | 46.6 | 38.5 | 1.00 (m) | 39.5 |
20 | 31.0 | — | 31.0 | 38.5 | 0.95 (m) | 39.4 |
21 | 33.9 | 1.62 (m) | 34.3 | 30.3 | 1.27 (m) | 31.1 |
22 | 33.2 | 1.30 (m) | 33.2 | 37.4 | 1.72 (m) | 37.4 |
23 | 28.0 | 1.00 (s) | 28.8 | 28.9 | 1.00 (s) | 28.8 |
24 | 16.8 | 0.79 (s) | 16.5 | 15.6 | 0.79 (s) | 16.5 |
25 | 15.3 | 0.93 (s) | 15.6 | 15.4 | 0.94 (s) | 15.7 |
26 | 17.1 | 0.79 (s) | 17.5 | 17.1 | 0.82 (s) | 17.5 |
27 | 26.0 | 1.16 (s) | 26.2 | 23.5 | 1.10 (s) | 24.0 |
28 | 180.0 | — | 180.0 | 179.6 | — | 179.7 |
29 | 33.1 | 0.92 (s) | 33.4 | 17.0 | 0.87 (d; |
17.5 |
30 | 23.7 | 0.94 (s) | 23.8 | 21.4 | 0.97 (d; |
21.4 |
Minimum inhibitory concentration (MIC) of
|
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Extracts | H37Rv | INHr | RMPr | |||
( |
( |
( |
||||
1st Collection | ||||||
Leaves DCM | S | 6.25 | S | 25 | S | ≤ 6.25 |
Leaves MeOH | R | >200 | R | >200 | S | 200 |
Branches DCM | S | 100 | S | 100 | S | 100 |
Branches MeOH | R | >200 | R | >200 | R | >200 |
| ||||||
2nd Collection | ||||||
Leaves DCM | S | 200 | S | 50 | R | >200 |
Leaves MeOH | S | 100 | S | 12.5 | S | 100 |
Branches DCM | S | 25 | S | 50 | R | >200 |
Branches MeOH | S | 100 | S | 100 | S | 100 |
DCM: dichloromethane, MeOH: methanol, R: resistant, S: sensible, H37Rv: sensible strain, INHr: isoniazid resistant strain, RMPr: rifampicin resistant strain. Extract with MIC > 200
The 1H-NMR spectrum from fraction 25–40.6.4.2 showed several signals at the shielded region, between
When analyzing the 13C-NMR spectral data one can find seven methyl carbons (CH3), nine methylene carbons (CH2), seven methine carbons (CH) and seven non-hydrogenated carbons (C), resulting in thirty carbons characteristic of pentacyclic triterpenes.
On the other hand, the 1H-13C NMR (HSQC) correlation map showed correlation of the hydrogen at 5.27 ppm with the carbon at 125.9, which were identified as the vinilic C-12 carbon and the multiplicity of the signals corresponding to H-18 and related CH3-29 and CH3-30 determined the ursolic acid.
In the two-dimensional 1H-1H NMR (COSY) correlation map, the following correlations are observed: hydrogen H-11 (
It is common to isolate the ursolic acid with oleanolic acid mixture due to molecule similarity, yet a few differences between them enable telling them apart through NMR, due to the difference between the H-18, C-18, C-12, C-13 and C-29 [
1H-NMR spectra and HSQC and HMBC NMR correlation maps overall analysis as well as comparison with literature data [
Structures of oleanolic and ursolic acids and their 1H-13C long-range correlations.
The mass spectra analysis of each triterpene isolated showed the molecular ion peak at
All extracts showed activity against
The wide variety of natural products chemical structures plays a major role on the development of new antimycobacterial drugs generations, as shown in the extensive literature revision made by Copp [
The highest activity of the dichloromethane extract from leaves (1st collection) in this work could be attributed to the presence of terpenes. Several studies, such as those performed by Newton et al. [
Extracts and compounds from other Rubiaceae species, such as
Out of the 27 assayed fractions present in this work, only fraction 63-65 was as active against
Minimum inhibitory concentration (MIC) of dichloromethane fractions from the leaves of
Fraction |
|
|||||
---|---|---|---|---|---|---|
H37Rv ( |
INHr ( |
RMPr ( |
||||
Fr 1–4 | R | >200 | R | >200 | R | >200 |
Fr 1–4.17–20 | R | >200 | R | >200 | R | >200 |
Fr 5 | R | >200 | R | >200 | R | >200 |
Fr 6–12 | S | 50 | S | 100 | S | 100 |
Fr 6–12.30 | R | >200 | R | >200 | R | >200 |
Fr 6–12.33–35 | R | >200 | R | >200 | R | >200 |
Fr 6–12.38–63 | R | >200 | R | >200 | R | >200 |
Fr14–16 | S | 100 | S | 50 | S | 100 |
Fr 17–21 | R | >200 | R | >200 | R | >200 |
Fr 17.21.1–5 | R | >200 | R | >200 | R | >200 |
Fr 25–40 | S | 200 | S | 200 | S | 200 |
Fr 25–40.2 | R | >200 | S | 200 | S | 200 |
Fr 25–40.6 | S | 200 | S | 200 | S | 200 |
Fr 25–40.6.32 | S | 100 | S | 100 | S | 100 |
Fr 41–44 | S | 100 | S | 50 | S | 100 |
Fr 46–56 | S | 200 | S | 200 | S | 100 |
Fr 46–56.5 | S | 200 | S | 200 | S | 200 |
Fr 46–56.8–10 | S | 50 | S | 50 | S | 50 |
Fr 46–56.13–17 | R | >200 | R | >200 | R | >200 |
Fr 57 | S | 100 | S | 200 | S | 200 |
Fr 57.6–12 | S | 100 | R | >200 | R | >200 |
Fr 63–65 | S | 100 | S | 25 | S | 100 |
Fr 66–68 | S | 200 | S | 100 | S | 200 |
Fr 70–74 | R | >200 | S | 200 | R | >200 |
Fr 76–86 | R | >200 | S | 200 | S | 200 |
Fr 87–92 | S | 200 | S | 50 | S | 100 |
Fr 94–99 | S | 200 | S | 200 | S | 100 |
Fr: fraction, R: resistant, S: sensible, H37Rv: sensible strain, INHr: isoniazid resistant strain, RMPr: rifampicin resistant strain. Fractions with MIC > 200
Studies conducted by Higuchi et al. [
Ge et al. [
The high lipophilicity of terpenes is probably the main factor that allows their penetration through the mycobacterial cell wall [
Other studies showed these substances inhibited 99% the growth of
According to Pauli et al. [
The authors are grateful to CT-Agro/CNPq/MCTI (520281/2007-1 and 562892/2010-9), PPBio/CNPq/MCTI (558321/2009-7), and CENBAM/CNPq/MCTI for the financial support.