Antimicrobial Triterpenoids and Ingol Diterpenes from Propolis of Semi-Arid Region of Morocco

The chemical composition and antimicrobial activity of propolis from a semi-arid region of Morocco were investigated. Fifteen compounds, including triterpenoids (1, 2, 7–12), macrocyclic diterpenes of ingol type (3–6) and aromatic derivatives (13–15), were isolated by various chromatographic methods. Their structures were elucidated by a combination of spectroscopic and chiroptical methods. Compounds 1 and 3 are new natural compounds, and 2, 4–6, and 9–11 are newly isolated from propolis. Moreover, the full nuclear magnetic resonance (NMR) assignments of three of the known compounds (2, 4 and 5) were reported for the first time. Most of the compounds tested, especially the diterpenes 3, 4, and 6, exhibited very good activity against different strains of bacteria and fungi. Compound 3 showed the strongest activity with minimum inhibitory concentrations (MICs) in the range of 4–64 µg/mL. The combination of isolated triterpenoids and ingol diterpenes was found to be characteristic for Euphorbia spp., and Euphorbia officinarum subsp. echinus could be suggested as a probable and new plant source of propolis.


Introduction
Plants and plant-derived products have a long history of use as therapeutic agents and sources of drug leads. Propolis (bee glue) is considered a plant-derived product since its main and biologically active ingredients are plant secretions. Bees collect resins and exudates from different parts of the plants, and after bringing them to the hive, they mix them with beeswax [1]. The resulting product called propolis is used by the bees as a protective barrier of the hive against pathogenic microorganisms and was recently shown as an essential element of the bee colony's social immunity [2]. Humans have recognized propolis as a healing substance since ancient times, and nowadays, it is still one of the most frequently used natural remedies [3,4]. Propolis has also found a place as an active ingredient in various cosmetic, food and pharmaceutical preparations [5][6][7], widely available on the market.
The broad application of propolis is due to its multiple biological activities, such as antimicrobial, antioxidant, antitumor, antiviral, anti-inflammatory and immunomodulatory [8,9], among others. Since propolis has been shown to possess both antiviral and anti-inflammatory activity, its perspective as a complementary treatment in patients with SARS-CoV-2 was also studied and underlined [10][11][12].
The beneficial effects of propolis are attributed to various plant metabolites as its chemistry depends strongly on the vegetation around the beehives [13]. In fact, the biodiversity

Structural Elucidation of Isolated Compounds
Detailed chemical analysis of Moroccan propolis sample collected in Sidi-Ifni province was performed. By using different chromatographic procedures, a total of 15 constituents ( Figure 1) were isolated: triterpenoids, macrocyclic diterpenes and aromatic derivatives. Their structures were elucidated by a combination of one-dimensional (1D) and twodimensional (2D) nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), optical rotation and comparison with the literature data.
Compound 2 was isolated as a light yellow solid and identified as 3α-hydroxy-tirucall-8,24-dien-21-al-26-oic acid (3-epi-isomasticadienolalic acid) by means of 1D and 2D NMR data ( Figure S3) and optical rotation. In fact, two identical to 2 structures, differing only in C-20 configuration (C-20 epimers), were previously isolated from Schinus molle L. [33,34]. Firstly, in 1978, Pozzo-Balbi et al. [33] characterized the 20S epimer (compound 2; tirucallane series) and named it 3-epi-isomasticadienolalic acid, based on selected 1 H NMR signals and reactions of reduction and tosylation. Later, Olafsson et al. [34] provided 13 C NMR data with many interchangeable assignments and claimed that the configuration at C-20 is R (euphol series). In terms of the limited 1 H NMR data and the fact that euphane and tirucallane triterpenes cannot be distinguished based on 13 C NMR data, because of very similar carbon resonances [30], we proceeded with detailed 1 H NMR assignments of 2 (Table 1), and subsequent interpretation of the data of a NOESY experiment. The latter was recognized as an essential approach for discrimination between tirucallanes and euphanes [30,31,[35][36][37]. NOE correlations between H-21 and H-16 were reported as Compound 2 was isolated as a light yellow solid and identified as 3α-hydroxy-tirucall-8,24-dien-21-al-26-oic acid (3-epi-isomasticadienolalic acid) by means of 1D and 2D NMR data ( Figure S3) and optical rotation. In fact, two identical to 2 structures, differing only in C-20 configuration (C-20 epimers), were previously isolated from Schinus molle L. [33,34]. Firstly, in 1978, Pozzo-Balbi et al. [33] characterized the 20S epimer (compound 2; tirucallane series) and named it 3-epi-isomasticadienolalic acid, based on selected 1 H NMR signals and reactions of reduction and tosylation. Later, Olafsson et al. [34] provided 13 C NMR data with many interchangeable assignments and claimed that the configuration at C-20 is R (euphol series). In terms of the limited 1 H NMR data and the fact that euphane and tirucallane triterpenes cannot be distinguished based on 13 C NMR data, because of very similar carbon resonances [30], we proceeded with detailed 1 H NMR assignments of 2 (Table 1), and subsequent interpretation of the data of a NOESY experiment. The latter was recognized as an essential approach for discrimination between tirucallanes and euphanes [30,31,[35][36][37]. NOE correlations between H-21 and H-16 were reported as characteristic for euphanes and between H-21 and H-12α for tirucallanes. For 2, NOESY correlations between protons of CHO-21/CH 2 -12α, CHO-21/CH-17, CH 3 -18/CH-20 and CH 3 -18/CH 2 -12α were observed ( Figure 2) along with correlations of CH 3 -30/H-17 and CH 3 -30/H12β that led to the determination of 2 as a tirucallane. Additionally, compound 2 showed [α] 20 D − 5.23 • (c 0.9, CHCl 3 ), and negative optical rotation is also associated with tirucallane triterpenes [30]. Similar optical rotation data for a number of tirucallanes, including related 3α-tirucallanes, were reported [30,35,37]. In this paper, we report for the first time the 13 C NMR and detailed 1 H NMR data of 2 (Table 1).
Compound 4 was isolated as a white amorphous powder. Its NMR data were partly similar to those of 3 (Tables 2 and 3; Figure S6). In the 1 H NMR spectrum of 4 in CDCl 3 (Table 2), the signals due to CH 2 -1 appeared at δ H 2.78 (1H, dd, J = 14.9 and 9.1 Hz) and 1.68 (1H, dd, J = 14.9 and 0.9 Hz), which together with J 2,3 = 8.5 Hz was an indication that 4 is a true ingol derivative [38]. The signals for protons of three acetyl groups were also observed, along with signals for a p-methoxyphenylacetyl group dd, J = 11.0 and 3.9 Hz) to acetyl carbonyls at δ C 170.2 and 170.4, respectively, were used to place two of the acetyl groups at H-8 and H-12. However, due to the small chemical shift differences (0.03 ppm) of H-3 (δ H 5.16, d, J = 8.5 Hz) and H-7 (δ H 5.13, d, J = 1.3 Hz), and of the rest two ester carbonyls (0.06 ppm), it was difficult to assign the position of the p-methoxyphenylacetyl group and/or of the third acetyl group unambiguously. Moreover, the NMR ( 1 H and 13 C) data of 4 are essentially the same as those of ingol diterpenes with C-3 or C-7 substituted phenylacetyl groups [42,43]. For this reason, the structure of 4 was further elucidated by NMR data recorded in acetone-d 6 , where well-shifted signals were observed (Table 2; Figure S7). In acetone-d 6 , H-3 and H-7 appeared at δ H 5.28 (d, J = 8.5 Hz) and δ H 5.06 (d, J = 1.6 Hz), respectively, and the 13 C NMR spectrum showed resonances of the four ester carbonyls at δ C 170.6, 170.8, 170.9 and 171.1. These data allowed clear assignment of the HMBC correlations, in particular for the position of the p-methoxyphenylacetyl group, which was placed at C-7 due to the HMBC cross-peaks of both H-7 and benzylic CH 2 to the same ester carbonyl at δ C 171.1 (C-1 ).   Figure 4). All these interactions were consistent with the configuration of ingol diterpenes [40]. Thus, 3 was determined as 2-epiingol-3,7,12-triacetate-8-isobutyrate ( Figures S4 and S5), a new natural compound.
Compound 4 was isolated as a white amorphous powder. Its NMR data were partly similar to those of 3 (Tables 2 and 3; Figure S6). In the 1 H NMR spectrum of 4 in CDCl3 (Table 2), the signals due to CH2-1 appeared at δH 2.78 (1H, dd, J = 14.9 and 9.1 Hz) and 1.68 (1H, dd, J = 14.9 and 0.9 Hz), which together with J2,3 = 8.5 Hz was an indication that 4 is a true ingol derivative [38]. The signals for protons of three acetyl groups were also observed, along with signals for a p-methoxyphenylacetyl group [δH 7.  The relative configuration of 4 was determined by a ROESY experiment and was identical to that of ingol derivatives [38,40,43].  Figure 4). Thus, the structure of compound 4 was determined as ingol-7-p-methoxyphenylacetyl-3,8,12-triacetate.
Compound 4 was previously isolated from Euphorbia resinifera [43,44], but only limited 1 H NMR data are provided. It was first found and only characterized by Hergenhanh et al. [44] in 1974 on the base of selected 1 H NMR signals and partial hydrolysis. In this paper, we report for the first time its 13 C NMR and detailed 1 H NMR data.
Compound 5 was isolated as a white amorphous powder. Its 1D and 2D NMR data are highly similar to those of 4 (Tables 2 and 3; Figure S8). The main difference was the absence of signals for aromatic methoxyl, and thus its 1D NMR spectra showed signals for the monosubstituted aromatic ring at δ H 7.26-7.32 and δ C 127. 8-135.4. Additionally, very small chemical shift differences were observed for H-3 (−0.02 ppm) and H-7 (+0.01 ppm) in CDCl 3 that resulted in their appearance as overlapping signals at δ H 5.14 (d, J = 8.5 Hz for H-3 and d, J = 1.5 Hz for H-7). In order to follow the HSQC and HMBC correlations of these protons, which also showed the direct H-C correlation to the same carbon resonances (δ C 76.9), the NMR spectra of 5 in acetone-d 6 were recorded ( Figure S9). In acetone-d 6 , the proton and carbon signals for both methine groups were shifted sufficiently and appeared at δ H 5.30/δ C 77.7 and δ H 5.08/δ C 77.8, respectively. Further, the HMBC correlations supported the position of two of the acetyl groups at C-8 (acetyl carbonyl at δ C 170.8) and C-12 (acetyl carbonyl at δ C 170.6). Unfortunately, it was difficult to assign the position of the benzyl and/or the third acetyl group to any of H-3 and H-7 due to extremely close carbon resonances (0.02 ppm) for the rest two ester carbonyls. For this reason, we resorted to the ROESY experiment, where identical to 4 correlations between protons of CH 2 -2 and both H-5 and H-7, as well as of H 3 -16/3-acetate and H 3 -20/12-acetate allowed assigning of the benzyl group at C-7. All remaining ROESY correlations were also consistent with those of 4. Thus, compound 5 was determined as ingol-7-phenylacetyl-3,8,12-triacetate.
Similar to 4, compound 5 was isolated by Hergenhanh et al. [44], and its structure has been elucidated on the base of selected 1 H NMR signals and partial hydrolysis. In this paper, we report for the first time its 13 C NMR and detailed 1 H NMR data.

Antimicrobial Activity
The total 70% ethanolic extract and selected isolated compounds were tested for antimicrobial activity against Staphylococcus aureus ATCC 29213, Methicillin-resistant Staphylococcus aureus (MRSA) 1337, Mycobacterium tuberculosis ATCC 27294, Escherichia coli ATCC 35218 (American Type Cell Culture Collection, Manassas, VA, USA), Pseudomonas aeruginosa ATCC 27853, and the fungus Candida albicans 562 by broth microdilution method. The results obtained are presented in Table 4. Table 4. Antibacterial and antifungal activities of the total 70% ethanolic extract and isolated compounds.

Discussion
The detailed chemical analysis of propolis from the semi-arid region of Morocco led to the characterization of 15 compounds, including new lanostane (1) and 2-epi-ingol (3) derivatives. Moreover, seven newly isolated from propolis compounds (2, 4-6, and 9-11) were also characterized as the full assignment of one-dimensional (1D) and twodimensional (2D) nuclear magnetic resonance (NMR) data for three of them (2, 4, and 5) was reported for the first time.
Further, suggestions for the botanical origin of the propolis sample analyzed were made based on the comparison between propolis chemistry and the literature data for the plants from which the compounds were previously isolated. The knowledge of the plant sources is, in fact, knowledge of the bees' choice and preference to certain appropriate resin sources [54], whose availability to the beehives is essential for the wellbeing of the bee colony, as well as for the high quality of propolis [2,55].
Furthermore, looking at the chemistry of studied Moroccan cactiforms, in the latex of the E. officinarum L. only triterpenoids and ingol diterpenes were found, until now [42,[64][65][66], while that of E. resinifera O. Berg is rich in triterpenes, and macrocyclic diterpenes with daphnane, tigliane, ingenane and lathyrane (including ingol derivatives) skeletons [43,56,67]. Based on the chemical comparison between propolis sample analyzed and the literature data of the latex of those species, E. officinarum rather than E. resinifera could be suggested as a botanical source of the propolis. This is also supported by the fact that E. resinifera O. Berg is endemic to the regions of Azilal and Beni Mellal (Middle Atlas) [68], whereas species belonging to the E. officinarum group E. officinarum L. and E. officinarum subsp. echinus (Hook.f. and Coss.) Vindt are distributed in the south-western regions of the country from the coast to Anti-Atlas Mountains [69,70]. The propolis sample analyzed was collected from an area of the Sidi-Ifni province (south-western Marocco), where the vegetation is of the infra-Mediterranean type, composed mainly of the succulent Euphorbia officinarum subsp. echinus (Hook.f. and Coss.) Vind, best known as Euphorbia echinus Hook.f. and Coss. [71] (basionym) [63,[72][73][74]. This species was also indicated as a source for propolis production by the local beekeepers. Unfortunately, only one article was published on the chemistry of the E. echinus latex, focusing on the triterpene composition, and lanostane derivatives were characterized [64].
Although at the current stage of research, we cannot provide unambiguous evidence for a particular cactiform, it is a first scientific insight for the Euphorbia spp. as a propolis source. It is also an example of a plant that is a source of materials for both honey [75,76] and propolis production. Only one document where Euphorbia spp. is mentioned as a source of propolis was found in the literature. In 2014, Faid [77] revealed the hepatoprotective effect of oil extract of the "Moroccan Euphorbia resinifera black propolis" in patients with chronic hepatitis C, but no chemical data or other evidence for the propolis sample tested and/or its botanical source was provided.
The 70% ethanolic extract and selected isolated compounds were tested in vitro for antimicrobial activity, which is a major driving bioactivity in propolis usage. Moreover, unless the triterpenes are known to possess antimicrobial activity, no data were published for the isolated diterpenes. The results (Table 4) showed that most of the compounds, especially the diterpenes 3, 4 and 6, exhibited very good activity against the different strains of bacteria and fungi, compared with the reference antibiotics gentamicin and amphotericin B. Compound 3 is the most active one against all tested bacteria and fungi, with minimum inhibitory concentration (MIC) values in the range of 4-64 µg/mL, and together with cycloartane triterpene 8 is markedly active against Staphylococcus aureus, but 2-3-fold less active against MRSA. On the whole, most of the compounds exhibited strong antimicrobial activity against Pseudomonas aeruginosa and Candida albicans as 3, 8, 9, and the mixture of 11 and 12 inhibited P. aeruginosa at lower concentrations (MICs 4 µg/mL). All tested compounds (except for 3; MIC 4 µg/mL) showed moderate activity against the Gram-negative bacteria Escherichia coli with MICs in the range 16-32 µg/mL and were non-efficient against Mycobacterium tuberculosis (MICs ≥ 64 µg/mL). Although the total extract displayed a weaker antibacterial effect against all tested bacteria in comparison to the tested compounds, similar to them appears as more active against the Gram-negative bacteria in comparison to the Gram-positive ones. This is an interesting and promising result considering that propolis from different regions is usually inactive against Gramnegative bacteria [78]. Moreover, taking into consideration that natural products (extracts) usually display MICs in the range 100-1000 µg/mL in the in vitro susceptibility tests, the Moroccan Euphorbia propolis can be considered as a promising antimicrobial agent [79] and as a good starting point for further in-depth research of its pharmacokinetics and other relevant properties.
The above-mentioned data are in good accordance with the fact that the latex of Euphorbia spp. is known to possess antimicrobial activities, along with other beneficial properties such as antiviral, anti-inflammatory, antiproliferative and cytotoxic activities [56]. This material is widely used in traditional medicine around the world [80,81] as E. officinarum and E. echinus, for example, are used for the treatment of ophthalmic and various skin diseases [59,63], and the dry latex of E. resinifera, called euphorbium, which is also commercially available in many countries, is used for the treatment of neurological problems, chronic pain, tuberculosis, etc. [82]. On the other hand, however, it should be mentioned that along with the beneficial properties, the Euphorbia latex is well known for its toxic and irritant effect on the skin and mucous membranes, and the requirements for usage of low quantities were underlined [83]. The adverse effects have been attributed to macrocyclic diterpenes in a great majority of tigliane (phorbol esters), daphnane and ingenane types [44,[83][84][85]. For the ingol diterpenes ingol-7-p-methoxyphenylacetyl-3,8,12-triacetate (4) and ingol-7-p-methoxyphenylacetyl-3,8,12-triacetate (5), in particular, Hergenhahn et al. [44,84] reported that they could be considered practically inactive as irritants, a conclusion based on studies on a mouse ear.

Propolis Sample
The propolis sample was collected by scraping from Apis mellifera bees' hives in Sidi-Ifni province (Guelmin-OuedNoun region) in Morocco in August 2018.

Extraction and Isolation
The raw propolis (40.0 g) was extracted with 70% ethanol (1:10, w/v) at room temperature (2 × 24 h). The extracts obtained were filtrated, combined and concentrated on a rotary evaporator. The total extract was suspended in water and subjected to liquid-liquid extraction successively with petroleum ether (PE, 3 times) and chloroform (CHCl 3 , 3 times) to provide 4.5 g and 3.6 g dry residue, respectively.
The PE extract (4.3 g) was then subjected to silica gel VLC, eluted with a gradient system of PE:EtOAc (1:0 to 0:1), to obtain twelve fractions A-L.

Minimal Inhibitory Concentration (MIC)
The in vitro antimicrobial activity of the total extract and selected isolated compounds was determined by the broth microdilution method according to ISO 20776-1:2006 [86]. Briefly, the bacterial inoculums with concentration 10 5 CFU/mL were added to 96-well plates containing MHB or BHIB loaded with two-fold serial dilutions of the tested samples. Plates were incubated at 37 • C for 18 h. According to EUCAST requirements, gentamicin for the test bacteria and amphotericin B for C. albicans were used. Experiments were performed in triplicate. In vitro antimycobacterial activity was assessed according to the EUCAST broth microdilution reference method for MIC determination [87]. Briefly, bacterial suspension was prepared at a concentration of about 2 × 10 6 cells/mL and further diluted 1:20 in Middlebrook 7H9 medium with 10% OADC (oleic acid-albumin-dextrosecatalase) (Becton Dickinson and Co., Sparks, MD, USA). Ninety-six-well microplates were used in which Middlebrook 7H9 medium was added dropwise with the appropriate concentration of test compounds range 0.125 µg/mL to 512 µg/mL and M. tuberculosis suspension. Ethambutol and isoniazid were used as controls. Reading was performed after 7, 14, and 21 days incubation at 37 • C using an inverted mirror. The MIC was the lowest concentration without visual growth and was expressed as µg/mL.

Conclusions
The detailed chemical analysis and antimicrobial evaluation of propolis from a semiarid region of Morocco were performed for the first time. The results revealed that it possesses specific chemical composition with triterpenoids and ingol diterpenes as characteristic and antimicrobial compounds. Euphorbia spp., most probably Euphorbia officinarum subsp. echinus, could be suggested as a plant source of the propolis. Further studies are needed in order to prove the particular botanical source, as well as to reveal the area of distribution of this specific propolis type. Special attention should be paid in respect to the possibilities for its application and safety use.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.