1. Introduction
Diarrhoea is an intestinal disorder characterised by the passage of frequent loose or watery stools in a period of 24 h [
1]. Infectious diarrhoea remains one of the leading causes of child mortality worldwide. In 2015 alone, diarrhoeal diseases caused an estimated 1.3 million deaths worldwide and were the fourth leading causes of death among children under the age of 5 years [
2]. Some of the prominent etiological agents of community and hospital-aquired diarrhoea include
Escherichia coli,
Enterococcus faecalis,
Klebsiella pneumonia and
Staphylococcus aureus [
3,
4]. Globally, medicinal plants are used to manage diarrhoeal symptoms [
5].
Acacia nilotica (seed powder),
Bacopa monnieri (leaf decoction),
Rheum palmatum (rhizome infusions),
Santalum album (rhizome infusion) are, for instance, listed in the Asian Pharmacopeia as traditional herbs with anti-diarrhoeal properties [
6,
7,
8]. In Europe, diarrhoea is managed traditionally using plants such as
Matricaria chamonilla (dry flowering decoctions),
Solanum tuberosum (tuber decoctions) and
Vaccinium myrtillus (fruit decoctions) [
9]. African herbs commonly used to treat the disease include
Elephantorrhiza elephantina (root decoctions),
Euphorbia hirta (leaf macerate),
Heinsia pulchella (root bark decoction),
Ozoroa insignis (bark decoctions),
Psidium guajava (leaf infusions),
Sclerocarya birrea (leaf and bark infusions),
Solanum supinum (root decoction),
Terminalia sericea (root infusions) and
Ximenia caffra (roof decoctions) [
10,
11,
12,
13]. In South Africa,
Protea caffra Meisn (Proteaceae) is among the common plants utilised by local herbalists for treating diarrhoea [
14].
Protea caffra is a dicotyledonous shrub (3 m) that grows in different parts of Mozambique, South Africa and Zimbabwe [
15,
16,
17]. The plant is native to South Africa where it is locally known as the common sugarbush, Natal sugarbush (English),
gewone suikerbos,
waboom (Afrikaans),
isadlunge,
indlunge,
isiqwanwe (Isixhosa),
uhlinkihane (isiZulu),
tshididiri, tshidzungu (TshiVhenda),
mahlako,
mogalagala,
segwapi,
sekila and
tshidzungu (Sotho) [
18,
19,
20]. As applicable with other members of the genus
Protea, it has characteristic large beautiful flower heads which have made it an important ornamental plant in southern Africa and other parts of the world [
17]. Apart from having important horticultural purposes,
Protea caffra has several ethnomedicinal applications. For instance, the aqueous infusions of the root and stem barkare used to manage bleeding stomach ulcers, diarrhoea or as enemas [
14,
20]. According to Zukulu [
21], the roots of the plant are used to prepare
umhlabelo, a decoction used to help heal broken bones. The fruit and stem bark are also used to manage dizziness, while decoctions of dried seeds are used to manage different psychological disorders [
22]. A study by Semenya et al. [
23], revealed that the Bapedi traditional healers of South Africa use
Protea caffra seed infusions to manage chlamydia, a sexually transmitted bacterial infection. However, the scientific basis for the majority of its traditional uses are currently lacking or limited. In addition, the phytochemical and toxicological evaluations of
Protea caffra remain pertinent to contribute toward its wider acceptance. The present study investigated the antibacterial (against drug-sensitive and -resistant strains), mutagenic and phytochemical properties of different parts of
Protea caffra.
2. Results
The antibacterial MIC values of evaluated plant extracts are presented in
Table 1. The extracts were classified as having significant (MIC ≤ 0.1 mg/mL), moderate (0.1 < MIC ≤ 0.625 mg/mL) or weak (MIC > 0.625 mg/mL) antibacterial properties [
24]. All aqueous extracts yielded MIC values >2.5 mg/mL (
Table S1). Generally, the evaluated plant parts demonstrated moderate antibacterial properties with MIC values ranging from 0.3–0.6 mg/mL. Interestingly, extracts from the seed (MeOH and petroleum ether) and twigs (MeOH) demonstrated noteworthy antibacterial activities against Gram-negative bacterial strains (
Escherichia coli and
Klebsiella pneumonia). It was also worth noting that the methanolic leaf and twig extracts of the plant demonstrated promising bacteriostatic properties (MIC = 0.63 mg/mL) against the penicillin-resistant
Staphylococcus aureus. However, the other drug-resistant bacterial strains were not susceptible to the plant extracts (MIC > 2.5 mg/mL) and hence were excluded from
Table 1.
The MeOH twig extract demonstrated extended-spectrum antibacterial properties (
Table 1), an observation that stimulated interest in determining its phytochemical profile. Acetone, cold ethyl acetate and hot ethyl acetate were used to partition the compounds in the biologically active extract and the resultant fractions were screened for antibacterial properties [
25]. As shown in
Table 2, the acetone and methanol sub-fractions demonstrated very weak antibacterial properties (MIC ≥ 2.5 mg/mL). The best antibacterial property was however, exhibited by the cold ethyl acetate sub-fraction which was active against both Gram-negative (
Escherichia coli, Klebsiella pneumoniae) and Gram-positive (
Enterococcus faecalis,
Staphylococcus aureus) bacterial strains (MIC range: 0.078–0.6 mg/mL). The hot ethyl acetate sub-fraction demonstrated moderate activities against
Enterococcus faecalis (0.3 mg/mL) and
Staphylococcus aureus (0.6 mg/mL). However, none of the fractions evaluated demonstrated noteworthy antibacterial activities against drug-resistant bacterial strains (
Table 2).
GC-MS data analysis revealed that the aqueous (70%) MeOH extract of
Protea caffra twigs consisted of 15 compounds (
Table 3,
Figures S1–S3). No peaks were obtained from the methanol sub-fraction. The major phytocompounds in the cold ethyl acetate sub-fraction were polygalitol (34.76%), phenol, 4-(1,1,3,3-tetramethylbutyl) (9.8%), Spiro-1-(cyclohex-2-ene)-2’-(5’-oxabicyclol) (8.2%), 1-adamantane carboxylic acid (8.07%) and carbamic acid (7.03%), which together accounted for approximately 60% of the compounds found in the sub-fraction (
Table 3). The hot ethyl acetate sub-fraction consisted of 1-heptacosanol (70.57%) as its major component (
Table 3). Oxalyl chloride (51.12%) and polygalitol (48.88%) were the only compounds detected in the acetone sub-fraction.
The UHPLC-MS/MS analysis revealed that
Protea caffra contained varying quantities of both hydroxybenzoic and hydroxycinnamic acids (
Table 4 and
Table 5). Overall, the most abundant hydroxybenzoic (
p-hydroxybenzoic acid, 374.55 µg/g DW) and hydroxycinnamic (caffeic acid, 266.37 µg/g DW) acids were detected in the leaves. The leaves also contained the least abundant hydroxycinnamic acids (sinapic acid, 0.08 µg/g DW), while the bark contained the least abundant hydroxybenzoic acid (salicylic acid, 0.1 µg/g DW).
None of the evaluated plant extracts demonstrated concentration-dependent increase in the number of His
+ revertants (
Table 6). The average TA98 revertants ranged from 6.4–29.0, while the TA102 revertants ranged from 109.7–284.3. The corresponding average number of TA98 and TA102 revertants in the negative control were 19.1 and 145.2, respectively. The evaluated extracts were therefore non-mutagenic against TA98 and TA102 tester strains [
26].
3. Discussion
Given that water is one of the most commonly used solvent in folklore medicine [
27], it was included in the present study to mimic traditionally prepared herbal medicines. However, water extracts have been widely reported to exhibit poor antibacterial activity. This is attributed to the fact that many antibacterial phyto-compounds are non-polar or have intermediate polarity and as such cannot be readily extracted from plant material using water [
28,
29]. In the current study, it is possible that the antibacterial compounds in
Protea caffra water extracts occurred in very low, sub-lethal concentrations resulting in poor antibacterial activity (
Table S1). It should, however, be noted that some phyto-compounds indirectly help patients fend off pathogenic infections by acting as immune boosters [
30]. Furthermore, some of the bioactive compounds in water extracts may exist as pro-drugs, which only become bactericidal once they are modified in the human body [
27]. The presence of immune stimulators and pro-drugs in plant extracts cannot be detected using the techniques employed in the current study, as such, further studies are warranted to determine the chemical profiles of traditionally prepared herbal medicines.
The current study revealed for the first time that
Protea caffra has potent and extended-spectrum antibacterial properties, which could be attributed to a wide range of biologically active compounds present in the plant. The twigs, for instance, contained 8 putative antibacterial compounds (caffeic,
p-coumaric, gallic, ferulic chlorogenic acids, adamantyl heterocycle, heptacosanol and nonadecanol,
Table 3,
Table 4 and
Table 5) and they demonstrated moderate antibacterial properties against the majority of the evaluated bacterial strains (
Table 1). In particular, they also exhibited significant antibacterial activities against
Enterococcus faecalis (MIC = 0.078 mg/mL,
Table 2). The antibacterial properties observed in the present study were generally comparable to those previously reported for some South African medicinal plants such as
Newtonia hildebrandtii,
Newtonia buchanannii,
Ozoroa insignis,
Syzgium cordatum,
Terminalia. sericea, and
Trichilia emetica (MIC range: 0.1–0.6 mg/mL) [
4,
12,
13,
31].
Relatively higher concentrations of caffeic and
p-coumaric acids (>10 µg/g DW) in the seed (
Table 5) could have contributed to the inhibitory effects MeOH seed extracts had on the growth of
Escherichia coli,
Klebsiella pneumoniae and
Staphylococcus aureus (
Table 1). The current observations could perhaps explain why some herbalists in South Africa use
Protea caffra seeds to manage bacterial infections including chlamydia [
32]. The presence of different types of antibacterial compounds in the leaves and twigs could also perhaps justify the observed moderate antibacterial properties these organs had against penicillin-resistant
Staphylococcus aureus (
Table 1).
The antibacterial compounds present in
Protea caffra worked in different synergistic combinations or individually to affect the observed bacteriostatic properties. Different groups of phenolic compounds often exhibit unique antibacterial mechanisms. Gallic and ferulic acids for example, exert their bactericidal effects by binding to and rupturing bacterial cell membranes [
33]. Chlorogenic acid on the other hand, binds to bacterial membranes and increases their permeability. This in turn causes the leakage of both cytoplasmic and nuclei material, eventually leading to bacterial cell death [
34].
P-coumaric acid also causes intracellular material leakages and interferes with bacterial DNA replication and gene expression [
35].
Apart from phenolic acids,
Protea caffra contained additional antibacterial compounds as revealed by GC-MS analysis. For instance, 1-Adamantyl heterocycle, which was detected in the twigs of
Protea caffra, is usually incorporated into anti-infectious molecules to improve their efficacy [
36]. Several potent antimicrobial and antiviral agents such as Rimantadine [
37], Oxadiazole [
38], Isoxazole [
39] and Thiadiazole [
40] are all 1-adamantanyl derivatives. It was of great interest to note that
Protea caffra produces levoglucosan (
Table 3), an important source of C1-C10 and C1-C13 carbon skeletons used to produce the antibiotics erythromycin A and B, [
41,
42]. 1-Heptacosanol, another compound detected in
Protea caffra, is a fatty alcohol present in plants [
43], marine algae [
44] and cuttlefish,
Sepiella inermis [
45]. The compound has potent antimicrobial properties [
46]. The presence of 1-heptacosanol in
Protea caffra suggests that the plant might have potent antioxidant [
44,
47], nematocidal [
48] and antidiabetic [
49] properties. Unlike 1-nonadecanol which is a known antibacterial phyto-compound [
50], polygalitol has not yet been demonstrated to have antibacterial properties, but has been detected in several plant extracts with potent antibacterial activities [
51]. Polygalitol was the most abundant compound in the cold ethyl acetate fraction which demonstrated broad-spectrum antibacterial activities in the present study (
Table 2). Further studies are warranted to determine the compound’s phytochemical properties. Given that 1-heptacosanol, an antibacterial compound, was the major phyto-chemical constituent in the hot ethyl acetate fraction, it is logical to suggest that it was probably the one that inhibited the growth of both
Enterococcus faecalis and
Staphylococcus aureus (
Table 2). Polygalitol and other phyto-compounds detected in this sub-fraction could also have contributed to the observed antibacterial activities. Oxalyl chloride detected in the acetone sub-fraction is a synthetic compound used in oxidative processes involved in manufacturing antibiotics, pesticides, herbicides and other organic products [
52,
53]. There are no indications in the current literature suggesting that the compound is produced by plants and/or that it is biologically active. Oxalyl chloride was therefore probably incorporated into
Protea caffra tissues from an external source. Further investigation would reveal which of the two compounds present in the sub-fraction (acetone) was responsible for the weak antibacterial activity observed against
S.
aureus (MIC ≥ 1.25 mg/mL). The principle antibacterial compounds in the
Protea caffra should, however, be unequivocally identified and their respective antibacterial mechanisms elucidated.
Given that some plants are inherently toxic [
54], the safety of traditional herbal remedies remains a serious cause of concern. It was encouraging to note that all evaluated plant extracts were non-mutagenic against both
Salmonella typhimurium tester strains (TA98 and TA102,
Table 6). Based on accessed literature, none of the plant species within the genus Protea have been reported to have potential toxic effects on humans. It is, however, important to note that while some medicinal plants exhibit non-mutagenic effects in vitro, they may possess cytotoxic effects [
55]. It should also be kept in mind that besides mutagenesis, carcinogens can also induce cancerous growth in animals through altering intracellular signals and gene expressions both of which are not detected by the Ames test [
56]. It is therefore imperative that further toxicological studies be conducted to ascertain the plant’s safety.