Antimicrobial, antioxidant activities, and total phenolic contents of Pycnanthus angolensis Sap and Cryptolepis sanguinolenta root extracts

Humans and animals have coexisted with plants since the beginning of time, using them for food and medicine for thousands of years [1]. According to the World Health Organization [2], the use of traditional medicine in treating diseases is based on health practices, knowledge, and beliefs in incorporating plants, animals, and mineral-based spiritual therapies that are applied solely in the prevention and treatment of diseases and illnesses [2].

Despite the significant advances observed in modern medicine, herbal medications have been used to relieve the symptoms of various diseases [3]. Over the years, the efficacy of synthetic antibiotics in treating microbial infections has been limited owing to the ability of microbes to mutate, adapting to harsh conditions and developing resistance towards antibiotics [3]. The interest in medicinal plants in treating diseases is due to their prolonged use by our forefathers in treating themselves, a term known as “traditional” or “indigenous” medicines [2, 4].

Pycnanthus angolensis (P. angolensis) is a nutmeg tree species in the Myristicaceae family. In Africa, it is widely known as “Ilomba” [5, 6] while Twi and Fante dialects in Ghana call it “Otie”. Among the numerous purposes it serves are; the dried fruits of P. angolensis used as spices for soups [4]. The seed yields a reddish-yellow brown fat known as Kombo butter or Angola tallow, used for illumination and soap making [7]. The bark is used in treating skin infections, as a purgative, in cleansing the milk of lactating mothers, and in treating chest and cough pains. In Ghana, the bark is used to treat anaemia while in Côte D’Ivoire, it is used as an antidote to ascites and leprosy [5, 6, 8]. In Congo DR, the bark is used to solve infertility problems and also to treat gonorrhoea and malaria. Antimicrobial and anthelminthic properties of the leaves, stem and roots have also been reported [6]. The leaves also possess antioxidant and anti-inflammatory activities [9] and this has necessitated the need to discover the antimicrobial and antioxidant activities of the crude sap extract of P. angolensis [5].

Cryptolepis sanguinolenta (C. sanguinolenta) is the most familiar plant of the species of trees belonging to the family Apocynaceae [10, 11]. It is commonly called “Nibima” in Ghana [11] and serves numerous medicinal purposes, including herbalists’ use in treating fever, urinary tract, and upper respiratory tract infections [12, 13]. The most well-known use of C. sanguinolenta roots is in the treatment of malaria [14, 15]. It is also historically used to treat insomnia, although the mechanism underlying this has yet to be fully understood [16]. Despite the prevalent use of P. angolensis and C. sanguinolenta since time immemorial, much knowledge has yet to be found in the literature evaluating their antimicrobial and antioxidative properties [6, 10, 17, 18]. The current study, therefore, seeks to evaluate in vitro the antimicrobial, phytoconstituents, total phenols, and antioxidant activity of the crude sap of P. angolensis and the ethanol and aqueous extracts of roots of C. sanguinolenta.

Antimicrobial Resistance (AMR) is a global health issue that causes significant mortality and morbidity. Multiple drug resistance in Gram-positive and Gram-negative bacteria has made treating common illnesses with standard medicines challenging [24]. The rapid proliferation of multiple drug resistance bacteria, along with a lack of effective medications and suitable preventative measures, has prompted the development of innovative treatment alternatives and alternative antimicrobial treatments that are both less expensive and more effective [24].

Bioactive plant constituents have been used in the treatment of both Gram-positive and Gram-negative bacterial infections [25] for centuries. Several studies in the West African sub-region have reported that C. sanguinolenta is an anti-malarial plant [10, 11] and there have been other reports on its antimicrobial properties by researchers such as Boakye-Yiadom, Mills-Robertson et al., and Paulo et al. [10‐12, 26].

The current study found that C. sanguinolenta was susceptible to S. aureus (Gram-positive bacteria) but resistant to Gram-negative organisms such as P. aeuroginosa, S. saprophyticus, P. mirabilis, and S. typhi (Tables 2 and 3). This finding was consistent with a study by Boakye-Yiadom [26] who revealed that less than 50 mg/mL of aqueous extract generates mild antibacterial activity, a finding similar to a study of Paulo et al. [12, 26]. Another study by Ramli et al., [27] studied Ambrosia maritima and Bituminaria bituminosa plants (plant extracts) from Algeria and found that the butanolic extract of Bituminaria bituminosa exhibited remarkable antimicrobial activity against Staphylococcus aureus and Candida albicans, highlighting their potential as sources of antimicrobial agents [27]. This observation could be due to the action of cryptolepine (a bioactive ingredient in C. sanguinolenta) on the bacterial cell wall of both Gram-positive and Gram-negative organisms. Gram-negative bacteria have an outer cell membrane, a lipopolysaccharide with low permeability [28]. Some Gram-negative microorganisms also express resistance to inducible cephalosporins or antibiotic efflux pumps that give them high intrinsic resistance to antibiotics; hence, such could be why the extracts of the C. sanguinolenta did not work successfully on the selected Gram-negative organisms [28]. These current findings contradict the conclusions from Mills-Robertson et al. [11], in which the plant extract worked against both Gram-positive and Gram-negative microorganisms used in their studies.

Pycnanthus angolensis showed high potency against both selected Gram-negative and Gram-positive organisms except P. mirabilis and the potency was concentration and dose dependent. This current study finding is consistent with a similar study by Chukwudozie and Ezeonu [18] who reported that the stem bark of P. angolensis showed higher inhibition when tested against Gram-positive and negative bacteria. This research complements study by Spengler et al. [29], on the essential oil of Juniperus oxycedrus L. ssp. macrocarpa, which showed high antibacterial activity against Salmonella spp and Gram-positive bacteria, indicating potential for combating antibiotic and antifungal resistance [29]. In those studies, the ethanol extracts of the plant extracts were more susceptible than the aqueous extracts when used against the selected microorganisms [18]. The resistance of P. mirabilis could be due to dose dependency and, therefore, will require a higher dose of the plant extract to be susceptible [24]. The current study shows that the phytochemicals in P. angolensis are potent against both Gram-negative and Gram-positive organisms in a dose-dependent pattern. It will be ideal to investigate this further in developing novel antimicrobial agents to tackle the growing threat of AMR [24].

Results obtained from P. angolensis has confirmed the early claims by Omobuwajo et al. [9] and Sofidiya and Awolesi [5] (Table 1), that P. angolensis is a remedy for chest pains and skin diseases such as boils, furuncles caused by S. aureus, wound healing, and gastrointestinal ailment which are usually caused by some of these microorganisms [5, 9]. Agyare et al. and Onocho and Otula [8, 14] also claimed that P. angolensis is a medicinal source for the management of food poisoning, bloody diarrhoea, and urinary tract infections caused by S. typhi and S. saprophyticus. In this current study, we did not evaluate the potential interactions between the drug compounds under investigation specifically, their synergistic, antagonistic, or additive effects. It is important to note that such assessments would require further experimentation to determine the extent to which the drugs interact with one another, and whether their combined effects result in a greater or lesser therapeutic outcome than anticipated based on their individual efficacy. Therefore, while our current findings are informative, they do not provide insights into the potential synergistic, antagonistic, or additive effects of the drugs, and further studies will be required to elucidate these potential interactions.

Phenols have been reported to have antiseptic, anti-inflammatory, antimicrobial, and anti-tumour properties, and tannins have also been reported to have anti-ageing properties as well as skin regeneration, anti-inflammatory and diuretic properties [30]. According to Agyare et al. [14], flavonoids have splendid antimicrobial and anticancer activities, while alkaloids are used as painkiller medications [31]. Phenolic compounds are known to possess potent antioxidant properties and are believed to be the primary contributors to the antioxidant activity of plant extracts [30]. Therefore, it is generally expected that plant extracts with higher total phenolic content will exhibit greater antioxidant activity [30]. Similarly, certain phytochemicals, such as flavonoids, alkaloids, and terpenoids, have been shown to possess antibacterial properties. Therefore, plant extracts that contain high levels of these phytochemicals are more likely to exhibit antibacterial activity [30].

Several studies have investigated the correlation between total phenolic content, phytochemical composition, and antioxidant or antibacterial activity of plant extracts. These studies have generally found a positive correlation between these variables, suggesting that plant extracts with high levels of phenolic compounds and specific phytochemicals tend to exhibit higher antioxidant and antibacterial activity. Krasteva et al. [32] conducted a study to evaluate the phenolic content, composition, antioxidant and antibacterial activities of four grape seed extracts (Cabernet Sauvignon, Marselan, Pinot Noir, and Tamyanka). The extracts exhibited high total phenolic content, with Pinot Noir having the highest antimicrobial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli [32, 33]. The extracts’ components were determined using HPLC, and high contents of catechin, epicatechin, and procyanidin B1 were found. The extracts showed high sensitivity to the tested bacteria, and a correlation was found between the phenolic content of the GSEs and their antibacterial potential [32]. Another study by Jawhari et al. [33] evaluated the mineral and chemical compositions, total phenolic and flavonoid contents, and antimicrobial and antioxidant activities of two varieties of Anacyclus pyrethrum (L.). The hydroalcoholic extracts from different parts of the plants (leaves, capitula, roots, and seeds) were analyzed [33]. The results revealed both varieties’ exciting mineral and chemical compositions, with specific active compounds detected in each. The antioxidant and antimicrobial activities of the extracts showed promising properties, with leaves, capitula, and seeds exhibiting similar activity patterns [33]. Results from this study (Table 7) revealed that the crude sap of P. angolensis contained the highest amount of total phenol content compared to the roots of C. sanguinolenta. In comparison, it was observed that the ethanol extract had a significantly higher total phenol content than the aqueous solution. The aqueous solution recorded a low value, possibly due to the inability of water to adequately extract non-polar polyphenols into the solution [34]. In the current study, 70% ethanol was used to prepare the ethanol extract. Ethanol when combined with water, has a much greater potential to extract polar and non-polar polyphenols into solution than when absolute concentration is used [34]. Low values were also recorded in the aqueous extract, possibly due to the action of the enzyme polyphenol oxidase, which works best in an aqueous medium and acts on polyphenols and degrades them, thereby reducing their presence in solution [34]. From the hypothesis test carried out, it was realised that a comparison of the three extracts produced a p-value of 0.0001, indicating that the various extracts were very different from each other and, as such, one extract could not be substituted for another for its usage in the manufacturing of potent drugs [35].

This study showed an increase in the mean percentage of antioxidant activity as the concentrations increased. This was reflected in all the extracts and the standard BHT to which the extracts were compared. It was observed that P. angolensis (sap), C. sanguinolenta (aqueous), and C. sanguinolenta (ethanol) recorded IC₅₀ values of 0.0674 mg/mL, 2.1609 mg/mL and 1.002 mg/mL, respectively, compared to the BHT of 0.0432 mg/mL (Figs. 3, 4, 5 and 6). Comparing the extracts for the study to the standard, P. angolensis (sap) recorded values comparable to the reference value [36]. Even though the IC₅₀ values of the aqueous and ethanol forms of C. sanguinolenta are not close to that of the standard, it can be said conclusively that they are good antioxidants as few amounts of these extracts can mop up 50% of free radicals [37]. It was also observed that C. sanguinolenta (ethanolic) recorded an IC₅₀ value much closer to the standard than C. sanguinolenta (aqueous). This indicates that the ethanol crude extract of C. sanguinolenta is a much better antioxidant than the aqueous extract. This is likely due to the percentage of ethanol (70%) used for the extraction. Coupled with some amount of water, ethanol had a more significant potential to dissolve more phenolic compounds than using only distilled water or ethanol [38]. It must be noted, that the closer an IC₅₀ value of an extract is to zero, the more likely it is for the extract to possess potent antioxidant capabilities [21]. Thus, the crude sap of P. angolensis is a more powerful antioxidant than the ethanol extract of C. sanguinolenta, which is also a better antioxidant than the aqueous extract of C. sanguinolenta. On the whole, all three extracts proved to be very effective antioxidants. The current study findings agree with study by Khanc et al. [39], where it was reported that in the nitric oxide scavenging experiment, the crude extract of P. angolensis showed astounding efficacy with a 99.0% Radical Scavenging Activity (RSA) compared to the reference, n-propyl gallate (90.3% RSA) [39]. Another study conducted by Oladimeji and Akpan [6], also showed that P. angolensis had a moderate antioxidant activity of 0.55 µg/mL when compared with the standard drug (Vitamin C) with an antioxidant activity of 0.45 µg/mL [6]. Furthermore, the antioxidant activities of P. angolensis were better than those of vitamins A and E at 0.57 and 0.59 µg/mL [6], respectively. The antioxidant capabilities of the extracts were instructive since the phytochemical analysis of the plants revealed the presence of terpenes, flavonoids, and tannins, all of which have antioxidant properties. Studies have shown a direct correlation between the total phenol content and extracts’ antioxidant activity [36, 40, 41]. It is therefore not surprising that the crude sap of P. angolensis, which recorded a higher IC₅₀ value of 0.0674 mg/mL, had a higher amount of phenol content (55.427 ± 4.248) compared to the ethanol extract of C. sanguinolenta which also recorded a higher IC₅₀ value of 1.002 mg/mL and an amount of 26.888 ± 4.248 g/100 g GAE of phenol content than its aqueous extract which recorded the least values [10, 11].

The present work revealed that the root extracts (aqueous and ethanolic) of C. sanguinolenta possess alkaloids, cardiac glycosides, and saponins. In contrast, the crude sap of P. angolensis possessed alkaloids, cardiac glycosides, tannins, saponins, terpenoids, and phenols. These secondary metabolites have been found to possess antimicrobial and anticancer properties. Alkaloids are mostly known for their toxicity against cells of foreign organisms, and these have the potential to eliminate and reduce human cancer cell lines [14]. Alkaloids are naturally occurring metabolites in plants and are mostly present as heterocyclic compounds containing nitrogen atoms (which are very essential for plant growth) and are in the form of salts coupled with organic acids [14]. It was therefore not surprising that they were found to be present in various extracts. Eleazu and Eleazu [42] reported that isolated alkaloids and their derivatives possess medicinal properties due to their antispasmodic, antibacterial, and analgesic properties [42].

Tannins are known to form irreversible complexes with proline-rich proteins [43]. Parekh and Chanda [44] also found that tannins react with proteins to produce essential effects for the treatment of inflamed or ulcerated tissues. Plants rich in tannins are astringent and may be used for treating intestinal disorders like dysentery and diarrhoea [44]. P. angolensis is a plant used to treat intestinal disorders like dysentery and diarrhoea diseases in West Africa. This may prove the antimicrobial and anticancer properties of P. angolensis based on its phytochemical constituents [10]. The absence of flavonoids in both aqueous and ethanolic extracts of the root extracts of C. sanguinolenta and crude sap of P. angolensis does not mean a lack of the bioactive constituents [11]. However, this may be due to the low levels of the bioactive compounds in the crude plant extracts used in this current study [11]. Saponins possess hypolipidemic and anticancer activities and are also important for co-functioning with cardiac glycosides to enable them to carry out their activities which include serving as cardiac drugs and promoting nitrogen retention in osteoporosis or with animals with wasting illness [45‐47]. Terpenoids also have a broad range of properties including antitumor, antiviral, bactericidal, fungicidal, analgesic, anti-inflammatory, spermicidal, and cytotoxic activities [48]. Phenolic compounds are most notable for their antioxidant action due to their high tendency to chelate metals and inactivate their actions [49]. All these medicinal effects of the various phytoconstituents make them possible for their usage in treating numerous diseases.

Results from this current study are consistent with the works carried out by others. Considering the sap of P. angolensis, the work done by Udeozo et al. [50], on the powdered stem revealed the presence of flavonoids, alkaloids, saponins, tannins, terpenoids, and glycosides. Oladimeji et al. [6], who also worked on the ethanolic extract revealed the presence of saponins, cardiac glycosides, and terpenoids except for alkaloids, tannins, and flavonoids. Akinyenye and Olatunya [51] also confirmed positive tests for alkaloids, saponins, tannins, terpenoids, flavonoids, and cardiac glycosides upon working on the aqueous extract of the plant. Their results were also compared with that of Udeozo et al. [50]. This study was consistent with the works by Mills-Robertson et al. [11], who worked on the cold and hot water extracts as well as the ethanol extracts and revealed the presence of alkaloids. Bunalema [52], worked on the crude extracts of the roots and revealed the presence of alkaloids, tannins, and flavones. Claude et al. [52] worked on the methanol extracts and obtained positives for alkaloids, tannins, and flavones just as obtained by Bunalema [52]. Mills-Robertson et al. [10], worked on the aqueous, ethanol, and chloroform extracts that revealed the presence of alkaloids and the absence of saponins and flavonoids in all three extracts. Chahar et al. [13], also worked on the aqueous, ethanol, and chloroform extracts and their study revealed the presence of alkaloids and terpenes (for only the aqueous extract) and the absence of saponins and flavonoids in all the three extracts. A study carried out by Chime [53] on the ethanol crude extracts of C. sanguinolenta revealed the presence of alkaloids, terpenoids and glycosides. Saponins, tannins, but flavonoids were absent. From the various studies on C. sanguinolenta, it was realized that alkaloids tested positive throughout and this confirms the work done by Gibbons et al. [54], who not only identified the alkaloid cryptolepine but also went a step further to isolate this potent alkaloid. The differences in results from this study as compared to the others could be genuinely due to their absence or the difference in the methods of preparation and the types or parts of crudes used in the various extracts [10].

This study provides valuable insight into the potential antimicrobial properties and total phenolic content of two selected West African plants, P. angolensis and C. sanguinolenta. The in vitro results suggest that these plant extracts may have promising applications in the treatment of microbial infections and other non-communicable diseases.