Iridoids

Iridoids are a group of monoterpenoids recognized by their basic structure in which a pyran is fused to a cyclopentane, and are usually grouped into glycosidic, non-glycosidic, seco- and bis-iridoids (Ludwiczuk et al. 2017). Their activities could be likened to those of adaptogens and immunomodulators, and they are considered as prodrugs that get easily converted to active pyridine monoterpene alkaloids (Ghisalberti 1998). Chemical structures of iridoids are quite diverse and might be confusing if not well looked at because they often look alike. They play a role in ants’ defence mechanism and are commonly found in plants infested with ants. Some patents have been filed for some natural iridoids in a recent review article for many biological activities (Hussain et al. 2019). However, many of them were in vitro investigation with most activities recorded at very high concentrations of iridoids which might be considered scientifically insignificant. For instance, in one of the patents recorded in Hussain et al. (2019) review article, an IC₅₀ value of 104.1 µM was presented for one of the phytoconstituents of Psydrax (21) as bioactive anti-SARS agent. But all hope is not lost since iridoids are projected as pro-drugs whose activities could be greatly enhanced in vivo. It is worthy of note that some iridoids have shown good activities including cardiovascular, antihepatotoxic, hypoglycemic, hypolipidemic, anti-inflammatory, antispasmodic, antitumor, antiviral, and purgative activities (Ghisalberti 1998; Hussain et al. 2019; Tundis et al. 2008) at low concentrations. They are found naturally in varied forms in different plant species, and a good number has been documented for Pysdrax species. The structures of this class of compounds are captured in Fig. 1.

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Fig. 1

Iridoids and their glycosides

In a recent study, involving the stem bark of P. subcordata, two non-gylcosidic iridoids (1 and 2) were isolated, alongside other constituents (Awantu et al. 2019). Another set of non-glycosidic iridoids (3–8), which were novel and two known ones (9 and 10) were isolated from leaf and bark extracts of P. subcordata (Zhou et al. 2019). The largest number of iridoids (11–21) was isolated from iso-butanol fraction of methanol extract of P. subcordata dried fruits, and out of the eleven iridoids, five (16–20) were novel compounds (Joubouhi et al. 2015). Compound (22) was characterized in an extract of P. montigena using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) (Yang et al. 2016), while (21 and 27) were obtained from the methanol extract of the stem bark of P. subcordata (Achenbach 1986; Achenbach et al. 1981), after an initial isolation of only (27) from the same extract (Achenbach et al. 1980). Out of the 22 iridoids so far isolated from P. subcordata, (21) is observed to be the most common and widely distributed iridoid of the species; it was isolated as the major component of the methanol extract of dry fruits and stem bark of P. subcordata. Another species that yielded iridoids is P. odorata; its leaf and bark produced four glycosidic iridoids (23–26), in company with other non-iridoid constituents (Coulerie and Poullain 2016; Sévenet and Pusset 1996). These data on iridoids suggest that the genus accumulates high amounts of this class of metabolites which may be of taxonomic relevance in plant systematics if further investigation is carried out.

Other terpenes and terpenoids

Terpenes are a class of compounds often found abundantly in essential oils because of their high volatility, and they are usually characterized using GC–MS technique. However, in this review, eight terpenes were isolated from three species of the genus and their structures and names are given in Fig. 2 and Table ​Table44 respectively.

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Fig. 2

Terpenes and their glycosides

A non-glycosidic terpenoid (28) was isolated and characterized with NMR technique from the stem bark of the methanol extract of P. subcordata (Awantu et al. 2019) as well as in a GC–MS analysis of the n-hexane fraction of the leaves of P. puberula (Castro et al. 2016). The presence of (28) in two different species of the genus indicates possible similarities in the biosynthetic gene clusters of Psydrax species. In other studies, four non-glycosidic terpenoids (29–32) and three non-glycosidic terpenoids (33–35) were obtained from methanol extract of the stem bark and fruit of P. subcordata (Achenbach et al. 1981; Awantu et al. 2019; Joubouhi et al. 2015).

Cyanogenic glycosides

Cyanogenic glycosides are often considered toxic due to the ease of transformation of their aglycones to hydrogen cyanides a poisonous chemical (Yulvianti and Zidorn 2021), and could be seen as unimportant therapeutic agents. However, a bioactivity of this class of compound was reported by Tan et al (2012) in their study of the neuroprotective effects of prunasin gallates against H₂O₂ induced oxidative damage of NG108-15 cells. They reported in vitro neuroprotective property of highly glycosylated trigallates and tetragallate of prunasin (36), one of the compounds isolated from Psydrax, at a concentration of 100 µM of the test samples and a positive control, catechin (Tan et al. 2012). It is obvious that this report was marred by the high concentration of test samples and the reference drug used. Also the bioavailability of these compounds are not guaranteed in vivo due to the high polarity of the glycosylated compounds and a possible loss of the sugar moiety in vivo. Apart from (36), two other cyanogenic glycosides (37 and 38) were isolated from methanol extracts of the leaf, stem and root of P. livida (Rockenbach et al. 1992) and the fourth cynogenic glycoside (40) was from a methanol extract of ripe fruits of P. schimperiana (Schwarz et al. 1996). The chemical structures of these compounds are shown in Fig. 3.

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Fig. 3

Cyanogenic glycosides

Alkaloids

It is observed in the course of this review that alkaloid is not a common constituent of this genus and there are some conflicting claims as to whether they are present or absent in the genus. Some researchers discovered zero alkaloid when they chemically profiled the leaf, stem and root barks of P. subcordata, P. peruviana, and P. acutiflora (Denise P Ilboudo et al. 2013; Goh et al. 1997; Achenbach 1986; Achenbach et al. 1981; Daanaa et al. 2018; Akoto et al. 2019). On the contrary, some other research groups identified alkaloids in a different part of P. subcordata, P. horizontalis, P. schimperiana and P. dicoccos, but they reported the alkaloid content of P. schimperiana and P. dicoccos to be small (Anokwah et al. 2016; Feenna et al. 2020; Ochwang’i et al. 2016; Umaiyambigai et al. 2016). To confirm the presence of alkaloids in Psydrax, three alkaloids: two monoterpene alkaloids (40 and 41) and a peptide alkaloid (42) were isolated from P. odorata bark and leaf (Coulerie and Poullain 2016) and their structures are given in Fig. 4. The observed variations in alkaloid content of Psydrax species could be attributed to different factors, such as geographical differences, different harvesting seasons and analytical techniques and methods adopted by different authors and/or differences in the genetic make-up of different species of the genus. This inconsistency in the alkaloid contents of the genus calls for further extensive phytochemical studies of the genus across different geographical locations where the species are endemic and adoption of a standard analytical technique and method that will provide a clearer perspective on the types of alkaloids and the alkaloidal content of the genus.

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Fig. 4

Alkaloids

Flavonoids

Flavonoids are relatively rare in Psydrax, out of the 55 compounds isolated from the genus, only three were flavonoids: a flavone (43) isolated from the fruit of P. subcordata (Joubouhi et al. 2015, 2017), and a flavonol (44) and its glycoside (45) from P. dicoccos leaves (Gunasegaran et al. 2001). The structures of the flavonoids are given in Fig. 5.

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Fig. 5

Flavonoids and their glycosides

Antimicrobial activity

European Committee on Antimicrobial Susceptibility Testing (EUCAST), and Clininal and Laboratory Standards Institute (CLSI) provide standard methods called antimicrobial susceptibility testing (AST) for conventional antimicrobials for rational comparison among antimicrobial assay results generated by researchers (Bubonja-Šonje et al. 2020). Unfortunately, there are  no such established methods for assessing the antimicrobial properties of plant materials due to the complexity of plant samples and this leads many researchers to adopting varied modified AST to achieve their desired goals (Bubonja-Šonje et al. 2020). The result of these of these incoherent test methods is the generation of incomparable, difficult-to-interpret and often times misinterpreted research outcomes (Bubonja-Šonje et al. 2020; Cos et al. 2006; Eloff 2019; Gertsch 2009). This is what was observed in the course of this review, different researchers adopted varying test methods; different concentrations of plant samples were used; test organisms and media were not similar in many cases; and endpoint parameters (MIC, IC₅₀) assessed were dissimilar (Cos et al. 2006).

Some of the articles reviewed reported sample concentrations far above the recommended MIC value for plant extracts and pure compounds ((0.1 mg/mL (100 µg/mL) for extracts/fractions or less than 25 µM for pure compounds), and acceptable maximum in-test concentration of 1 mg/mL for plant mixtures and 0.1 mg/mL for pure plant constituents (Ríos and Recio 2005); many articles reported activity as minimum inhibitory concentration (MIC) values instead of concentration at 50% inhibition (IC₅₀), which is adjudged to be scientifically significant at a value less than 0.1 mg/mL (100 µg/mL) for extracts/fractions or less than 25 µM for pure compounds; some authors used concentration of microbial inoculum greater than the acceptable standard of 10⁵ CFU/mL for bacteria and 10³ or 10⁴ CFU/mL for fungi (Cos et al. 2006). Another crucial observation made in this review is that, some authors adopted agar diffusion method which is not a generally acceptable standard method for determining the MICs of plant extracts/fractions because of the low polarity of most plant’s antimicrobial constituents which often hinders their diffusion through the polar agar medium used for the tests and ultimately leads to false negative result (Eloff 2019). Eloff also pointed out that an MIC value less than 0.1 mg/mL is scientifically relevant for further antimicrobial studies, that anything above this benchmark should not be pursued for further investigation (Eloff 2019). Being guided by these recommendations, we hereby enumerate only the in vitro antimicrobial assay results of extracts and pure compounds of the genus whose IC₅₀/MIC value is less than 100 µg/mL for plant extract/fraction and less than 25 µM or<10 µg/mL for pure compounds, as well as those that used microbial inoculum concentration of 10⁵ CFU/mL for bacteria and 10³ or 10⁴ CFU/mL for fungi. To the best of our knowledge, no antimicrobial in vivo assay nor clinical trial has been reported for either the extracts nor phytoconstituents of this genus.

Antimicrobial assay conducted by Awantu et al. (2019) for eight compounds (1, 2, 28, 29, 30, 34, 35 and 51) they isolated from the stem bark of P. subcordata, against three Gram positive bacteria (Staphylococcus aureus, Staphylococcus saprophiticus, Streptococcus faecalis), three Gram negative bacteria (Klebsiella pneumonia, Pseudomonas aeruginosa) and two fungi species (Candida albicans and Candida krusei) using broth dilution method showed only (1, 2 and 30) to be effective. They reported the following results for the three active compounds using gentamicin (IC₅₀ 2.4 µg/mL) and nystatin (IC₅₀ 4.8 µg/mL) as positive control for bacteria and fungi, respectively: (1) was active against only C. albicans (IC₅₀ 9.7 µg/mL); (2) was active against C. albicans (IC₅₀ 4.8 µg/mL), C. krusei (IC₅₀ 9.7 µg/mL) and S. aureus (IC₅₀ 9.7 µg/mL); (30) was active against C. albicans (IC₅₀ 9.7 µg/mL), K. pneumonia (IC₅₀ 9.7 µg/mL) and P. aeruginosa (IC₅₀ 4.8 µg/mL) (Awantu et al. 2019). In another study conducted by Joubouhi et al.( 2017) for the fourteen compounds (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 31, 32 and 43) they isolated from the fruit extract of P. subcordata using micro dilution method, only (13) displayed antibacterial activity against a Gram-positive bacterium, S. aureus, with an MIC value of 8 µg/mL; they used two reference drugs – ampicillin (MIC 8 µg/mL) and ciprofloxacin (MIC 2 µg/mL). None of the compounds was active against any of the Gram-negative bacteria strains (four strains of Vibrio cholerae, and Shigella flexneri) used by the authors (Joubouhi et al. 2017).

In a preliminary study to support the use of P. livida leaves in the traditional treatment of wound myiasis in animals, a group of researchers Mukandiwa et al. (2012b) carried out an in vitro antibacterial assay of four extracts (hexane, dichloromethane, acetone and methanol) of P. livida leaves against two Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), which are implicated in the pathogenesis of wound myiasis, by adopting microdilution assay method. They discovered that the dichloromethane extract was the most active among the extracts. However, the dichloromethane extract was active against only S. aurus with MIC values of 78 µg/mL as against the MIC value (0.78 µg/mL) for gentamycin, the reference drug.

Antimicrobial assay conducted by Castro et al. (2016) for crude extract and three fractions (hexane, chloroform and butanol) of P. puberula leaf against S. aureus, P. aeruginosa and E. coli, using microdilution method, showed varying antimicrobial activity. The crude extract and three fractions were all active against only S. aureus with MIC values ranging from 31.25 – 62 µg/mL as against the MIC value of about 3.91 µg/mL of ciprofloxacin, the positive control (Castro et al. 2016). An antifungal assay of P. subcordata mature fruits’ essential oil against A. niger showed activity with an MIC value of 39 µg/mL in comparison to a positive control, amphotericin B (MIC value of 0.61 µg/mL) (Essien et al. 2015); the essesntial oil showed no significant activity against any of the bacteria species (Bacillus cereus, S. aureus, E. coli and P. aeruginosa) tested. From these enumerated antimicrobial properties, Psydrax could hold a promise in the discovery of antibacterial and antifungal agents with broad spectrum of activity. However, more research works, especially in vivo study of the active samples and cytotoxic assays, is needed to substantiate this knowledge-informed hypothesis. These few promising antimicrobial properties observed for the extracts and constituents of the genus support some of its traditional applications in the treatment of diarrhoea and wound myiasis.

Anti-inflammatory activity

It is recommended that every biological assay should be conducted by adopting a standardized procedure for the assay, using as small an amount of plant sample as possible and a standard positive control (Cos et al. 2006). Whenever a too low or too high concentration is used, a false negative or positive result will prevail. For anti-inflammatory assays, Chukwujekwu et al. (2005) rightly observed that recording a relatively good in vitro prostaglandin synthesis inhibition for a non-polar extract as against more polar extracts does not corroborate the traditional use of the aqueous extracts in the traditional management of inflammation (Chukwujekwu et al. 2005). The possibility of aqueous herbal extracts to elicit anti-inflammatory effect in folkloric medicine could be linked to the high phenolic content of aqueous extracts which are mainly antioxidants and could give anti-inflammatory effect by improving the immune system of the patient (Eloff 2019). For the Psydrax genus, different in vitro and in vivo anti-inflammatory assays have been carried out for different extracts of P. subcordata and P. acutiflora, but we will only report results that used maximum in-test concentration of 1 mg/mL of plant extracts/fractions or 0.1 mg/mL of pure plant constituents in in vitro assays, and in vivo oral concentration of not more than 100 mg/kg for extracts.

Unfortunately, only the in vivo study done by Anokwah et al.( 2016) fell within our chosen concentration range. In an in vivo per oral (p.o.) anti-inflammatory assay they conducted for the methanol stem bark extract of P. subcordata using carrageenan-induced footpad oedema in seven-day-old chicks, the authors reported dose-dependent percentage oedema inhibitions of 61.21, 53.00 and 49.34% at 300, 100 and 30 mg/kg body weight p.o. doses, respectively, of the extract using two positive controls, dexamethasone (0.3, 1 and 3.0 mg/kg) and diclofenac (10, 30, and 100 mg/kg) given intraperitoneally (Anokwah et al. 2016). Though they recorded anti-inflammatory activity for the plant extract, the concentrations used for the extract were higher than those of the positive control coupled with different routes of administration used for the test sample and the controls.

Antiparasitic activity

Antiparasitic assay like other anti-infection assays should follow some recommended standards previously mentioned to guarantee the authenticity of its results. Psydrax species have found applications in traditional management of malaria and fever, and in attempts to support this ethnomedicinal claim, some researchers conducted in vitro antiplasmodial assay of different extracts and constituents of the genus and they came up with varied antiplasmodial activities for the genus. However, it was observed that majority of these in vitro studies investigated either susceptible or resistant strains of Plasmodium falciparium and not both strains as deemed appropriate for a primary in vitro antiplasmodial assay (Cos et al. 2006). For studies that determined the IC₅₀ of test samples (extracts), the IC₅₀ values they obtained were within the acceptable range of below 100 µg/mL for crude extracts, but toxicity assays were not conducted for these active extracts to confirm that the activities reported were not due to non-specific activity and potential toxicity of the extracts (Cos et al. 2006).

In a study involving stem bark methanol extract of P. subcordata against a chloroquine resistant P. falciparum W2, there was an inhibition against the parasite with an IC₅₀ of 3.044 µg/mL, however, fractions and constituents (1, 2, 28, 29, 30, 34, 35 and 51) from the extract showed no activity against the parasite strain (Awantu et al. 2019). However, the authors did not give the IC₅₀ value for the positive control. In another study, 80% methanol extract of P. palma, showed parasitic inhibition against a chloroquine-sensitive P. falciparum strain (positive control: chloroquine 2H₃PO₄ – IC₅₀ 0.08 µg/mL), Trypanosoma brucei brucei (positive control: suramin – IC₅₀ 0.08 µg/mL), and Trypanosoma cruzi (positive control: suramin – IC₅₀ 0.05 µg/mL), with the same IC₅₀>64 µg/mL, using 96-well tissue culture plates method (Mesia et al. 2008). They also conducted cytotoxicity test for the extract against the MRC-5 human cell-line and got the same IC₅₀ value, but they reported the extract as inactive at this IC₅₀ comparing its activity with the activities of extracts from other plants having IC₅₀ as low as 0.7 µg/mL and did not calculate the selectivity index (SI) for the extract.

In another study involving different extracts (hexane, diisopropyl ether, dichloromethane, ethyl acetate, and methanol–water) of P. acutiflora leaf/twig, tested against chloroquine susceptible and resistant strains of P. falciparum, using the parasite lactate dehydrogenase assay method, the exracts displayed variable IC₅₀ values against the two strains of the parasite. The diisopropyl ether extract showed highest activity against the parasite strain (IC₅₀ 8.8 µg/mL and IC₅₀ 9.5 µg/mL) against the susceptible and resistant strains respectively, while the ethyl acetate extract was the least active (IC₅₀ 61.3 µg/mL) against the sensitive strain and methanol–water extract showed least activity (IC₅₀ 64.8 µg/mL) against the resistant strain. Chloroquine used as the positive control for this study had an IC₅₀ of 0.20 µg/mL and IC₅₀ 0.009 µg/mL against the sensitive and resistant strains respectively (Ilboudo et al. 2013). (Ilboudo et al. 2013) also tested the in vitro antiplasmodial activity of the most abundant constituent (50) of the diisopropyl extract and reported less activity (IC₅₀ 18.1 µg/mL and IC₅₀ 43.4 µg/mL) for (50) against the sensitive and resistant strains, respectively, in comparison with the diisopropyl extract. In addition to these antiplasmodial tests, (Ilboudo et al. 2013) also conducted cytotoxicity tests and calculated SI for the aqueous extract of P. acutiflora with IC₅₀ 80.0 and 46.91 µg/mL against the sensitive and resistant strains of organism, respectively, and obtained a CC₅₀ value of 615.4 µg/mL and SI values of 7.7 and 13.1 for the sensitive and resistant strains, respectively.

Anticonvulsant activity

In the quest to support the folkloric use of ethanol extract of P. subcordata in the management of epileptic seizures, Daanaa and co-workers used varying animal models in in vivo anticonvulsant assays of 70% ethanol extract of the leaves of P. subcordata. They explored the following animal models and reference drugs: pentylenetetrazole-induced seizure (reference drug: diazepam (0.3, 1 or 3 mg/kg, i.p.)); picrotoxin-induced seizure (reference drug: diazepam (0.3, 1 or 3 mg/kg, i.p.)); maximal electroshock (reference drug: carbamazepine (3, 10 or 30 mg/kg, p.o)); strychnine-induced seizure (reference drug: diazepam (1 mg/kg, i.p.)); 4-aminopyridine-induced seizure (reference drug: sodium valproate, (100, 300 or 600 mg/kg, p.o.)); and lithium/pilocarpine-induced status epilepticus (reference drug: diazepam (1 mg/kg, i.p.), and discovered that the extract worked by delaying the onset of seizure and reducing the duration and frequency of induced convulsions in most of the animal models, at study doses of 30, 100 and 300 mg/kg (p.o.). However, in electroshock and strychnine-induced test models, the hydroethanol extract failed to delay the onset of tonic hind limb extensions and the onset of seizure, respectively (Daanaa et al. 2018). Though the doses of the extract used in this study were ten times those of the reference drugs, this report by Daanaa et al. gives a pharmacological insight to the use of ethanol extract of P. subcordata leaves in the ethnomedicinal management of epileptic convulsion and presents the plant as a possible source of anticonvulsant agents if further investigated to find out if there could be any synergistic effect of using the extract in a combination therapy, since herbal medicines are often administered as a mixture of many extracts. Another way to ascertain the use of this extract in traditional medicine is by screening and isolating the active constituent(s) of the extract and carrying out extensive assays (in vitro and in vivo) on the isolated compound(s) and ultimately conduct clinical studies to substantiate their activities. This study by Daanaa et al. (2018) is the only anticonvulsant assay so far done for this genus.

Antihyperglycemic/antidiabetic activity

In a bid to support or refute the ethnomedicinal use of P. subcordata bark in the management of diabetes, Zhou et al. (2019) carried out an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay on eight iridoids (3–10) isolated from ethanol extracts of P. subcordata leaves and bark. Only (3), one of the six novel iridoids (3–10) showed relavant enzyme inhibition with an IC₅₀ value of 22.2 µM in comparison with the positive control, oleanolic acid (IC₅₀ 4.3 µM). Though this is not very fantastic, but chemical modification of the compound could lead to a better activity. To prove this assertion, when the structures of two enantiomers, subcordatanol III (5) and subcordatanol IV (6) were transformed to subcordatalactons A and B, respectively, their antihyperglycemic property dramatically improved from IC₅₀ value of>80 µM to 8.9 and 9.8 µM for A and B, respectively (Zhou et al. 2019). We recommend in vitro cytotoxicity assay and calculation of SI for (3) as well as in vivo assays to determine its bioavailability and mechanism of action.

Antioxidant activity

Since the antioxidant activity recorded for this genus was from in vitro assay, which is not a sufficient prove of a possible in vivo efficacy of the samples as antioxidants, we hereby present the in vitro radical scavenging results as an indication of the phytoconstituents of the species rather than as a bioactivity. Many a time, extracts that exhibit in vivo antioxidant property are usually high in phenolic content, But, the phytochemical analysis of the eight studied species of Psydrax gave only three flavonoids as phenolic compounds which might be considered too small to accord the genus antioxidant properties. Surprisingly, some of the extracts and isolated compounds of the genus exhibited in vitro radical scavenging properties. We decided to present in vitro radical scavenging results of extracts with IC₅₀ or EC₅₀ values less than 10 or 100 µg/mL for compounds or extracts/fractions and to exclude results with very high concentrations of no scientific relevance. In a study conducted by Awah et al. (2012), 80% methanol root extract of P. subcordata displayed DPPH radical scavenging property with an EC₅₀ value of 23.9 µg/mL in comparison to ascorbic acid of EC₅₀ value of 4.9 µg/mL. Akoto et al. (2019) reported that the methanol extract of P. peruviana stem bark also displayed DPPH radical and hydrogen peroxide scavenging activities with IC₅₀ values of 12.20 and 24.26 μg/ml, respectively. Two iridoids (13 and 17) isolated from the fruit extracts of P. subcordata reportedly displayed DPPH radical scavenging activity with EC₅₀ values of 1.12 µg/mL and 2.03 µg/mL for (13) and (17), respectively in comparison with EC₅₀ of 1.74 μg/mL for vitamin C, the positive control (Joubouhi et al. 2017). (13) and (17) are not polyphenolic compounds with inherent antioxidant properties, neither are they glycosidic, yet they exhibited radical scavenging properties. Thus, their radical scavenging properties needs further investigation – probably in vivo studies – to confirm the activity and elucidate the mechanism of action of these extracts and compounds.