Comprehensive Review of the Components in Cat’s Claw (Uncaria tomentosa) and Their Antibacterial Activity

Abstract

Cat’s claw (Uncaria tomentosa (Willd. ex Schults) DC.), a plant that is exceptionally rich in phytochemicals, has been used for centuries by the indigenous people of South and Central America as a therapeutic and is currently widely exported for medicinal purposes. Extracts and individual components have shown considerable potential as antibacterials in the literature. The purpose of this review is twofold: first, to provide a substantiated, comprehensive collection of the known chemical constituents of U. tomentosa, including their detailed structures; second, to identify those components that offer some promise as antibacterials based on the research to date. Bacterial resistance to currently available antibiotics continues to increase and is widely recognized as an impending, potentially catastrophic, problem. There is research to suggest that U. tomentosa components may have antibacterial potential individually or synergistically with established antibiotics against microbes, including Borrelia burgdorferi, the causative agent of Lyme disease. It is our intention that this review will provide a valuable resource to investigators in search of new antimicrobials to meet the daunting challenge of antibiotic resistance.

1. Introduction

Medicinal plants and their natural products have been a source of important drug discoveries for decades. Since 1981, of the 1394 small-molecule drugs approved by the FDA, 930 of them were derived, to some extent, from a natural product, with fourteen being direct isolates from medicinal plant sources [1]. Many of these medicinal plants continue to be a primary therapeutic source for a large portion of the population in developing countries in Asia, South America, and Africa. The family Rubiaceae contains many of these plants, of which the genus Uncaria is prominent. This review focuses on Uncaria tomentosa (Willd. Ex Schults) DC., commonly referred to as “cat’s claw”, a plant that is native to South and Central America. The name of this woody vine was derived from the long, cat claw-like thorns protruding from the stem at its leaf junctions. The phytochemical composition of the plant has been the subject of many studies that pre-date this review [2,3,4,5,6,7,8,9,10,11,12], with one of these studies purporting more than 50 phytochemicals originating from the plant [13].

The interest in the specific phytochemical composition of U. Tomentosa comes from the plant’s long history of medicinal use by indigenous people in the region. Peruvian tribes such as the Ashaninka, Aguarurna, Cashibo, and Shipibo have used the plant for centuries to treat a multitude of ailments. Documentation from these cultures have indicated its use as a therapeutic for allergies, arthritis, asthma, diabetes, cancer, bacterial and viral infections, and other medicinal uses [14]. Scientific investigations into U. tomentosa extracts and constituents have uncovered a range of biological activities, such as antioxidant, anti-inflammatory, antimicrobial, antiviral, and immunomodulating activity [11,12,13].

Of the biological activities and potential medicinal purposes, this review focuses on its antibacterial properties. As a traditional medicine, U. tomentosa has been documented in wound treatment, indicating potential as an antibacterial agent [14]. Recent investigations of the plant and its extracts have produced in vitro evidence of antibacterial activity [15,16,17,18,19]. Antimicrobial activity has been observed against both the latent and active forms of the bacterium Borrelia burgdorferi, the bacterium responsible for Lyme disease [20,21]. Lyme disease is a tick-borne ailment brought on by the introduction of Borrelia burgdorferi to a host through a tick bite. Recent Centers for Disease Control (CDC) investigations have indicated that as many as 300,000 Americans are affected by Lyme disease every year [22,23]. Improved antibiotic therapies capable of not only treating the initial symptoms of B. burgdorferi infections, but also preventing and treating persistent infections are needed. The in vitro anti-borrelia efficacy demonstrated by U. tomentosa warrants an investigation into the possibility of identifying the constituent or a combination of constituents responsible for this effect.

To date, several reviews have been published providing general information on the genus Uncaria, including U. tomentosa, which contain focused, but limited, information on constituents and/or bioactivity [11,12,13]. In addition, none of these reviews have focused specifically on U. tomentosa constituents and their applications as antibacterial agents. The purpose of this review is to present a comprehensive collection of molecular structures from U. tomentosa, as well as consolidate research and literature regarding these components’ potential as antibacterial candidates. The information provided in this review will hopefully highlight promising candidates for the possible development or isolation of antibacterial agents from the species U. tomentosa, and specifically those related to anti-borrelia efficacy.

2. Methodology

This review focuses on reporting a comprehensive list of all known phytochemical structures of U. tomentosa, while also providing relevant information on antibacterial activity. Structures were sourced and verified from the literature, Reaxys, PubChem, Google Scholar, and SciFinder. A number of existing reviews were referenced citing the entire or multiple plant species within the Uncaria genus [11,12,14,24,25,26,27,28,29,30,31], and one recent review focused directly on U. tomentosa constituents and bioactivity [13]. The information in these reviews were corroborated before being reported here, and minor errors in which constituents were wrongly assigned to the species U. tomentosa are also noted in this review. Antibacterial activity was investigated via a literature search for every identified phytochemical component. Few examples of antibacterial activity exist from compounds directly sourced from U. tomentosa. As a result, instances where the same constituents were extracted from other natural products are included. The phytochemical constituents of U. tomentosa were divided into three classes: alkaloids, polyphenols, and terpenoids (Supplementary Materials). The compounds identified in these classes were then further divided into groups and subclasses, based on structural commonality.

3. Alkaloids

Alkaloids are heterocyclic compounds that contain one or more nitrogen atoms in the structure. They are secondary metabolites generally derived from amino acids and found in a wide range of plants, with an estimated 12,000 molecules identified from natural sources [32]. The structural definition of alkaloids allows for the inclusion of structurally diverse compounds in the class, resulting in a wide range of bioactivities [33].

There are various systems of nomenclature used to discuss the structural diversity found in alkaloids. Biological sources, structural motifs, and biogenesis pathways are a few ways to classify alkaloids [34,35]. In this review, the grouping and discussion of alkaloids focus on using a method of chemo-molecular classification based on the structural motifs surrounding the nitrogen-containing heterocyclic portion of the molecule. The alkaloids of Uncaria tomentosa contain a variety of structures that were classified into two groups: indole alkaloids and oxindole alkaloids. These groups were further classified into subclasses defined as tetracyclic and pentacyclic indoloquinolizidine, β-carboline-type, and tetracyclic and pentacyclic oxindole alkaloids.

3.1. Indole Alkaloids

Indole alkaloids contain an indole group, a heterocyclic structure that consists of a five-member pyrrole ring fused to a benzene ring, within the heterocyclic structure of the molecule. The indole alkaloids of U. tomentosa were classified into three subclasses: tetracyclic indoloquinolizidine, pentacyclic indoloquinolizidine, and β-carboline. The indole alkaloids are biosynthesized from the precursor compounds tryptophan and tryptamine [32,36]. These indole alkaloids are known for possessing a range of bioactivity, which includes antimicrobial activity [37]. The indole moiety is present in twenty alkaloid constituents identified in U. tomentosa (Figure 1 and Figure 2).

3.1.1. Tetracyclic Indoloquinolizidine Alkaloids

There are six tetracyclic indoloquinolizidine alkaloids that have been identified in U. tomentosa (Figure 1, 1–4). These compounds are distinguished by a quinolizidine ring fused to an indole to form a tetracyclic indoloquinolizidine highlighted in blue in Figure 1. These alkaloids are commonly identified as corynanthe-type monoterpenoid alkaloids in other literature sources [38].

The six compounds share several similar structural motifs besides the tetracyclic indoloquinolizidine structure. An alkoxy and carboxylate ester within the monoterpene skeleton exists for all compounds within this subclass. Compounds 1a–2b were isolated and quantified in a study of sixteen plant samples utilizing high-performance liquid chromatography (HPLC) retention times, mass spectrometry (MS), proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectroscopy, and ultraviolet-visible (UV–Vis) spectroscopy, with 2b reported in trace amounts [10]. Of the citations from this source that reference structural determination of compounds 1–4, a single chromatographic method utilizing retention time and mass spectroscopy is the only report that confirmed structural identification of these molecules [39]. Molecules 1a–2b are structurally related. Hirsuteine 1a and corynantheine 1b are epimers with a vinyl group at C-20, while the compounds hirsutine 2a and dihydrocorynantheine 2b are epimers with ethyl substitutions at C-20. The structural determination of these compounds was completed utilizing—chromatographic method with the addition of MS [39].

N-oxide derivatives of alkaloids are known to naturally occur in plants [40]. Two N-oxide tetracyclic indoloquinolizidine compounds have been identified from the leaves of U. tomentosa [41]. The N-oxide tetracyclic indoloquinolizidine alkaloids 3 and 4 possess a structure identical to 1a and 2b, respectively, with the addition of an oxygen atom bound to the nitrogen atom of the quinolizidine structure. Heterocyclic N-oxides are currently being investigated as an emerging class of therapeutic agents with antibacterial activity [42,43]. However, antibiotic data for 3 and 4 have not been reported to date. Examples of N-oxides exhibiting antibacterial activity are the quinoxaline 1,4-di-N-oxide derivatives [42]. The exact mechanism of antibacterial activity is unknown; however, it has been established that the N-oxide moiety improves antibacterial activity [44], and that the mechanism proceeds though oxidative damage of bacterial DNA [45].

3.1.2. Pentacyclic Indoloquinolizidine Alkaloids

Pentacyclic indoloquinolizidine alkaloids contain one additional heterocyclic ring (Figure 1, 5a–d). Like tetracyclic indoloquinolizidine alkaloids, these compounds are also referred to as monoterpene alkaloids (i.e., heteroyohimbine alkaloids, a type of corynanthe-type alkaloid) [46,47]. Four of the pentacyclic indoloquinolizidine alkaloids are stereoisomers. Compound 6 is structurally related to both the tetracyclic and pentacyclic indoloquinolizidine subclasses but does not contain the indoloquinolizidine moiety. Instead, 6 contains a hydroxy substituted 7-membered ring in addition to a β-linked glucose molecule. The glycosylated indole alkaloid 6 has been identified from root cells grown under oxidative stress alongside the production of oxindole alkaloids [48,49,50]. This compound possesses biological activity including hypotensive, lipid lowering, and antioxidant activity; however, no antibacterial evidence has been tested or implicated [48,49].

While investigating literature sources, it was discovered that several compounds within this subclass were incorrectly described as being constituents of U. tomentosa [12]. In a 2005 review, compounds 7a, 7b, 8, and 9 were incorrectly attributed to U. tomentosa via a study on constituents from Uncaria rhynchophylla (Miq.) Miq. ex Havil. a separate species of Uncaria, native to Southeast Asia (Figure 1) [51]. These structures are highlighted in red in Figure 1, and to date, this is the first correction of the error.

3.1.3. β-Carboline Alkaloids

Indole alkaloids possessing the β-carboline subclass have been more recently isolated from U. tomentosa, with the first study identifying and structurally determining their presence in plant extracts established by Kitajima and colleagues in 2000 [4,5]. This group of indole alkaloids have a tricyclic structure highlighted in blue in Figure 2. This characteristic scaffolding is composed of an indole structure fused to a pyridine ring, where primary structural differences within the subclass arise from the saturation of this pyridine-type ring. In all cases, a carbon skeleton branches from C-3.

The simplest of these alkaloids found in U. tomentosa is harman (10). This molecule represents the core scaffolding of the β-carboline alkaloid subclass, with a fully aromatic ring system and a simple methyl substitution at C-3. Compound 10 was first isolated by Kitajima and colleagues along with other β-carboline structures 11, 12, and 13 [4]. Compound 10 has demonstrated antibacterial activity against both Gram-negative and Gram-positive bacteria, exhibiting minimum inhibitory concentration (MIC) values of 500 µg/mL against Escherichia coli and Bacillus subtilis, and 1000 µg/mL for Staphylococcus aureus [52]. Compound 10 has also been utilized as a scaffold for the development of antibacterial candidates, with some analogues exhibiting MIC values below 10 µg/mL for S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), B. cereus, B. subtilis, and Ralstonia solanacearum [53]. Substitutions of the harman structure that improve antibacterial activity include substituting C-6 with a methoxy group, C-1 with a benzo[d][1,3]dioxle group, and the quaternization of N-2 with a para-substituted benzyl group and a complexed bromine anion [53].

The gluco-β-carboline-type alkaloids 11–13 were also isolated by Kitajima and colleagues in a previous study [5]. The structures were determined utilizing UV, ¹H and ¹³C NMR spectroscopy, and MS, with the absolute configuration of compound 12 solved through a total synthesis based upon the biosynthetic precursors L-tryptophan and secologanin [4,5,7]. Compounds 12 and 13 differ only by the bond between C-3 and N-4, which is saturated in 13 and unsaturated in 12. The saturation of this bond produces a stereocenter at C-3 for 13, in the S configuration. Compound 11 possesses both the same skeletal structure and β-linked glucose molecule as 12 and 13, with the fully conjugated β-carboline core, but lacks the carboxylic acid.

There is only one study confirming the isolation of an additional glycosylated β-carboline alkaloid 14 from micropropagated plantlets and root cultures [50]. This structure possesses some unique features, including a methyl-substituted nitrogen and a stereocenter at C-3 in the S configuration. The glycosyl portion of the molecule is identical to that of compounds 11–13. Compound 14 and a derivative of compound 13 have been identified as precursor molecules in the biosynthesis of tetracyclic indoloquinolizidine and pentacyclic indoloquinolizidine alkaloids [47]. Antibacterial activity has been noted for compound 13. When isolated from Psychotria nuda (Cham. and Schltdl.) Wawra, 13 exhibited in vitro antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv with a reported MIC value of 26.3 µg/mL [54].

3.2. Oxindole Alkaloids

The first extraction of an oxindole derivative from a natural source was acquired from U. tomentosa [55]. Oxindole alkaloids, like indole alkaloids, are derived from L-tryptophan [34,56]. The biogenesis of these compounds has been studied using molecular and isotopic labeling in Uncaria guianensis (Aubl.) J.F.Gmel. It was determined that the formation of the oxindole moiety proceeds along a pinacol-pinacolone rearrangement reaction from an indole precursor [57]. The oxindole moiety is comprised of a benzene ring fused with a pyrrole ring, substituted at C-2 with a carbonyl [55]. Oxindole derivatives have been shown to have biological properties that are useful in drug development. There are several positions on the oxindole molecule that can be selectively modified to enhance antibacterial activity. Oxindole derivatives with substitutions at the N-1, C-3, C-4, and C-5 positions have exhibited antibacterial activity. For a comprehensive review of these compounds, please refer to references [58,59,60].

3.2.1. Tetracyclic Oxindole Alkaloids

Tetracyclic oxindole alkaloids contain a tetracyclic structure linked to the methyl 3-methoxyacrylate skeleton (Figure 3, 15–20). The structures depicted in Figure 3 were isolated from U. tomentosa and were reported in several research papers focused on structural determination [10,41,50,61]. The structural differences in the compounds reside in the substitution of an ethyl or vinyl group on the indolizidine ring. Additional structural differences exist at the spiro carbon stereocenter. The presence and verification of these compounds were established through HPLC methods employing reference material and comparison of UV–Vis absorbance curves and retention time [62,63]. Other studies validated the structural identification of these compounds only through HPLC-MS analyses [39].

Two N-oxide variations have also been reported [41]. Structures 17 and 18 represent the N-oxide versions of compounds 15a and 15b, respectively. The N-oxide moiety resides on the nitrogen at position 4 on the indolizine. Compound 19 was isolated and characterized as a minor alkaloid during an investigation into the phenolic composition of U. tomentosa [2]. This compound contains an ethyl substitution at C-20 in the R configuration. In addition, another unique structural characteristic is located on the methyl 3-methoxyacrylate, where the ester group has undergone demethylation into a carboxylic acid. The structure of this compound was confirmed via ¹H and ¹³C NMR spectroscopy [64].

Compounds 20a and 20b are diastereomers with ethyl substitutions at C-20; however, their distinguishing feature is the presence of a hydroxy group on the oxindole ring. These compounds are attributed to U. tomentosa through older work contributed by Hemingway and Phillipson [39,65]. More recent investigations into the alkaloid components of U. tomentosa, such as those reported by Laus and coworkers, have not identified these compounds as present [10,27].

3.2.2. Pentacyclic Oxindole Alkaloids

The pentacyclic oxindole alkaloids present in U. tomentosa are shown in Figure 3 (21–22). These compounds possess the identical oxindole group linked to an indolizidine with an additional di-hydropyran structure fused to the six-member ring of the indolizidine structure. This ring structure is substituted with a methyl group at C-19 and an acetyl group branching from the unsaturated bond from the pentacyclic heterocycle. These compounds have been identified in alkaloid studies of U. tomentosa [10,50]. Structural determinations were established by comparison with NMR datasets [66,67]. A single N-oxide exists from these groups of compounds, Uncarine F N-oxide (22), which is like other N-oxides of U. tomentosa (17 and 18).

The bioactivity of these compounds has been the focus of many investigations. A recent review focusing on the bioactivity of components and extracts from Uncaria tomentosa cites a number of sources [13]. A few of the bioactivities referenced include anticancer activity for compound 21e [68], antineoplastic activity for 21c [69], antioxidant activities for 21f and 21g [70,71], anti-inflammatory activity for 21f, and immunomodulating properties for 21b [72]. The antimicrobial activity of these compounds has not been as thoroughly investigated as other bioactivities, although a few examples of antibacterial evidence were found during the literature search and are presented below.

The stereoisomers 21f and 21g demonstrated in vitro activity against two tuberculosis causing mycobacteria strains resistant to the antibacterial medication isoniazid. Isolated from the leaves of Hallea rubrostipulata (K.Schum.) J.-F.Leroy and tested using a broth microdilution method, the results indicated moderate antimycobacterial activity. Compound 21f demonstrated MIC values of 800 and 400 µg/mL against Mycobacterium madagascariense and M. indicus pranii, respectively. Compound 21g demonstrated MIC values of 800 µg/mL for both M. madagascariense and M. indicus pranii [73].

One of the few examples of compounds directly isolated from Uncaria tomentosa displaying antibacterial activity was reported by Garcia and colleagues. The oxindole 21d isolated from a methanolic extraction of U. tomentosa demonstrated moderate in vitro antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Gram-negative B. subtilis, with MIC values of 150 and 250 µg/mL [74].

4. Polyphenols

The term polyphenol is a broad term that encompasses a large range of compounds. Structurally, all polyphenols contain hydroxyl groups attached to aromatic rings; however, the number of hydroxyl groups as well as the size of these compounds could range from a single benzene ring to a series of fused rings up to 4000 Daltons. This class of compounds is structurally diverse with a variety of carbon skeletal arrangements that make up several different groups of compounds including flavonoids, proanthocyanidins, and phenolic acids.

Polyphenols are found in all types of plants and can vary widely in structure and content. These compounds are all bio-generated through the shikimate/phenylpropanoid or the acetate/malonate secondary metabolic pathways [75]. The role of these compounds range from defensive functions such as antimicrobial and antifungal activities to cell wall strengthening and repair [76]. Polyphenols exhibit antioxidative capabilities as well as solubility in water [75]. As a result, these compounds have a diverse array of bioactivities and are generally considered key components to a healthy diet when consumed from natural plant sources [77,78]. The polyphenol fraction of U. tomentosa includes a variety of compounds. The compounds will be categorized and discussed in relation to antibacterial activity in the following subsections.

4.1. Flavonoids

Flavonoids make up a large portion of the polyphenols present in U. tomentosa. Flavonoids are a group of secondary metabolites found in an array of natural plant materials including fruits, vegetables, grains, bark, roots, stems, and flowers [79]. Flavonoids contain a simple skeleton consisting of three rings commonly referred to as rings A, B, and C depicted in Figure 4, Figure 5 and Figure 6. Flavonoids can be divided into several subclasses based on the saturation, linkage, and substitution of these ring moieties. The subclasses identified in U. tomentosa include flavone, flavonol, and flavan-3-ol structures.

4.1.1. Flavones

The structural composition of flavones can be seen highlighted in structures 23–27 in Figure 4. The key structural components of flavones are localized to the aromatic C ring, also characterized by a ketone substitution at C-4. The position of hydroxy groups can vary; however, the flavones present in U. tomentosa have been noted to contain hydroxy groups in C-5 and C-7 of ring A and C-3′, -4′, -5′, and -6′ of ring B. Flavones putatively identified from U. tomentosa are apigenin (26) and luteolin (27). These compounds were identified upon analysis of an ethanol extraction of U. tomentosa using UV absorption peaks and retention time compared to standard references; however, no efforts were made to spectroscopically characterize the compounds [80]. These basic structures contain the same components of flavones coupled with hydroxy groups at C-5 and -7 of ring A and C-4′ of ring B (highlighted in blue in Figure 4), with an additional hydroxy group at C-3′ for compound 27.

Three additional flavones—artochamin C (23), 5′-hydroxycudraflavone A (24), and dihydrocudraflavone B (25)—were identified via a bioactivity-guided isolation of a methanol extract of U. tomentosa. They were identified utilizing MS and ¹H and ¹³C NMR spectroscopy [81]. These compounds possess additional substitutions when compared to the previously discussed flavones.

Compound 23 contains a dimethyl 2H-pyran structure fused to the A ring via C-7 and -8, with an additional hydroxy on C-3′ of ring B. Compounds 24 and 25 are structurally related, where 24 contains a dimethyl substituted 2-H pyran structure fused at C-6 and -7 of ring A, while 25 contains a saturated pyran. Structures 24 and 25 also contain an oxygen bound to C-6′ of the B ring. In 25, the oxygen is present as a hydroxy moiety, while 24 has an ether that links to the 2 methylpent-2-ene group emanating from C-3 of ring C, forming a 2H-pyran heterocycle.

The compounds 23–25 exhibited antibacterial activity when isolated via a methanol extraction. They were analyzed to determine MIC values for S. aureus, B. subtilis, E. coli, and Klebsiella pneumoniae. Of the compounds tested, 23 produced the best results with MIC values ranging from 4.1 to 6.7 µg/mL, indicating an antibacterial effect on both Gram-positive and Gram-negative bacteria. The MIC values of 25 ranged from 5.1 to 8.3 µg/mL and 24 was found to be least efficacious with values ranging from 9.8 to 16.9 µg/mL [81].

4.1.2. Flavonols

Like flavones, flavonols also have an aromatic C ring with a C-4 ketone; however, they also contain a hydroxy group at C-3 on the C ring (Figure 5, 28–34). Several constituents of U. tomentosa have been identified that possess these characteristics. The simple structures are kaempferol (28) and quercetin (29). Both these compounds possess the structural characteristic of the flavonol subclass, with additional hydroxy substitutions at C-5 and C-7 of ring A (highlighted in blue in Figure 5). Differences in their structures are localized to ring B, which is monosubstituted in 28 and disubstituted in 29. These compounds were identified in an ethanol extraction of U. tomentosa bark utilizing a HPLC with diode-array detection setup to compare retention times and spectral analysis to stock standards [80]. Larger structures have been identified that are products from biosynthetic pathways involving 29 [80,82,83]. Rutin (31) and quercitrin (32) are both glycosides of 29. These three related structures have been identified and structurally characterized in several studies [80,82,83].

When isolated from other plant sources, such as Dicranopteris linearis (Burm.f.) Underw., 31 and 32 exhibited antibacterial activity [84]. When tested against E. coli, B. subtilis, S. aureus, Erwinia carotovora, and B. cereus, 31 produced MIC values of 31.25 µg/mL, 15.5 µg/mL, 31.25, 15.5, and 31.25 µg/mL, respectively, while 32 produced MIC values of 31.25, 15.5, 15.5, 15.5 and 31.25 µg/mL, respectively. These values indicate a moderate level of antibacterial efficacy. Additional cytotoxicity experiments indicated 32 as a strong cytotoxic agent for human leukemia cells with IC₅₀ values of 4.5 and 9.0 µg/mL, while 31 was not found to be cytotoxic (>50 µg/mL).

Compound 30 is not a component in U. tomentosa but has been mistakenly attributed to the plant in prior instances due to mistaken identity with close relative, U. guianensis [85]. In addition, there has been no isolation or structural characterization reported directly from U. tomentosa. Compounds 33 and 34 were incorrectly cited as being sourced from U. tomentosa in a review by Heitzman [12], which references a paper from Aimi and coworkers that isolate and characterize these compounds from U. rhynchophylla rather than U. tomentosa [51]. This correction is also directly related to the previously described error for compounds 7a, 7b, 8, and 9. We have not been able to find a source during our review that demonstrates the isolation or characterization of these compounds from a U. tomentosa plant source.

4.1.3. Flavan-3-ols

Like flavonols, flavan-3-ols of U. tomentosa contain a hydroxy group at C-3 of ring C. However, unlike the previous subclasses, flavon-3-ols contain a C ring that is saturated and therefore possesses stereochemistry. The stereochemistry of C-2 that links the B and C ring is in the S configuration for all flavan-3-ols identified from U. tomentosa, while the stereochemistry of the hydroxy group can alternate (Figure 5, 35–37). Procyanidins formed from monomers of flavan-3-ols have been isolated in U. tomentosa [2,3,86]. The monomers identified in U. tomentosa are the compounds (−)–epicatechin (35) and (+)–catechin (36) (Figure 5). These compounds are stereoisomers. Several sources have indicated the presence of these monomers in U. tomentosa [2,3,82,86,87].

Procyanidin Type

Proanthocyanidins are polymeric structures composed of flavan-3-ol monomer subunits (Figure 6). The most prevalent types of proanthocyanidin structures present in U. tomentosa are procyanidins. Procyanidins are comprised of 35 and/or 36 flavan-3-ol monomers. These monomer units are described above and consist of a hydroxylated B ring, with the orientation of the C ring and hydroxy groups being in the cis or trans conformation. The prefix epi is used to indicate a cis configuration as indicated by (−)–epicatechin (35).

The number of monomers in a procyanidin structure can vary. Structures ranging from 2 to 10 monomers have been identified from polyphenolic assessment of U. tomentosa utilizing mass spectrometry [88]. The molecular weight of procyanidin structures has been indicated to affect bioactivity when consumed, with lower molecular weights (degree of polymerization (DP) < 3) leading to absorption into the gastrointestinal tract and higher molecular weights (DP >3) possessing a lower bioavailability [86].

The monomers that comprise procyanidins can be joined via two different types of interflavan linkages, known as A- and B-type linkages. A-type linkages have two linkages: one between C-4 of the C ring and C-6 or C-8 of the B ring and an ether linkage from a hydroxy group on ring A to C-2 of ring C. B-type linkages occur between C-4 of the C ring and C-6 or C-8 of the A ring. The types of interflavan linkages present in procyanidins of U. tomentosa include C-4→C-6 B-type and C-4→C-8 B-type linkages. The most common type of procyanidins found in nature consist of 35 with B-type linkages, which corresponds to those found in U. tomentosa [3,87,88,89].

Procyanidins from extractions of plant material are known to possess antibacterial activity. The procyanidin extraction of bark from the larch birch tree, Larix gmelinii (Rupr.) Rupr., was characterized via Fourier-transform infrared (FTIR) spectroscopy analysis to possess dimers comprised of 35 and 36 monomers joined via C-4→C-8 linkages. These dimer structures are similar to those identified in U. tomentosa, particularly procyanidins 38–41 (Figure 6). This extract exhibited a weak inhibitory effect on S. aureus with an MIC value of 1.75 mg/mL. There is supporting evidence that the extract’s mode of action affects the integrity and permeability of the cell wall and membrane, protein synthesis, and binds to the grooves of DNA [90].

Procyanidin Dimers

The procyanidin dimer structures present in extracts of U. tomentosa have been identified extensively by Navarro and colleagues [2,3,86]. The dimer structures identified include procyanidin B1–B7 (Figure 6, 38–44). These compounds can be more easily identified as reference material exists for these compounds unlike larger procyanidin structures. Some of the dimers have been indicated to possess antibacterial activity when isolated from other plant sources and tested for MIC. Evidence has been found to support the antibacterial activity of 38–40 when extracted from plants. Procyanidin 38 and 39 were isolated from Antidesma bunius L. and tested against a variety of bacteria. MIC values ranging from 0.12 to 125 µg/mL were reported against Gram-positive bacteria and less potent MIC values ranging from 7.25 to 125 µg/mL against Gram-negative bacteria [91]. This study also proposes that the mechanism of action can be attributed to an effect on the cellular membrane, referencing the stronger antibacterial effect on Gram-positive bacteria and previous investigations into the antimicrobial effect of other flavan-3-ols [92]. Additionally, 40 was isolated from Quercus ilex L. and demonstrated weak antibacterial activity ranging from 98.3 to 393.176 µg/mL [93].

Procyanidin Trimers

Three procyanidin trimers have also been identified by Navarro and colleagues. Two of the trimers were characterized using MS/MS alongside commercial standards. Procyanidin trimer T and C1 were identified, while the third trimer remained uncharacterized as reference material could not be obtained [2]. It should be noted that trace amounts of compound were obtained and reported, but not enough for full characterization. This compound was given the generic name procyanidin trimer B in the study.

Propelargonidin Type

Propelargonidins are polymers composed of flavan-3-ol monomers of the afzelechin type. Afzelechin (37, Figure 5) is similar in structure to 36 but with a single hydroxy substitution on the B ring. The polymeric structures have been identified in extractions of U. tomentosa [88,89]. The monomeric subunits of 37 have not been characterized independently in any reported instances. Propelargonidin dimers have been identified in extracts of U. tomentosa by ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) and triple quadrupole (TQD) tandem mass spectrometry (UPLC/TQD–ESI–MS). These structures have been identified as existing in dimers, trimers, and larger polymeric states, based on mass spectrometry results, but no information relating to linkage type or stereochemistry was provided due to a lack of reference material [89].

Proanthocyanidins Polymeric Structures (DP > 3)

Polymeric structures comprised of both 35 and 37 monomers are also known to exist up to a DP of 11 in ethanol extracts of U. tomentosa [3,88]. These compounds are entirely formed with B-type linkages. The composition of these polymeric structures varies from being completely comprised of 35 to entirely made up of 37, allowing the proanthocyanidins polymers to range from a procyanidin to propelargonidin polyphenol. The detection of the larger structures and their composition is attributed to the m/z charge through MS methods [3,88].

4.2. Miscellaneous Polyphenols

Two stereoisomeric cinchonain-type compounds 45a and 45b have been identified in U. tomentosa with their flavone core highlighted in blue in Figure 7. These compounds were identified in a constituent study investigating bioactive procyanidins of U. tomentosa [94]. The compound 45b was later misrepresented in a 2005 review of the genus Uncaria [12], we have presented the original structure from the Wirth and Wagner study referenced herein. These compounds are flavonoids that possess the epicatechin monomer with a fused δ-valerolactone group replacing the hydroxy group of C-7 on ring A. The stereocenter of these two compounds exists at the site where a catechol structure is linked to the beta carbon of the lactone structure, with 45a in the R configuration and 45b with the S configuration.

Ellagic acid (46) is a common phenolic constituent of flowering plants. This polyphenol is formed from the dimerization of two gallic acid molecules in a head to tail orientation, and subsequent oxidation, to form the two lactone groups present in the molecular structure [95]. The structure of 46 contains four-fused six member rings, two δ-valerolactone and two catechol rings. This molecule was isolated from U. tomentosa utilizing HPLC methods. The structure was characterized by comparing spectral analysis from 200–600 nm and comparisons of retention times with standard references [80]. This isolation and characterization were the only source that identified ellagic acid from an extraction of U. tomentosa. Compound 46 is known to possess antimicrobial activities and the mechanism for these activities include the hyper acidification of the plasma membrane, disruption of electron transport and ATP generation, as well as destabilization of the bacterial membrane [96].

4.3. Phenolic Acids

Several phenolic acids have been identified and extracted from U. tomentosa [2,3,86,89]. The term phenolic acid is used to describe phenolic structures with a variety of hydroxy and methoxy substitutions around a single carboxylic acid substitution on a benzene ring. The two main types of phenolic acids identified from extractions of U. tomentosa are hydroxybenzoic and hydroxycinnamic acids (

Table 1. Structures of hydroxybenzoic acid derivatives (47–53).

Compound	R1	R2	R3	R4	R5
Benzoic, 47	H	H	H	H	H
Salicylic acid, 48	OH	H	H	H	H
4-Hydroxbenzoic, 49	H	H	OH	H	H
Protocatechuic acid, 50	H	OH	OH	H	H
Gallic acid, 51	H	OH	OH	OH	H
Syringic, 52	H	OMe	OH	OMe	H
Vanillic acid, 53	H	OMe	OH	H	H

and

Table 2. Structures of hydroxycinnamic acid derivatives (54–58). Quinic acid is highlighted in olive.

Compound	R1	R2
p-Coumaric acid, 54	H	OH
Caffeic acid, 55	OH	OH
Ferulic acid, 56	OMe	OH
Isoferulic, 57	OH	OMe

, respectively). Ethanolic and aqueous extractions of U. tomentosa leaves gave a concentration of hydroxycinnamic acids from 33.6 to 188.3 µg/g and hydroxybenzoic acids from 113.2 to 983.0 µg/g. Extractions of bark from the same plants provided similar levels of hydroxycinnamic acids, but much higher concentrations of hydroxybenzoic acids ranging from 568.5 to 6206.3 µg/g [89].

These phenolic acids are common natural products, found in a wide variety of plant life including fruits and vegetables [97]. These compounds are typically produced in plants through the shikimic pathway, a pathway used for the biosynthesis of aromatic compounds from precursors of simple carbohydrate molecules [98]. They are used by plants to perform a wide array of functions, some of which are not yet fully characterized or described [98]. Some of the functions are related to responses initiated by microbial contact. There is evidence to suggest that these compounds are utilized by plants in microbial interactions, for both symbiotic relationships and to combat infectious strains [99,100].

4.3.1. Hydroxybenzoic Acids

The hydroxybenzoic acids present in U. tomentosa are described in Table 1 (47–53). These compounds consist of a similar core structure to 47, they then differ in amount and position of hydroxy or methoxy substitutions around the benzene ring. Compounds 48 and 49 are monohydroxylated in an o- and p-position with respect to the carboxylic acid. Compounds 50 and 53 have two substituents in addition to the carboxylic acid and is dihydroxylated in the m- and p-position for 50 or has a m-methoxy and a p-hydroxy substitution for 53. Compounds 51 and 52 have three substituents in addition to the carboxylic acid, with 51 possessing three hydroxy groups, two in the m-positions and one in the p-position and 52 containing two m-groups and a one p-hydroxy group.

There are some structural trends that dictate antibacterial activity in these acids. It has been found that as the number of hydroxy and methoxy substitutions increases around the benzene ring, the antibacterial activity decreases [101,102,103]. The least substituted acid, benzoic acid (47), is commonly utilized as a food preservative due to its antibacterial activity as well as general safety at high dosages. In addition, it was noted in a comparative study that substitution of hydroxy groups with methoxy groups slightly increased the antibacterial activity with 52 performing better than 51 on E. coli and B. subtilis [101].

4.3.2. Hydroxycinnamic Acids

Hydroxycinnamic acids (54–58) are described in Table 2. It should be noted that chlorogenic acid (58) is structurally related to caffeic acid (55). Compounds 54–57 feature the cinnamic acid core, with substitutions meta and para to the short chain, unsaturated carboxylic acid. Compound 54 features a single p-hydroxy. Compounds 55, 56, and 57 have m- and p-substitutions with 55 containing two hydroxy groups, 56 a m-methoxy and p-hydroxy, and 57 a m-hydroxy and p-methoxy. Compound 58 differs in that a quinic acid is linked via esterification of caffeic acid. The double bond of the propene side chain can alternate between the cis and trans positions under increased energetic conditions; however, the trans configuration is more stable.

When compared to hydroxybenzoic acids these compounds generally have a stronger antimicrobial activity [101]. This has been attributed to the propene side chain leading to the carboxylic acid. It is assumed that the increased lipophilicity of this structure makes it more suitable for penetrating bacterial membranes [104]. The addition of hydroxy and methoxy groups also reduces the antibacterial activity of these compounds, but less than observed with hydroxybenzoic acids [101].

Transformation of cinnamic acid from trans- to cis-configuration raises a notable bactericidal and synergistic activity against multiple-drug resistant Mycobacterium tuberculosis [105]. The mechanism for antibacterial activity among these compounds is also well defined. Described as weak organic acids, these compounds’ antimicrobial activity is strongly linked to their ability to diffuse across cell membranes, causing an acidification of the cytoplasm, which leads to cell death [103].

Several of the MICs for compounds 47–58 have been reported for common pathogenic bacteria. In general, these compounds do not possess strong antibacterial activity, with MIC values typically above 500 µg/mL [101,102]. Lower MIC values have been reported in some instances, such as 56 exhibiting an MIC value of 100 µg/mL for E. coli and P. aeruginosa [104]. In addition, 58 (3-0-caffeoylquinic acid or chlorogenic acid) produced MIC values of 100 µg/mL for S. aureus and E. coli and 200 µg/mL for S. enterica and Vibrio parahaemolyticus [106].

Additional sources also indicate a synergistic effect when used in combination with established antibacterial drugs. Compounds 54–56 and 58 all exhibit an enhancement of antibacterial activity when paired with commonly used antibiotics [107,108]. It was also suggested that this effect could be due to these compounds’ ability to damage cell membranes and increase permeability of the antibiotics [108].

5. Terpenes

Terpenes or terpenoids are a large and diverse group of naturally occurring compounds found in a wide variety of plants. These secondary metabolites have a history of medicinal use, as their plant sources have been utilized as folk medicines for hundreds of years [109]. Terpenes continue to be used for medicinal applications to this day with various bioactivities reported due in part to the wide structural diversity of these natural compounds [110]. Antibacterial activity has been reported for this class, especially against Gram-positive bacteria [111]. The mechanism for the antibacterial efficacy is likely due to their lipophilic features which allows these compounds to penetrate microbial cell walls [112]. However, their lipophilic features make them relatively insoluble and difficult to work with respect to medicinal applications.

All terpenes are comprised of isoprene units, a 5-carbon structural unit. These units are assembled via condensation reactions of dimethylallyl diphosphate (DMAPP) with one or more iso-pentenyl diphosphates (IPP) to afford a 10 carbon intermediate geranyl diphosphate (GPP), 15 carbon farnesyl diphosphate (FPP), or 20 carbon geranylgeranyl diphosphate (GGPP) [113]. Larger carbon chains can then be formed from a condensation reaction of FPP and GGPP to form intermediates for terpenoids with up to 40 carbons [113]. The resulting terpenoid is typically classified as a mono-, sesqui-, di-, tri-, or tetra-terpenoid based on the number of isoprene units that comprise its carbon skeleton. Acyclic terpenoids formed from these reactions are then formed into cyclic structures via a cascade reaction catalyzed by plant enzymes [114]. The diversity of the structures created from the biosynthesis reactions is a current topic of research and investigation. More than 100 cyclical scaffolds of triterpenes have been identified, demonstrating the large structural diversity this class of compounds has to offer [115]. In this section, we will discuss the terpenoid structures identified from extracts of U. tomentosa as well as any indications of antibacterial activity.

The types of terpenoids identified and extracted from U. tomentosa to date include pentacyclic acid triterpenes of various skeletal structures including ursolic, oleanolic, quinovic, cincholic, saponins, phytosterols, and a single monoterpene. The references in which these compounds were isolated and characterized will be cited, as well as any instances of antibacterial activity noted for these compounds whether sourced from U. tomentosa or other plant sources.

5.1. Phytosterols

Phytosterols or plant sterols are a group of terpenoids that fall into the triterpene family. The core structure of phytosterols consists of a 29-carbon structure formed from the joining of six isoprene monomers (Figure 8). The core structure consists of four fused rings, three hexacyclic and one pentacyclic, all with trans ring junctions forming a planar structure, and an 8-carbon side chain emanating from C-17. There are three substitutions on the planar ring system, all with stereochemistry above the cyclic plane, a methyl group at C-18, a methyl group at C-19, and a hydroxy group at C-3 [116]. Variation in phytosterol structure can be found at C-4 which may or may not feature methyl or di-methyl substitution, the presence of a double bond at C-5, and/or C-7, methyl or ethyl substitution at C-24 with variable stereochemistry, and a double bond at C-22 with variable stereochemistry. The most abundant type of phytosterols found in nature feature no methyl substitution at C-4, a double bond between C-5, and a methyl or ethyl substitution at position C-24, similar to those found in U. tomentosa [117].

An important chemical attribute of sterols is their amphiphilicity due to the large planar non-polar regions interacting with hydrophobic membranes and the small, polar hydroxy substitution. Phytosterols play an important role in the regulation of plant cell membranes, affecting the fluidity and permeability [116]. In addition, phytosterols play roles in plant cell proliferation and development, as well as defense against biotic and abiotic stressors [118].

The types of phytosterols found in U. tomentosa consist of three of the most common phytosterols; β-sitosterol 59, stigmasterol 60, and campesterol 62 (Figure 8) [119]. These sterols are all 4-desmethyl sterols that feature a double bond at position C-5. Variation to the structure can be seen in substitutions to C-22 of the side chain. Compounds 59 and 60 feature an ethyl substitution, while compound 62 features a methyl substitution. These compounds also differ by the double bond at position C-22, with compound 60 possessing a double bond in the trans configuration and 59 and 62 being fully saturated at this position. The characterization of these compounds was determined via ¹H NMR spectroscopy and mass spectrometry [119].

Minor plant sterols found in U. tomentosa include a sterol glycoside. In this compound, the hydroxyl group at C-3 becomes a target for glycosylation. Compound 61, or β-sitosterol-D-glucopyranoside, has a D-glucose moiety, which has been attached via the hydroxyl at C-3. The core structure of compound 59 is identical to β-stigmasterol. These compounds make up a small percentage of the steroidal fraction of U. tomentosa [120].

Phytosterols are bioactive compounds, with the most prominent activity related to their cholesterol lowering effect, due to the structural similarity between these compounds and cholesterol [118]. Phytosterols also exhibit antibacterial activity. Compound 59 exhibited antibacterial activity against S. aureus and E. coli producing zones of inhibition 17.83 ± 0.58 mm and 14.5 ± 1.84 mm [121], and 13 mm and 14 mm, respectively [122]. Compound 60 exhibited antibacterial activity against seven bacterial strains with MIC values between 12.5 and 25 µg/mL and minimum bactericidal concentration (MBC) values between 25 and 50 µg/mL [123]. Compound 61 indicated antibacterial activity against a variety of microbes with MIC and MBC values reported [124].

5.2. Pentacyclic Triterpene Acids

Pentacyclic triterpenes are a group of terpenoids identified in extracts of U. tomentosa. The core structure of pentacyclic triterpenes consists of five fused six-member rings. Common substitutions to the ring structure include a hydroxy group at C-3, methyl groups at C-10, C-8, C-27, C-19, and C-20, dimethyl groups at C-4 and C-20, carboxylic acids groups at C-17 and C-14, and a double bond at C-12. In general, these compounds are not very soluble in water giving them a low bioavailability and imparting challenges for biomedical applications. The plant biosynthesis of these compounds is thought to proceed through the intermediate β-amyrin [114]. It is known that these compounds accumulate in surface regions of stems and leaves, with evidence pointing toward a possible role in protection from dehydration or herbivores [114].

The plant U. tomentosa contains four varieties of pentacyclic triterpenes. The types of pentacyclic triterpenes include oleanolic, cincholic, ursolic, and quinovic acid (Figure 9). The pentacyclic triterpene families identified in U. tomentosa include a hydroxy group at C-3, a dimethyl substitution at C-4, methyl substitutions at C-10 and C-8, and a carboxylic acid at C-17. Variations to the core structure that distinguish each type of pentacyclic triterpene exist at C-14, C-19, and C-20. Compound 63 contains a methyl group at C-14, and a dimethyl group at C-20, whereas 64 contains a carboxylic acid at C-14 and dimethyl group at C-20. Compound 65 contains a methyl group at C-14, C-19, and C-20, whereas 66 contains a methyl group at C-19 and C-20 and a carboxylic acid at C-14.

Several of the above pentacyclic triterpene acids above have been investigated for antibacterial activity. Compound 65 is the most extensively studied of the group as an antibacterial, with both reviews and individual studies available. Antibacterial evidence collected on 65 reveals a stronger inhibitory effect on Gram-positive bacteria than Gram-negative bacteria [125]. There has been specific interest in testing 65 and its derivatives to combat MRSA, as well as other drug resistant bacteria [126]. The mechanisms of antibacterial activity can be attributed to several factors including its ability to adhere to the cell membrane, reducing the bacteria’s ability to adhere to epithelial cells, as well as its ability to disrupt and impair the bacterial cell membranes both inside and outside the cell [125]. In addition to targeting the bacterial membrane, 65 also exhibits antibiofilm activity [125], with some evidence supporting its ability to reduce expression of genes related to biofilm production [127,128]. Additional evidence supporting the synergistic effect of these compounds when used in combination with existing antibiotics also exists [126]. The issue surrounding the utilization of 65 is its low in vivo bioavailability, due to its poor solubility; however, these attributes can likely be improved by structural modifications, which has become a topic of several investigative studies [129,130,131].

The antibacterial activity of 63 has also been investigated. Like 65, activity seems to increase in Gram-positive bacteria compared to Gram-negative bacteria. MIC values obtained from a microbroth dilution test were 64 µg/mL for S. aureus (ATCC 25923), 32 µg/mL for S. aureus (ATCC 29213), and 8 µg/mL for E. faecalis (ATCC 29212), while reported values of 256 µg/mL and above were found for E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) [132]. A review on the antibacterial activity of 63 against several bacterial strains can be found in reference [133]. A synergistic effect has also been noted when compound 63 and 65 were utilized with β-lactam-type antibiotics [134]. Antibacterial activity of compounds 64 and 66 was not found during the literature search.

5.2.1. Ursolic Acid Derivatives

Derivatives of 65 were also found in U. tomentosa, specifically compounds 67–77 in

Table 3. Ursolic Acid Derivatives (67–77).

Compound	R1	R2	R3	R4	R5	R6	R7	R8	R9	R10
(3β)-3-Hydroxy-7-oxo-urs-12-ene-27,28-dioic acid, 67	OH	Me	Me	H	=O	CO2H	H	Me	H	H
(3β,16α)-16-Hydroxy-3-methoxy-ursa-12,19(29)-diene-27,28-dioic acid, 68	MeO	Me	Me	H	H	CO2H	OH	=		H
(3β,4α)-3,19-Dihydroxy-6,23-dioxo-urs-12-en-28-oic acid, 69	OH	Me	CHO	=O	H	Me	H	Me	OH	H
(3β,4α)-3,19,23-trihydroxy-6-oxo-urs-12-en-28-oic acid, 70	OH	Me	CH2OH	=O	H	Me	H	Me	OH	H
(6β)-6,19-dihydroxy-3-oxo-urs-12-en-28-oic acid, 71	=O	Me	Me	OH	H	Me	H	Me	OH	H
(3β,6β)-3,6,19-Trihydroxyurs-12-en-28-oic acid (uncaric acid), 72	OH	Me	Me	OH	H	Me	H	Me	OH	H
(3β,4α,6β)-3,6,19-trihydroxy-23-oxo-norurs-12-en-28-oic acid, 73	OH	=O		OH	H	Me	H	Me	OH	H
(3β,4α,6β)-3,6,19-trihydroxy-23-oxo-urs-12-en-28-oic acid, 74	OH	Me	CHO	OH	H	Me	H	Me	OH	H
(3β,6β)-3,6,19-Trihydroxy-24-norursa-4(23),12-dien-28-oic acid (floridic acid), 75	OH	=		OH	H	Me	H	Me	OH	H
(3β,4α,6β)-dimethyl ester-3,6,19-trihydroxy-urs-12-ene-23,28-dioic acid, 76	OH	Me	CO2Me	OH	H	Me	H	Me	OH	Me
(3β,4α,6β)-3,6,19,23-tetrahydroxy-urs-12-en-28-oic acid, 77	OH	Me	CH2OH	OH	H	Me	H	Me	OH	H

. These compounds contain various substitutions to the core structure of ursolic acid (65). The following section will discuss these substitutions.

Compound 67 contains a ketone at R₅ and a carboxylic acid at R₆. Compound 68 contains a methoxy substitution at R₁, a carboxylic acid at R₆, hydroxy group at R₇, and an ethene at R₈. These compounds were identified in a methanol extract of dried root bark, separated using reverse phase HPLC (RP-HPLC), and characterized with ¹H and ¹³C NMR spectroscopy [135]. Compounds 69 and 70 are similar in structure, both contain a ketone at R₄ and a hydroxy at R₉. However, at R₃, compound 69 possesses an aldehyde substituent while 70 possess a hydroxymethyl group. Compound 71 includes a ketone at R₁ and a hydroxy at R₄ and R₉. The characterization of compound 69, 70, and 71 was completed utilizing MS, ¹H, and ¹³C NMR spectroscopy, on a methanol extraction of dried plant material, with 69 and 71 first characterized in the paper cited and 70 confirmed by comparison to previously reported data from another plant source [8]. Compound 72 possesses a hydroxy at R₄ and R₉. Antiviral activity for this compound against SARS-CoV-2 was investigated in silico [136]. The findings indicate the compound was a possible candidate of interest due to its binding affinity for an interface between the virus and human receptor cells and its druggability according to the Lipinski rules [136]. Compound 73 contains hydroxy groups at R₄ and R₉, as well as a ketone at R₂.

Compound 74 possesses an aldehyde at R₃ and hydroxy substituents at R₄ and R₉. Compound 75 possesses an ethene group at R₂ and hydroxy groups at R₄ and R₉. Compound 75 was also investigated in silico for potential antiviral activity against SARS-CoV-2 [136]. Compounds 72, 74, and 75 were all characterized using ¹H and ¹³C NMR spectroscopy from a CHCl₃ extraction of plant materials [137]. Compound 75 was also identified in subsequent research by Kitajima and coworkers, and again characterized with ¹H and ¹³C NMR spectroscopy [8]. Compound 76 contains a unique carbomethoxy group at R₃, as well as hydroxy groups at R₄ and R₉, and a methyl group at R₁₀. This compound was characterized from a CHCl₃-MeOH (9:1) extraction using ¹H and ¹³C NMR [138]. Compound 77 contains a hydroxymethyl group at R₃, as well as hydroxy groups at R₄ and R₉. This compound was identified in U. tomentosa by Aquino and colleagues using an extraction of dried plant material in methanol and ¹H and ¹³C NMR spectroscopy to structurally identify the compound [135]. The characterization was compared to established literature [139]. Compound 77 was also identified as possessing moderate antibacterial activity when tested against bacterial strains B. subtilis (ATCC6633), S. aureus (ATCC12600), E. coli (ATCC25922), K. pneumoniae (ATCC13883) and P. stutzeri (ATCC17588), with MIC values of 78 µg/mL for S. aureus (ATCC12600), 156 µg/mL for P. stutzeri (ATCC17588), and 312 µg/mL for all other bacterial strains reported [140].

5.2.2. Glycosylated Pentacyclic Triterpene Acids

The glycosylated versions of pentacyclic triterpenes are also commonly referred to as saponins. These compounds are comprised of a pentacyclic aglycone with various glycones attached at specific positions. These compounds have been identified as being much more soluble, due to their increased polarity, which increases the probability of bioactivity [114]. The glycosylation of the triterpenes is controlled by a family of enzymes known as carbohydrate-active-enzymes family 1 [141].

Several pentacyclic acid glycosides have been found in U. tomentosa, of which the aglycone has been identified as cincholic, pyrocincholic, quinovic, or pyroquinovic acid, and the glycones have been identified as the monosaccharides β-D-glucose, β-D-fucose, β-D-quinovose, β-D-galactose, α-L-rhamnose or the disaccharides of these sugars. It should be noted that in the literature, rhamnose, fucose, and quinovose can also be referred to as 6-deoxy-mannose, -galactose, and -glucose, respectively. The glycones are bound to the hydroxy group at C-3, as well as the hydroxy groups of the carboxylic acids at C-14 and C-17. All pentacyclic acid glycosides contain either one or two glycone additions.

Cincholic Acid Derivatives

Three cincholic acid derivatives have been isolated and characterized from an extract of U. tomentosa. The structure of compounds 78–80 were identified utilizing MS and ¹³C NMR spectroscopy [6] and are described in

Table 4. Cincholic acid derivatives (78–81).

No.	Compound Name
78	Cincholic acid 27-O-β-D-fucopyranosyl-28-O-β-D-glucopyranoside
79	Cincholic acid 27-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside
80	Cincholic acid 27-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranosyl-28-O-β-D-glucopyranoside
81	Pyrocincholic acid 27-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranosyl-28-O-β-D-glucopyranoside (Tomentoside B)

. These compounds only differ by the type of glycones attached to locations R₁ and R₂.

Quinovic Acid Derivatives

Fourteen quinovic acid glycosides have been identified from U. tomentosa and make up most of the glycosides isolated to date [135,138,142,143]. Compounds 82–94 from

Table 5. Quinovic Acid Glycosides (82–95).

No.	Compound
82	Quinovic acid 3-O-β-D-quinovopyranosyl-27-O-β-D-glucopyranoside
83	Quinovic acid 3-O-β-D-quinovopyranosyl-28-O-β-D-glucopyranoside
84	Quinovic acid 3-O-α-L-rhamnoside
85	Quinovic acid 3-O-β-D-fucopyranosyl-27-O-β-D-glucopyranoside
86	Quinovic acid 3-O-β-D-fucopyranosyl-28-O-β-D-glucopyranoside
87	Quinovic acid 27-O-β-D-glucopyranoside
88	Quinovic acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-quinovopyranoside
89	Quinovic acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside
90	Quinovic acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranosyl-27-O-β-D-glucopyranoside
91	Quinovic acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranosyl-28-O-β-D-glucopyranoside
92	Quinovic acid 3-O-α-L-rhamnopyranosyl-(3→1)-β-D-glucopyranoside
93	Quinovic acid 3-O-α-L-rhamnopyranosyl-(3→1)-β-D-glucopyranosyl-27-O-β-D-glucopyranoside
94	Quinovic acid 3-O-β-D-galactopyranosyl-(1→3)-β-D-quinovopyranoside
95	Pyroquinovic acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranosyl-28-O-β-D-glucopyranoside (Tomentoside A)

describe the structures of these compounds. Structural differences are reserved to the glycones attached to the hydroxyl group at C-3 and the hydroxyl group of the two carboxylic acid groups. Eight different glycones have been identified in the research to date, the monosaccharides that make up these glycones are listed in Table 5 and distinguished with individual colors. Improved methods for quantification and identification have been developed since the initial isolation and characterization [144]. Instances of antibacterial activity for 83 and 84 were reported. Compound 83 was investigated in silico for antibacterial activity utilizing a docking simulation with Staph GyraseB 24 and results indicated a comparable binding energy to novobiocin (a known antimicrobial used as the control for the study) [145]. Compound 84 demonstrated antibacterial activity against Haemophilus influenzea when isolated from Nauclea latifolia Smith with a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL [146].

Pyroquinovic and Pyrocincholic Acid Derivatives

A pyroquinovic acid glycoside and a pyrocincholic acid glycoside were identified using ¹H and ¹³C NMR spectroscopy [9]. These compounds interestingly, have only been identified in their glycosidic forms. The core structure of pyroquinovic and pyrocincholic acid have yet to be identified as independent structures in U. tomentosa. The pyroquinovic and pyrocincholic glycosides differ from quinovic and cincholic acid by the lack of a carboxylic acid group at C-14. Both compound 95 and 81 (Table 5), identified in U. tomentosa contain a disaccharide bound to the hydroxy group at C-3 and a monosaccharide bound to the carboxylic acid at C-17.

5.3. Miscellaneous Terpenoid

An additional terpenoid that does not fit into any of the classifications above has also been identified from extractions of U. tomentosa. The monoterpene glycoside, 7-deoxyloganic acid (96), has been identified using ¹H and ¹³C NMR data (Figure 10) [120]. Compound 96 features a cyclopentapyran substituted with a carboxylic acid, the C-7 position with a β-D-glucose, and a methyl group. This compound is an intermediate of the secologanin and iridoid biosynthesis in other plant species; however, neither of these compound types have been identified directly in U. tomentosa in our research [147].

6. Conclusions

This review is unique in that it provides a substantiated, comprehensive collection of the known chemical constituents of Uncaria tomentosa, including their detailed structures. We have also provided corrections from the literature where compounds were accidentally attributed to U. tomentosa. This also demonstrates that extracts and several of the plant’s isolated molecules have exhibited considerable potential as antibacterial agents, warranting further investigation. The significance of increasing bacterial resistance to the currently available array of antibiotics cannot be overstated. New antibiotic drugs are needed and medicinal plants such as cat’s claw are a rich resource to provide new insights and inspiration.