Walsura robusta Roxb. (Meliaceae), a little-known tree with a rich limonoid profile

The plant Walsura robusta Roxb. (Meliaceae) is a robust tree largely distributed in south-east Asia, including provinces of southern China. A few traditional usages of the plant have been mentioned, notably for the treatment of microbial infections. But experimental studies using different types of plant extracts only revealed modest antibacterial effects, and no major antiparasitic activity. Walsura robusta Roxb. is a rich source of secondary metabolites. Several series of limonoids have been isolated from the leaves or the fruits of the plant, such as walsuronoid A-I, walsurins A-E, walsunoids A-I, walrobsins A-R and other cedreloneor dihydrocedrelone-type limonoids, in addition to a few other terpenoids. All information about Walsura robusta Roxb. have been collated in this brief review. The analysis underlines the presence of two limonoids endowed with significant anticancer activities, walsuronoid B and cedrelone. They both activate the production of reactive oxygen species in cancer cells, modulate mitochondrial activities and induce apoptosis of cancer cells. Their molecular targets and mechanism of action are discussed. Walsura robusta Roxb. has a potential for the development of anticancer natural products. The use of the plant extracts could be further considered for the treatment of diseases with a cell proliferation component.

Bailly name refers in fact to W. yunnanensis, as cited in another study (Luo et al., 2006). W. robusta seem to be used in traditional medicine in diverse countries in Asia, but there is a lack of robust information about this traditional usage. Moreover, there is also little experimental evidence to support the traditional medicinal use of W. robusta. Initially, a modest antibacterial activity was reported with an ethanolic plant extract found to inhibit the growth of a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) and the S. aureus ATCC strain 25923 with a minimum inhibitory concentration (MIC) of 1.6 mg/ml Kitpip, 2005a, 2005b). A better level of activity was observed using wood extracts of the plant against the enterohaemorrhagic Escherichia coli strain O157:H7, with MIC of 0.09 and 0.19 mg/ml for the aqueous and ethanolic extracts, respectively (Voravuthikunchai et al., 2004) but a later study reported a marginal bacteriostatic and bactericidal activities with a similar extract (Voravuthikunchai and Limsuwan, 2006). In another study, an aqueous extract of W. robusta was found to mildly inhibit the growth of Grampositive and some Gram-negative bacteria, including E. coli. In the same study, the plant extract was tested against trophozoites of the intestinal parasite Giardia intestinalis but no anti-giardial activity was observed (Voravuthikunchai et al., 2010). Clearly, there is not enough experimental data to support the traditional use of the plant as an anti-infectious agent.

PHYTOCHEMICAL ANALYSES OF WALSURA ROBUSTA ROXB.
Diverse types of secondary metabolites have been isolated from W. robusta, in particular limonoids which are heavily oxygenated, modified triterpenes frequently encountered in Meliaceae and Rutaceae (Citrus species) and to a lesser extent in Cneoraceae and Simaroubaceae (Tan and Luo, 2011;Tundis et al., 2014;Zhang and Xu, 2017). The major limonoids in W. robusta are named walsuronoids A-C, initially isolated from the leaves of the plant (Yin et al., 2007). Walsuronoid A (Fig. 2) bears a seco limonoid skeleton with a 3,4-peroxide bridge and walsuronoids B and C possess an 18(13→14)-abeo-limonoid skeleton. Walsuronoid C has been considered as an oxidation product of walsuronoid B because its furyl ring is easily oxidized when exposed to air in chloroform. A modest growth inhibition of the malaria parasite Plasmodium falciparum was observed with walsuronoids A and B (40% inhibition at the dose of 40 µM) (Yin et al., 2007). Walsuronoid B was also identified from the fruits of the plant together with a series of linonoids, known as walsuronoid F-I and walsurins A-E (Fig. 2) and many other terpenoids .
Walsuronoid B deserves a special mention because it has been shown to exert potent anticancer effects in vitro and in vivo (Geng et al., 2017). The compound dose-dependently inhibited the proliferation of several types of cancer cells, with IC 50 in the range of 3-4 µM. Walsuronoid B blocked cell cycle progression of HepG2 and Bel-7402 liver cancer cells (G 2 /M arrest), with a concomitant up-regulation of cyclin B1 and down-regulation of (phospho)-cdc55C. The compound was found to trigger a massive apoptosis of these malignant cells, with activation of caspases and a drug-induced dysfunction of mitochondria and lysosomes (Fig. 3a). The molecular target of the drug is not known at present, but the compound enhanced the production of reactive oxygen species (ROS) and elevated the expression of the tumor-suppressor protein p53. Interestingly, walsuronoid B (at the intraperitoneal dose of 4 mg/kg) displayed a marked anticancer activity in mice with xenografted HepG2 liver tumors, with an efficacy comparable to that of the standard drug cisplatin (Geng et al., 2017). The activation of the ROS/  (Geng et al., 2017). Walrobsin M represses the expression of the phosphorylated signaling proteins ERK (extracellular signal-regulated kinase) and p38 in THP-1-like macrophages (stimulated with the skin pathogen Propionibacterium acnes), thereby inhibiting the MAPK signaling pathway  b a p53 signaling pathway is central to the mechanism of action of walsuronoid B, as it is the case for other anticancer plant natural products like the cardiac glycoside odoroside A from the plant Nerium oleander Linn. (Chen et al., 2019), betulinic acid (Shen et al., 2017) and gambogic acid (Liang and Zhang, 2016) for examples. There is no doubt that walsuronoid B warrants further studies to better define its mode of anticancer action, tumor selectivity and molecular targets.
Another small group of limonoids isolated from W. robusta has been named walsunoids A-I, isolated together with different cedrelone-type limonoids such as 11β-hydroxyisowalsuranolide and 11β-hydroxydihydrocedrelone ). These compounds were tested against the human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), the enzyme which reduces inert cortisone into active cortisol, and which is frequently implicated in metabolic diseases (obesity, diabetes). A modest inhibition of human 11β-HSD1 was observed with walsunoid H (Fig. 2) in vitro (IC 50 = 9.9 µM) .
Two other analogues with an atypical 5-oxatricyclo[5.4.1 1,4 ] hendecane ring system have been isolated from the root bark of the plant. They were named walrobsins A and B (Fig. 2). These two compounds showed no cytotoxic activity, but a modest anti-inflammatory activity was reported with walrobsin A. The compound was found to inhibit the release of the proinflammatory cytokine interleukin-1β from lipopolysaccharideactivated RAW264.7 macrophages and to dose-dependently inhibit the expression of the inducible nitric oxide synthase (iNOS) enzyme, the enzyme responsible for nitric oxide (NO) production . A subsequent phytochemical analysis from the root bark of W. robusta led to the identification of other compounds with a 5-oxatricyclohendecane ring system and named walrobsins C-to-R . The bioactivity of these compounds has not been deeply investigated but a preliminary evaluation indicated that walrobsin M (Fig. 3b) presents an interesting anti-inflammatory profile, with a druginduced down-regulation of the expression of protein phospho-ERK and phospho-p38 in inflamed THP-1 macrophage cells .
The fruits of the plant also contain a series of compounds structurally similar to cedrelone (Fig. 2) and dihydrocedrelone, which were found to potentiate the cytotoxicity of the anticancer drug doxorubicin in human MCF-7/DOX breast cancer cells, normally resistant to the drug. These compounds could efficiently reverse the multidrug resistance (MDR) phenotype in vitro . Cedrelone limonoids have been isolated from W. robusta, W. yunnanensis (Ji et al., 2014), Toona sinensis (Jiang et al., 2020) and from the leaves of Trichilia americana, another plant of the Meliaceae family (Ji et al., 2015). Acetylation of the cedrelone core affords cedrelone acetate, a synthetic molecule presenting enhanced cytotoxic properties and efficient to revert the malignant phenotype of cancer cells (Becceneri et al., 2020). Cedrelone itself is an interesting anticancer agent because this compound was found to activate the expression of the protein PBLD (phenazine biosynthesis-like domain-containing protein) which is often down-regulated in hepatocellular carcinoma (HCC) (Wu et al., 2019). PBLD functions as a tumor suppressor. An increase of the expression of PBLD could reduce HCC cell growth and invasion via inactivation of several tumorigenesisrelated signaling pathways (Li et al., 2016). Through the activation of PBLD in cells, cedrelone regulates the Ras and Ras-proximate-1 (Rap1) signaling pathways and this signaling action triggers apoptosis of cancer cell, while reducing cell proliferation and the epithelialmesenchymal transition (Wu et al., 2019). The antitumor capacity of cedrelone has been characterized in different experimental models, notably using drug-resistant human glioma cells  and leukemia cells . In both cases, the compound selectively affected the ERK/MAPK signaling pathway. A direct interaction of cedrelone with the ERK1 kinase has been postulated, based on a computational docking analysis. The compound was predicted to bind to a small pocket on the protein, leading to the kinase activation (Fig. 4). Similarly, a combination of docking and molecular dynamic simulations has suggested that cedrelone could bind to the multi domain ceramide transfer protein (CERT), a protein implicated in sphingolipid metabolism and which allows the transport of ceramide from the endoplasmic reticulum to the Golgi apparatus (Fig. 4). Cedrelone would bind to the linked PH and START domains of CERT, thereby inhibiting the protein (Ghoula et al., 2020). Inhibition of CERT is considered as an appropriate mechanism to re-sensitize cancer cells to chemotherapy (Palau et al., 2018;Kumagai and Hanada, 2019). The anticancer effects of cedrolone have been evidenced using other cell types, including MDA-MB-231 breast cancer cells, NCI-H460 non-small cell lung cancer cells and A375 melanoma cells (Cazal et al., 2010;Fuzer et al., 2013). Cedrolone is an interesting natural product which also displays anti-fungal and insecticidal activities (Govindachari et al., 1995(Govindachari et al., , 2000. It exhibits a sub-micromolar activity against the protozoan parasite Cryptosporidium parvum in vitro (EC 50 = 0.27 µM) (Jin et al., 2019) and causes lethal and sublethal effects on the armyworm Spodoptera frugiperda (Giongo et al., 2016).
Other terpenes and sesquiterpenes have been isolated from W. robusta, such as the carotane sesquiterpene 10-oxo-isodauc-3-en-15-al (Fig. 2) and its nitro derivative, both isolated from a methanolic extract of the plant leaves. These two compounds showed no antimicrobial activity against Staphylococcus aureus and different MRSA strains (Hou et al., 2013). It is worth to note that this isodaucane type sesquiterpenoid can also be found in the leaves of the sunflower crop (Helianthus annuus L.) and it has revealed a significant herbicidal activity. Indeed, 10-oxo-isodauc-3-en-15-al was found to inhibit coleoptile elongation and is considered as a useful allelopathic compound (Fuentes-Gandara et al., 2019). It has been found in diverse plants, such as Senecio crassiflorus (Pox.) De Candolle (Jares and Pomillo, 1989), Uvaria lucida (Moriyasu et al., 2012), Chromolaena laevigata (Misra et al., 1985) and Conza linifolia (Hussein et al., 1995). Surprisingly, this compound and its C-10 epimer which is known as sinulin A, can be found also in a marine organism, the Xisha soft coral Sinularia sp. (Qin et al., 2018).

Plants of the Walsura genus have been little studied thus far.
For examples, the PubMed data bank comprises 30 references with the name Walsura, compared to >2000, 300 and 150 for the genera Trichilia, Aglaia and Xylocarpus, which also belong to the Meliaceae family (as of April 2021). Moreover, in the Walsura genus, the specie W. pinnata Hassk (synonyms: W. cochinchinensis (Baill.) Harms and W. yunnanensis C.Y. Wu) is more frequently studied than W. robusta Roxb. This is perhaps not surprising because the plant is not so frequently used in traditional medicine, except in very local areas. W. robusta trees should be investigated further, for the usefulness of their woods and the diversity of the secondary metabolites which can be isolated from the bark, leaves and fruits of the plant.
As presented here, W. robusta Roxb. provides a rich reservoir of limonoids, a huge family of natural products widely distributed in nature. Limonoids can be isolated from many plants, and some of them can be (semi) synthesized (Zhang and Xu, 2017;Fu and Liu, 2020). They are particularly abundant in Meliaceae (Tan and Luo, 2011). The great majority of the new natural products isolated from W. robusta over the past few years are limonoids, such as the walsuronoids, walrobsins, walsurins, and walsunoids. Beyond the isolation and characterization of these compounds, the bioactivity measurements have been limited mainly to a few preliminary in vitro tests, notably to evaluate their antimicrobial activity. No outstanding activity has been reported, but occasionally a few compounds revealed a modest antibacterial or anti-inflammatory effect. Two compounds emerge from this literature survey: walsuronoid B and cedrelone, because they both present noticeable anticancer properties against multiple cancer cell lines. Walsuronoid B stands as a potent anticancer agent active in a xenograft model in vivo and cedrelone also displays marked anticancer activities. They both impact mitochondrial activities, cause an increase of ROS in cells, and trigger apoptosis of cancer cells (Geng et al., 2017;Cao et al., 2019). They could be considered as prototypes for the design of novel anticancer derivatives (Becceneri et al., 2020).
Plants of the Walsura genus, and the specie W. robusta Roxb. in particular, have received little attention thus far. This robust tree, well distributed in south Asia, should be considered further in the search of natural products of medicinal interest. We can certainly recommend the investigation of extracts of the plant and its abundant fruits, for the treatment of cancers, and possibly other diseases with a cell proliferation component. The specie deserves its named "robusta", as a robust provider of bioactive limonoids.

FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

DECLARATION OF COMPETING INTEREST
The author declares no conflict of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Figure 4:
Two potential protein targets of the limonoid cedrelone, which can be isolated from Walsura robusta Roxb. The molecular model (left) shows a drug fragment bound to the ERK1/2 kinase, to modulate the phosphorylation and catalytic activity of the enzyme (PDB: 6G91) (Heightman et al., 2018). A molecular modeling analysis has predicted that cedrelone can bind to the same site of the protein, thereby activating the kinase . Molecular modeling has also predicted that cedrelone can bind to the PH domain of the multi domain ceramide transfer protein (CERT) to inhibit its transport activity (Ghoula et al., 2020). Binding of the compound to ERK1/2, CERT and other proteins likely contributes to the anticancer activity of this limonoid