From Ethnomedicine to Plant Biotechnology and Machine Learning: The Valorization of the Medicinal Plant Bryophyllum sp.

pharmaceuticals Review From Ethnomedicine to Plant Biotechnology and Machine Learning: The Valorization of the Medicinal Plant Bryophyllum sp. Pascual García-Pérez 1,2,†, Eva Lozano-Milo 1,2,†, Mariana Landin 3,4 and Pedro P. Gallego 1,2,* 1 Applied Plant & Soil Biology, Plant Biology and Soil Science Department, Biology Faculty, University of Vigo, E-36310 Vigo, Spain; [email protected] (P.G.-P.); [email protected] (E.L.-M.) 2CITACA—Agri-Food Research and Transfer Cluster, University of Vigo, E-32004 Ourense, Spain 3Pharmacology, Pharmacy and Pharmaceutical Technology Department, Grupo I+D Farma (GI-1645), Pharmacy Faculty, University of Santiago, E-15782 Santiago de Compostela, Spain; [email protected] 4Health Research Institute of Santiago de Compostela (IDIS), E-15782 Santiago de Compostela, Spain *Correspondence: [email protected] †Both authors contributed equally in this manuscript. Received: 10 November 2020; Accepted: 2 December 2020; Published: 4 December 2020   Abstract: The subgenus Bryophyllum includes about 25 plant species native to Madagascar, and is widely used in traditional medicine worldwide. Different formulations from Bryophyllum have been employed for the treatment of several ailments, including infections, gynecological disorders, and chronic diseases, such as diabetes, neurological and neoplastic diseases. Two major families of secondary metabolites have been reported as responsible for these bioactivities: phenolic compounds and bufadienolides. These compounds are found in limited amounts in plants because they are biosynthesized in response to different biotic and abiotic stresses. Therefore, novel approaches should be undertaken with the aim of achieving the phytochemical valorization of Bryophyllum sp., allowing a sustainable production that prevents from a massive exploitation of wild plant resources. This review focuses on the study of phytoconstituents reported on Bryophyllum sp.; the application of plant tissue culture methodology as a reliable tool for the valorization of bioactive compounds; and the application of machine learning technology to model and optimize the full phytochemical potential of Bryophyllum sp. As a result, Bryophyllum species can be considered as a promising source of plant bioactive compounds, with enormous antioxidant and anticancer potential, which could be used for their large-scale biotechnological exploitation in cosmetic, food, and pharmaceutical industries. Keywords: Bryophyllum; traditional medicine; secondary metabolism; bioactive and phenolic compounds; bufadienolides; antioxidants; cytotoxic activity; plant tissue culture; artificial intelligence 1. Introduction The genus Kalanchoe (Adanson, 1736 [ 1 ]) belongs to the Crassulaceae family and comprises 150 to 200 succulent species native to Madagascar and naturalized across Africa, South America, and Asia [ 2 , 3 ]. Kalanchoe constitutes a complex genus with an intricate taxonomy, not yet clearly elucidated. Two different trends have been remarkable throughout the published literature concerning bothitsnomenclatureandsystematics[ 4 ].Authorsdisagreewhethertheclassificationisbasedonasingle genus called Kalanchoe (sensu lato) or three separate sections: Kalanchoe (sensu stricto), Bryophyllum Kahl. (Salisbury, 1805 [ 5 ]), and Kitchingia (Baker, 1881 [ 6 ]). However, other authors propose a three-subgenera classification of the genus Kalanchoe, due to different evolutive arguments, morphological traits [ 7 ] and molecular analyses [ 8 ], including Kalanchoe,Bryophyllum and Calophygia [ 4 ]. Amongst the different Pharmaceuticals 2020,13, 444; doi:10.3390/ph13120444 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020,13, 444 2 of 23 subgenera, the subgenus Bryophyllum includes around 25 species, endemic to Madagascar [ 9 ] that gained much interest on plant science research, as they are considered model plants for different physiological features: the Crassulacean Acid Metabolism (CAM) [ 10 ], vegetative reproduction [ 11 ], plant cell regeneration [ 12 ], and a source of therapeutical compounds [ 13 ]. Nevertheless, the most relevant feature associated to this subgenus is the use of their constitutive species in the traditional medicine worldwide, thus considering Bryophyllum sp. as medicinal plants, due to their associated bioactivities [13]. CAM photosynthesis is an advantageous adaptative strategy that enables plant adaptation to arid ecosystems, as it is the case of the whole Kalanchoe genus [ 14 ]. Bryophyllum species present a flexible CAM regime, with no time restriction on CO 2 uptake, which is fixed at night [ 15 ]. On the other hand, Bryophyllum sp. present a highly specialized asexual reproductive mechanism, based on the symmetric plantlet development along the leaf margins or leaf tips of adult plants (Figure 1) [ 12 , 16 ]. Such clonal-spreading reproductive mechanism is driven by a complex phenomenon that combines both embryogenic and organogenetic events that has not been fully elucidated to date [ 17 – 21 ]. Both the metabolic and reproductive patterns found on Bryophyllum sp. contribute to the invasiveness of these species. It allows them a rapid colonization of unexplored territories with high adaptative efficiency, which has contributed to their worldwide naturalization [22,23]. Figure 1. In vitro -cultured plants of B. daigremontianum ( left ); B. × houghtonii ( center ); and B. tubiflorum (right). Bars =1 cm; arrows indicate plantlets formed asexually on leaf margins. Original figure. Bryophyllum and other Kalanchoe species have been widely used in the traditional medicine of vast regions throughout Africa, South America, and Asia [ 24 ]. Because of its wide distribution and ubiquitous medicinal use, much research on this subgenus has focused on Bryophyllum pinnatum (Lam.) Oken [ 25 – 27 ]; however, there is an extensive variety of other species that have also been exploited in Ethnomedicine, such as: B. daigremontianum (Raym.-Hamet et Perr.) Berg. [ 28 ], B. tubiflorum Harv. [ 29 , 30 ], and B. × houghtonii D.B. Ward (syn. B. daigremontianum × tubiflorum) [ 31 ]. Leaf and root-derived formulations have been mostly used for the treatment of several common illnesses such as burns, wounds, insect bites, skin diseases, cough, fever or several infections, and chronic diseases, such as diabetes, and neurological and neoplastic diseases (Table 1). Pharmaceuticals 2020,13, 444 3 of 23 Table 1. Ethnobotanical uses of Bryophyllum species. Species Ethnobotanical Uses Plant Organ Locations 1References B. crenatum (Andr.) Baker Wounds, smallpox, otitis, cough, asthma, palpitations, headache, abscesses, convulsions, general debility, diabetes, obstetrics and gynecology, vermifuge, abortion, antimicrobial treatment Leaves Roots Africa [32–35] B. daigremontianum Raym.-Hamet et Perr. Leucorrhea, dysmenorrheal, carminative, psychic agitation, anxiety, restlessness Leaves Bangladesh [28,36] B. fedtschenkoi Raym.-Hamet et Perr. Analgesic, cytotoxic, antimicrobial treatment Leaves Aerial parts Woody stems Brazil [37–39] B. mortagei (Raym.-Hamet et Perr.) G.E. Wickens Digestive disorders, neoplastic diseases, vermifuge, antimicrobial treatment Aerial parts Flowers Roots Mexico, Colombia, Indonesia [37,40–42] B. pinnatum (Lam.) Oken Wounds, burns, coughs, earache, headache, muscle pain, asthma, bronchitis, pneumonia, arthritis, rheumatism, ulcers, diabetes, urinary bladder stones, dysentery, diarrhea, vermifuge, antibacterial, insect bites, fevers, menstrual disorders, nausea, tumors, gynecology Leaves Roots Nigeria, Uganda, Madagascar, India, China, Vietnam, Bangladesh, Australia, Brazil, Peru, Trinidad and Tobago [43–52] B. serratum (Mann. and Boit.) Blanco Pain, inflammation, fever, antiviral Stems Taiwan [53,54] B. tubiflorum Harv. Wounds, epilepsy, vermifuge, neoplastic diseases Leaves Brazil, Ethiopia [29,30] 1Locations where the ethnobotanical uses have been reported. Pharmaceuticals 2020,13, 444 4 of 23 The great therapeutic potential reported on Bryophyllum sp. [ 39 ] has promoted in-depth phytochemical analysis to adequately evaluate its biological and pharmacological properties [ 55 , 56 ]. Several authors have demonstrated the whole bioactive potential of Bryophyllum-derived extracts, acting as multifaceted agents. The anti-inflammatory activity of Bryophyllum extracts has been determined by different methods using both in vivo and in vitro models. For instance, aqueous extracts from B. pinnatum were shown to exert a relevant effect against croton oil-induced ear edema and carrageenan-induced paw edema in murine models, driven by a decrease in pro-inflammatory cytokines [ 57 ]. Moreover, different flavonoids produced by B. tubiflorum showed an inhibitory effect on nitric oxide production by lipopolysaccharide-induced macrophage in vitro RAW264.7 cell line [58]. The antimicrobial activity attributed to Bryophyllum extracts was shown to present a high effectiveness against a wide range of both bacterial and fungal activities. In this sense, hydroethanolic extracts from B. fedtschenkoi showed a strong inhibitory effect against different antimicrobial resistant strains from the ESKAPE complex, including both Gram-negative and Gram-positive bacteria [ 37 ]. Similarly, the bactericidal effect of B. crenatum leaf juice against Bacillus subtilis and Klebsiella pneumoniae was also reported, as well as high effectiveness of methanol extracts from B. pinnatum to Gram-positive bacteria [ 34 ]. Moreover, different isolated fractions from B. daigremontianum ethanolic extracts promoted a potent activity against Safase S-04 yeast strain, fungi, such as Candida albicans and Aspergillus niger, and bacteria, including Staphylococcus aureus and Escherichia coli [ 59 ]. Furthermore, the antiviral activity of Bryophyllum extracts has been also assessed for relevant viral diseases. It is the case of the antiviral activity of kaempferol derivatives from B. daigremontianum against Herpes Simplex Virus (HSV) types 1 and 2 [ 60 ] and bryophyllin B from B. pinnatum as a potent inhibitor of Human Immunodeficiency Virus (HIV) [61]. Additionally, the analgesic and sedative properties of Bryophyllum extracts were evaluated using in vivo murine models, indicating that leaf extracts from B. crenatum showed a protective effect against formalin and acetic acid-induced pain and inhibited the manifestation of seizures under convulsant agents application [32]. The antioxidant properties of Bryophyllum extracts have been widely reported by a plethora of different methods. The radical scavenging activity against 2,2-diphenyl-picryl-hydrazyl (DPPH), superoxide anion and nitric oxide of B. daigremontianum,B. tubiflorum,B. × houghtonii, and B. pinnatum leaf and aerial part extracts was reported [ 62 , 63 ]. The inhibition of lipid peroxidation by hydromethanolic extracts from aerial parts of B. daigremontianum,B. tubiflorum, and B. × houghtonii, cultured in vitro was also determined [ 64 ]. Moreover, cell-based in vitro antioxidant assays have been performed for the inhibition of lipid peroxidation of root extracts from B. daigremontianum [24]. Bryophyllum extracts have been also shown to present insecticidal properties, as a consequence of bufadienolide production, as reviewed later. In this sense, methanolic leaf extracts from B. daigremontianum,B. pinnatum, and B. × houghtonii showed an intense effect against silkworm larvae (Bombyx mori) [65–67]. Moreover, cardioprotective and antihypertensive properties were attributed to different Bryophyllum sp. [ 68 ]. For instance, the aqueous extracts of B. pinnatum have been shown to exhibit in vivo antihypertensive activity on high salt-loaded rats models [ 69 ]. Furthermore, isolates from B. daigremontianum root extracts developed an in vitro anti-thrombotic activity [70]. Against all the bioactivities associated with Bryophyllum sp., the cytotoxic activity gained much interest during the phytochemical characterization of these species [ 71 ]. A great variety of in vitro models have been employed for the determination of cytotoxic and anti-cancer activities on different Bryophyllum species, whose extracts have been tested against a high number of cancer cell lines [ 13 , 68 ]. Due to the relevance of this bioactivity, the cytotoxic properties of Bryophyllum extracts are included during this review. Finally, there are additional health-enhancing properties related to Bryophyllum sp., as it is the case of hepatoprotective, antidiabetic activities. Thus, the leaf juice and aqueous of B. pinnatum showed Pharmaceuticals 2020,13, 444 5 of 23 a marked in vivo hepatoprotective effect on carbon tetrachloride-induced hepatotoxicity in rats [ 72 ], as well as hypoglycemic and hypocholesterolemic effects in streptozotocin-induced diabetic rats [73]. As a result, the combination of all bioactivities attributed to Bryophyllum sp. aroused the interest in the study of their great therapeutic potential, which is a challenge, as it is an unexplored subgenus with countless potential as a health promoter. This is a systematic review in which general search engines, including PubMed, the Web of Science, and Google Scholar were employed, according to Preferred Reported Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 2. Bryophyllum sp. Secondary Metabolites as Antioxidants and Anticancer Agents It is now well-known that the full set of bioactivities attributed to Bryophyllum sp. is developed by a plethora of phytoconstituents, including phenolic compounds, bufadienolides, organic salts, terpenoids and fatty acids [ 55 ]. Phytoconstituents are considered secondary metabolites, since they are biosynthesized by induction of secondary metabolism, which is responsible for the defensive and adaptative plant response against environmental threads and biotic stress [ 74 , 75 ]. Phenolic compounds and bufadienolides are considered the two main families of secondary metabolites of Bryophyllum sp., widely distributed throughout the subgenus [ 13 ]. Furthermore, they are responsible for the bioactivity associated with Bryophyllum sp. and, consequently, a deeper insight into these compounds will be provided. 2.1. Phenolic Compounds Two major subfamilies of phenolic compounds have been widely reported for Bryophyllum sp.: phenolic acids and flavonoids [ 76 , 77 ], which have been recently found to accumulate inside highly specialized leaf cells, called idioblasts [78]. The antioxidant activity of Bryophyllum phenolic compounds, focused on the free-radical scavenging activity, has been largely determined [ 63 , 79 ]. Recently, the antioxidant capacity of Bryophyllum extracts for preventing the lipid oxidation of omega-3 enriched fish oil emulsions was reported, thus conferring a valuable approach for the application of Bryophyllum-derived by-products in the food and pharmacological industries [ 64 ]. In the same way, the polyphenols from Bryophyllum-derived extracts may be efficiently purified using environmental-friendly procedures, like the use of activated carbon [ 80 ]. These approaches have been developed in order to allow the industrial exploitation of Bryophyllum polyphenols, due to the increasing interest in the research of these medicinal plants. The great diversity of bioactivities described for these compounds places the phenolic compounds of Bryophyllum sp. as one of the main families of plant secondary metabolites that boost the phytochemical potential of this subgenus [62,64,81]. 2.1.1. Phenolic Acids Three species of Bryophyllum present high content in phenolic acids: B. pinnatum,B. daigremontianum, and B. tubiflorum, mostly located in leaf tissues (Table 2) [ 76 , 82 ]. Both subfamilies of phenolic acids have identified compounds in either free or glycosylated forms. Caffeic acid and ferulic acid are the most abundant cinnamic acids, while within the benzoic acids it is protocatechuic acid. β -resorcylic and γ-rosorcylic acids have also been referenced, although these are more unusual. [63]. Concerning bioactivities, phenolic acids are considered powerful antioxidants whose activity depends on the number, position, and combination of hydroxyl groups within their structure [ 83 ]. Potential therapeutic properties for them have also been reported, as they promote antimicrobial, antiviral, cytotoxic, and anti-inflammatory activities [ 84 – 87 ]. Phenolic acids from Bryophyllum-derived extracts have already been related to the development of antibacterial and antifungal activity against a series of pathogenic microorganisms [ 88 ], antioxidant activity, and cytotoxicity against human lymphoblastic leukemia J45 and H9 T-cell lines [63]. Pharmaceuticals 2020,13, 444 6 of 23 Table 2. Phenolic acids reported in Bryophyllum sp. Subfamily Compound 1Species 2References Cinnamic acids p-Coumaric acid BD, BP, BT [63,82,88–90] Caffeic acid BD, BP, BT [63,79,88,91] Chlorogenic acid BD, BT [63,92] Ferulic acid BD, BP, BT [26,63,82,92,93] Benzoic acids p-Hydroxybenzoic acid BD, BP, BT [91] Protocatechuic acid BD, BP, BT [26,63,82,91,93] Vanillic acid BT [58,78] Gallic acid BD, BP, BT [63,78,82,88,90,91,93] Syringic acid BD, BP, BT [63,78,90] 1 Compounds are named as their free-form to simplify the identification. 2 BD: B. daigremontianum; BP: B. pinnatum; BT: B. tubiflorum. 2.1.2. Flavonoids Flavonoids are universally found in Bryophyllum sp. in O-glycosylated form. To a large extent, they have been reported in three species, namely: B. pinnatum,B. daigremontianum and B. tubiflorum (Table 3). The flavonol glycosides were shown as the most abundant subfamily of flavonoids, showing a restricted accumulation on leaf tissues [ 13 , 76 , 90 ]. Both kaempferol and quercetin glycosides were found in Bryophyllum species [ 39 , 94 , 95 ]. Other flavonoid subfamilies, such as flavones and catechins, have also been reported, and a number of anthocyanins have been isolated from the flowers of different species [ 39 , 96 ], which are stored in the foliar idioblasts of B. daigremontianum [ 82 ] and B. tubiflorum [ 78 ]. The antioxidant activity of flavonoids is directly proportional to the number and position of hydroxyl groups in their structure [ 97 ], that assist in the dissipation of electrons generated after UV-overexposure [ 98 ]. Additionally, they also prevent lipid peroxidation [ 99 ] (by decomposing lipid peroxides and scavenging harmful free-radicals) and develop an effective metal chelation activity [ 100 ]. The free-radical scavenging [ 62 , 101 , 102 ] and lipid oxidation preventing activities [ 64 ] of Bryophyllum-derived extracts rich in flavonoids have already been reported. Other bioactivities, such as antibacterial [ 103 ], antiviral [ 104 ], cytotoxic [ 105 ], anti-inflammatory [ 106 ], cardioprotective [ 107 ], sedative and anti-diabetic activities [ 108 ] have been associated to flavonoids. These bioactivities have been extensively studied for Bryophyllum sp. and have also been related to flavonoid content, mainly using B. pinnatum as a plant model [88,89,94,95,109,110]. Table 3. Flavonoids reported in Bryophyllum sp. Subfamily Compound 1Species 2References Flavanones Naringenin BT [92] Flavones Luteolin BP [89,94,111] Apigenin BP, BT [50,78] 4’,5-dihydroxy-3’,8-dimethoxyflavone BP [109,112] Acacetin BP [90] Diosmetin BP [90] Afzelin BP [102] Galangustin BT [58] Hispidulin BT [92] Flavonols Quercetin BD, BP, BT [58,77,78,88,89,92,94,95,109] Kaempferol BD, BP, BT [77,78,88–90,92,102,109,112] Quercitrin BP [109,112] Myricetin BD, BP, BT [77,90,92] Rutin BP [89,94] Isorhamnetin BD, BP [77,88] Kaempferitrin BP [102] Herbacetin BT [58] Patuletin BD [77] Isoquercetin BT [92] Aromadendrin BT [92] Galangin BT [92] Pharmaceuticals 2020,13, 444 7 of 23 Table 3. Cont. Subfamily Compound 1Species 2References Flavanols Catechin BP [89] Epicatechin BT [92] Epigallocatechin BP [111] 1Flavonoids are named as their free-form to simplify the identification. 2BD: B. daigremontianum; BP: B. pinnatum; BT: B. tubiflorum. 2.2. Bufadienolides Bufadienolides constitute a subfamily within cardiac glycosides family of secondary metabolites and they are considered polyhydroxy C-24 steroids, presenting an α -pyrone ring at the C-17 β position (Figure 2) [ 113 ]. Bufadienolides presence in Bryophyllum species is genotype and organ dependent [ 68 ], being four species the most representative sources of these compounds: B. daigremontianum, B. ×houghtonii, B. tubiflorum, and B. pinnatum (Table 4). Universally-distributed bufadienolides, such as bersaldegenin and bryophyllin derivatives [ 77 , 114 ], can be found together with genotype-specific compounds, such as kalanchosides [115] and kalanhybrins [71]. Figure 2. Basic molecular structure of bufadienolides. As cardiac glycosides, the original bioactivity attributed to bufadienolides is their cardiotonic activity, acting as inhibitors of the sodium pump at the myocardial tissue [ 116 ]. However, its reduced therapeuticwindow conditionsits efficacy, allowing eventualcardiotoxicevents duetooverdosage[ 117 ]. In fact, the accidental consumption of Bryophyllum species by different mammals is one of the leading causes of cattle mortality in Africa [ 118 ], with reporting episodes of stroke, subendocardial hemorrhages, and heart tissue necrosis [ 119 ]. The biosynthesis of bufadienolides is a plant defensive mechanism against insect and herbivore attacks. They have already been reported as effective insecticidal compounds [31]. Bufadienolides have also been described as potent anticancer agents, as demonstrated by a number of in vitro studies with multiple cancer cell lines (Table 4) [ 120 ]. Nevertheless, their inherent toxicity difficult their administration in animal and human models [ 121 ]. Current research on these compounds is focused on finding effective and safer semi-synthetic derivatives [122]. Table 4shows the associated bioactivities of identified bufadienolides in Bryophyllum sp., with a special focus on the cytotoxic activity of these compounds, being effective against relevant cancer cell lines, mainly those derived from breast, ovarian and lung carcinomas [71,115]. The bioactivity of phenolic and bufadienolides compounds reveals an unexploited phytochemical potential associated with Bryophyllum sp. However, research on these secondary metabolites is still very limited, since their concentration and activity depend on adaptive responses of plants, which is why low-yield extraction protocols have been reported [ 61 , 123 ]. Consequently, in order to explore the phytochemical properties of these medicinal plants, the establishment of efficient biotechnological approaches is required to achieve the valorization of Bryophyllum subgenus. Pharmaceuticals 2020,13, 444 8 of 23 Table 4. Bufadienolides identified in Bryophyllum sp. and their associated bioactivities. Species 1Plant Organ Bufadienolides Bioactivities 2References BD Roots 11 α ,19-dihydroksytelocinobufagin, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate, 19-(acetyloxy)-3β,5β,11α,14-tetrahydroxyl-12oxo-bufa-20,22-dienolide and 19-(acetyloxy)-1b,3b,5b,14tetrahydroxyl-bufa-20,22-dienolide Moderate antioxidant activity using in vitro blood plasma model under peroxynitrite-induced oxidative stress. Effective for prevention of lipid hydroperoxides generation and thiobarbituric acid-reactive substances (TBARS) [24] BP Leaves Bryophyllin A and C Insecticidal against silkworm larvae [66] BH Leaves Bryophyllin A and C, bersaldegenin-1-acetate, bersaldegenin-3-acetate, bersaldegenin-1,3,5-orthoacetate, daigremontianin, methyl daigremoniate Insecticidal against silkworm larvae, except for bersaldegenin-1-acetate. Cytotoxic effect of bersaldegenin-1,3,5-orthoacetate and daigremontianin against induced Raji cell line (Burkitt’s lymphoma); inhibition of Epstein–Barr virus [31,67] BH Whole plant Kalanhybrins A, B and C, bersaldegenin-1-acetate, bersaldegenin-3-acetate Cytotoxic activity of bersaldegenin derivatives against human breast MCF-7 cancer cell line, human lung carcinoma NCI-H460 and glioblastoma SF-268 cell line [71] BD Roots Kalandaigremosides A-H nd [124] BP Whole plant Bryophyllin A and B, bersaldegenin-3-acetate Cytotoxic effect against keratin-forming tumor KB cell line, adenocarcinomic human alveolar basal epithelial A-549 cell line and human ileocecal carcinoma HCT-8 cell line [125] BP, BD, BT Leaves (BD, BP) and stems (BT) BP, BT: bersaldegenin-1-acetate, bersaldegenin-3-acetate, bersaldegenin-1,3,5-orthoacetate, bryophyllin A. BD: Bersaldegenin-1,3,5-orthoacetate nd [114] BD Leaves Bersaldegenin-1,3,5-orthoacetate, daigremontianin Insecticidal against silkworm larvae [65] BP Leaves Bersaldegenin-1-acetate, bersaldegenin-3-acetate, bersaldegenin-1,3,5-orthoacetate, bryophyllin A nd [90] Pharmaceuticals 2020,13, 444 9 of 23 Table 4. Cont. Species 1Plant Organ Bufadienolides Bioactivities 2References BD, BP Leaves BD: Bersaldegenin-1-acetate, bersaldegenin-2-acetate, bersaldegenin-1,3,5-orthoacetate, bryophyllin A, daigremontianin. BP: Bersaldegenin-1-acetate, bersaldegenin-2-acetate, bersaldegenin-3-acetate, bersaldegenin-4-acetate, bersaldegenin-5-acetate, bersaldegenin-1,3,5-orthoacetate, bryophyllin A Cytotoxic activity against human ovarian cancer SKOV-3 cell line, cervical adenocarcinoma HeLa S3 cell line and malignant melanoma A375 cell line. Antimicrobial activity against Corynebacterium diphtheriae, Staphylococcus aureus,Staphylococcus epidermidis, and Enterococcus hirae [77,126] BT Whole plant Kalantubosides A and B, bryophyllin A, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate Cytotoxic effect against adenocarcinomic human alveolar basal epithelial A-549 cell line, promyelocytic leukemia HL-60 cell line, oral adenosquamous carcinoma Cal-27 cell line, and melanoma A2058 cell line [127] 1BD: B. daigremontianum; BH: B. ×houghtonii; BP: B. pinnatum; and BT: B. tubiflorum.2nd: not determined. Pharmaceuticals 2020,13, 444 16 of 23 22. Herrera, I.; Nassar, J.M. Reproductive and recruitment traits as indicators of the invasive potential of Kalanchoe daigremontiana (Crassulaceae) and Stapelia gigantea (Apocynaceae) in a Neotropical arid zone. J. Arid Environ. 2009,73, 978–986. [CrossRef] 23. 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