Narrative review of production of antioxidants and anticancer compounds from spp. () using plant cell tissue culture
Review Article

Narrative review of production of antioxidants and anticancer compounds from Bryophyllum spp. (Kalanchoe) using plant cell tissue culture

Eva Lozano-Milo1,2#, Pascual García-Pérez1,2#, Pedro P. Gallego1,2

1Applied Plant & Soil Biology, Plant Biology and Soil Science Department, Biology Faculty, University of Vigo, Pontevedra, Spain; 2CITACA—Agri-Food Research and Transfer Cluster, University of Vigo, Ourense, Spain

Contributions: (I) Conception and design: All authors; (II) Administrative support: PP Gallego; (III) Provision of study materials or patients: All authors; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Correspondence to: Pedro P. Gallego. Applied Plant & Soil Biology, Plant Biology and Soil Science Department, Biology Faculty, University of Vigo, Pontevedra E-36310, Spain; CITACA—Agri-Food Research and Transfer Cluster, University of Vigo, Ourense E-32004, Spain. Email:

Abstract: For centuries, plants have been widely used in traditional medicine worldwide for the treatment of many diseases. The subgenus Bryophyllum (genus Kalanchoe) have been used in ethnobotanic medicine across America, Africa and Asia. Traditionally, some formulations derived from leaves and roots of Bryophyllum spp. have been applied for the treatment of common illness such as coughs, fever, infections, insect bites, wounds and burns. Phenolic compounds and bufadienolides are the two major families of secondary metabolites identified in several species of the subgenus Bryophyllum. These compounds have gained much attention due to their associated antioxidant and cytotoxic activity, but they are synthetized by plants in fairly limited amounts. In this sense, plant tissue culture (PTC) technology provides a powerful methodology, able to overcome the limitations of low yields associated with conventional open field cultivation of medicinal plants. Several types of PTC methods are routinely employed in plant in vitro propagation, although micropropagation and cell culture are the most common. While micropropagation provides a reliable multiplication procedure, enabling a continuous in vitro production of great amounts of whole medicinal plantlets or just their organs producing the bioactive metabolites, such as their leaves and/or roots; the cell suspension culture procedure allows for the massive production of secondary metabolites using huge bioreactors. In both cases, the addition of biotic and abiotic elicitors and metabolic precursors trigger the bioaccumulation of secondary metabolites through the induction of plant defense mechanisms.

Keywords: Ethnomedicine; bioactive compounds; oxidative stress; bufadienolides; plant in vitro culture

Received: 29 October 2020; Accepted: 23 November 2020; Published: 30 December 2020.

doi: 10.21037/lcm-20-46


The term “Ethnobotany” refers to the study of the practical uses of plants, which has been perpetuated over time thanks to the traditional knowledge of the local population (1). The knowledge of plants as sources of medicines, known as Ethnobotanic Medicine (2), has a long history in the treatment of many diseases worldwide (3) and it dates back about 60,000 years ago (4). Currently, the use of plants for therapeutical purposes is more extended in countries with poor economic development (5) and rural areas (6), where the modern medicine is difficult to access (7) and the indigenous knowledge about these plants is still preserved (8). According to the World Health Organization (WHO), approximately the 80% of world’s population from economically developing nations depends on plants and their natural derived by-products for their primary healthcare (9). In this sense, the ethnobotanical study of medicinal plants and their uses in local regions, is not only useful for the conservation of biodiversity and cultural traditions (10), but also could serve at the starting point for the development of novel drugs (11). Among the 500,000 species of plants estimated in the world (12), only around the 5% have been studied from a pharmacological point of view (13), conferring a broad territory of unexplored plants with potential medicinal properties.

The subgenus Bryophyllum (genus Kalanchoe) comprises approximately 25 succulent species endemic to Madagascar (14) naturalized across South America, Africa and Asia (15). Bryophyllum spp. were widely studied in the field of Plant Science, as they are considered plant models for the Crassulacean Acid Metabolism (CAM) (16), plant cell regeneration (17) and vegetative asexual reproduction (18). However, Bryophyllum subgenus gained much interest in the last decades for the use of different species in traditional medicine worldwide (19-27). Concerning their properties as medicinal plants, several formulations from leaves and roots of Bryophyllum spp. have been used traditionally for the treatment of common illnesses such as cough, fever, infections, insect bites, wounds and burns, among others (28).

Due to therapeutic properties widely reported on Bryophyllum spp. (29), several phytochemical analyses were performed with the objective of determining the compounds responsible for the pharmacological potential associated to this subgenus (30). In this way, plant extracts obtained from Bryophyllum spp. have demonstrated to be an efficient source of analgesic (31), antidiabetic (32), anti-inflammatory (33), antimicrobial (34), antioxidant (35), antiviral (36), cardioprotective (37), cytotoxic (38), hepatoprotective (39) and sedative (40) agents. Much of the research on this subgenus has focused on a single species, Bryophyllum pinnatum (Lam.) Oken, due to its ubiquitous distribution and wide use in ethnomedicine worldwide (41-46). In this context, the limited knowledge about its phytoconstituents, along with its reported therapeutic effects, makes Bryophyllum an unexplored subgenus with a promising phytochemical potential.

On this review, we performed an overview of the peer-reviewed published literature on this topic, using the Web of Science, PubMed and Scholar Google, according to PRISMA (Preferred Reported Items for Systematic Reviews and Meta-Analyses) guidelines.

We present the following article in accordance with the Narrative Review reporting checklist (available at

Bryophyllum as a source of secondary metabolites with phytochemical potential

Different authors have already identified several families of Bryophyllum spp. bioactive compounds, including bufadienolides, phenolic acids, flavonoids, organic salts, terpenoids and fatty acids (29,47-50). All of them are considered bioactive metabolites, biosynthesized by the induction of secondary metabolism, in response to different stimuli, collectively known as stresses (51). Therefore, these bioactive compounds are the result of the adaptative and/or defensive responses to different biotic and abiotic threats, such as infections and attacks by microorganisms, insects and herbivores (52) and drought, extreme temperatures or UV-radiation (53), respectively. Among the large number of secondary metabolites that have been reported in Bryophyllum subgenus, bufadienolides and phenolic compounds, are considered the two major families (28), because of two reasons: (I) they are ubiquitously distributed among all the subgenus (54); and (II) both families are responsible for the development of the bioactivities associated to these species (55). However, much attention has been paid to the bufadienolides from Bryophyllum sources, and several reports have focused on these compounds exclusively, as it is the case of Bryophyllum daigremontianum (Raym.-Hamet et Perr.) Berg. (48,56-59).

Phenolic compounds as potent antioxidant compounds

Phenolic compounds constitute the largest family of secondary metabolites with more than 8,000 compounds described, ubiquitously distributed in the Plant Kingdom (60). Due to the large number of compounds and the structural heterogeneity found on this family, a large number of subfamilies have been established (61). The great importance of phenolics as bioactive compounds, lies in their wide demonstrated antioxidant activity, developed by multiple mechanisms, acting as: (I) anti-radical agents (62), (II) modulators of antioxidant enzyme activity (63), (III) preservatives of lipid oxidation (64) and (IV) regulators of oxidative stress (65). Because of their preserving effect against oxidative stress, phenolic compounds have been reported as effective for several diseases promoted by this phenomenon, such as cancer (46), cardiovascular and neurodegenerative diseases (66) and diabetes (67). Besides antioxidant activity, phenolic compounds present additional bioactivities, acting as anti-inflammatory (68), antimicrobial (69), antiviral (45) and cytotoxic (70) agents, which may develop beneficial effects on the above-mentioned diseases.

In the case of the medicinal Bryophyllum spp., two major subfamilies of phenolic compounds has been described: phenolic acids and flavonoids. Caffeic acid, ferulic acid and protocatechuic acid are the major representatives of phenolic acids, as reported in B. daigremontianum and B. pinnatum (49) and flavonol glycosides, including kaempferol and quercetin glycosides, such as more abundant compounds of flavonoids (48,71).

The antioxidant activity of Bryophyllum extracts has been already assessed in terms of radical-scavenging activity (72,73) and the inhibition of lipid peroxidation (74). Particularly, the use of these extracts as preservatives of fish oil emulsions was recently assessed, leading to the prevention of omega-3 acids oxidation (74). Furthermore, the use of environmental-friendly procedures for the purification of phenolic compounds from in vitro-cultured Bryophyllum extracts, using activated carbon was also reported recently (75). In conclusion, Bryophyllum subgenus can be considered as a promising source of phenolic compounds and the PCT as new procedures which allow the large-scale production of those extracts.

Bufadienolides as potent anticancer compounds

Bufadienolides are secondary metabolites belonging to the cardiac glycosides family (76). Chemically, these secondary metabolites are polyhydroxy C-24 steroids characterized by the presence of a six-membered lactone ring at the C-17β position (77). Their synthesis is very complex, and it is known that their structures and conformation may play an important role in determining the potency of their biological activity (78). Some bufadienolides, such as bersaldegenin-3-orthoacetate and kalandaigremosides, are restricted to specific organs on discrete species, being identified in the leaves and roots of B. daigremontianum, respectively (48,79); meanwhile other bufadienolides, like bryophyllin derivatives, are ubiquitously distributed across Bryophyllum subgenus (58).

As cardiac glycosides, bufadienolides act as Na+/K+-ATPase inhibitors at the myocardial tissue, promoting cardiotonic effects and used in pharmacology for this purpose (80). However, several complications arise from the application of these compounds, since the overdosage may lead to a significant cardiotoxicity (81), thus causing a limitation for their therapeutic application. In fact, it has been reported that the accidental consumption of Bryophyllum spp. by cattle, causes severe cardiac symptoms leading to death (82,83). In order to overcome their inherent toxicity, synthetic analogues of these compounds are being developed to ensure their safety on the administration of bufadienolides-derived drugs in humans (84,85).

Nonetheless, besides their role as cardiotonic agents, bufadienolides gained much attention because of their associated cytotoxic activity, by the promotion of several mechanisms, including: (I) the induction of apoptosis and autophagy of malignant cells; (II) the arrest of cell cycle, tumor invasion and metastasis; and (III) the modulation of cancer-related intracellular signaling pathways (28,78). A wide variety of bioactivities, have been associated to bufadienolides extracted from Bryophyllum sources. For instance: (I) bersaldegenin-1-acetate has been proved to be cytotoxic against astrocytoma U-373, breast MCF-7, colorectal HT-29, glioma Hs683, lung A-549, melanoma SKMEL-28 and prostate PC-3 cancer cell lines and antiviral against Epstein-Barr virus (58,79,86,87); (II) bersaldegenin-1,3,5-orthoacetate was reported as effective against several cancer lines such as adenocarcinoma HeLa, ovarian SKOV-3 and melanoma A-375, and as anti-influenza, insecticidal and sedative agent (48,58,59,86,87); (III) bersaldegenin-3-orthoacetate showed a cytotoxic effect against MCF-7, NCI-H460 and SF-268 cancer lines (48,77); (IV) bryophyllins A–C were shown to be effective against several cancer lines such as gastric cancer KB and A-549, and antivirals against Epstein-Barr virus and human immunodeficiency virus (48,57,58,77,86,87); (V) daigremontianin was proved to exert a cytotoxic effect against different cancer cell lines such as MCF-7 and A-549, acting also as anti-influenza, insecticidal and sedative agent (57-59,88); and (VI) kalantubosides A and B demonstrated a cytotoxic effect against HL-60 leukemia cells (76).

The knowledge derived from the possible use of the secondary metabolites identified in Bryophyllum spp. as phytosanitary products has aroused great interest in their valorization by traditional medicine, but also by the biopharmaceutical industry. This carries the risk of depletion of wild plants in order to obtain sufficient quantities of plants to satisfy the demand for natural compounds. In this sense, the implementation of new and highly efficient systems of plant propagation will be required to increase the amount of plant raw material and adjust it to market demand, which would allow exploring all the phytochemical potential of the Bryophyllum subgenus in a sustainable way. In this regard, the use of plant tissue culture (PTC) technology could increase the biotechnological production of these compounds, without putting wild populations of Bryophyllum at risk. The use of this technology is reviewed below.

PTC as a biotechnological system for the production of secondary metabolites with phytochemical potential

Since the beginning of agriculture during Neolithic period, humanity has had a long history of dependence on plants: an early period, for food and nutrition purposes, and a late period in which other purposes, such as medicine, were established (89). Currently, the great global demand of medicinal plants for different commercial purposes and their derived products (90), makes of crucial importance the development and implementation of efficient approaches with the aim to obtain great amounts of plant material (91). During last decades, Plant Biotechnology has focused on the development of new strategies for the industrial production of plant-derived products (92). In this context, PTC technology provide a powerful way, able to overcome the difficulties and disadvantages associated to conventional agricultural production of secondary metabolites (93). For instance, PTC prevents from challenging environmental conditions, by conferring a reliable system with controlled growing conditions (94). Additionally, taking advantage of plant cell totipotency, PTC makes possible the establishment of different culture types, including the culture of plant organs, tissues, cell, protoplasts, but also plantlets (94). This fact, facilitates the scalability of cultures, thus promoting the large-scale production of bioactive compounds (95). In all cases, the development of axenic cultures is required in order to preserve the integrity and viability of the cultured materials, thus assuring the absence of pathogenic microorganisms (96). However, the implementation of PTC methodology requires the application of specialized knowledge and technologies, thus increasing its investment (97,98).

Thanks to the great applicability of PTC, a detailed overview of the current aspects of PTC will be addressed along this section, with particular attention to the methodology applied to Bryophyllum spp.: plant in vitro propagation (micropropagation) and cell culture.


Because of its advantages as a multiplication system over conventional plant macropropagation methods, micropropagation has become an important platform for the exploitation of many plant species (99). Some of the advantages offered by micropropagation are: (I) a continuous annual production due to their independence of seasonal changes (100); (II) controlled culture conditions, making possible the adjustment of most of the factors involved in growth and multiplication, such culture media (nutrients, plant growth regulators, vitamins, sugars, etc.) and growth conditions (temperature, light intensity and photoperiod, etc.) (101); (III) the possibility to produce clones from plants difficult to propagate vegetatively and to choose desirable specific traits (102); and (IV) the less energy and space required to maintain a large-scale stock of plant material (103).

Among the widely available micropropagation methods, they can be classified into: (I) organogenesis by meristematic tissue of apical and axillary buds (104-106); (II) organogenesis by adventitious shoots and roots (107-109) including: direct adventitious organogenesis (106) and indirect adventitious shoots from callus (109); and (III) somatic embryogenesis including direct embryogenesis (110) and indirectly-initiated somatic embryos from callus (111).

Despite of the whole diversity of micropropagation methods, this technique involves some common steps along the multiplication process. An initial stage (stage 0) is essential to select healthy mother plant material, followed by a second stage (stage I) to initiate the establishment of the culture in which plant material is disinfected for the establishment of axenic cultures. After that (stage II) it takes place a stage of multiplication for obtaining new buds, propagules or embryos formation, capable of giving rise to complete plants. Sometimes, depending on the final objective, the multiplication stage can be followed by rooting (stage III) and acclimatization (stage IV), prior to the transference of micropropagated plants into natural environment (ex vitro) (112).

Concerning Bryophyllum spp. micropropagation and plant regeneration protocols, the available published research is limited. Nevertheless, some authors have achieved the multiplication of some Bryophyllum spp. by using Murashige and Skoog (MS) formulation (113-116). MS medium is considered the most relevant formulation designed to date, being considered as universal medium for multiple PTC applications with more than 82,000 citations on literature. Nevertheless, as individual species show specific nutritional requirements, MS may cause several physiological disorders in some cases (117,118), and MS modifications are usually applied (119). In the case of Bryophyllum spp., the reduction in macronutrient concentration to half-strength, improves the in vitro growth and multiplication in B. daigremontianum, Bryophyllum × houghtonii D.B.Ward and Bryophyllum tubiflorum Harv. species (73-75). On the other hand, the organogenetic responses in Bryophyllum spp. by the addition of several concentrations of plant growth regulators have been reported by several authors, by focusing on the design of indirect organogenesis protocols (115,116,120). Furthermore, it was recently reported that cytokinin 6-belzylamonipurine (BAP) drives the organogenetic process on B. tubiflorum showed prevalence for indirect organogenesis via callus formation, whereas B. daigremontianum and B. × houghtonii developed organogenetic responses from direct organogenesis (121).

Plant cell suspension cultures (PCSCs)

In recent years, the commercial importance of secondary metabolites resulted in a growing interest of the enhancement in the production of bioactive plant molecules, through different plant biotechnology methodologies (122). Plant cell culture systems have been successfully applied for the production of secondary metabolites (119). This way, PCSCs have emerged as a reliable biotechnological system, offering a robust productive system, in comparison with the production and extraction of bioactive molecules from whole plants and tissues (123). PCSCs are established from the transference of callus-derived cell aggregates to liquid medium, thus enabling their promotion to large-scale systems, such as bioreactors (124). However, PCSCs methodologies, is not exempt of their own limitations and difficulties. The fact that plant material in this type of culture is based on dedifferentiated cells, may cause genetic instability and somaclonal variation, which can result in epigenetic changes (125). Moreover, concerning productivity, PCSCs are composed by heterogenous cell populations, showing high-yielding, low-yielding and non-producing cell lines, which may negatively affect the production of secondary metabolites (95).

As a solution, different strategies can be applied to PCSCs, with the aim of improving the production of secondary metabolites, such as: (I) the selection of stable and high-yielding cell lines (126); (II) the implementation of two-phase culture systems to prevent cell auto-toxicity, by the accumulation of produced secondary metabolites (127); (III) the optimization of cell culture conditions (nutrients, plant growth regulators, temperature, light, etc.) (128); (IV) the addition of elicitors and metabolic precursors, to induce the biosynthetic pathways involved in the production of secondary metabolites (129); (V) the use of metabolic engineering to overexpress target pathways, inhibiting competing pathways and preventing the degradation of the final product (92) and; (VI) the selection of appropriate operational aspects and configuration of bioreactors, according to the nature of cultured cells (130).

Among the multiple strategies for the enhancement of the production of secondary metabolites, elicitation has been traditionally used for this purposes (131), as widely reported in countless successful cases (132). As defined by Narayani and Srivastava (2017), an elicitor is a “physical or chemical agent capable of inducing or stimulating defense response in plant/cell tissues via production of secondary metabolites” (133). Depending on their nature, elicitors are classified as biotic, deriving from biological sources (bacteria, fungal, algae, plant, animal-derived compounds), or abiotic, if are derived from non-biological sources (physical agents, such as UV-radiation, temperature, light, salinity, etc.; or chemical agents such as heavy metals) (134). Thus, the addition of elicitors into culture medium is a potent biotechnological strategy, which can lead to an increase in the accumulation of secondary metabolites in cell suspension cultures (135), through the induction of plant defense mechanisms leading to the biosynthesis of secondary metabolites.

Concerning PCSCs from Bryophyllum spp., very limited information is available. PCSCs from B. × houghtonii were subjected to elicitation with cyclodextrins (73). The results demonstrated that only 7 days were needed to achieve a stable cell growth and production of phenolic compounds. Moreover, it was shown that the elicitation by cyclodextrin addition into culture medium, improved the production of phenolic compounds and their associated radical scavenging activity (73).


Funding: This study was funded by Xunta de Galicia through “Red de Uso Sostenible de los Recursos Naturales y Agroalimentarios” (REDUSO, grant number ED341D 2017/2018), “Cluster of Agricultural Research and Development” (CITACA Strategic Partnership, grant number ED431E 2018/07), and the FPU grant awarded to Pascual García-Pérez from the Spanish Ministry of Education (grant number FPU15/04849).


Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ethical approval was not required for this study design.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See:


  1. Albuquerque UP, Hurrell JA. Ethnobotany: one concept and many interpretations. Recent Dev Case Stud Ethnobot 2010;1:87-99.
  2. Fabricant DS, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 2001;109:69-75. [PubMed]
  3. Salmerón-Manzano E, Garrido-Cardenas JA, Manzano-Agugliaro F. Worldwide research trends on medicinal plants. Int J Environ Res Public Health Int J Environ Res Public Health 2020;17:3376. [Crossref] [PubMed]
  4. Efferth T, Greten HJ. Traditional medicine with plants present and past. Med Aromat Plants 2014;3:e151.
  5. Chen G, Sun W, Wang X, et al. Conserving threatened widespread species: a case study using a traditional medicinal plant in Asia. Biodivers Conserv 2019;28:213-27. [Crossref]
  6. Sheng-Ji P. Ethnobotanical approaches of traditional medicine studies: some experiences from Asia. Pharm Biol 2001;39:74-9. [PubMed]
  7. Oyebode O, Kandala NB, Chilton PJ, et al. Use of traditional medicine in middle-income countries: a WHO-SAGE study. Health Policy Plan 2016;31:984-91. [Crossref] [PubMed]
  8. Liu B, Guo ZY, Bussmann R, et al. Ethnobotanical approaches of traditional medicine studies in Southwest China: a literature review. J Ethnopharmacol 2016;186:343-50. [Crossref] [PubMed]
  9. Jamshidi-Kia F, Lorigooini Z, Amini-Khoei H. Medicinal plants: Past history and future perspective. J Herb Med Pharmacol 2018;7:1-7. [Crossref]
  10. Silva HCH, Caraciolo RLF, Marangon LC, et al. Evaluating different methods used in ethnobotanical and ecological studies to record plant biodiversity. J Ethnobiol Ethnomed 2014;10:48. [Crossref] [PubMed]
  11. Talalay P, Talalay P. The importance of using scientific principles in the development of medicinal agents from plants. Acad Med 2001;76:238-47. [Crossref] [PubMed]
  12. Singh R. Medicinal plants: a review. J Plant Sci 2015;3:50.
  13. Cragg GM, Newman DJ. Natural products: A continuing source of novel drug leads. Biochim Biophys Acta 2013;1830:3670-95. [Crossref] [PubMed]
  14. Descoings B. Le genre Kalanchoe, structure et définition. Journal de Botanique de la Société Botanique de France 2006;33:3-28.
  15. Herrando-Moraira S, Vitales D, Nualart N, et al. Global distribution patterns and niche modelling of the invasive Kalanchoe × houghtonii (Crassulaceae). Sci Rep 2020;10:3143. [Crossref] [PubMed]
  16. Cushman JC. Crassulacean Acid Metabolism: recent advances and future opportunities. Funct Plant Biol 2005;32:375-80. [Crossref] [PubMed]
  17. Garcês HMP, Koenig D, Townsley BT, et al. Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana. Plant Physiol 2014;165:196-206. [Crossref] [PubMed]
  18. Garcês H, Sinha N. The “mother of thousands” (Kalanchoe daigremontiana): a plant model for asexual reproduction and CAM studies. Cold Spring Harb Protoc 2009;2009:pdb.emo133.
  19. Abebe W. An overview of ethiopian traditional medicinal plants used for cancer treatment. European J Med Plants 2016;14:1-16. [Crossref]
  20. Malan DF, Neuba DFR. Traditional practices and medicinal plants use during pregnancy by Anyi-Ndenye women (Eastern Côte d’Ivoire). Afr J Reprod Health 2011;15:85-93. [PubMed]
  21. Rahmatullah M, Mollik MAH, Ali M, et al. An ethnomedicinal survey of vitbilia village in Sujanagar sub-district of pabna district, Bangladesh. Am J Sustain Agric 2010;4:302-8.
  22. Lans CA. Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus. J Ethnobiol Ethnomed 2006;2:45. [Crossref] [PubMed]
  23. Bhowmik R, Saha MR, Rahman MA, et al. Ethnomedicinal survey of plants in the Southern District Noakhali, Bangladesh. Bangladesh Pharm J 2015;17:205-14. [Crossref]
  24. Sen P, Dollo M, Choudhury MD, et al. Documentation of traditional herbal knowledge of Khamptis of Arunachal Pradesh. Indian J Tradit Knowl 2008;7:438-42.
  25. Tariq A, Sadia S, Pan K, et al. A systematic review on ethnomedicines of anti-cancer plants. Phytother Res 2017;31:202-64. [Crossref] [PubMed]
  26. Budi V, Sihotang L. Ethnomedicinal study of the Sundanese people at the Bodogol area, Gede Pangrango Mountain National Park, West Java. Gard Bull Singapore 2011;63:527-34.
  27. Namukobe J, Kasenene JM, Kiremire BT, et al. Traditional plants used for medicinal purposes by local communities around the Northern sector of Kibale National Park, Uganda. J Ethnopharmacol 2011;136:236-45. [Crossref] [PubMed]
  28. García-Pérez P, Barreal ME, Rojo-De Dios L, et al. Bioactive natural products from the genus Kalanchoe as cancer chemopreventive agents: a review. Studies in Natural Products Chemistry 2019;61:49-84. [Crossref]
  29. Prasad AK, Kumar S, Iyer SV, et al. Pharmacognostical, phytochemical and pharmacological review on Bryophyllum pinnata. Int J Pharm Biol Arch 2012;3:423-33.
  30. Fürer K, Simões-Wüst AP, Von Mandach U, et al. Bryophyllum pinnatum and related species used in anthroposophic medicine: constituents, pharmacological activities, and clinical efficacy. Planta Med 2016;82:930-41. [Crossref] [PubMed]
  31. Afzal M, Gupta G, Kazmi I, et al. Anti-inflammatory and analgesic potential of a novel steroidal derivative from Bryophyllum pinnatum. Fitoterapia 2012;83:853-8. [Crossref] [PubMed]
  32. Aransiola EF, Daramola MO, Iwalewa EO, et al. Anti-diabetic effect of Bryophyllum pinnatum leaves. Int J Biol Life Sci Eng 2014;8:89-93.
  33. Gupta R, Lohani M, Arora S. Anti-inflammatory activity of the leaf extracts/fractions of Bryophyllum pinnatum Saliv.Syn. Int J Pharm Sci Rev Res 2010;3:16-8.
  34. Tatsimo SJN, Tamokou JDD, Havyarimana L, et al. Antimicrobial and antioxidant activity of kaempferol rhamnoside derivatives from Bryophyllum pinnatum. BMC Res Notes 2012;5:158. [Crossref] [PubMed]
  35. Sharma A, Bhot M, Chandra N. In vitro antibacterial and antioxidant activity of Bryophyllum pinnatum (Lam.) Kurz. Int J Pharm Pharm Sci 2014;6:558-60.
  36. Al-Snafi A. The chemical constituents and pharmacological effects of Bryophyllum calycinum. A review. Int J Pharma Sci Res 2013;4:171-6.
  37. Adekunle A, Adelusi T, Oyewo E, et al. Antihypercholesterolemic, Cardioprotective and vitamins E and C sparing properties of Bryophyllum pinnatum in rabbits. European J Med Plants 2016;11:1-13. [Crossref]
  38. Mahata S, Maru S, Shukla S, et al. Anticancer property of Bryophyllum pinnatum (Lam.) Oken. leaf on human cervical cancer cells. BMC Complement Altern Med 2012;12:15. [Crossref] [PubMed]
  39. Afzal M, Kazmi I, Anwar F. Antineoplastic potential of Bryophyllum pinnatum Lam. on chemically induced hepatocarcinogenesis in rats. Pharmacognosy Res 2013;5:247-53. [Crossref] [PubMed]
  40. Lambrigger-Steiner C, Simões-Wüst AP, Kuck A, et al. Sleep quality in pregnancy during treatment with Bryophyllum pinnatuM: AN observational study. Phytomedicine 2014;21:753-7. [Crossref] [PubMed]
  41. Quazi Majaz A, Tatiya AU, Khurshid M, et al. The miracle plant (Kalanchoe pinnata): a phytochemical and pharmacological review. Int J Res Ayurveda Pharm 2011;2:1478-82.
  42. Fernandes JM, Cunha LM, Azevedo EP, et al. Kalanchoe laciniata and Bryophyllum pinnatum: an updated review about ethnopharmacology, phytochemistry, pharmacology and toxicology. Rev Bras Farmacogn 2019;29:529-58. [Crossref]
  43. Muzitano MF, Tinoco LW, Guette C, et al. The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata. Phytochemistry 2006;67:2071-7. [Crossref] [PubMed]
  44. Chibli LA, Rodrigues KCM, Gasparetto CM, et al. Anti-inflammatory effects of Bryophyllum pinnatum (Lam.) Oken ethanol extract in acute and chronic cutaneous inflammation. J Ethnopharmacol 2014;154:330-8. [Crossref] [PubMed]
  45. Ürményi FG, Saraiva GD, Casanova LM, et al. Anti-HSV-1 and HSV-2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana. Chem Biodivers 2016;13:1707-14. [Crossref] [PubMed]
  46. Carocho M, Ferreira I. The role of phenolic compounds in the fight against cancer – a review. Anticancer Agents Med Chem 2013;13:1236-58. [Crossref] [PubMed]
  47. Milad R, El-ahmady S, Singab AN. Genus Kalanchoe a review of its ethnomedicinal, botanical, chemical and pharmacological properties. European J Med Plants 2014;4:86-104. [Crossref]
  48. Stefanowicz-Hajduk J, Asztemborska M, Krauze-Baranowska M, et al. Identification of flavonoids and bufadienolides and cytotoxic effects of Kalanchoe daigremontiana extracts on human cancer cell lines. Planta Med 2020;86:239-46. [Crossref] [PubMed]
  49. Bogucka-Kocka A, Zidorn C, Kasprzycka M, et al. Phenolic acid content, antioxidant and cytotoxic activities of four Kalanchoë species. Saudi J Biol Sci 2018;25:622-30. [Crossref] [PubMed]
  50. Katrucha EM, Lopes J, Paim M, et al. Phenolic profile by HPLC-ESI-MS/MS and enzymatic inhibitory effect of Bryophyllum delagoense. Nat Prod Res 2020. [Epub ahead of print]. [Crossref] [PubMed]
  51. Yang L, Wen KS, Ruan X, et al. Response of plant secondary metabolites to environmental factors. Molecules 2018;23:762. [Crossref] [PubMed]
  52. Mazid M, Khan TA, Mohammad F, et al. Role of secondary metabolites in defense mechanisms of plants. Biol Med 2011;3:232-49.
  53. Moore BD, Andrew RL, Külheim C, et al. Explaining intraspecific diversity in plant secondary metabolites in an ecological context. New Phytol 2014;201:733-50. [Crossref] [PubMed]
  54. Hamburger M, Potterat O, Fürer K, et al. Bryophyllum pinnatum - Reverse engineering of an anthroposophic herbal medicine. Nat Prod Commun 2017;12:1359-64. [Crossref]
  55. Stefanowicz-Hajduk J, Hering A, Gucwa M, et al. Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content. Pharm Biol 2020;58:732-40. [Crossref] [PubMed]
  56. Kolodziejczyk-Czepas J, Nowak P, Wachowicz B, et al. Antioxidant efficacy of Kalanchoe daigremontiana bufadienolide-rich fraction in blood plasma in vitro. Pharm Biol 2016;54:3182-8. [Crossref] [PubMed]
  57. Supratman U, Fujita T, Akiyama K, et al. Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana × tubiflora. Biosci Biotechnol Biochem 2001;65:947-9. [Crossref] [PubMed]
  58. Moniuszko-Szajwaj B, Pecio Ł, Kowalczyk M, et al. New bufadienolides isolated from the roots of Kalanchoe daigremontiana (Crassulaceae). Molecules 2016;21:243. [Crossref] [PubMed]
  59. Maharani R, Fajriah S, Hardiawan R, et al. Insecticidal bufadienolides from the leaves of Kalanchoe daigremontiana (Crassulaceae). Proceeding Int Semin Chem 2008;11:236-9.
  60. Tungmunnithum D, Thongboonyou A, Pholboon A, et al. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. Medicines 2018;5:93. [Crossref] [PubMed]
  61. Haminiuk CWI, Maciel GM, Plata-Oviedo MSV, et al. Phenolic compounds in fruits - an overview. Int J Food Sci Technol 2012;47:2023-44. [Crossref]
  62. Huyut Z, Beydemir Ş, Gülçin I. Antioxidant and antiradical properties of selected flavonoids and phenolic compounds. Biochem Res Int 2017;2017:7616791. [Crossref] [PubMed]
  63. Ruiz JM, Romero L. Bioactivity of the phenolic compounds in higher plants. Studies in Natural Products Chemistry 2001;25:651-81. [Crossref]
  64. Maqsood S, Benjakul S, Abushelaibi A, et al. Phenolic compounds and plant phenolic extracts as natural antioxidants in prevention of lipid oxidation in seafood: a detailed review. Compr Rev Food Sci Food Saf 2014;13:1125-40. [Crossref]
  65. Zhang H, Tsao R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr Opin Food Sci 2016;8:33-42. [Crossref]
  66. Albarracin SL, Stab B, Casas Z, et al. Effects of natural antioxidants in neurodegenerative disease. Nutr Neurosci 2012;15:1-9. [Crossref] [PubMed]
  67. Lin D, Xiao M, Zhao J, et al. An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules 2016;21:1374. [Crossref] [PubMed]
  68. Ambriz-Pérez DL, Leyva-López N, Gutierrez-Grijalva EP, et al. Phenolic compounds: Natural alternative in inflammation treatment. A Review. Cogent Food Agric 2016;2:1131412.
  69. Cetin-Karaca H, Newman MC. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia coli. Food Biosci 2015;11:8-16. [Crossref]
  70. Kuete V, Mbaveng AT, Nono ECN, et al. Cytotoxicity of seven naturally occurring phenolic compounds towards multi-factorial drug-resistant cancer cells. Phytomedicine 2016;23:856-63. [Crossref] [PubMed]
  71. Fürer K, Raith M, Brenneisen R, et al. Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology. Planta Med 2013;79:1565-71. [Crossref] [PubMed]
  72. García-Pérez P, Lozano-Milo E, Landín M, et al. Combining medicinal plant in vitro culture with machine learning technologies for maximizing the production of phenolic compounds. Antioxidants (Basel) 2020;9:210. [Crossref] [PubMed]
  73. García-Pérez P, Losada-Barreiro S, Gallego PP, et al. Cyclodextrin-elicited Bryophyllum suspension cultured cells: Enhancement of the production of bioactive compounds. Int J Mol Sci 2019;20:5180. [Crossref] [PubMed]
  74. García-Pérez P, Losada-Barreiro S, Bravo-Díaz C, et al. Exploring the use of Bryophyllum as natural source of bioactive compounds with antioxidant activity to prevent lipid oxidation of fish oil-in-water emulsions. Plants (Basel) 2020;9:1012. [Crossref] [PubMed]
  75. García-Pérez P, Losada-Barreiro S, Gallego PP, et al. Adsorption of gallic acid, propyl gallate and polyphenols from Bryophyllum extracts on activated carbon. Sci Rep 2019;9:14830. [Crossref] [PubMed]
  76. Huang HC, Huang GJ, Liaw CC, et al. A new megastigmane from Kalanchoe tubiflora (Harvey) Hamet. Phytochem Lett 2013;6:379-82. [Crossref]
  77. Kuo PC, Kuo TH, Su CR, et al. Cytotoxic principles and α-pyrone ring-opening derivatives of bufadienolides from Kalanchoe hybrida. Tetrahedron 2008;64:3392-6. [Crossref]
  78. Zhong Y, Zhao C, Wu WY, et al. Total synthesis, chemical modification and structure-activity relationship of bufadienolides. Eur J Med Chem 2020;189:112038. [Crossref] [PubMed]
  79. Kolodziejczyk-Czepas J, Stochmal A. Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use. Phytochem Rev 2017;16:1155-71. [Crossref] [PubMed]
  80. Xu Y, Liu X, Schwarz S, et al. Inhibitory efficacy of bufadienolides on Na+,K+-pump activity versus cell proliferation. Biochem Biophys Rep 2016;6:158-64. [Crossref] [PubMed]
  81. Li W, Lin X, Yang Z, et al. A bufadienolide-loaded submicron emulsion for oral administration: Stability, antitumor efficacy and toxicity. Int J Pharm 2015;479:52-62. [Crossref] [PubMed]
  82. Botha CJ, Penrith ML. Poisonous plants of veterinary and human importance in southern Africa. J Ethnopharmacol 2008;119:549-58. [Crossref] [PubMed]
  83. McKenzie RA, Franke FP, Dunster PJ. The toxicity to cattle and bufadienolide content of six Bryophyllum species. Aust Vet J 1987;64:298-301. [Crossref] [PubMed]
  84. Wu PL, Hsu YL, Wu TS, et al. Kalanchosides A-C, new cytotoxic bufadienolides from the aerial parts of Kalanchoe gracilis. Org Lett 2006;8:5207-10. [Crossref] [PubMed]
  85. Hayes RA, Piggott AM, Dalle K, et al. Microbial biotransformation as a source of chemical diversity in cane toad steroid toxins. Bioorg Med Chem Lett 2009;19:1790-2. [Crossref] [PubMed]
  86. Supratman U, Fujita T, Akiyama K, et al. New insecticidal bufadienolide, bryophyllin C, from Kalanchoe pinnata. Biosci Biotechnol Biochem 2000;64:1310-2. [Crossref] [PubMed]
  87. Oufir M, Seiler C, Gerodetti M, et al. Quantification of Bufadienolides in Bryophyllum pinnatum Leaves and Manufactured Products by UHPLC-ESIMS/MS. Planta Med 2015;81:1190-7. [Crossref] [PubMed]
  88. Wanka L, Iqbal K, Schreiner PR. The lipophilic bullet hits the targets: Medicinal chemistry of adamantane derivatives. Chem Rev 2013;113:3516-604. [Crossref] [PubMed]
  89. Purugganan MD, Fuller DQ. The nature of selection during plant domestication. Nature 2009;457:843-8. [Crossref] [PubMed]
  90. Schippmann U, Leaman DJ, Cunningham AB. Biodiversity and the ecosystem approach in agriculture, forestry and fisheries. satellite event on the occasion of the ninth regular session of the commission on genetic resources for food and agriculture. Rome: FAO, 2002:12-3.
  91. Akin-Idowu PE, Ibitoye DO, Ademoyegun OT. Tissue culture as a plant production technique for horticultural crops. African J Biotechnol 2009;8:3782-8.
  92. Verpoorte R, Memelink J. Engineering secondary metabolite production in plants. Curr Opin Biotechnol 2002;13:181-7. [Crossref] [PubMed]
  93. Smetanska I. Production of secondary metabolites using plant cell cultures. In: Stahl U, Donalies UEB, Nevoigt E. editors. Food biotechnology. Heidelberg: Springer, 2008:187-228.
  94. George EF, Hall MA, De Klerk GJ. Plant tissue culture procedure-background. In: George EF, Hall MA, De Klerk GJ. editors. Plant propagation by tissue culture. Dordrecht: Springer, 2008:1-28.
  95. Yue W, Ming QL, Lin B, et al. Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites. Crit Rev Biotechnol 2016;36:215-32. [Crossref] [PubMed]
  96. Iliev I, Gajdosova A, Libiaková G, et al. Plant micropropagation. Davey MR, Anthony P. editors. Plant cell culture: essential methods. Hoboken: John Wiley & Sons, Inc., 2010:1-23.
  97. Kane ME, Kauth PJ, Stewart SL. Micropropagation. In: Beyl CA, Trigiano RN. editors. Plant propagation concepts and laboratory exercises. 2nd ed. Boca Raton: CRC Press, 2016:359-70.
  98. Loberant B, Altman A. Micropropagation of plants. In: Flickinger MC. editor. Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology. Hoboken: John Wiley & Sons, Inc., 2010:1-17.
  99. Debnath AJ, Gangopadhyay G, Basu D, et al. An efficient protocol for in vitro direct shoot organogenesis of Sesamum indicum L. using cotyledon as explant. 3 Biotech 2018;8:146.
  100. Chandran H, Meena M, Barupal T, et al. Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep (Amst) 2020;26:e00450. [Crossref] [PubMed]
  101. Silva ST, Bertolucci SKV, da Cunha SHB, et al. Effect of light and natural ventilation systems on the growth parameters and carvacrol content in the in vitro cultures of Plectranthus amboinicus (Lour.) Spreng. Plant Cell Tissue Organ Cult 2017;129:501-10. [Crossref]
  102. Preece JE, Read PE. Novel methods in micropropagation. Acta Hortic 2003.71-6. [Crossref]
  103. Espinosa-Leal CA, Puente-Garza CA, García-Lara S. In vitro plant tissue culture: means for production of biological active compounds. Planta 2018;248:1-18. [Crossref] [PubMed]
  104. Arumugam G, Sinniah UR, Swamy MK, et al. Micropropagation and essential oil characterization of Plectranthus amboinicus (Lour.) Sprengel, an aromatic medicinal plant. Vitr Cell Dev Biol - Plant 2020;56:491-503.
  105. Rosal LF, Pinto JEBP, Bertolucci SKV, et al. Micropropagation of the medicinal plant Eremanthus erythropappus (DC.) MacLeish. HortScience 2007;42:1420-4. [Crossref]
  106. Chen S, Xiong Y, Yu X, et al. Adventitious shoot organogenesis from leaf explants of Portulaca pilosa L. Sci Rep 2020;10:3675. [Crossref] [PubMed]
  107. Ahire ML, Ghane SG, Lokhande VH, et al. Micropropagation of Uraria picta through adventitious bud regeneration and antimicrobial activity of callus. Vitr Cell Dev Biol - Plant 2011;47:488-95.
  108. Rahmat E, Kang Y. Adventitious root culture for secondary metabolite production in medicinal plants: a review. J Plant Biotechnol 2019;46:143-57. [Crossref]
  109. Matand K, Shoemake M, Li C. High frequency in vitro regeneration of adventitious shoots in daylilies (Hemerocallis sp) stem tissue using thidiazuron. BMC Plant Biol 2020;20:31. [Crossref] [PubMed]
  110. Saha PS, Sarkar S, Jeyasri R, et al. In vitro propagation, phytochemical and neuropharmacological profiles of Bacopa monnieri (L.) wettst.: a review. Plants (Basel) 2020;9:411. [Crossref] [PubMed]
  111. Dehestani-Ardakani M, Hejazi M, Aliabad KK. Indirect somatic embryogenesis of purple coneflower (Echinacea purpurea (L.) Moench): a medicinal-ornamental plant: evaluation of antioxidant enzymes activity and histological study. Mol Biol Rep 2020;47:6621-33. [Crossref] [PubMed]
  112. George EF, Hall MA, De Klerk GJ. Micropropagation: uses and methods. In: George EF, Hall MA, De Klerk GJ. editors. Plant propagation by tissue culture. Dordrecht: Springer, 2008:29-64.
  113. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 1962;15:473-97. [Crossref]
  114. Kaviani B. The effect of different concentrations of plant growt regulators on micropropagation of Kalanchoe blossfeldiana cv. White. J Ornam Plants 2014;4:101-6.
  115. Kim TR, In JG, Yang DC, et al. Plant regeneration from leaf explants of Kalanchoe daigremontiana Hamet & Perrier. Korean J Med Crop Sci 2006;14:293-8.
  116. Naz S, Javad S, Ilyas S, et al. An efficient protocol for rapid multiplication of Bryophyllum pinnatum and Bryophyllum daigremontianum. Pakistan J Bot 2009;41:2347-55.
  117. Gamborg OL, Murashige T, Thorpe TA, et al. Plant tissue culture media. In Vitro 1976;12:473-8. [Crossref] [PubMed]
  118. Nezami-Alanagh E, Garoosi GA, Landín M, et al. Combining DOE with neurofuzzy logic for healthy mineral nutrition of pistachio rootstocks in vitro culture. Front Plant Sci 2018;9:1474. [Crossref] [PubMed]
  119. Phillips GC, Garda M. Plant tissue culture media and practices: an overview. Vitr Cell Dev Biol - Plant 2019;55:242-57.
  120. Kulus D. Micropropagation of Kalanchoe tubiflora (Harvey) Hamet. Nauk Przyr Technol 2015. doi: 10.17306/J.NPT.2015.1.14. [Crossref]
  121. García-Pérez P, Lozano-Milo E, Landín M, et al. Machine learning technology reveals the concealed interactions of phytohormones on medicinal plant in vitro organogenesis. Biomolecules. 2020;10:1-21. [Crossref] [PubMed]
  122. Marchev AS, Yordanova ZP, Georgiev MI. Green (cell) factories for advanced production of plant secondary metabolites. Crit Rev Biotechnol 2020;40:443-58. [Crossref] [PubMed]
  123. Xu J, Ge X, Dolan MC. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnol Adv 2011;29:278-99. [Crossref] [PubMed]
  124. Moscatiello R, Baldan B, Navazio L. Plant cell suspension cultures. In: Maathuis FJM. Editor. Plant mineral nutrients. Totowa: Humana Press, 2013:77-93.
  125. Hussain MS, Fareed S, Ansari S, et al. Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 2012;4:10-20. [Crossref] [PubMed]
  126. Rao SR, Ravishankar GA. Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 2002;20:101-53. [Crossref] [PubMed]
  127. Malik S, Hossein Mirjalili M, Fett-Neto AG, et al. Living between two worlds: two-phase culture systems for producing plant secondary metabolites. Crit Rev Biotechnol 2013;33:1-22. [Crossref] [PubMed]
  128. Chattopadhyay S, Farkya S, Srivastava AK, et al. Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures. Biotechnol Bioprocess Eng 2002;7:138-49. [Crossref]
  129. Zhao J, Davis LC, Verpoorte R. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 2005;23:283-333. [Crossref] [PubMed]
  130. Kim Y, Wyslouzil BE, Weathers PJ. Secondary metabolism of hairy root cultures in bioreactors. Vitr Cell Dev Biol - Plant 2002;38:1-10.
  131. Bourgaud F, Gravot A, Milesi S, et al. Production of plant secondary metabolites: A historical perspective. Plant Sci 2001;161:839-51. [Crossref]
  132. Ramirez-Estrada K, Vidal-Limon H, Hidalgo D, et al. Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 2016;21:182. [Crossref] [PubMed]
  133. Narayani M, Srivastava S. Elicitation: a stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Phytochem Rev 2017;16:1227-52. [Crossref]
  134. Thakur M, Bhattacharya S, Khosla PK, et al. Improving production of plant secondary metabolites through biotic and abiotic elicitation. J Appl Res Med Aromat Plants 2019;12:1-12. [Crossref]
  135. Zhang B, Zheng LP, Wang JW. Nitric oxide elicitation for secondary metabolite production in cultured plant cells. Appl Microbiol Biotechnol 2012;93:455-66. [Crossref] [PubMed]
doi: 10.21037/lcm-20-46
Cite this article as: Lozano-Milo E, García-Pérez P, Gallego PP. Narrative review of production of antioxidants and anticancer compounds from Bryophyllum spp. (Kalanchoe) using plant cell tissue culture. Longhua Chin Med 2020;3:18.