Brassinolide

Brassinolide: A Comprehensive Reference

Identity and Chemical Character

Brassinolide is a naturally occurring plant steroid and the founding member of the brassinosteroid (BR) class of phytohormones. It is a natural steroid that promotes cell elongation and division in plants. It was discovered in rape (Brassica napus) pollen in 1979, and its structure was determined by X-ray crystallography and spectroscopic analysis to be (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan-6-one.

The CAS registry number of brassinolide is 72962-43-7. Its molecular formula is C₂₈H₄₈O₆ and its molecular weight is 480.7 g/mol. In pure form, it presents as a white crystalline powder.

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids. Brassinolide is the first and the most active member of the family of the brassinosteroids (BS), which by the year 2000 was formed by more than 40 fully characterised members isolated from plants from both the terrestrial and marine kingdom. To date, more than 70 related compounds called brassinosteroids (BS) have been found in natural sources.

Common structural characteristics are A/B cis-fused steroidal skeletons with oxygenated functions in ring A, the lateral chain, and with very few exceptions ring B. The magnitude of biological activity has been found to depend on the position and spatial orientation of the hydroxyl groups in ring A, the nature of the oxygenated function present in ring B, the configuration of chiral centres C22 and C23, and the substitution pattern and configuration of the chiral centre C24. A particularly critical biosynthetic step is the C6 oxidation: the most biologically significant of these reactions is the C6 oxidation, where a ketone is formed at the C6 carbon position. This single reaction increases the biological activity of the molecule by a factor of 200.

Natural Sources and Distribution

Naturally occurring brassinolide steroids are found in nearly all plants, algae, ferns, gymnosperms and angiosperms. It is found in highest concentrations in the pollen, immature seeds, flowers and roots of plants. More than 40 brassinolide analogues, collectively known as BRs, have been identified and characterized from many different plant species, including 37 angiosperms, 5 gymnosperms, a pteridophyte (Equisetum arvense), and an alga (Hydrodictyon reticulatum).

Taking into account their vanishingly small content in plants, chemical synthesis is the only practical source of brassinosteroids both for scientific and practical purposes. For research and supplemental use, brassinolide and its analogues are therefore typically obtained through chemical synthesis or semi-synthesis rather than direct extraction.

Key Analogues

Several closely related analogues are discussed in the scientific literature alongside brassinolide itself:

• 28-homobrassinolide, 28-epihomobrassinolide, 24-epibrassinolide, and 22,23,24-trisepibrassinolide are sterol substances obtained by chemical synthesis.
• Castasterone — the immediate biosynthetic precursor of brassinolide, possessing partial activity.
• All naturally occurring brassinosteroids exert the same qualitative effects as brassinolide.

Biosynthesis

The production of brassinolide begins with a closely related sterol called campesterol, which is found in the cell membrane. Initially, it is reduced by an enzyme called DET2. This is followed by a series of oxidation reactions, facilitated by cytochrome P-450 enzymes, which add hydroxyl groups to the molecule. The enzymes BR6ox1 and BR6ox2 then convert an intermediate to castasterone, and BR6ox2 can further catalyze formation of a seven-membered lactone ring from the C6 ketone, yielding brassinolide. Brassinolide homeostasis is regulated by several catabolic enzymes, including the P450 enzyme BAS1, the UDP-glycosyltransferase UGT73C5, and sulfotransferases. Brassinolide biosynthesis occurs, at least in part, within the endoplasmic reticulum.

Historical Discovery and Context

The discovery of brassinolide, the most active natural BR currently identified, was preceded by three decades of experiments at the United States Department of Agriculture (USDA) in which organic extracts of pollen from over 60 species were applied to a variety of crop plants to identify new compounds with growth-promoting properties. Extracts of Brassica napus L. (rape) pollen (family Cruciferae) had been reported to produce a novel growth-promoting effect when applied to young pinto bean plants. Fractions active in the bean second-internode assay give rise to both increased cell elongation and cell division. In 1979, M. D. Grove and colleagues isolated it from Brassica napus (rapeseed) pollen and characterized it, publishing the structural determination in Nature. The isolation of brassinolide in 1979 opened a new era in research on phytohormones.

Traditional and Historical Use of Pollen-Containing Plants

Brassinolide as a discrete chemical entity was unknown prior to 1979, and no traditional medicinal system explicitly employed brassinolide itself as a recognized compound. Traditionally, plants containing brassinosteroids have been utilized in various cultures for their reputed health-promoting qualities, although the specific identification of brassinolide itself as a medicinal compound is a more recent scientific advancement.

Pollen, the male gametophyte of flowering plants, is a high energy material collected by insects and stored as food reserve. Pollen has been used traditionally by humans for religious purposes and as supplementary food. Pollen is a concentrated, energy and vitamin-rich food that in contemporary times is not only consumed as a dietary component but also used in alternative medical treatments. Pollen has potential importance as a supplementary and survival food, and for conditioning of athletes. Pollen has been used medically in prostatitis, bleeding stomach ulcers and some infectious diseases, although such use has been questioned by the medical profession.

The use of pollens in folk medicine also indicates the scope of steroids of plant pollens in medicines. It is important to note, however, that the attribution of any traditional therapeutic effect specifically to brassinolide — as opposed to other pollen constituents — is a retrospective inference made in the scientific literature and was not part of the original folk or traditional knowledge. No specific documented traditional preparation or dosing regimen for isolated brassinolide has been found in the peer-reviewed ethnobotanical record.

Since brassinolide and its congeners are natural products, and are abundant in the vegetable kingdom, they are not excluded from the usual diet of all living organisms and therefore do not constitute an "unnatural" additive.

Key Constituents and Mechanisms of Action

Plant Signalling Mechanism (BRI1 Receptor Pathway)

The intracellular signalling pathway for brassinolide in plants is among the best-characterized steroid-hormone transduction pathways in biology. While animal steroid hormones are perceived by nuclear receptor family of transcription factors, brassinosteroids in plants are perceived by a cell surface receptor kinase, BRI1.

In Arabidopsis thaliana, brassinolide is sensed by the extracellular domains of the receptor kinases BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and its homologs BRL1 and BRL3. Brassinolide binding promotes their interaction with co-receptors, triggering a phospho-transfer cascade that permits nuclear accumulation of downstream transcription factors to regulate target genes.

The active BR, brassinolide (BL), binds to the extracellular domain of the BRI1 receptor kinase, promoting a basal BRI1 kinase activity that phosphorylates the negative regulator BKI1 on Y211, releasing it from the membrane and allowing BRI1 to associate with BAK1 or its homologs BKK1 and SERK1. Activated BRI1 or BAK1 then regulate, possibly indirectly, the activities of BIN2 kinase and/or BSU1 phosphatase, which directly regulate the phosphorylation status and nuclear accumulation of two homologous transcription factors, BZR1 and BES1. BZR1 and BES1 directly bind to promoters of BR responsive genes to regulate their expression.

The BR signaling pathway has become a paradigm for both receptor kinase signaling in plants and steroid signaling by cell surface receptors in general. Studies of mutants with defects in BR biosynthesis or signalling demonstrated that BR plays essential roles in nearly all phases of plant development, as these mutants show multiple developmental defects, such as reduced seed germination, extreme dwarfism, photomorphogenesis in the dark, altered distribution of stomata, delayed flowering and male sterility.

Mechanisms Proposed in Animal and Human Cell Systems

Brassinosteroids, a class of plant-specific steroid hormones, control many developmental and physiological processes like their animal counterparts, including regulation of gene expression, cell division and expansion, differentiation, programmed cell death, and homeostasis. In animal and human cell systems, the mechanisms described in preclinical research differ from the plant BRI1 pathway and primarily involve:

• PI3K/Akt pathway activation: 28-Homobrassinolide (HB), a steroidal lactone with potent plant growth-promoting property, stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC₅₀ 4 μM) mediated in part by PI3K/Akt signaling pathway. Stimulation of protein synthesis by HB depends on PI3K/Akt and PKC but not on MAPK signaling. Pretreatment with Akt inhibitor triciribine (20 μM), PI3K inhibitor LY294002 (25 μM), and PKC inhibitor GO6976 (10 nM) inhibited HB-mediated (3 μM) protein synthesis in L6 rat skeletal muscle cells, while MEK1 inhibitor PD98059 (2 μM) and p38 MAPK inhibitor SB203580 (50 nM) had no effect.
• Apoptosis induction in cancer cells: Brassinolide has been observed to promote apoptosis of human prostate cancer cell line, PC-3, by enhancing caspase-3 activity and downregulating the expression of Bcl-2 (an anti-apoptotic protein).
• Antioxidant enzyme modulation: 24-Epibrassinolide reduces the levels of intracellular reactive oxygen species and modulates superoxide dismutase, catalase, and glutathione peroxidase activities.
• P-glycoprotein inhibition: Brassinolide could effectively reverse the resistance of human T lymphoblastoid cell line CCRF-VCR 1000 by inhibiting the effusion of drug transported by P-glycoprotein.
• Antiviral interference with viral replication: Brassinosteroid compounds have been shown to be capable of disturbing the synthesis of viral proteins as well as the maturation of the viral capsule.
• Cell migration and Akt phosphorylation: There was a direct correlation between a compound's ability to promote cell migration and to induce Akt phosphorylation. Akt is a key enzyme in signal transduction pathways involved in cell survival, cell-cycle progression, and migration. Increasing evidence suggests that Akt may play a role in repair and collagen production by activated fibroblasts.

Scientific Evidence by Area of Use

The following sections survey the preclinical and, where available, early clinical evidence. Unless otherwise stated, all evidence is preclinical (in vitro or animal models); no published completed randomized clinical trial (RCT) in humans specifically for brassinolide supplementation has been identified in the peer-reviewed literature at the time of writing.

1. Skeletal Muscle Anabolism and Body Composition

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids, with no known function in mammals. Despite this, the following preclinical evidence has emerged:

28-Homobrassinolide (HB), a steroidal lactone with potent plant growth-promoting property, stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC₅₀ 4 μM) mediated in part by the PI3K/Akt signaling pathway. Oral administration of HB (20 or 60 mg/kg/d for 24 days) to healthy rats fed a normal diet (protein content 23.9%) increased food intake, body weight gain, lean body mass, and gastrocnemius muscle mass as compared with vehicle-treated controls. The effect of HB administration increased slightly in animals fed a high-protein diet (protein content 39.4%).

Both oral (up to 60 mg/kg) and subcutaneous (up to 4 mg/kg) administration of HB showed low androgenic activity when tested in the Hershberger assay. This is a standardized rodent assay for androgenic side effects. The observation of low androgenic activity in this assay is a preliminary safety signal but does not substitute for human clinical data.

Evidence strength: Preliminary; in vitro and rodent studies only. No human clinical trials exist for this indication.

2. Glucose Metabolism and Metabolic Effects

Oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. The aim of a subsequent study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in the liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50–300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 hours of treatment. Daily chronic administration of HB (50 mg/kg for 8 weeks) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition.

Regarding brassinolide itself (rather than the homobrassinolide analogue), brassinolide had a similar function of reducing blood glucose levels as phenformin, but without dose-dependent manner. Blood glucose levels showed significant differences after brassinolide treatment with different doses (200, 100, and 50 mg/kg). These results indicated that brassinolide could reduce blood glucose levels without toxicity. As of the time of reporting, no clinical study in humans had been reported.

Evidence strength: Animal (rodent) models only. All dose ranges reported are in mg/kg body weight in mice/rats and cannot be directly extrapolated to human dosing. No human trials exist.

3. Anticancer and Antiproliferative Activity

The first evidence that natural brassinosteroids can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells came from in vitro structure-activity relationship studies.

Regarding brassinolide specifically in prostate cancer cells: The study investigated the effect of brassinolide on androgen-independent human prostate cancer PC-3 cell viability. Results showed that brassinolide could induce a time- and concentration-dependent cytotoxicity in PC-3 cells. The mode of cell death appeared to be predominantly apoptosis, as shown by flow-cytometric analysis, fluorescence and transmission electron microscopy. Caspase-3 activity was obviously increased after brassinolide treatment. Western blot studies indicated that treatment with brassinolide triggered a time-dependent decrease in the expression of anti-apoptotic protein Bcl-2.

For 24-epibrassinolide in colon cancer: Application of 24-EpiBL in colon carcinoma cells (HT-29 and HCT 116) has been found to upregulate Foxo3a (Forkhead/Winged Helix Box Class O) and protein tyrosine kinase Src p38, after the activation of PI3K/AKT, hence leading to mitochondria-regulated cell death in colon cancer cells.

Brassinosteroids are plant hormones that stimulate their growth and resistance. BRs have anticancer, antiviral, and antipsoriatic activity, among others. Brassinosteroids, a class of plant-specific steroid hormones, are considered as new potential anticancer agents for the treatment of tumors of different origin, including hormone-dependent cancers.

Evidence strength: In vitro (cell line) studies only. No animal tumor model or human clinical trial data for brassinolide as a cancer treatment has been identified. These findings are hypothesis-generating only.

4. Antiviral Activity

Natural brassinolide (BL) and 28-homocastasterone, and a synthetic BR display antiviral activity against poliovirus (PV), herpes simplex virus (HSV) type-1 and HSV-2, measles virus (MV), vesicular stomatitis virus (VSV), and the arenaviruses. BR analogues have been reported to have antiviral activity against herpes simplex virus type 1 (HSV-1), arenaviruses as well as against replication of vesicular stomatitis virus (VSV) in Vero cells. Antiherpetic activities have also been reported both in a human conjunctive cell line (IOBA-NHC) and murine herpetic stromal keratitis (HSK) experimental models.

Brassinosteroids have inhibitory effects on multiplication of viruses specifically in human cell lines, at times with a large selectivity index (SI), including cytotoxic consequences in a plethora of cancer cells without affecting normal human cells. Further studies are needed to elucidate the precise in vitro antiviral mechanism(s) of these BR analogues and structural features that are associated with their bioactivity.

Evidence strength: Primarily in vitro and animal (murine) models. No completed human antiviral trial has been identified for brassinolide.

5. Wound Healing

Brassinosteroids could accelerate wound healing by positively eliminating inflammation and stimulating reepithelialization of the reparation stage. A study from Rutgers University investigated the wound-healing properties of brassinosteroid analogues in cell and animal models (published in Wound Repair and Regeneration, 2013). All four brassinosteroids tested for their ability to induce cell proliferation at 5 μM showed moderate biological activity. There was no correlation between a compound's ability to induce cell proliferation and stimulation of cell migration, as both R,R- and S,S-24-epibrassinolides promoted cell proliferation but not migration, while HB treatment resulted in significant increase in both parameters.

Evidence strength: In vitro and rodent models. No human clinical wound healing trial for brassinolide has been identified.

6. Neuroprotection

Researchers analyzed the ability of 24-epibrassinolide (24-Epi), a natural BR, to protect neuronal PC12 cells from MPP⁺-induced oxidative stress and consequent apoptosis in dopaminergic neurons. Results demonstrated that 24-Epi reduces the levels of intracellular reactive oxygen species and modulates superoxide dismutase, catalase, and glutathione peroxidase activities. This was the first time that the potent antioxidant and neuroprotective role of 24-Epi was shown in a mammalian neuronal cell line.

A subsequent structure-activity study found that: 6 of the 9 BRs and analogs tested protected neuronal PC12 cells against MPP⁺ toxicity. The structure-activity study suggests that the steroid B-ring and lateral chain play an important role for their neuroprotective action.

Evidence strength: In vitro cell line studies (PC12 cells) only. The MPP⁺ model is a recognized in vitro surrogate for Parkinson's disease-relevant oxidative stress, but these findings remain distant from human clinical utility. No human neuroprotection trial exists.

7. Antigenotoxic Activity

Mutagenic studies using the Ames assay, carried out at the Scientific Research Center of Toxicologic and Hygienic Regulation of Bio-preparations of Russia, showed negative results either without or with metabolic activity, using Salmonella typhimurium (TA1534, TA1537, TA1950, TA98, and TA100) as the tester strain. This finding is considered a preliminary indicator of non-mutagenicity.

Castasterone showed antigenotoxicity in human blood lymphocytes employing comet assay, and 10⁻⁹ M was found most effectual in suppressing DNA injuries. This work applies specifically to castasterone, a biosynthetic precursor to brassinolide, not to brassinolide itself.

Evidence strength: In vitro and microbial assay data only. Results are preliminary and cannot be extrapolated to human use.

8. Immunomodulation

Brassinosteroids have been found to be a novel category of steroids which possess anti-inflammatory activities. Recent studies have indicated that these hormones have antiviral, antifungal, antiproliferative, antibacterial, neuroprotective, and immunomodulatory properties in animal systems. Research on a synthetic brassinosteroid analogue published in Journal of Steroid Biochemistry and Molecular Biology examined in vitro immunomodulatory activity that was associated with improvement of herpetic stromal keratitis in a mouse model.

Evidence strength: Preclinical (in vitro and murine models) only. No human immunomodulation trial exists.

Body Systems Associated with Brassinolide Research

• Musculoskeletal system: Anabolic effects on skeletal muscle (protein synthesis, lean mass, muscle fiber size) studied in rodent models via PI3K/Akt signaling.
• Endocrine/metabolic system: Hypoglycemic and insulin-sensitizing effects observed in obese mice; potential gluconeogenesis modulation.
• Oncology: Antiproliferative and pro-apoptotic effects observed in prostate, colon, breast, and other cancer cell lines in vitro.
• Virology/immunology: Antiviral effects against HSV, VSV, arenaviruses, and others in cell culture; putative immunomodulatory action.
• Neurological system: Antioxidant and neuroprotective effects in dopaminergic cell line models relevant to Parkinson's disease research.
• Integumentary system (skin/wound repair): Cell proliferation and migration effects relevant to wound healing studied in cell culture and rodent models.
• Genomic integrity: Antigenotoxic effects characterized in microbial and plant-based assays.

Dosage Forms and Reported Study Dosages

Brassinolide and its analogues are not approved pharmaceutical drugs, and there are no established human clinical dosages. The following doses are drawn exclusively from animal and in vitro research as reported in peer-reviewed sources:

• In vitro (cell culture) — protein synthesis / muscle cells: EC₅₀ of 4 μM for 28-homobrassinolide in L6 rat skeletal muscle cells.
• Rat oral — anabolic effect: Oral administration of HB at 20 or 60 mg/kg/d for 24 days in healthy rats.
• Rat oral — blood glucose reduction: Blood glucose levels showed significant differences after brassinolide treatment with different doses of 200, 100, and 50 mg/kg (in a rodent model).
• Mouse oral — hypoglycemic chronic dosing: Acute oral administration of 50–300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 hours. Daily chronic administration of HB (50 mg/kg for 8 weeks) ameliorated hyperglycemia and improved oral glucose tolerance.
• Subcutaneous — androgenicity safety test: Subcutaneous administration up to 4 mg/kg was used in the Hershberger androgenicity assay in rats.
• In vitro — wound healing / cell proliferation: Brassinosteroids were tested for ability to induce cell proliferation at 5 μM.
• Developmental toxicity in rats (HBL): HBL was administered by oral gavage at doses of 0, 100, and 1000 mg/kg of body weight in water during gestation days 6 to 15, in groups of 20 mated females.

As dietary supplement products, brassinolide preparations — including 24-epibrassinolide and 28-homobrassinolide — are marketed in powder, capsule, and liquid solution forms. Taking into account their vanishingly small content in plants, chemical synthesis is the only source of brassinosteroids both for scientific and practical purposes, meaning commercially available forms are synthetic or semi-synthetic.

Safety Considerations

Mutagenicity

Mutagenic studies using the Ames assay showed negative results either without or with metabolic activity using Salmonella typhimurium (TA1534, TA1537, TA1950, TA98, and TA100) as the tester strain. This standard bacterial reverse mutation assay is used as an early-stage screen for mutagenic potential; a negative result is generally considered a favorable indicator, but does not constitute a complete safety assessment.

Developmental and Teratogenic Toxicity

HBL was administered by oral gavage at doses of 0, 100, and 1000 mg/kg of body weight during gestation days 6 to 15 in groups of 20 mated female Wistar rats. Maternal and embryo-fetal toxicity was analyzed by studying clinical signs, mortality/morbidity, abortions, body weight, feed consumption, pregnancy data, gravid uterine weights, implantation losses, litter size, and external, visceral, and skeletal malformations. No treatment-related effect was observed on any of the maternal/fetal end points in any dose group. It was concluded that HBL is non-teratogenic at doses as high as 1000 mg/kg body weight in Wistar rats. This was a study of the homobrassinolide analogue, not of brassinolide itself; results cannot be directly extrapolated to brassinolide or to humans.

Androgenic Activity

Because brassinosteroids structurally resemble animal steroid hormones, androgenic side effects represent a theoretical concern. Both oral (up to 60 mg/kg) and subcutaneous (up to 4 mg/kg) administration of HB showed low androgenic activity when tested in the Hershberger assay. This finding, obtained in rats, suggests low androgenic potential at studied doses; however, this cannot be generalised to humans in the absence of clinical data.

Cytotoxicity Considerations

Replacement of the two 2α,3α-vicinal hydroxyl groups by an α-fluoro group decreased but did not abolish bioactivity; however, the cytotoxicity of these compounds against 3T3-NIH murine fibroblast cells was increased. This structure-activity finding indicates that different brassinolide analogues carry different cytotoxicity profiles, and that structural modifications can increase off-target cytotoxicity.

Selectivity Against Normal Cells

A key observation from in vitro anticancer studies is that: natural BRs can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells. While this is a favorable finding in vitro, it does not constitute evidence of selectivity or safety in vivo or in humans.

Absence of Human Clinical Data

Most of the scientific validation for the reported effects of brassinolide is still in the preclinical stage, primarily involving in vitro studies and animal models. Currently, clinical studies in humans evaluating the efficacy and safety of brassinolide as a dietary ingredient remain limited. At present, knowledge of the effects of BRs in animals or humans is still rather fragmentary.

Pollen Allergenicity

For products derived from or marketed alongside pollen preparations, because some individuals are allergic to pollen, and various pollen species contain specific allergens, individual sensitivities must be tested before pollen is used as a treatment or as a supplementary food. This applies to any pollen-sourced product but does not directly implicate synthesised brassinolide.

Known Interactions

No documented pharmacokinetic or pharmacodynamic drug–drug interactions for brassinolide in humans have been identified in the peer-reviewed literature at this time. The finding that brassinolide could effectively reverse the resistance of human T lymphoblastoid cell line CCRF-VCR 1000 by inhibiting the effusion of drug transported by P-glycoprotein raises a hypothetical concern about interactions with drugs that are substrates of P-glycoprotein (a major drug efflux transporter), but this is based solely on in vitro cell line data and has not been evaluated clinically.

Overall Evidence Assessment

Over the last decade, brassinosteroids have attracted worldwide interest due to their diverse biological activities in animal systems. Recent studies have indicated anticancerous, antiangiogenic, antiviral, antigenotoxic, antifungal, and antibacterial bioactivities of BRs in animal test systems. Despite this breadth of preclinical activity, current investigations of brassinosteroids are greatly focused on understanding the molecular and cellular basis of their effects both in plants and in animal systems, and the field has not yet advanced to systematic human clinical evaluation. The totality of evidence for brassinolide as a dietary supplement ingredient in humans must therefore be characterised as very early-stage and preliminary, resting primarily on in vitro and rodent model data without human clinical trial support.

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