Repurposing of Bryophyllum pinnatum for dysmenorrhea treatment: a systematic scoping review and case series

Dysmenorrhea affects women throughout their reproductive years but there has been a lack of effective and well-tolerated treatment options. Pain symptoms mainly result from inflammatory processes and increased contractile activity in the myometrium. The reported use of Bryophyllum pinnatum preparations against inflammation and pain in ethnomedicine as well as current pharmacological data on their inhibition of myometrial contractility led us to hypothesize that this medicinal plant might be a new treatment option for dysmenorrhea. In the first part of the present work, clinical, in vivo, and in vitro studies on the anti-nociceptive and anti-inflammatory, as well as on myometrium relaxing properties of B. pinnatum are reviewed. In the second part, cases of five women with dysmenorrhea who were tentatively treated with a B. pinnatum product are described. The review revealed thirty-three experimental in vivo and in vitro studies, but no clinical study, reporting anti-nociceptive and anti-inflammatory effects of B. pinnatum extracts and compounds in a wide range of conditions. Moreover, sixteen publications on smooth muscle contractility revealed relaxing effects. The latter consisted of clinical evidence, as well as of in vivo and in vitro data. The evidence reviewed therefore provided a rational basis for the use of B. pinnatum in the treatment of dysmenorrhea. We subsequently set out to tentatively treat patients with a well-tolerated B. pinnatum product that is registered (without indication) and commonly used in obstetrics and gynecology in Switzerland. All five treated patients reported a reduction in pain symptoms and 4 out of 5 indicated a reduced intake of painkillers during menstruation. Taken together, the reviewed information on the pharmacological properties and clinical evidence of B. pinnatum extracts and compounds as well as the outcomes of all five patients in the case series support our hypothesis in favor of B. pinnatum as a new, well-tolerated therapeutic approach for dysmenorrhea. Prospective clinical studies are urgently needed.


Introduction 1.Dysmenorrhea
Dysmenorrhea-derived from the Greek, meaning "difficult menstrual flow" (Deligeoroglou, 2000)-is defined as painful uterine cramps shortly before and/or during menstruation that typically last for 8-27 h.Abdominal pain is usually most severe on the first day of menstruation, may radiate to the back and thighs and may be accompanied by other symptoms like nausea, vomiting, diarrhea and fatigue (Hofmeyr and Bassin, 1996;Ruoff and Lema, 2003).Dysmenorrhea has a major impact on the everyday life of affected women, leading to decreased work productivity and a drop in social activities (Ortiz et al., 2009;Eryilmaz et al., 2010;Wong and Khoo, 2010;Pitangui et al., 2013;Smith and Kaunitz, 2022).The disorder is divided into two subtypes: primary dysmenorrhea, where there is no underlying uterine pathology, and secondary dysmenorrhea with similar pain symptoms but due to disorders like endometriosis, adenomyosis or uterine fibroids (Smith and Kaunitz, 2022).First occurrence of primary dysmenorrhea takes place at or shortly (6-24 months) after menarche and the disorder often affects women throughout their reproductive years (Dawood, 2006;Smith and Kaunitz, 2022).
Risk factors for experiencing primary dysmenorrhea are early menarche, nulliparity, irregular menstrual cycle, prolonged and heavy menstrual bleeding, family history of dysmenorrhea, and smoking.Due to diverse definitions and a lack of assessment tools, data on prevalence ranges from 45% to 95% of menstruating women (Jamieson and Steege, 1996;Proctor and Farquhar, 2006;Unsal et al., 2010) whereby in 5%-15% of cases the symptoms interfere with normal daily activities (Pinkerton, 2020).Part of the prevalence data are thought to be underestimated since healthcare professionals as well as women affected themselves disregard the disorder and think of it as normal part of the menstrual cycle, regardless of the severe distress it causes (Jamieson and Steege, 1996;Proctor and Farquhar, 2006;Unsal et al., 2010).
The etiology of pain symptoms that women with primary dysmenorrhea experience mainly results from uterine contractions, the increased contractile activity being caused by various biological processes that are depicted in Figure 1 (Iacovides et al., 2015).The non-rhythmic and uncoordinated contractions lead to high uterine pressure, which leads to reduced blood flow.Furthermore, myometrial blood flow is also impaired due to vasoconstriction in the myometrial tissue.Thus, uterine ischemia develops, leading to stimulation of type C pain neurons and to the typical painful sensation of abdominal menstrual pain (Dawood, 2006).The overproduction of prostaglandins (PG), especially F2α and E2, in the dysmenorrheic uterus is believed to play a major role in the pathophysiology of dysmenorrhea.These inflammatory mediators are responsible not only for inflammation, but also for pain, the increase in body temperature, and sleep dysregulation.Physiologically, prostaglandins are produced from arachidonic acid in a reaction catalyzed by the enzyme cyclooxygenase 2 (COX-2) (Iacovides et al., 2015).The arachidonic acid itself is cleaved from phospholipids by phospholipase A. The decrease in progesterone in the late luteal phase of the menstrual cycle leads to an increased release of phospholipids, as does the tissue trauma due to rejection of mucous tissue in the uterus (Iacovides et al., 2015).Although all women have elevated PGF2α levels during menstruation, dysmenorrheic women have even higher levels and the level of PGF2α correlates with pain intensity (Lundström and Green, 1978).Additionally, regular perception of pain bears the risk of chronification of pain symptoms, which is presumed to occur due to structural and functional changes in the brain, and neuroinflammation (Vergne-Salle and Bertin, 2021; Schrepf et al., 2023).These changes are especially important in primary dysmenorrhea as it typically starts at a very young age.
Treatment options for dysmenorrhea include pain medication such as paracetamol or non-steroidal anti-inflammatory drugs (NSAID) or-especially but not only if contraception is desired-hormonal contraceptives.A cross-sectional study among female university students revealed that 70.5% take NSAID to manage pelvic pain during menstruation (Karout et al., 2021).In a review including 80 randomized controlled trials, NSAID were found to be almost 5 times more effective than placebo to relieve abdominal pain and more effective than paracetamol (Marjoribanks et al., 2003).However, NSAID are not a solution for every patient due to the frequent occurrence of NSAID-resistant dysmenorrhea.Up to 18% of women report non-existent or insufficient pain relief, a phenomenon in which underlying mechanisms are still not fully understood (Oladosu et al., 2018).Furthermore, even if painkillers like ibuprofen are effective, they carry the risk of side effects, especially if taken frequently.In the meta-analysis mentioned before, (severe) adverse effects like gastrointestinal problems, headaches and drowsiness underlined the limitation of this treatment option (Marjoribanks et al., 2003).Due to the lack of safe and effective therapy options in conventional medicine, patients with pelvic pain seek help via complementary and alternative therapies like acupressure, heat application (Karout et al., 2021) and herbal medicine (Slavin et al., 2010;Luo et al., 2023).New well-tolerated and effective therapies are urgently needed.
The traditional use of B. pinnatum and related species in tropical countries goes far back and its range of indications is wide: treatment of wounds, bruises and insect bites (Agoha, 1973); gastrointestinal diseases such as diarrhea, flatulence and vomiting due to its astringent effects (Kamboj and Saluja, 2009); hypertension and urinary disorders (Lans, 2006).Anti-inflammatory, anti-bacterial and anti-viral effects of preparations from B. pinnatum seem to support some of these uses (Kolodziejczyk-Czepas and Stochmal, 2017).In the integrative approach of Anthroposophic Medicine, B. pinnatum was introduced in the treatment of preterm contractions in the 1970s (Hassauer et al., 1985;Daub, 1988;Fürer et al., 2016).Today, B. pinnatum preparations are also used in conventional settings in Switzerland, mainly in gynecology and obstetrics; besides preterm contractions, also overactive bladder syndrome, nocturia and sleeping disorders are common indications (Fürer et al., 2015b;Schenkel et al., 2018;Gantner et al., 2021).
The reported use of B. pinnatum preparations against pain and inflammation in ethnomedicine as well as in the treatment of preterm contractions led us to hypothesize that B. pinnatum might be a new treatment option for primary dysmenorrhea.In the first part of the present work, clinical and experimental data on anti-inflammatory and anti-nociceptive properties of B. pinnatum as well as on its myometrium-relaxing effects, are reviewed according to an a priori defined protocol.In the second part, the first five cases of women with dysmenorrhea who were tentatively treated with a well-tolerated B. pinnatum product that is registered and commonly used in Switzerland (Bryophyllum 50% chewable tablets by Weleda AG) are described.Finally, the reviewed evidence is discussed in view of dysmenorrhea pathophysiology.

Search strategy
The literature review was split into two search strategies: a search of records on anti-inflammatory and anti-nociceptive effects of B. pinnatum was performed using the search terms "("Bryophyllum pinnatum" OR "Kalanchoe pinnata") AND ((inflamm* OR (cyclooxygenase OR COX) OR prostaglandin OR MAPK OR NFkB) OR (antinocicep* OR pain OR analgesic))"; the search regarding the smooth muscle relaxing effects of B. pinnatum was carried out using the search terms "("Bryophyllum pinnatum" OR "Kalanchoe pinnata") AND ("smooth muscle" OR myometr* OR bladder OR vessel OR relax* OR contract* OR tocolys*)".Databases searched were PubMed, Embase and Cochrane.The final search was conducted on 10 July 2023.

Study selection
The study selection was performed independently by LZ and MGS; different classification of the studies was solved by discussion with APSW.Citations were listed and duplicates were removed.Since only full-text articles were taken into consideration, congress abstracts and entries in trial registries were also removed.The remaining records were screened for relevance first on the level of the title, followed by the abstract and then full-text whereas ineligible publications were removed due to the following exclusion criteria: species other than B. pinnatum; non-original articles; no quantitative data on pharmacological or clinical outcome measures; in silico data only; general anti-oxidative effects only; not focus of scoping review.An Excel list and Endnote 20 ® were used for data management during the screening process.

Data extraction
Data extraction was first performed by LZ and in a second step complemented/verified by APSW.The following information was extracted for each publication: year of publication, type of study, plant material, pathophysiological focus and major findings.Retrieved data was grouped into clinical, in vivo and in vitro data.

Case series
In this consecutive case series, five patients with dysmenorrhea were experimentally treated with Bryophyllum 50% chewable tablets; brief case descriptions are presented below.The typical sequence of events was as follows: 1) patient presented at the medical practice with dysmenorrhea complaints; 2) treatment with Bryophyllum 50% chewable tablets was initiated after patients' informed consent; 3) clinical follow-up, allowing for a comparison of the outcomes before and after the Bryophyllum 50% chewable tablets treatment.Patients were asked to rate their pain using a numeric rating scale (NRS; from 1 = no pain, to 10 = worst pain) twice and to report on the standard pain medication used and absences from work due to dysmenorrhea symptoms.

Medication
Patients were treated with Bryophyllum 50% chewable tablets (à 350 mg, each corresponding to 170 mg of fresh leaf press juice) manufactured by Weleda AG, Arlesheim, Switzerland with B. pinnatum leaves provided by Weleda, Brazil.Medication was dosed upon physicians' choice whereby previous studies on longterm treatments served as orientation points (2-2-2 for overactive bladder (Betschart et al., 2013), and 0-0-2-2 for nocturia (Mirzayeva et al., 2023)).A voucher specimen (No.ZSS 29717) has been deposited at the Zurich Succulent Plant collection, Switzerland.The chewable tablets are registered at the Swiss Agency for Therapeutic Products (Swissmedic; without indication).

Ethics
Written consent for publication of their case was obtained from all five patients.A waiver for specific ethics authorization was given for the study publication of this case series, as it does not fall under the jurisdiction of the Swiss Federal Law on data protection (Human Research Act, Article 2)

Results
3.1 Literature review 3.1.1Overview of the current literature and of the included articles Despite a marked relative increase of the literature on B. pinnatum during the last decade (34 publications found with the keywords " Bryophyllum AND pinnatum" between 1971and 2012and 69 between 2013and 2023), a manageable number of publications was found.This encouraged us to proceed with the initially planned two searching strategies on the (i) anti-nociceptive and anti-inflammatory and (ii) relaxant/contractility-inhibitory properties of B. pinnatum (Figure 2A, B, respectively).Approximately a quarter of the initially found publications were reviews.Since there was no limit regarding publication year, only original publications-no systematic reviews-were included in the scoping review.No case report was identified in the search process.The search strategy restricted the work to publications in English or at least with an English summary.There were distinctly more articles on in vitro and in vivo data but only a limited amount on clinical studies.Due to the variety of approaches, no classical risk/quality assessment was performed.
Thirty-three experimental studies reporting on anti-nociceptive and anti-inflammatory effects and sixteen publications reporting on relaxing effects on smooth muscle contractility of B. pinnatum extracts and compounds were included in the review after the selection process.In the following subsections, the data found from the two searches have been synthesized (see Tables 1, 2).For each search, data have been grouped by topic/indication and paragraphs mentioning clinical evidence first, followed by results from animal models and in vitro experimental evidence.

Anti-nociceptive and anti-inflammatory effects
Neither retrospective nor prospective clinical studies were encountered.Indications for clinical evidence consist of reported use in ethnomedicine (cf.Introduction) and have not been included in the present review.In vivo and in vitro studies cover a wide range of pathophysiological topics related to inflammatory and nociceptive processes and are described in the following.
An often used in vivo set up to assess the systemic antiinflammatory effect of B. pinnatum is the paw or ear edema model.In this, edema is induced by various substances administered in the ear or paw of the test animal, and inflammation is assessed by measuring weight or volume of the body part.B. pinnatum leaf extracts inhibited inflammation induced by carrageen (Olajide et al., 1998;Pandurangan et al., 2019) and formaldehyde (Gupta et al., 2010).Moreover, an extract from B. pinnatum stem reduced croton oil-induced edema in rat ear (Chaturvedi et al., 2012).Also, after topical application, another B. pinnatum extract was able to inhibit croton oil-, arachidonic acid-, phenol-and capsaicin-induced ear edema in mice, with histopathologic evaluation showing reduced tissue infiltration by inflammatory cells (Chibli et al., 2014).In one publication, the inhibitory effect of a flower extract of B. pinnatum on croton-oil induced ear edema was shown (Ferreira et al., 2014).Quercetin 3-Oα-L-arabinopyranosyl (1→2) α-L-rhamnopyranoside, the most abundant flavonoid in B. pinnatum leaves and flowers, was shown to have anti-inflammatory and anti-nociceptive effects in vivo (Coutinho et al., 2012;Ferreira et al., 2014).This flavonoid with a rather unusual glycosylation pattern was further shown to inhibit the activity of COX-1 as well as COX-2 enzymes (Ferreira et al., 2014).
Local administration of an aqueous extract of B. pinnatum resulted in the reduction of inflammation in the ear and paw edema model (de Araújo et al., 2019).This led to investigations regarding wound healing properties of B. pinnatum, since the treatment of wounds is also a frequent use in ethnomedicine.The topical application of a cream containing an aqueous leaf extract and another containing the major flavonoid (quercetin 3-O-a-L-arabinopyranosyl-(1→2)-a-L-rhamnopyranoside) showed better re-epithelization and denser collagen after 12 days of application in a rat excision model (Coutinho et al., 2021).After showing that B. pinnatum applied via a gel formulation achieved wound healing properties by reducing wound area, authors of a recent publication also aimed to find the mechanism of action.They found out that besides reducing inflammatory infiltrate in the   wound area, angiogenesis was also improved based on increased expression of vascular endothelial growth factor (VEGF) (de Araújo et al., 2023).
In an in vivo acute gastric lesion induction model, B. pinnatum press juice (de Araújo et al., 2019) and a methanolic extract (Pal and Nag Chaudhuri, 1991) showed gastro protective and ulcer-healing effects, and inhibited inflammatory reaction by reducing the levels of interleukin 1β (IL-1β), tumor necrosisfactor α (TNF-α) and the expression of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB-p65) (de Araújo et al., 2018;de Araújo et al., 2021).In an experimental study focusing on the use of B. pinnatum for treatment of Colitis ulcerosa and Crohn's disease, hydroethanolic leaf extract showed downregulation of toll-like receptor and NF-κB-p65 in vivo (rats and mice), improved cytoarchitecture of colon tissue, as well as mucosa protection, and in vitro reduction in pro-inflammatory mediators (Andrade et al., 2020).Some identified publications focused on the immunomodulatory effect of B. pinnatum extracts and compounds.In lupus mice, B. pinnatum reduced T cell activation (Indriyanti et al., 2018b) and B cell maturation, decreased production of TNF-α (Dantara et al., 2021) and improved histopathological nephritis markers (Indriyanti et al., 2018a).In addition, the aqueous extract and the flavonoid quercetin reduced in vitro mast cell activation and in vivo airway inflammation in allergic airway models (Cruz et al., 2012).
In myometrial cells, oxytocin (OT) is known to trigger inflammatory signaling pathways by leading to activation of mitogen activated protein kinases (MAPK) stress-activated protein kinase or c-jun N-terminal (JNK/SAPK) and extracellular-signal regulated kinases (ERK1/2), which are known to influence the expression of many downstream enzymes such as COX-2, and play important roles in calcium-independent contractility regulation.Data on substances from B. pinnatum leaves revealed that press juice, a flavonoide-enriched fraction and a bufadienolideenriched fraction inhibited OT-induced JNK/SAPK and ERK1/ 2 activation by phosphorylation (see (Santos et al., 2021) and references therein).Further, the effects of B. pinnatum extracts on various enzymes involved in inflammatory processes were investigated.In these studies, leaf extract as well as root extract inhibited xanthinoxidase (Latif et al., 2020;Morais Fernandes et al., 2021).3-O-α-L-Arabinopyranosyl-(1→ 2)-O-α-L-rhamnopyranoside was shown to inhibit phosphodiesterase 4 (PDE4) (Lourenço et al., 2020) and the antiplatelet effect of the aqueous extract indicated an effect on the arachidonic acid pathway (as does the inhibition of COX) (Naqvi et al., 2019).

Relaxant/contractility-inhibitory effects
Clinical evidence for the use of B. pinnatum in tocolysis can be found in a retrospective matched-pair study from 2006.It was shown that pregnancy prolongation under treatment with B. pinnatum was comparable to beta-agonists, but with notably fewer side effects (Plangger et al., 2006).An effort was made to gain data from randomized clinical trials; however, the two initiated trials had to be stopped early due to difficulties in patient recruitment.Both trials showed good tolerability of B. pinnatum although data were not sufficient to support statements on clinical efficacy (Simões-Wüst et al., 2018).The use of B. pinnatum in preterm labor management has been reported since the 1970s, but corresponding studies were not identified in the present review since they were published exclusively in German.The latter consisted of an initial retrospective analysis showing that B. pinnatum was as effective as fenoterol, the standard treatment at that time, and that the dosage of fenoterol could be reduced when combined with B. pinnatum preparations (Hassauer et al., 1985).These results were supported by comparable retrospective analyses conducted in the following years (Daub, 1988;Vilàghy, 2002).
Two clinical trials concerned the use of B. pinnatum in women with overactive bladder syndrome (one pilot, double-blind and placebo-controlled, the other prospective but not randomized).In the case of the pilot study, a trend in reduction of micturition frequency and a positive trend for efficacy were apparent (Betschart et al., 2013).In the before-after comparison, a reduction in nocturia and a beneficial effect on sleep quality were observed (Mirzayeva et al., 2023).
The use of B. pinnatum preparations as a tocolytic, as mentioned above, is supported by experimental evidence.The first experimental data on the effect of B. pinnatum on myometrial contractility were published in 2004.In an ex vivo organ-bath model, myometrium biopsies gained from women undergoing term caesarean section were used to assess the relaxing effect of B. pinnatum aqueous leaf extract, and the effect was compared to the β-agonist fenoterol.The results showed that B. pinnatum reduced the strength of contractions, which was confirmed by later work and supporting its use as a tocolytic (Gwehenberger et al., 2004;Wächter et al., 2011;Santos et al., 2019).To gain more information on the mode of action, the effect of B. pinnatum (press juice) on the OT-triggered free intracellular calcium [Ca 2+ ] i increase was thereafter examined on a cellular level.This was done in vitro using a human myometrial cell line obtained by transformation from the uterus of a (non-pregnant) woman undergoing hysterectomy (hTERT-C3 cells).Data showed a concentration-dependent inhibition of [Ca 2+ ] i increase (Simões-Wüst et al., 2010).More recently, the effect of B. pinnatum press juice was investigated using an additional human myometrial cell line obtained from a pregnant uterus (PHM1-41 cells) and the inhibitory effect on the [Ca 2+ ] i increase could be confirmed (Santos et al., 2021).In an attempt to determine which secondary plant compounds are responsible for the OT-induced inhibition of myometrial contractility, the organ-bath model was used again to assess the effects of a bufadienolide-and a flavonoid-enriched fraction.All test substances led to a decrease in myometrial contractility, but the bufadienolide-enriched fraction was most effective (Santos et al., 2018).Both fractions also inhibited the OT-driven increase of [Ca 2+ ] i in myometrial cells in vitro and most interestingly, appeared to have a synergistic effect (Santos et al., 2021).
Besides myometrial tissues, other smooth muscle types were tested with similar approaches using the organ bath model to investigate the relaxant effect of B. pinnatum.In porcine bladder strips (detrusor muscle) B. pinnatum press juice and a flavonoidenriched fraction inhibited electrically induced contraction and also carbachol pre-contracted strips showed reduced contractility (Schuler et al., 2012;Fürer et al., 2015a), whereby a doseindependent initial increase in contractility was observed in one study (Fürer et al., 2015a).In a later study where porcine detrusor muscle strips were stimulated by potassium chloride a highly purified bufadienolide-enriched fraction was the most effective and led to a concentration-dependent lowering of the contraction force.B. pinnatum press juice alone however increased the contraction force of muscle strips (Bachmann et al., 2017).When comparing eight different plants known to be used owing to their spasmolytic potential to reduce the histamine-induced contracting force of guinea pig ileum, a B. pinnatum leaf extract was able to inhibit it progressively from 40% to 95% (Mans et al., 2004).Finally, ex vivo experiments based on an organ bath model revealed that B. pinnatum extract reduces the contractility of tracheal rings of guinea pigs (Ozolua et al., 2010;Mans et al., 2015).These latter experiments are in line with the already mentioned use of B. pinnatum in ethnomedicine for inflammatory gastrointestinal diseases and as a cough remedy, respectively.

Case series
In this consecutive case series, five patients suffering from dysmenorrhea were treated with Bryophyllum 50% chewable tablets over 3-6 months.All five patients (aged 14-27 years) reported a reduction in pain symptoms (shown by a reduction of the NRS score by at least 2) and 4 out of 5 indicated a reduced intake of painkillers during menstruation.None of the patients mentioned adverse reactions.Cases are described in more detail below and a summary of patients and outcomes can be found in Table 3.

Patient 1
An 18-year-old virgin patient presented with dysmenorrhea and hypermenorrhea.She complained about menstruationassociated migraine.The patient had had her menarche when she was 14-years old; family-history revealed only migraine on the paternal side.She did not lose workdays during menstruation, but on the first and second period days, she reported an NRS score of 7.
Her usual analgesic requirement per period was 2 times ibuprofen 400 mg per day for 2 days.Abdominal examination was inconspicuous, transabdominal ultrasound scan was difficult with empty bladder, but a uterus malformation was suspected (European society of gynecological endoscopy U2a dd U3a), both ovaries (and both kidneys) unremarkable, no evidence of endometriosis.Iron levels were determined (no iron deficiency).The patient started treatment with Bryophyllum 50% chewable tablets (3-0-3).Three months after start of the Bryophyllum 50% chewable tablets treatment, at follow-up, the patient mentioned poor compliance during the first 30 days, but regular use thereafter with strong improvement in symptoms.Her NRS score was reduced to 3-4, she used ibuprofen during menstruation only sporadically and perceived a subjective improvement in migraine.Transabdominal ultrasound examination confirms previous findings.

Patient 2
A 27-year-old woman presented with primary dysmenorrhea on the first and second days of her period.She reported an NRS score of 6, and subjective hypermenorrhea and analgesic treatment on the first day of her period (1x ibuprofen 400 mg), but no lost work days during menstruation.At anamnesis, she reported menarche at 11 years of age, a known mammary cyst on the left side (US 2021: breast imaging reporting and data system 2), never being pregnant and an in conspicuous family anamnesis.As contraception, she uses condoms; vaginal and transvaginal ultrasound examinations inconspicuous.The patient started treatment with Bryophyllum 50% chewable tablets (2-2-2).
Three months later, at follow-up, the patient reported an NRS score of 3 and commented "Before the treatment, the pain woke me up in the night, now I hardly know that I have my period".However, she was still taking one ibuprofen 400 mg tablet on the first day of her period; "I take it preventively" she said.

Patient 3
A 21-year-old woman presented with primary dysmenorrhea and a desire to have children.Gynecological examination was unremarkable.Apart from smoking (10 cigarettes/day) she was otherwise healthy; family anamnesis inconspicuous.Current complaints comprised a severe dysmenorrhea-related NRS score of 10, requiring analgesics (ibuprofen 400 mg 2 times per day for the first 2 days of the period).Patient was advised to take Bryophyllum 50% chewable tablets 3x daily (2-2-2) from the 16th day of the cycle until the end of menstruation; in addition, the patient had to start taking folic acid.
Six months later, the patient presented with a positive pregnancy test (pregnancy week 5 and 3 days).A transvaginal ultrasound revealed a uterus with gestational sac 7.2 × 4.5 mm and yolk sac 1.3 mm, no embryo visible yet.When asked about the effect of Bryophyllum 50% chewable tablets on dysmenorrhea, she reported that the NRS score was 6 or even lower with no need for analgesics any longer.

Patient 4
A 20-year-old woman presented with dysmenorrhea that lasted for approximately 2 years and a desire to have children for the last approximately 6 months.Vaginal examination was unremarkable, but transvaginal ultrasound revealed a retroflected uterus, an endocervical polyp 11.7 × 3.9 mm and a myoma of 8.4 × 8.8 mm.Endometrium was 12 mm secretive and both ovaries were unremarkable.The patient had had her menarche when she was 13 years old and is otherwise healthy; family anamneses inconspicuous.Current complaints comprised a dysmenorrhearelated NRS score of 3-5, and a partial analgesic requirement (paracetamol 500 mg if needed).Patient was advised to take Bryophyllum 50% chewable tablets 3x daily (2-2-2) from the 16th day of the cycle until the end of menstruation; in addition, she had to start taking folic acid.
Three months later the patient presented with a positive pregnancy test (pregnancy week 5 and 4 days).A transvaginal ultrasound revealed a uterus with gestational sac 11.1 × 6.2 mm and yolk sac 1.1 mm, no embryo visible yet; intracervical intramyometrical anterior wall myoma stable, no polyp could be visualized; both ovaries were inconspicuous.Asked about the effect of Bryophyllum 50% chewable tablets on dysmenorrhea (two

Patient 5
A 14-year-old female patient presented with hypermenorrhea on day 1 and 2 of menstrual cycle (duration of menses 3-4 days) and severe dysmenorrhea on the first day of menstruation (reporting an NRS score of 8).She reported that an intake of analgesics (not specified) was always necessary on the day in question.She further reported that on these days she was sometimes unable to attend school and was prevented from pursuing other activities.Patient was prescribed Bryophyllum 50% chewable tablets 3 times a day 2 tablets (2-2-2).
After 3 months of following the procedure, patient presented with an NRS score of 6 on first day of menstruation and reported a reduced intake of analgesics (only occasionally necessary).Furthermore, she was no longer prevented from carrying out everyday activities.

Discussion
Taken together, the pathophysiology of dysmenorrhea syndrome points towards inflammatory events, uterine spasms and vasoconstriction, as the three pillars of dysmenorrhea symptomatology (see Introduction).The reviewed in vitro, in vivo and clinical data suggest that at least anti-nociceptive/antiinflammatory effects and uterine relaxation could be promoted by B. pinnatum preparations.In the following, the evidence most relevant for estimating potential effects of B. pinnatum on dysmenorrhea-related pathophysiological processes is discussed.
A variety of studies demonstrated the analgesic effect of B. pinnatum extracts and compounds (Olajide et al., 1998;Pal et al., 1999;Igwe and Akunyili, 2005;Ojewole, 2005), explaining its use for pain treatment in ethnopharmacy and suggesting that they could attenuate dysmenorrhea-associated pain.Moreover, the reported anti-inflammatory effects of B. pinnatum make it an interesting candidate for the treatment of dysmenorrhea, since inflammatory events are an important part of its pathophysiology.Besides the data on systemic anti-inflammatory effects shown in various rodent models (Olajide et al., 1998;Gupta et al., 2010;Chibli et al., 2014;Pandurangan et al., 2019), the inhibitory effects on specific regulators of the inflammatory cascade are of special interest.Leaf juice and extracts inhibited the activation of NF-κB-p65, MAPKs JNK/SAPK and ERK1/2, and the activity of the downstream enzymes COX-1 and COX-2 (Ferreira et al., 2014;de Araújo et al., 2018;Andrade et al., 2020;Santos et al., 2021).Quercetin 3-O-α-L-arabinopyranosyl (1→2) α-L-rhamnopyranoside showed to be at least partly responsible for the inhibition of COX-1 and COX-2 (Ferreira et al., 2014).
Further rational for the exploration of B. pinnatum as a new treatment option for dysmenorrhea is given by the identified publications regarding the smooth muscle relaxing effect observed in in vitro and ex vivo studies in the context of other gynecological as well as non-gynecological indications like preterm labor, overactive bladder syndrome, and gastrointestinal disease.Especially in the pregnant myometrium, the hormone OT plays a major role in the induction of contractions.In vitro research related to the use of B. pinnatum as a tocolytic showed the direct relaxation of myometrial muscle strips, and the inhibition of OT-triggered calcium-signaling in myometrial cell lines from both pregnant and non-pregnant uterus (Gwehenberger et al., 2004;Simões-Wüst et al., 2010;Santos et al., 2018;Santos et al., 2019;Santos et al., 2021).
In this context, the research efforts on an arginine-vasopressin (AVP) related approach for treatment of dysmenorrhea are worth mentioning.The approach is based on the high secretion of AVP in the menstruating uterus, where this hormone is a relevant factor for myometrial contractility and decreased uterine blood flow (Åkerlund et al., 1979;Strömberg et al., 1984;Arrowsmith, 2020).Furthermore, the approach has to do with the high expression of the AVP receptor 1a (AVPR1a) in the non-pregnant myometrium [approximately five times higher than the very similar OT receptor (OTR) (Åkerlund, 2002)].Therefore, AVPR1a antagonists could be a potential new treatment option for primary dysmenorrhea.One example of an intensively studied AVPR1a-antagonist is relcovaptan, which was investigated in a prospective clinical study with non-pregnant women.The administration of relcovaptan preceding a bolus injection of AVP or OT showed a dose related reduction in intrauterine pressure after AVP, but not after OT application (Steinwall et al., 2004).While this supports AVPR1a as a therapeutic target, adverse reactions (e.g., headaches, nausea and hypotension) might limit future application (Lemmens-Gruber, 2006).Whether the inhibitory effect of B. pinnatum on myometrial contractility is mediated through inhibition of the OTR or also AVPR1a, is currently unknown.Due to the high receptor homology of OTR and AVPR1a and the extensive cross talk between OT and AVP (Åkerlund et al., 1999;Arrowsmith, 2020), it is conceivable that B. pinnatum extracts also inhibit AVP-mediated signaling.Which contractility-associated pathways B. pinnatum press juice inhibits and, most importantly, whether it only inhibits OT-induced signaling or, in addition, AVPinduced increase of [Ca 2+ ] i in myometrial cells deserves further investigations.
The effectiveness observed in the first five patients treated supports our suggestion to repurpose B. pinnatum for the treatment of dysmenorrhea: in all five women, the NRS score decreased by at least 2 (and up to 4) and 4 out of 5 patients reported a reduced intake of pain medication during menstruation (Table 3).Case series can be seen as a low-cost and feasible method for a preliminary proof of concept for a certain treatment in a real-world setting.However, results have to be interpreted with caution, given the low number of patients involved and the absence of a placebo-control group.In our case, this might be particularly relevant as previous studies have revealed that patients with primary dysmenorrhea respond favorably to placebo.In a study from 1989, a third of patients treated with placebo experienced symptom improvement in the first treatment cycle.The study was conducted over four consecutive cycles over which the efficacy of placebo treatment decreased continuously, with only 10% of patients still reporting an effect in the fourth cycle (Fedele et al., 1989).The initial decrease of pain intensity in women with primary dysmenorrhea treated with placebo emphasizes the importance of assessing efficacy of treatment options over several cycles (for comparison, in the present case series Bryophyllum 50% tablets were usually taken for 3 months before reassessment).
Repurposing B. pinnatum for the treatment of dysmenorrhea presupposes that it is safe and well tolerated.With a view to safety, especially bufadienolides should be considered, since they are known for their cardiotoxic activity.However, cardiotoxicity of B. pinnatum and related plant species was only observed in animals that ingested a large amount of plants (McKenzie et al., 1987;Reppas, 1995).With a daily dose of 6 tablets, the intake of bufadienolides is very low (0.035 μg/kg of bersaldegenin-1,3,5aceate) and below the amount shown to have toxic effects [see (Bachmann et al., 2017) and references therein].In experiments using fresh myometrium and detrusor muscle, tissue viability was confirmed after the exposure to test compounds by assessing contractile activity after a wash out phase (Fürer et al., 2015b;Santos et al., 2018;Santos et al., 2019).Additionally, toxic effects were found in myometrial cell lines only when very high doses of press juice or fractions enriched with bufadienolides and flavonoids were used (Santos et al., 2018;Santos et al., 2019).In an in vivo study, the treatment of mice at supratherapeutic levels did not show any effect on liver enzymes, urea nitrogen, or alkaline phosphatase (Torres- Santos et al., 2003;Hosomi et al., 2022).Also the treatment of pregnant wistar rats with B. pinnatum showed no toxicity at therapeutic levels and merely reduced glucose levels (Hosomi et al., 2022).All clinical studies performed so far showed that treatment with B. pinnatum preparations can be considered as safe and well-tolerated, without severe adverse events and only occasional adverse events that included stomachache, diarrhea, and exanthema (Betschart et al., 2013;Simões-Wüst et al., 2018;Mirzayeva et al., 2023).Gastrointestinal side effects upon treatment with the best-investigated preparations of B. pinnatum, namely, Bryophyllum 50% tablets, are most probably related to the contained lactose in patients with lactose intolerance.This product is widely used in Switzerland, especially during pregnancy (more than 30% of pregnant women take Bryophyllum at some point during their pregnancy) (Fürer et al., 2015b;Gantner et al., 2021).
In a next step, the effectiveness of Bryophyllum 50% tablets for the treatment of dysmenorrhea should be investigated in a prospective clinical study with an appropriate patient number.While doing so, the following aspects should be taken into consideration: Firstly, the dosage of six tablets daily (chosen in our case series) seems to be suitable for treatment.Secondly, it is important to cover at least three cycles to avoid over-evaluation of placebo effects.Thirdly, and in view of the anti-inflammatory effects of B. pinnatum preparations, changes in inflammationassociated symptoms such as lower back pain, diarrhea, sleeping disorders and other psychological symptoms should be considered.Such symptoms are experienced by many women with dysmenorrhea and are likely to be associated with the high occurrence of prostaglandins in the body of menstruating women (Iacovides et al., 2015).Finally, it would be advisable to directly monitor quality of life.In a study conducted among university students in Spain, women with dysmenorrhea had a lower mean quality of life score than women who did not suffer from dysmenorrhea, even if no correlation between the pain score and the perceived quality of life was found (Fernández-Martínez et al., 2019).
The review of the literature described in the first part of the present work showed that the pharmacological and clinical effects of B. pinnatum preparations support its use in the treatment of dysmenorrhea.In the second part of this work, first cases of women tentatively treated with Bryophyllum 50% chewable tablets revealed symptom improvement and very good tolerability.Taken together, our work encourages further clinical investigation of the use of Bryophyllum 50% in the treatment of dysmenorrhea.As a next step, a prospective clinical study with higher patient number is under preparation.
extension for Scoping Reviews (PRISMA-ScR) guidelines according to the a priori protocol described below.The flowchart of the selection process is presented in Figure 2A, B. The research question addressed in the review was: do B. pinnatum extracts or compounds have anti-nociceptive/anti-inflammatory and smooth muscle relaxing effects?

FIGURE 2
FIGURE 2 Flow chart of the review selection process on (A) anti-nociceptive and anti-inflammatory effects and (B) anti-contractility and smooth muscle relaxing effects of B. pinnatum.

TABLE 1
Methodological characteristics, plant material and main results of included publications on anti-nociceptive and anti-inflammatory effects of B. pinnatum.

TABLE 1 (
Continued) Methodological characteristics, plant material and main results of included publications on anti-nociceptive and anti-inflammatory effects of B. pinnatum.

TABLE 1 (
Continued) Methodological characteristics, plant material and main results of included publications on anti-nociceptive and anti-inflammatory effects of B. pinnatum.

TABLE 1 (
Continued) Methodological characteristics, plant material and main results of included publications on anti-nociceptive and anti-inflammatory effects of B. pinnatum.

TABLE 1 (
Continued) Methodological characteristics, plant material and main results of included publications on anti-nociceptive and anti-inflammatory effects of B. pinnatum.

TABLE 2
Methodological characteristics, plant material, main results of included publications on anti-contractility and smooth muscle relaxing effects of B. pinnatum.

TABLE 2 (
Continued) Methodological characteristics, plant material, main results of included publications on anti-contractility and smooth muscle relaxing effects of B. pinnatum.

TABLE 2 (
Continued) Methodological characteristics, plant material, main results of included publications on anti-contractility and smooth muscle relaxing effects of B. pinnatum.Only in case of clinical studies p-values are shown. a

TABLE 3
Patient characteristics and objective outcomes before starting treatment with Bryophyllum 50% tablets and thereafter.