Toxicological Evaluation of the Liver and Kidneys of Rats Exposed to Myrothamnus Flabellifolius Crude Aqueous Extract

Abstract

Background

Myrothamnus flabellifolius (M. flabellifolius) herb is widely used across Southern Africa, often with poorly defined or unrestricted dosing and limited safety considerations across its diverse ethnomedicinal applications.

Objective

This study investigated the effects of M. flabellifolius crude aqueous extract on rat liver and kidneys.

Methods

M. flabellifolius crude aqueous extract was screened for its bioactive components. Safety was assessed using single and 28-day repeated oral dose toxicity studies. In the former, male and female Sprague–Dawley rats (n = 12 per group; 6 per sex) received M. flabellifolius stem and leaf crude aqueous extract at doses of 0, 500, 1000, 2000, 5000 and 10000 mg/kg and were observed for 14 days for clinical signs, body weight changes, and mortality. Based on the absence of clinical signs and lethality, the same dose range was applied in the 28-day repeated oral dose toxicity study in which female rats (n = 6 per group) received treatment. At the end of the study, blood samples were collected for biochemical analysis, while liver and kidney samples were harvested for relative organ weight and histopathological examination.

Results

Phytochemical screening confirmed the presence of tannins, flavonoids, coumarins and terpenoids in the extract. Treatment in both single and 28-day repeated oral dose toxicity studies showed no clinical signs or mortality; body/relative organ weights changes were non-significant (p > 0.05). The oral no-observed-adverse-effect level (NOAEL) was 10000 mg/kg in both sexes. Biochemical indices showed non-significant changes (p > 0.05), whereas liver and kidney histoarchitecture remained unremarkable.

Conclusion

M. flabellifolius crude aqueous extracts exhibited a wide safety margin with no detectable hepatorenal toxicity. Its confirmed bioactive phytochemicals valorizes ethnomedicinal use.

Introduction

Myrothamnus flabellifolius is a plant species that belongs to the family Myrothamnae, which is common in Southern Africa in countries such as Botswana, Namibia, Malawi, South Africa, Zambia and Zimbabwe.^1,2 This drought-resistant plant is sensationally called the “resurrection bush” due to its remarkable ability to swiftly regenerate from a complete drying state when it receives water or rain.^3,4 In Zimbabwe, the plant is called Mufandichimuka or Umfavuke in the local languages of Shona and Ndebele, respectively, both of which also vividly describe its extraordinary regeneration capacity. M. flabellifolius is widely used to treat numerous conditions such as dysmenorrhea, hypertension, diabetes mellitus, gingivitis, stroke, epilepsy and pain in communities where the plant is endemic.^5–7 It also has a long ethnomedicinal record in Zimbabwe and the wider Southern African region for respiratory indications, which include colds, chest ailments and asthma, for which the herb is often prepared as a decoction or infusion.⁸ Correspondingly, during the COVID-19 pandemic in Zimbabwe when there was a heightened public reliance on traditional home remedies and herbal preparations, M. flabellifolius (Mufandichimuka/Umfavuke) was among the commonly used botanicals for managing COVID-19-related respiratory symptoms particularly in community self-care contexts.⁹

Therefore, M. flabellifolius joins the league of many such herbs that have gained popularity in the management of medical conditions in recent years.^8,10 In fact, numerous studies have shown that over 80% of people globally now prefer using natural herbs and fruits for medicinal purposes.^5,8–12 This is because herbs are generally believed to be safer and, in some cases, have proven to be effective in the management of various ailments.¹³ However, some herbs have also been shown to cause severe toxic reactions since they are composed of a mixture of bioactive compounds.^13,14 It appears in most instances, herb users are not privy to the limitations of medicinal plants compared to approved conventional medicines or intentionally overlook them because of their historic relevance.¹³

Additionally, traditional herbal practice is frequently characterized by non-standardized, experiential dosing often prescribed in household measures such as one to several handfuls of plant material prepared in boiled water with doses administered as fractions of a cup, a full cup or a glass of the decoction one to three times daily resulting in substantial inter-user variability.¹⁵ These realities highlight long-standing calls for strengthened herb pharmacovigilance and more systematic safety monitoring of traditional medicines.^16–18 These concerns are amplified in the context of sexual stamina/aphrodisiac use, where demand for rapid effects has been associated internationally with a high-risk market segment of unregulated sexual-enhancement products and herbal/polyherbal mixtures.^16,19 M. flabellifolius is among the species reported for male and female reproductive or sexual-health indications,^5,20 and it may also be taken as decoctions or multi-herb preparations – contexts in which over-consumption or co-exposure to other bioactive plants can plausibly increase safety uncertainty. Accordingly, a safety framework for M. flabellifolius requires robust in-vivo dose-range exploration to establish safety margins, careful documentation of preparation/dosing practices reflective of community use to capture adverse event signals linked to real-world patterns of use and misuse.

The liver and kidneys are usually among the major target organs affected by xenobiotics, including herbal bioactive compounds.^21,22 For the liver, this is due to its central role in metabolism, portal location within the circulation, anatomical structure and physiological function making it highly prone to xenobiotic-induced injury.²¹ Meanwhile, the kidneys are equally susceptible to toxic alterations because they filter high volumes of blood; hence, large concentrations of toxins can accumulate in the tubules.²³ Toxicity in kidneys compromises their function rendering them unable to effectively excrete body waste, maintain fluid and electrolyte homeostasis and secrete metabolic hormones.²³ This condition results in systemic toxicity, leading to multiple organ failure and even death. Therefore, exploration of the status of the liver and kidneys is key to establishing the safety profile of herbs such as M. flabellifolius.

As the tide in the uptake of natural herbs grows globally, M. flabellifolius has become quite lucrative due to some proven medicinal properties that go beyond its traditional use. For instance, crude aqueous extracts of the herb have been scientifically proven to possess antioxidant, antibacterial and antiviral properties.^2,24,25 Recent studies have shown that M. flabellifolius has high nutritional value^2,3 and appreciable total phenolic and condensed tannin contents comparable to those reported for renowned herbal teas such as rooibos tea (Aspalathus linearis).²⁵ Moreover, since decoctions of M. flabellifolius are largely taken as teas, studies indicate great potential commercial value because of the pleasant aroma and taste of the herb.^6,25 Thus, all this makes it even more pertinent to establish the safety profile of M. flabellifolius which is quite scarce in the literature. Therefore, this study aimed to evaluate the effects of M. flabellifolius crude aqueous extract on the liver and kidneys using experimental rats as an in-vivo model.

Materials and Methods

Plant Material

Stems and leaves of M. flabellifolius used in this study were harvested from the Domboshava communal lands, Zimbabwe (Coordinates: Latitude −17.609196/S 17° 36’ 33.104”, Longitude 31.174119/E 31° 10’ 26.829”). The plants were harvested in May 2021. Prior to harvesting, the plant samples were taken to the National Herbarium, Botanical Gardens in Harare, Zimbabwe, and were verified by Zivisai Mano’mbe the resident plant specialist/research officer. The samples were processed and stored for record keeping (Voucher specimen number: T.N.0001).

Preparation of Extract

Crude aqueous extract of M. flabellifolius was prepared as described by Ashafa et al²⁶ with minor modifications. Fresh M. flabellifolius was air-dried at room temperature for 14 days, and the leaves and stems were ground to a grain size of 2 mm using a Fritish Pulverisette 14 Rotor-speed mill (Germany). The plant powder (100 g) was extracted in 1500 mL of distilled water for 24 hours on an orbital shaker (SO1 Stuart Scientific, Stone, UK). The extract was first filtered with muslin cloth and then through Whatman no.1 filter paper (Maidstone, UK). The filtrate was then concentrated by rotary evaporation (1760 mmHg) at 35–40°C then freeze-dried using a Savant refrigerated vapor trap (RVT104, California, USA) to yield 35 g of brown powder. The dried extracts powder was stored in tightly stoppered sample bottles and stored at –20°C awaiting further analysis.³ The required M. flabellifolius crude aqueous extract was then prepared by dissolving the powder in distilled water to give the required doses assigned in the single and repeated oral dose toxicity studies before administration. Also in both studies, the extract was given at a volume of 10 mL/kg body weight (BW).^27,28 The constitution of the extract into the required doses took into account the solubility of the extract in distilled water^2,25,29 as precedent in the literature for herbal extracts with similar properties.³⁰ All doses were freshly prepared in distilled water and gently vortexed immediately before administration to maintain a homogeneous aqueous preparation and support consistent dose delivery. This procedure aligned with standard expectations for stability and homogeneity of oral test-substance formulations in preclinical toxicity studies.²⁷ All dose preparations remained uniform and readily flowable, with no visible sedimentation, phase separation or undissolved particulate matter observed before dosing.

Phytochemical Screening

Standard qualitative methods were used to test for the presence of major phytocompounds such as tannins, flavonoids, coumarins, quinones, anthraquinones, terpenoids and glycosides, as previously described.^29,31,32

Experimental Animals

One hundred and eight healthy Sprague Dawley (SD) rats (36 males and 72 nulliparous females), aged 6–8 weeks, were acquired and maintained at the University of Zimbabwe, College of Health Sciences Animal House, Mount Pleasant, Harare, Zimbabwe. The rats were individually housed under standard laboratory conditions in Perspex cages. The bedding was changed twice a week to prevent infection and to maintain hygiene. A 12-hour light/12-hour dark regimen (lights on at 07:00) was maintained throughout the study. Ambient temperature of 24 ± 2°C was maintained with adequate ventilation. The rats were fed standard commercial rat chow (National Foods Stock Feeds, Harare, Zimbabwe) and provided with water ad libitum. The care and handling of animals used in this study followed international laws and guidelines on Animal Care and Use (European Community Guidelines, EEC Directive of 1986; 86/609/EEC and NIH guidelines, 1986) and the Zimbabwean legislation (Scientific Animal Experiment Act, 1963). Treatment techniques and protocols were prepared according to ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines.³³ In addition, the treatment techniques, handling of experimental animals and diets were supervised by a qualified veterinary/experimental animal expert stationed at the animal house. All rats were acclimatized to the experimental conditions for 1 week before the commencement of the study.

Single Oral Dose Toxicity Study

A single oral dose toxicity study was conducted as described by Adeyemo-Salami and Makinde,³⁴ Simfukwe et al,³⁵ Bikri et al,²⁸ Ali et al,³⁶ Lakmichi et al,³⁰ Shin et al,³⁷ Liu et al,³⁸ Ye et al,³⁹ Ovuakporie-Uvo⁴⁰ with modifications. Male and female Sprague–Dawley rats were randomly allocated to six groups (n = 12 per group; 6 males and 6 females) in accordance with established toxicological study designs.^41,42 The control group received distilled water (10 mL/kg BW), which served as the vehicle. Animals in the second to sixth groups received a single oral administration of M. flabellifolius crude aqueous extract by gavage at doses of 500, 1000, 2000, 5000 and 10000 mg/kg BW, respectively.²⁸ The broadened dose range was explored considering the long-standing use of M. flabellifolius and the low cytotoxic nature of the herb^6,24 indicating unlikely lethality. All rats were fasted for 12 hours before the administration of treatment but had access to water ad libitum. After treatment, the animals were rigorously observed for mortality, signs of acute toxicity (bleeding, itching, urination, sweating, writhing, laxation), and behavioral changes (aggression, unusual vocalization, agitation, sedation, somnolence, convulsions, tremors, ataxia, catatonia, paralysis, fasciculation, prostration and unusual locomotion, abnormality in food consumption, and asphyxia) for the first 4 hours and then every 2 hours for 24 hours. The rats were further monitored for behavioral changes and individually observed daily for general behavioral and body weight changes, severe toxicity symptoms, and mortality for up to 14 consecutive days after treatment. At the end of the experimental period, all animals were fasted for 12 hours, weighed then euthanized with sodium pentobarbitone (Euthanaze^®, South Africa); 200 mg/kg body weight intra-peritoneal injection.⁴³ The liver and kidneys were assessed for gross anatomical changes and weighed.

28-Day Repeated Oral Dose Toxicity Study

A 28-day repeated oral dose toxicity study was conducted as described by Liu et al,³⁸ Kim et al,⁴⁴ Chan et al,⁴⁵ Deng et al,⁴⁶ Ali et al,³⁶ Min et al,⁴⁷ Ji,⁴⁸ with modifications. Based on the absence of clinical toxicity signs and mortality in the single oral dose toxicity study, the same M. flabellifolius crude aqueous dose range was used for the repeated oral dose toxicity study as precedent in previous studies.^38,48,49 This facilitated the exploration of upper bound margins and comprehensive safety characterization to guide future dose-setting.^38,50 Accordingly, female SD rats were randomly assigned to 6 groups each with 6 rats according to Ayembilla et al.⁵¹ For this study, only female rats were used because of better sensitivity compared to male rats, as described by Murwanti et al.⁵² The first group served as the control and rats in the group were each treated with distilled water (10mL/kg BW), which served as the vehicle for treatment. Rats in the second to sixth groups were treated with M. flabellifolius crude aqueous extract at doses of 500, 1000, 2000, 5000 and 10000 mg/kg BW, respectively. All doses were administered once daily for 28 days by oral gavage. During treatment, rats were monitored daily for mortality, behavioral and toxicological signs consistent with those described under the single oral dose toxicity study but without the intensive early post-dosing observation intervals. These observations ensured humane endpoints and animal wellbeing.³³ At the end of the experimental period, all rats were fasted for 12 hours and weighed, before being euthanized with sodium pentobarbitone (Euthanaze^®, South Africa); 200 mg/kg body weight intra-peritoneal injection. The blood, liver and kidneys were collected according to standard procedures.

Body and Relative Organ Weight Changes

In both the single- and 28-day repeated oral dose toxicity studies, body and relative organ weight changes were measured (Radwag, Germany) and calculated as follows:⁵³

Rat body weight percentage changes were calculated as follows:

and

Relative organ weight was calculated as follows:

Blood Collection, Serum Preparation and Biochemical Analyses

At the end of the study, all rats in the 28-day repeated oral dose toxicity study were anesthetized using sodium pentobarbitone (Euthanaze^®, South Africa) and blood was collected by direct cardiac puncture using a 21-gauge needle mounted on a 5 mL syringe plunger. Blood samples for serum biochemical assays were collected in plain sample bottles and allowed to clot for 2 hours. The clotted blood samples were then centrifuged at 4000 rpm for 10 minutes using a bench centrifuge (Hettich Rotofix, Germany) to obtain serum. The levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), total protein (TP), albumin (ALB), creatinine (CR), and urea (UA) in the serum samples were measured spectrophotometrically using commercially available rat diagnostic kits (Sigma Aldrich, GmbH, Germany) according to the manufacturer’s protocol.

Histopathological Analyses

Tissue preparation was performed as described by Oduola et al,¹⁴ Khalaf et al,²² and others^43,54 with minor modifications. Rat liver and kidney samples were fixed in 10% buffered formalin, processed using an automatic tissue processor (Shandon Citadel 1000, Germany) and embedded in paraffin wax. For consistency of the methods and accuracy of the results, liver midsections and the right kidneys of each rat were used for all light microscopic evaluations. Transverse liver and longitudinal kidney sections were cut using a rotary microtome (Leica, Germany) at 5 µm thickness and stained with Mayer’s hematoxylin and eosin (HE) for histological analysis. Histological assessments of both hepatic and renal tissues from all slides were performed blinded to reduce observer bias and to ensure objective scoring of lesions. For hepatic tissue, alterations in architecture, portal triads, hepatocytes, sinusoids, inflammation, the presence of degeneration, necrosis, and fatty changes were evaluated. Tissue lesions were assessed^54,55 and scored according to the following categories:

1. Hepatitis: acute, chronic, cholestatic, granulomatous.

2. Steatosis: steatohepatitis: microvesicular or macrovesicular.

3. Cholestasis: bland or chlolestatic hepatitis.

4. Vascular abnormalities: sinusoidal obstruction syndrome.

Renal tissues were evaluated for inflammation, necrosis, apoptosis and vascular changes such as congestion. Tissue lesions were assessed⁵⁶ and reference was made according to the following categories:

1. Grade 0 = unremarkable renal tubules.

2. Grade 1 = renal tubular epithelial cell degeneration without significant apoptosis or necrosis.

3. Grades 2–5 = renal tubular epithelial cell degeneration including apoptosis and necrosis comprising <25%, <50%, <75%, and ≥75% of the renal tubules.

All tissue sections were analyzed using an Axioscope microscope (Carl Zeiss Microscopy, Gmbh, Jena, Germany) equipped with a digital camera (Axiocam HRc). Images were captured at (X400) magnifications and saved in JPEG format.

Data Analysis

Statistical analysis was performed using IBM SPSS version 28, data were managed, and graphs were constructed using GraphPad Prism version 5.0 and Microsoft Excel 365. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Data are expressed as mean ± standard error of the mean (SEM) and the p-value was set at 0.05.

The sample size was determined based on established practices in similar rodent models and precedent in the literature^41,51 guided by the resource equation approach. This approach is appropriate when formal power analysis parameters are not available. Therefore, six animals per group in both the single (12 per group, balanced sex) and repeated oral dose toxicity studies (females only) provided acceptable degrees of freedom for ANOVA.⁵⁷

Ethical Consideration

The study protocol was approved by the National Animal Research Ethics Committee of Zimbabwe (NAREC) [Reference code: 010/22] and Midlands State University Ethics Committee (Reference code: MSUFMEC 0004/10/20).

Results

Phytochemical Screening

The results of the phytochemical analysis of M. flabellifolius crude aqueous extract highlighted several bioactive compounds such as tannins, phenols, flavonoids, coumarins and terpenoids, whereas quinones, glycosides and anthraquinones were absent (Table 1).

Table 1.

Results of M. flabellifolius Crude Aqueous Extract Phytochemical Screening

Compound	Test	Result
Tannins	FeCl3 test	+
Flavonoids	Ammonia and H2SO4	+
Coumarins	NaOH test	+
Quinones	HCL acid test	–
Anthraquinones	Ammonium hydroxide test	–
Terpenoids	Salkowski test	+
Glycosides	Keller-Killiani test	–

Notes: (+) represents present; (−) represents absence of a particular phytochemical.

Single Oral Dose Toxicity Study

During treatment, no symptoms of toxicity or behavioral changes, such as increased salivation, emaciation, diarrhea, vomiting, lacrimation and irritability, were observed among rats in all treatment groups in the first 24 hours after treatment and up to 14 consecutive days. The general appearance, grooming, posture and gait were normal during the study period. No deaths were recorded up to the highest dose of 10000 mg/kg after the oral administration of the M. flabellifolius crude aqueous extracts. The mean body weight changes in rats 14 days after single oral dose treatment are shown in Figure 1. No significant difference in body weight change was recorded between the control and treated groups (both male and female). The relative weights of the terminal liver and kidneys are shown in Figure 2. There were no significant differences in the relative weights of the liver and kidneys between the control and treatment groups.

Figure 1.

Body weight percentage changes of rats treated orally with a single dose of M. flabellifolius crude aqueous extracts and observed for 14 days. All values were analyzed by One-way ANOVA, significant at p < 0.05 and data is presented as mean ± SEM.

Figure 2.

Relative organ weight of rats treated orally with a single dose of M. flabellifolius crude aqueous extracts and terminated after 14 days. All values were analyzed by One-way ANOVA significant at p < 0.05 and data is presented as mean ± SEM.

28-Day Repeated Oral Dose Toxicity Study

Clinical Signs, Mortality, Body and Organ Weight Changes

Repeated oral dose administration of M. flabellifolius crude aqueous extract did not cause any symptoms of toxicity, signs of behavioral changes or mortality among rats in any of the treatment groups throughout the 28 days of treatment. The mean body weight changes in the rats after 28 days of treatment are shown in Figure 3. No significant differences in body weights were observed between the control and treatment groups. This was also consistent with observations in the liver and kidney relative organ weights of rats in the control compared to the treatment groups, as shown in Figure 4.

Figure 3.

Body weight percentage changes in female rats treated once daily with M. flabellifolius crude aqueous extracts for 28 days. All values were analyzed by One-way ANOVA, significant at p < 0.05 and data is presented as mean ± SEM.

Figure 4.

Relative organ weight of female rats treated once daily with M. flabellifolius crude aqueous extracts for 28 consecutive days. All values were analyzed by One-way ANOVA, significant at p < 0.05 and data is presented as mean ± SEM.

Blood Serum Biochemical Analyses

The effects of M. flabellifolius crude aqueous extracts on biochemical parameters is presented in Table 2. No significant changes were recorded in either the liver or kidney function tests after 28 days of treatment. All indices for liver and kidney function were within the ranges reported in the historical control and baseline dataset references for healthy Sprague-Dawley rats, corresponding well with the age and sex of animals.^58–61

Table 2.

Effect of Once-Daily M. flabellifolius Crude Aqueous Extract Intake on Rat Liver and Kidney Function Profiles After 28 Days

Parameters	Control	M. flabellifolius (mg/kg)					Reference Interval
		500	1000	2000	5000	10000
ALT (IU/L)	104.51 ± 11.10	100.23 ± 13.10	102.74 ± 10.37	87.58 ± 19.60	86.29 ± 15.91	85.77 ± 12.35	2.1–426.9
AST (IU/L)	150.5 ± 10.11	148.7± 23.24	151.0 ± 13.91	145.8 ± 11.96	144.0 ± 13.38	141.8 ± 15.76	57–258
ALP (IU/L)	327.26 ± 11.66	329.72 ± 34.17	334.25 ± 11.11	324.76± 22.51	329.02 ± 17.66	330.37 ± 12.08	195–724.2
TBIL (µmol/L)	4.15 ± 0.31	3.93 ± 0.18	4.10 ± 0.28	3.883 ± 0.17	3.78 ± 0.22	3.91 ± 0.40	0.6–8.0
ALB (g/L)	29.67 ± 0.92	31.20 ± 0.97	31.00 ± 0.37	30.00 ± 0.82	28.67 ± 1.50	30.83 ± 0.79	26.85–34.55
TP (g/L)	53.33 ± 1.98	54.50 ± 1.26	51.67 ± 1.49	54.67 ± 1.12	55.67 ± 1.79	54.85 ± 1.82	51.10–64.55
UA (mmol/L)	11.83 ± 0.85	11.533 ± 0.57	10.13 ± 0.77	9.52 ± 0.93	8.433 ± 0.82	8.87 ± 0.92	5.56–12.67
CR (µmol/L)	51.67 ± 2.62	49.67 ± 2.53	50.17 ± 1.45	48.17 ± 3.75	46.10 ± 3.83	47.83 ± 1.97	34.91–59.67

Notes: All values were analyzed by One-way ANOVA, significant at p < 0.05, and data is presented as mean ± SEM [n =6]. Reference intervals for all biochemical tests were given as described in baseline studies by Delwatta et al,⁵⁸ He et al,⁵⁹ and Wang et al.⁶¹

Abbreviations: ALB, Albumin; ALP, alkaline phosphatase; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; CR, Creatinine; TBIL, Total bilirubin; TP, Total protein; UA, Urea.

Histopathological Analyses

Gross anatomy assessments of the liver and kidneys did not show any abnormalities in color, size, texture or structure compared with the organs from the control group. Light microscopy transverse and longitudinal sections of the liver and kidney are shown in Figures 5 and 6, respectively. Histopathological assessment of the tissue specimens from the control and treatment groups revealed normal hepatic tissue composed of hexagonal lobules and acini. The hexagonal lobules had central veins (CV) and peripheral portal triads containing branches of the portal vein (PV), hepatic artery (HA) and bile duct (BD) (Figure 5). In all treatment groups, the histological structure of the liver was normal, with cords of polygonal and regularly arranged hepatocytes radiating from the central veins. In addition, the central and portal veins were normal and appeared white indicating an absence of congestion. A normal sinusoidal space was also observed between the hepatocyte cords. Hepatocytes had well-defined normochromic nuclei, with conspicuous nucleoli and peripheral chromatin. Some cells even had two nucleoli each. The cytoplasm of these cells was clear with no vacuolization. Therefore, no hepatitis or vascular abnormalities were observed in any treatment group. The histological assessments of the liver are summarized in Table 3. Unremarkable histological structure (grade 0) of the kidneys was observed in the control and treatment groups. Under low magnification, pale staining of the cortical tissue and deep staining of the medullary tissue were observed. The proximal convoluted tubules (PCT) had a large, simple cuboidal epithelium with centrally located nuclei and eosinophilic cytoplasm. In all treated tissues, PCT could be easily distinguished from the distal convoluted tubules (DCT), which had a larger lumen and mildly basophilic epithelium made up of smaller cuboidal cells (Figure 6). Generally, the integrity of the renal tubules in both the control and all treatment tissues was intact with no hypertrophy or degradation of cells and no proteinaceous casts in the tubular lumen. There was also no monoclonal cellular infiltration or dilatation of glomeruli (G). Renal histological assessments are summarized in Table 4.

Figure 5.

Effect of once-daily oral administration of M. flabellifolius crude aqueous extracts for 28 days on rat liver tissues (HE, X400; scale bar=50 µm, transverse sections). Photomicrographs show unremarkable tissue sections from Control, 500 mg/kg, 1000 mg/kg, 2000 mg/kg, 5000 mg/kg and 10000 mg/kg treatment groups. Notice the clear sinusoids indicated by red arrow between well-organized hepatic cords indicated by asterisk (*).

Note: The black arrows point to the structures represented by the following abbreviations:.

Abbreviations: CV, central vein; HA, hepatic artery; BD, Bile duct, PV, Portal vein.

Figure 6.

Effect of once-daily oral administration of M. flabellifolius crude aqueous extracts for 28 days on rat kidney tissues (HE, X400; scale bar=50 µm, longitudinal sections). Notice the unremarkable kidney tissue photomicrograph from Control, 500 mg/kg, 1000 mg/kg, 2000 mg/kg, 5000 mg/kg and 10000 mg/kg treatment groups. All tissues have a well-defined renal corpuscle with glomerulus (labelled G).

Abbreviations: PCT, Proximal convoluted tubule is encircled by dotted line (dotted line circle); DCT, Distal convoluted tubule is encircled by continuous line (continuous line circle); MD, high columnar cells of the macula densa indicated by yellow arrow within the distal convoluted tubule shown that is encircled by continuous line; G, indicates the glomerulus.

Table 3.

Effect of Once Daily M. flabellifolius Crude Aqueous Extract Intake on Rat Liver Tissues After 28 Consecutive Days

Hepatic Lesions	Control	M. flabellifolius (mg/kg)
		500	1000	2000	5000	10000
Cytoplasmic vacuolation	–	–	–	–	–	–
Necrosis	–	–	–	–	–	–
Microagiopathy	–	–	–	–	–	–
Activated Kupffer Cell	–	–	–	–	–	–
Inflammation with infiltrate and aggregates of Leucocyte and lymphocyte	–	–	–	–	–	–
Ballooning degradation	–	–	–	–	–	–
Hepatocellular necrosis\degradation	–	–	–	–	–	–
Sinusoidal obstruction syndrome	–	–	–	–	–	–
Inflammation lobular\portal	–	–	–	–	–	–
Congestion	–	–	–	–	–	–

Notes: (−) represents absence of a particular lesion.

Table 4.

Effect of Once Daily M. flabellifolius Crude Aqueous Extract Intake on Rat Kidney Tissues After 28 Consecutive Days

Kidney Lesions	Control	M. flabellifolius (mg/kg)
		500	1000	2000	5000	10000
Hypertrophy of epithelial cells of renal tubules	–	–	–	–	–	–
Degeneration of tubular epithelia with simultaneous infiltration of mononuclear cells	–	–	–	–	–	–
Hyperaemia of medullary and cortical part with mononuclear cell infiltration	–	–	–	–	–	–
Dilation of renal glomeruli	–	–	–	–	–	–

Notes: (−) represents absence of a particular lesion.

Discussion

In recent years, the term “herbal” has gained widespread use and attention in various products such as functional foods, fitness diets, beverages, cosmetics, and personal care products.^62–64 This is due to the incorporation of medicinal plants into everyday products to promote health and well-being.^63,65 However, this increased use poses a risk as herbal plants such as M. flabellifolius are sometimes assumed to be safe owing to their natural origin, despite potential adverse reactions.¹³ Therefore, in the current study, we focused on establishing the toxicological profile of M. flabellifolius crude aqueous extract by firstly confirming the presence of key bioactive phytochemicals and then evaluating the effects of the extracts on the liver and kidney structure and function in an animal model.

The most widely reported use of M. flabellifolius in Southern Africa, beyond nutritional and cosmetic applications, relates to its acclaimed medicinal value.⁶⁶ These effects are largely attributed to naturally occurring bioactive compounds, collectively referred to as phytochemicals.²⁵ Consequently, the identification and characterization of the phytochemical profile of M. flabellifolius, as with other medicinal herbs, is essential for understanding its biological activity and safety. In this study, the qualitative phytochemical screening of crude aqueous extracts identified tannins, flavonoids, coumarins and terpenoids. Comparable metabolite profiles have been reported for M. flabellifolius plants collected in Namibia²⁹ and South Africa,⁶⁷ indicating a broad regional consistency. Notably, Namibian aqueous extracts have also been reported to contain phlobatannins and saponins, whereas the coumarins detected in the present study were not reported in materials from South Africa. Despite these minor regional differences, all studies have consistently reported the absence of anthraquinones across M. flabellifolius populations in South Africa, Namibia, and Zimbabwe. This pattern suggests substantial conservation of the core phenolic profile across Southern African ecotypes, with only limited qualitative variation, as also observed in comparative phytochemical studies.⁶⁶ Importantly, the biological activities commonly attributed to the absent class, particularly anti-inflammatory and antioxidant effects, are functionally overlapped by the flavonoids, tannins, and terpenoid-related compounds consistently identified in this and other studies.⁶⁸

Toxicological assessment of M. flabellifolius crude aqueous extract with a broad dose range of 500–10000 mg/kg through a single oral dose toxicity study did not result in any mortality or clinical signs of toxicity in the present study. This implies that the approximate lethal dose of M. flabellifolius crude aqueous extract is greater than 10000 mg/kg in male and female rats. The exploration of single-dose treatment of up to 10000 mg/kg M. flabellifolius crude aqueous extract in the present study was also comparable to that used for Sanghuangporus vaninii (S. vaninii) [10000 mg/kg],³⁸ Gancao Xiexin [10g/kg],⁶⁹ Somina [10000 mg/kg],⁷⁰ Haloxylon scoparium Pomel [10 g/kg]⁷¹ and Toxicodendron vernicifluum [10000mg/kg]⁷² using rodents as experimental models. Higher doses were also used in the studies by Lakmichi et al³⁰ and Rachmawaty et al,⁷³ establishing LD₅₀ values of 14000 mg/kg and 18000 mg/kg, respectively.

Similar to the present study on M. flabellifolius, the study by Lakmichi et al³⁰ gave an invaluable safety profile for Corrigiola telephiifolia Pourr, a Moroccan herb whose roots continue to record increased use for the management of inflammation with limited studies on its safety. Therefore, exploration of higher single-oral dose toxicity studies is critical for defining the upper limit of biological tolerance, establishing practical safety margins, and identifying potential target organ effects that might not be observed at lower exposures. This is especially essential for some herbs that have already been commercialized or formulated into supplements, beverages and polyherbal formulations such as Gancao Xiexin.⁶⁹ Much the same applies to the M. flabellifolius herb, which is already being sold as herbal tea, functional beverage infusions, cosmetic formulations and is even being considered as an animal feed additive.² It also informs risk characterization and dose selection for M. flabellifolius crude aqueous extract subsequent repeated-dose studies, where even higher dose groups can be selected though with care on animal health and nutritional balance.⁴⁶

Changes in general health and body weight are some of the most critical parameters used to index the signs of toxicity caused by xenobiotics. In fact, animals that lose 10% of their initial body weight after treatment with a substance are reported to have poor survival, which is an indicator of toxicity.^54,63 As such, repeated oral dose administration of M. flabellifolius for 28 days with no significant changes in body weight compared with the control recorded in this study provided confidence in the safety of the plant. This was further reinforced by similar observations of no significant changes in the relative organ weights of both the liver and kidney in rats from all treatment groups compared to the control.

Relative organ weight (ROW) is a pragmatic, cost-effective, sensitive screening endpoint for detecting potential target organ effects in general toxicology studies and is therefore routinely recommended alongside gross pathology and histopathology, particularly for organs such as the liver and kidneys.^43,54 Treatment-related shifts in ROW may reflect as hypertrophy, swelling, or atrophy and can precede, accompany, or help contextualize macroscopic and microscopic lesions, especially when interpreted against terminal body weight and the overall dataset.⁷⁴ This sensitivity was reported by Khalaf et al,²² where 60-day daily dosing with widely used Mediterranean herbs (Lepidium sativum and Eruca sativa) altered liver weight and was accompanied by biochemical and histological evidence of hepatorenal injury, supporting ROW as an early signal of organ toxicity.²² In contrast, several herbal safety assessments have reported no treatment-related changes in liver or kidney ROW even at comparatively high repeated-dose exposures, consistent with wide safety margins when systemic tolerance is high.

For instance, in studies comparable to the present study, Saposhnikoviae Radix⁴⁴ and Panax ginseng⁴⁵ were evaluated using 5-dose and 6-dose ladders respectively (OECD TG 407)⁷⁵ up to 5000 mg/kg and both showed no relative organ weight changes or deleterious histopathological effects. Similarly, Gancao Xiexin decoction showed no organ-index or histopathology findings in a 28-day repeated oral toxicity study, with a NOAEL of 3.83 g/kg, a low observed adverse effect level (LOAEL) of 10 g/kg, and an absence of acute toxicity at 30 g/kg/day.⁶⁹ Collectively, these studies highlight the value of ROW assessment’s ability to identify (or credibly exclude) target-organ toxicities across a broader exposure window while aligning dose selection with both scientific relevance and animal-welfare considerations.⁷⁶ This approach was particularly relevant for M. flabellifolius, whose human dose remains poorly defined across its diverse ethnomedicinal indications. This poorly defined human dose has also been reported by Deng et al⁴⁶ and Liu et al³⁸ in their studies. However, in a comparable study by Wang et al,⁶¹ the highest dose used in rodents (30 g/kg/day) was approximately 20 times that used in clinical practice, whereas Ru et al⁶⁹ selected their top dose based on a proposed human intake of 11 g/kg/day, reflecting the need to model potential real-world exposure. Therefore, exploration of a broader repeated-dose range interpreted using integrated endpoints such as ROW (including biochemical and histopathological assessments) provides a more defensible safety index that better predicts plausible use and potential misuse in real-world settings.^61,69,76

In this study, serum clinical biochemistry was performed to evaluate possible alterations in hepatic and renal functions due to repeated daily exposure to M. flabellifolius crude aqueous extracts. Liver and kidney function analyses are essential for the toxicity evaluation of drugs and plant extracts as both organs are necessary for the survival of an organism.⁵⁴ Usually, high levels of serum ALT, AST and ALP are indicators of liver damage and toxicity.^14,63 Therefore, since no significant changes in these parameters were recorded after treatment, it follows that M. flabellifolius crude aqueous extract does not cause hepatocellular damage or affect hepatocyte function. Meanwhile, a decrease in total protein, albumin and globulin is a sign of reduced synthetic function and/or hepatocellular function.^14,77 It may also suggest infection resulting in continuous loss of albumin.⁵⁴ Therefore, the non-significant change in serum concentrations of total protein, albumin and total bilirubin in M. flabellifolius crude aqueous extract-treated groups relative to the control group further confirmed that the extract did not alter the secretory function of the liver or cause hepatobiliary dysfunction to the maximum dose tested.⁷⁸ Renal dysfunction can be assessed by evaluating changes in urea and creatinine markers of the kidney.^14,77 Since serum levels of these parameters were within normal ranges and no significant changes were observed in M. flabellifolius crude aqueous extract-treated groups relative to the control group, it follows that renal filtration capacity was not compromised by treatment.

Qualitative macroscopic assessments of the liver and kidney of rats treated with various doses of M. flabellifolius did not show any morphological changes compared to the control group. This was consistent with the microscopic assessment of liver and kidney sections observed under a light microscope. Generally, damage to the liver parenchyma results in elevated blood transaminase levels.^63,77 This was the case in a study by Fall et al⁷⁹ where significantly high serum levels of ALT were recorded after treatment with Leptadenia Hastata (L. Hastata) methanolic extract. Just like M. flabellifolius, L. Hastata is a herb which is anecdotally used in several African countries to treat stomach pain, hypertension and reproductive health conditions.⁷⁹ In their study, Fall et al⁷⁹ recorded elevated ALT levels which were associated with vacuolar degradations, hepatocyte necrosis, angiopathy and inflammation. In our study though, M. flabellifolius treated and control rats had comparable transaminase levels. In fact, there was a general decline in transaminase levels with increasing M. flabellifolius treatment doses, although the difference was not statistically significant. Tannins and flavonoids identified in M. flabellifolius crude aqueous extracts in the current and previous studies have been reported to enhance enzymatic antioxidant activities, including superoxide dismutases, glutathione peroxidase and catalase1.^25,80 Therefore, it is possible that the slightly lower values of transaminase recorded with increase in extract doses in our study emanate from these phytochemicals in the herb that has general hepatoprotective properties with chronic use. This may rationalize the traditional use of M. flabellifolius in the management of chronic conditions such as diabetes mellitus and hypertension.

Xenobiotic toxicity to the kidney parenchyma is usually characterized by elevated levels of creatinine and urea, which are associated with kidney pathologies, including detachment of tubular cells from the basement membrane and tubular necrosis.⁸⁰ In such cases, these deleterious changes in renal tissues indicate kidney injury, which in both acute and chronic cases results in impaired glomerular filtration rate and associated spikes in creatinine and urea from muscle metabolism.⁸¹ In such cases again, treatment with herbs such as Ligustrum lucidum⁸⁰ and cinnamon (Cinnamomum burmanii)⁸² have been reported to reduce serum levels of creatinine thereby limiting and, in some cases, reversing tissue injury. This has been attributed to phytochemicals such as flavonoids, vitamins, resveratrol, anthocyanins, curcumin and phenolic acids, which are potent antioxidant and anti-inflammatory agents.^82,83 Curiously, as observed in the present and previous studies, M. flabellifolius is rich in some of the phytochemicals identified in Ligustrum lucidum and cinnamon.^2,25,29,67 Therefore, the action of these phytochemicals in M. flabellifolius is probably the most plausible reason for the slightly lower serum creatinine levels recorded in the treatment groups compared to the control as a possible index of renoprotective properties of the herb, warranting further investigation.

Conclusion

To our knowledge, this is the first study to provide a wide safety margin profile for M. flabellifolius crude aqueous extracts with NOAEL of 10000 mg/kg in male and female rats and no detectable hepatic or renal toxicity. Generally, the results of this study suggest that the resurrection bush, M. flabellifolius, may be safe when administered orally to SD rats. This is premised on the absence of mortality, clinically toxic behavioral changes, and alterations in the function of the liver and kidney with single- and 28-day repeated oral dose toxicity tests. Moreover, the lack of significant changes in animal body weight and relative organ weight of the liver and kidney, coupled with the absence of deleterious changes in the histoarchitecture of both organs further supports a safer chemical prolife for M. flabellifolius crude aqueous extracts. These findings, together with the confirmed availability of essential bioactive compounds may provide safety assurance for the use of M. flabellifolius in traditional African medicine. This also provides a promising basis for further exploration of the therapeutic and nutraceutical potential of M. flabellifolius, given the scarcity of such studies on this plant. Future efficacy studies should, however, move beyond general bioactivity screening and assess its effects in relevant disease- or condition-induced experimental models to better define its therapeutic relevance and underlying mechanisms of action.

Nevertheless, some limitations should be acknowledged. Although rodent safety findings provide useful preliminary evidence of tolerability, they do not fully predict human safety in all contexts. This is important particularly where M. flabellifolius may be co-administered with conventional medicines, incorporated into polyherbal formulations, or used under conditions that may alter absorption, metabolism, exposure or herb–drug interaction risk. Animal toxicity data have recognized limitations in predicting human responses, and herbal medicines may interact with drugs through pharmacokinetic or pharmacodynamic mechanisms. Also, since only female rats were used in the 28-day repeated oral dose toxicity study, the generalizability of the findings on the chronic administration of M. flabellifolius is limited, particularly with respect to possible male-specific toxicological responses. Future confirmatory studies should therefore include both male and female animals with robust sampling (formal power analyses) and randomization. In addition, longer-duration studies, such as 90-day sub-chronic toxicity assessments, should be accompanied by quantitative phytochemical profiling of the extract to better relate any toxicological outcomes to its chemical composition. Such studies would be valuable for evaluating hazards associated with prolonged repeated exposure, identifying potential target organs, and assessing the possibility of cumulative toxicity. This should also include the impact of the herb extract on the brain, gastrointestinal tract, reproductive organs and incorporate reproductive hormones, estrous-cycle monitoring, inflammatory markers and oxidative-stress markers to provide a more comprehensive safety profile.

Funding Statement

Midlands State University, Indigenous Fruits and Herbs Research Fellowship program financially supported this study.

Abbreviations

%, Percentage; <, Less than; =, Equal to; ≥, Greater than or equal to; °C, Degrees Celsius; ALB, Albumin; ALP, alkaline phosphatase; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; BD, Bile duct; BW, Body weight; CR, Creatinine; CV, Central vein; DCT, Distal convoluted tubule; g, Grams; G, Glomerulus; HA, Hepatic artery; HE, Hematoxylin and Eosin; kg, Kilograms; L. hastata, Leptadenia hastata; LD₅₀, The amount of a chemical that is lethal to one-half (50%) of the experimental animals exposed to it; LOAEL, Lowest observed adverse effect level; M. flabellifolius, Myrothamnus flabellifolius; MD, Macula densa; mg, Milligrams; mL, Milliliters; mm, Millimeters; NOAEL, No observed adverse effect level; PCT, Proximal convoluted tubule; PV, portal vein; ROW, Relative organ weight; TBIL, Total bilirubin; TP, Total protein; UA, Urea.

Data Sharing Statement

Data is available from the corresponding author on request.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Author contribution is highlighted as follows: Study concept and design: T. N., M. B, and J. M. Methodology and acquisition of data: T. N., M. B., E. N., A. R. N. Analysis and interpretation, T. N. M. B., A. R. N. Data Curation: T. N., M. B., J. M., A. R. N., and F. M. Original Draft preparation: T. N., E. N., A. R. N., and M. B. Review and Editing: T. N., E. N., S. C., F. M., A. R. N., M. B., and J. M. Supervision: A. R. N., M. B., and J. M.

Disclosure

An abstract of this paper was presented at the Pan Africa Chemistry Network Congress 2024 as a conference talk with interim findings. The authors declare no competing interests. Mr Trust Nyirenda reports Financial support in procurement of lab chemicals and reagents from Ministry of Higher and Tertiary Education (Zimbabwe), during the conduct of the study.