Introduction
Supplements have gained the attention of those that are trying to improve their quality of life through nutrition (Bailey et al., 2003). Global market of dietary supplements were estimated at USD 140.4 billion in 2020 (GVR, 2021), and 81 % of U.S. people over 55 years of age reported dietary supplement usage in the same year (CRN, 2020). Although there is no global consensus on how to define and regulate dietary supplements, they are products that are meant to supplement diets, and some of them may also have health-improving effects because they are not regarded to be medications and are not intended to treat illnesses (Dwyer, Coates, & Smith, 2018). Each country has specific components that can be included in dietary food supplements, but in general they can be vitamins, minerals, carbohydrates, plant nutrients, bioactive substances, enzymes, and probiotics (ANVISA, 2018).
Among dietary supplements, botanical dietary supplements (BDS) are awakening a great interest, reaching USD 5.26 billions in market value in 2017, globally (GVR, 2021), and 44 % of Americans reported supplementation with botanical or herbal components in 2020 (CRN, 2020). Often, BDS is presented as plant products in the form of fluid extract, dry extract powders, decoctions, infusions, tinctures, syrups, herbal tea bags, plant powders, granules, capsules, tablets, and oils (WHO, 2018). Since there may be antagonism between bioactive components in mixtures, isolated molecules from plants can occasionally be sold in place of a complex mixture such as an extract, but they may overlook potential synergisms (Caesar & Cech, 2019).
BDS have different health-related potential and nutritional benefits that depend on the daily intake and the amount of the botanical bioactive compound ingested, which provide different degrees of effectiveness and can influence their safety (Krochmal et al., 2004). Therefore, these dietary supplements should be scientifically tested to determine their safety and quality as well as their health benefits (Breemen, Fong, & Farnsworth, 2008). According to the National Toxicology Program of the U.S. Department of Health and Human Services, six botanical dietary supplements are now the subject of investigations; one of them is the Usnea lichen, which is frequently used for weight loss. Twelve more have been the subject of study; one substance is the popular anxiety-relieving herb Kava Kava (NTP, 2020). Obesity (Choi et al., 2020), oxidative stress (Hung et al., 2012), abnormal lipid profiles and glycemia (Eleazu, Maduabuchi, & Eleazu, 2018), fatigue (C.-S et al., 2016), and inflammation (Rosillo et al., 2012) are a few health-related disorders that the ongoing use of BDS seems to resolve.
Indigofera suffruticosa Mill., popularly known as “wild indigo”, “anil” or “anileira”, is a member of Fabaceae family, and was originally found in Tropical America regions (Matos et al., 2011; Paulino, Pessine, Teixeira, & De, 2010). In Brazil, it is found in all states (Do, 2021). I. suffruticosa is used in popular medicine as antispasmodic, antiepileptic, purgative, antipyretic, sedative (RB, 1990), in colic and kidney diseases (Matos, 1999), as a stomach enhancer and diuretic (Agra, Baracho, Nurit, Basílio, & Coelho, 2007). Studies proved that it has anti-inflammatory activity (Leite et al., 2003; Nascimento et al., 2022), antioxidant (Arriaga et al., 2013), chemopreventive (Chen et al., 2013), and gastroprotective (Luiz-Ferreira et al., 2011). Although there is scientific proof for some of the health advantages of I. suffruticosa, studies on the impact of supplementing a regular diet are lacking (Campos et al., 2018). Thus, this study aimed to evaluate the subacute toxicity and behavior of health-related parameters in mice supplemented with I. suffruticosa leaves aqueous extract for 11 days.
Materials and Methods
Collection of plant and preparation of I. suffruticosa aqueous extract (IsAE)
Indigofera suffruticosa Miller was collected in São Caetano, Pernambuco, Brazil (Latitude: 8° 19' 33'' S; Longitude: 36° 04' 21'' W). Plant was identified by Dr. Marlene Carvalho de Alencar Barbosa from the Department of Botany - UFPE (Federal University of Pernambuco) and a specimen was deposited in the herbarium UFP - Geraldo Mariz (number 45,217), at UFPE. In addition, there is a registration in the National System to Traditional Knowledge and Genetic Heritage (SisGen, Brazil) under the identification A149769. Leaves were separated and dried in a hot-air oven at 38 ° C for 72 hours. After drying, leaves were crushed in an industrial blender (Metalurgic Vithory Ltda., Brazil) to obtain the powder. I. suffruticosa leaves powder (50g) was mixed with 200 ml of distilled water (1:4), and left under constant stirring for 16 hours. These were then filtered with the assistance of a vacuum pump (Exipump, Brazil) and concentrated by freeze-drying (Liotop, Brazil).
Oral supplementation
The supplementation test was performed according to the previous supplementation methodology with some modifications (Araújo et al., 2016) and approved by the UFPE Animals Use Ethics Committee (CEUA) (protocol: 23076.042305/2017-67). Three experimental groups of six adult mice male albino Swiss (12-14 weeks) were obtained from Laboratório de Imunopatologia Keizo Asami (LIKA). After the one-week acclimatization period, they orally received 100 µl of water containing 50 mg/kg of IsAE, 100 µl of water containing or 100 mg/kg of IsAE, or just 100 µl of water (control group) for eleven days with the support of gavage needle (p.o.). This dose was established according to previously unpublished works regarding the acute toxicity of I. suffruticosa performed in our laboratory. During this period, animals were in a vivarium under ideal conditions of temperature (22 ± 2 ºC), light cycles of 12h light/ 12h dark, and suitable humidity (55 ± 5 %), besides water and standard rodent food (Presence, Neovia group, Brazil) ad libitum.
Subacute toxic analysis
During eleven days of supplementation, mice behavior and any signal of toxicity or death were observed accordingly to Organization for Economic Co-operation and Development (OECD, 2001). Motor alterations, reflex loss, breathing alterations, contortions, piloerection, bleeding, edema, aggressiveness, or enervation were observed and recorded on all days for all groups.
Body parameters
Before administrations, animals were weighed with an analytical digital balance (BEL Engineering® s.r.l., Italy) and measured their nose to root tail length with a measuring tape (expressed in centimeters). On the tenth day of supplementation, data was collected again. With the initial and final weight and length of each animal, body mass index (BMI) was calculated as following the formula below and expressed in grams per cubic centimeter (g/cm2).
Forced swimming test
Mice were put through a forced swimming test on the tenth day, an hour after receiving oral IsAE, following a previously published methodology with some modifications, following weight evaluation (Can et al., 2012). Mice's tails accounted for 10 % of their entire body weight. Individual mice were placed on a recipient that measured 35 centimeters in height by 25 centimetres in diameter and filled with water to a depth of 18 centimeters. The swimming time was recorded until the mice could not return to the water's surface for 5 seconds. At this point, mice were eliminated from the recipient and dried in a warm towel. We estimated the mean forced swimming time (minutes) and standardized it to final body weight.
Blood collection and biochemical analysis
Blood samples were collected via retro-orbital plexus using a capillary without anticoagulant after 24 hours of the forced swimming test. After the collection, blood was centrifuged at 550xg for 10 minutes (FANEM® Ltda., Brazil) and the serum was separated for biochemical quantification (Santos et al., 2009). Parameters analyzed were total cholesterol (TC), triglycerides (TG), cholesterol HDL (HDL-c) and non-HDL-c cholesterol [LDL cholesterol (LDL-c), and cholesterol VLDL (VLDL-c)] and glucose by commercial in vitro diagnostic kit (Labtest Diagnóstica S.A., Brazil) and expressed in mmol/l. VLDL-c was calculated using TG values, and LDL-c according to Friedewald formula, as follows:
Values of LDL-c, total cholesterol, and HDL-c were used for calculating Castelli index 1 and 2 as follows in the formulas below:
Analysis of body fat quantity
Anesthetized animals were submitted to surgery, in which the perirenal, peritoneal, and gonadal fats were removed and weighed in an analytical digital balance (BEL Engineering® s.r.l., Italy). After this procedure, animals were euthanized. Besides the mean of quantity of this body fats, the percentage of body fat in respect to the final body weight was also calculated.
Statistical analysis
All the result were presented as a mean ± SEM. GraphPad PRISM® 5 (GraphPad Software Inc., USA) was used to assess the significance between the groups using one-way analysis of variance (ANOVA), followed by Bonferroni post-test. For statistical significance, p < 0.05 was considered.
Results and Discussion
Supplementation of aqueous extract of I. suffruticosa leaves for 11 days can also assess its subacute toxicity. During these days, none of the animals showed any visible signs of toxic effects, and death was not recorded, which shows that 50 mg/kg and 100 mg/kg of leaves aqueous extract by oral administration does not have subacute toxicity, or lethality.
Botanical dietary supplements are emerging as a viable natural option in the daily dietary complement, due to their widespread reputation for being safer, more affordable, and having a huge diversity of phytochemicals that benefit health (Breemen, 2015; Raskin et al., 2002). I. suffruticosa is known for its toxicity when the whole plant is ingested, or when its aerial parts (in natura) are served ad libitum or orally forced in bovines, sheeps and goats. The most reported signals of toxicity are anemia, hemoglobinuria, and kidney and liver damage (Assis et al., 2009; Figueiredo et al., 2012; Neto et al., 2001; Salvador et al., 2010; Silva et al., 2006). Aqueous fruit extract, intraperitoneally administered, showed liver damage with a significant number of cells with aberrations in Balb C mice (Ribeiro et al., 1991). Leaves and branches (in natura) daily administred to guinea pigs (for 15 days) in all the animals presented apathy and blue urine and anemia. Necropsy revealed liver damage and authors associated the haemolityc anemia due to a possible presence of aniline (Salvador et al., 2011).
However, when intraperitoneally administred (for 7 days), IsAE not only treated sarcoma 180 infected mice, but also during the treatment time did not affect renal and hepathic morphology, indicating its safety use as a anticancer agent (Santana et al., 2015; Silva et al., 2014; Vieira et al., 2007). In liver, leaves methanolic extract demonstrated a hepatoprotective effect on paracetamol-induced liver damage and also indigan (a purified compound from I. suffruticosa) do not showed toxicity to liver when administred intraperitoneally (Lima et al., 2014; Lima et al., 2019).
The security of IsAE as a potential BDS has been assessed and its subacute toxicity results corroborated with previous in vitro toxicity tests that showed that different extractions types and parts of the plant I. suffruticosa did not demonstrate high toxicity, and in vivo tests demonstrated only low signs of toxicity, but when intraperitoneally administered, which suggested that some possible toxic compounds are metabolized during digestion (Silva et al., 2019; Vieira et al., 2007). As BDS are commonly ingested, the present and previous studies add important information on the safe use of IsAE for its multiple purposes.
Results of initial and final body weight of the mice in supplementation with aqueous extract of I. suffruticosa are shown in Table 1. There is no significant difference in weight in these two moments. Mice Body Mass Index (BMI) in the two moments also does not show a significant difference between groups.
Table 1
Initial and final body weight and BMI of mice inIsAE oral supplementationfor 10 days.
As shown in a study that administered Rhodiola rosea aqueous and fermented extract for 15 days at a concentration of 1.5 g/kg body weight (Kunming mice), the results of which were different from those of this study in that they showed an increase in body weight over time, but there was no statistically significant difference between the groups (Kang, Hong, Kim, & Choi, 2015). Another study evaluated a 20 days supplementation (Kunming mice) with aqueous extract of Millettia speciosae rhizomes, which also showed an increase in the body weight over time in doses of 500, 1,000, and 2,000 mg/ kg, but not statistically significant when compared to the control group. This indicates that unlike I. suffruticosa aqueous extract, aqueous extract can increase body weight (Zhao et al., 2015). In a longer supplementation time (28 days), it was seen a gain of weight after administration of Anisomeles indica whole plant aqueous extract, however, this was also not statistically significant when compared to the control group in doses of 125 mg/ kg, 250 mg/ kg, and 500 mg/ kg (C.-S et al., 2016). Even in administrations for longer periods and at higher doses, the weight of the animals seems to not significatly change, however, these studies did not evaluate the animals body fat and also did not determine the fat quantity percentage, as can be seen in the previous review, methodological and study coverage differences to assess body weight reduction can reveal different levels of potential (Ríos-Hoyo & Gutiérrez-Salmeán, 2016).
Table 2
Fat concentration in mice after IsAE supplementation for 11 days (p.o).
Body fats analysis is shown in Table 2, no significant difference between the treated groups and control was found for perirenal and peritoneal fat, only gonadal fat weight had a significant decrease for both dosages. Besides that, when the fat percentage was calculated, it was not possible to perceive a significant difference between the groups. Even if there is no significant difference, the results show a mild decrease in all fat counts (Table 2).
During the supplementation period with aqueous extract of I. suffruticosa, body weight, body fat, and fat quantity percentage could be gradually decreased (not significant, except for gonadal fat), this may be due to an increase in the energy expenditure, to an appetite suppressant, to lipase inhibitor effect, or due to a regulation in lipid metabolism and the differentiation of adipocytes, as previously summarized in a review as the possible mechanisms of anti-obesity (Sun, Wu, & Chau, 2016). It is observed in this study that a reduction in body weight and fat is reflected in an increase in swimming time, especially when this increase is normalized by the animals final weight.
As shown in Fig 1a, there is an increase in swimming time as IsAE concentration increases, but this difference was not significant. However, when the swimming time was normalized by the final animal weight as shown in Fig.1b, significant differences were found between the control group and the animals in the 100 mg/kg IsAE group.
Many studies did not find prolonged swimming times even when the administration period and the dose were greater or when the weight placed in its tail was smaller. Administering aqueous and fermented extract of R. rosea (1.5 g/ kg body weight for 15 days) and submitting the mice to swimming (7 % of body weight in the tail), researchers found 197.2 ± 41.85 s (approximately 3.29 minutes) as the longest swimming time. The antifatigue effect was associated with the antioxidant activity, increase in serum lactate dehydrogenase and decrease in blood urea, nitrogen, and lactic acid observed, mainly for the fermented extract (Kang et al., 2015). Another study, revealed swimming time similar (81.3 ± 8.3 min) to our dose of 100 mg/ kg on the 28th day of supplementation with ethanolic extract of Rubus coreanus in a dose of 1 g/ kg and performing the swimming test without load (ICR mice). A decrease in lactate, better use of energy sources like glycogen, increase in fat use, and oxidative stress protection were the main factors associated with this activity (You et al., 2015). And another study did not find a swimming time (with 5 % or 10 % body weight in the tail in ddY mice) bigger than 300 seconds (5 min) for 10 % of load and 25 minutes for 5 % load in doses of 150 and 300 mg/ kg body weight of Trigonella foenum graecum (Fenugreek) seed ethanolic extract in 4 weeks of administration. The increase in fatty acids utilization as an energetic source and increase in hepatic and muscle glycogen were the main explanations for this effect (Ikeuchi, Yamaguchi, Koyama, Sono, & Yazawa, 2006). In twenty days of supplementation with M. speciosae aqueous extract, it was not found swimming time (with 5 % body weight in the tail of Kunming mice) was higher than 60 minutes in any doses (500, 1,000, and 2,000 mg/ kg body weight). M. speciosae aqueous extract supplementation showed to decrease triglycerides, improve fat metabolization, delay blood urea nitrogen (BUN) accumulation, decrease creatine phosphokinase (CK), and improvement of glycogen in the muscle (Zhao et al., 2015). The highest swimming time value for the highest dose (500 mg/ kg) of another study was 692.5 ± 96.8 s (approximately 11.54 minutes) in 28 days of administration of A. indica aqueous extract for Balb/c female mice. Their results are attributed to the decrease in plasma triglycerides and ammonia levels and an increase in liver and muscle glycogen (C.-S et al., 2016). However, no study performed normalization for the forced swimming time by the animal final weight, which is important because the animal weight influence the swimming performance, and this influence is discounted after normalization (Bogdanova, Kanekar, D’anci, & Renshaw, 2013).
In this study, specific exercise-related analytes or pathways were not tested, but the trend for reduction in lipids and glucose, even though not significant, and the reduction in the fat quantity (significant for gonadal fat) may be a signal that IsAE supplementation can improve exercise endurance in the forced swimming test through consuming of energy sources to sustain the extensive exercise, as seen in previous studies (Ikeuchi et al., 2006; You et al., 2015). I. suffruticosa already demonstrated antioxidant activity (Arriaga et al., 2013) and anti-inflammatory properties (Leite et al., 2003), these two activities are essential to enhance exercise performance as reported by other botanical supplements (Harty, Cottet, Malloy, & Kerksick, 2019). Thus, IsAE supplementation can be acting scavenging reactive species and controlling the exercise-related inflammation generated during strenuous exercise as reported for other botanical supplements, attenuating muscular fatigue and improving animal performance during swimming.
Evaluating the lipid and glucose metabolism, significant differences in lipid profile and glucose levels between the groups tested were not observed. Only a mild lowering activity (not significant) in triglycerides, total cholesterol, and glucose was observed. Determination of Castelli 1 and 2 indexes also corroborate with those results (Table 3).
Table 3
Lipid andglucose profile of mice after IsAE oral administration for 11 days.
Figure 1
Results of swimming forced test of mice after IsAE oral administration for 10 days. (a) crude values; (b) time of swimming normalized for the body weight of mice.
Results are expressed as mean ± SEM, n = 6. *p<0.05 compared to control, one-way ANOVA followed by Bonferroni's test.
Evaluation of biochemical indicators following daily dosing did not appear to significantly affect the animals metabolism, indicating safety. A mild lowering activity (not significant) in triglycerides, total cholesterol, and glucose was observed. Similar results occurred in previous studies, in Wistar rat supplementation for 21 days with Cornus mas hydro-methanolic extract intraperitoneally administered (doses of 50, 200, and 400 mg/kg) found a not significant decrease in the same analytes of the present study and a mild increase and decrease (also not significant) in the HDL-c and TC, respectively (Abdollahi et al., 2014). In a more recent study supplementing for 28 days with the ethanol extract of boiled Treculia africana seed there was not significant difference in the lipid profile of Wistar rats supplemented with 100 and 200 mg/kg of the extract, additionally was found an increase in the LDL-c in only the dose of 100 mg/kg and no glycemic status improvement (Eleazu et al., 2018). In a high-cholesterol diet supplemented for 4 weeks with Ajuga iva boiled aqueous extract (0.5 % in the diet) a significant improvement in the lipid profile and antioxidant status was observed (Chenni et al., 2007). Also, significant improvement in lipid, glucose and antioxidant status was found in supplementation with 1% of Lepidium meyenii (Maca) in a high sucrose diet (hereditary hypertriglyceridemic rats) for 2 weeks (R et al., 2007). There is a clear relationship between antioxidant properties and the improvement of glucose and lipid metabolism that these botanical supplements have, combating oxidative stress, with some of them even testing the antioxidant status to determine this relationship as seen in some studies that tested antioxidant enzymes and lipid peroxidation status (Chenni et al., 2007). (R et al., 2007).
The not significant changes in weight (Table 1), visceral fats (Table 2), and biochemical parameters (Table 3) show that IsAE is not toxic when daily administered for a short period of time. It is possible that IsAE optimized energy expenditure and maintainance of life basic processes, because of IsAE seemed to not add significant caloric deposits, nor glycemic elevations as can be seen in Table 3, but improved exercise (Figure 1) possibly due to the improvement of the glycogen metabolism and, as the forced-swimimng time is an extenuant exercise test, IsAE might enhance lipid pathways to provide energy (Hanhineva et al., 2010; Pérez-Torres et al., 2021).
Indigofera suffruticosa has already some of its phytochemical constituents clarified, showing different classes of fatty acids found in leaves ethanolic extract (D & S, 2014), acyclic diterpene alcohol (phytol), methyl linoleate, monoterpenes, sesquiterpenes, phenylpropanoids as main constituent in leave oil (Arriaga et al., 2013; Silva et al., 2019), alkaloids, flavonoids, phenylpropanoids, triterpenoids and volatile oils in different degrees of presence in ether, chloroform and acetone leaves extracts (A.T.B et al., 2015), aqueous and ethanolic extracts that showed presence of various phenolic compounds (C.-S et al., 2016) , and also saponins, triterpenes, coumarins and flavonoids (Govín et al., 2006). Some of these phytochemicals are well known to directly or indirectly influence in the same health-related activities observed in this study.
Conclusions
Results of this study suggest that low doses of IsAE are safe for usage. IsAE seems to have the potential to diminish fat in a short period, and body weight in a longer supplementation period, and this does not disturb the homeostasis of analytes related to energetic metabolism, but pathways are still not clear. For muscle function, IsAE enhances muscle capacity for endurance tasks, and this might be due to the reduction of oxidative stress. Therefore, future studies should be focused on determining the precise mechanism of body weight and fat reduction. Thus, besides its popular medicinal use, IsAE seems to be a promising supplement.
Author contributions
R. X. da C., performed the experiments, designed the project and wrote the manuscript; W. M. N., J. R. S. de O. and A. N. de M. M. performed the experiments; A. P. S da S. and V. L. de M. L. designed the project and revised the final manuscript.
