English (pdf)
Article in xml format
Article references
Automatic translation
Send this article by e-mail
Cited by SciELO 
Similars in
SciELO 
Permalink
Introduction
Carapichea ipecacuanha (Brot.) L. Andersson (Ipecac) is a medicinal herb from South and Central America. The syrup prepared with Ipecac extract is used as an expectorant, amebicide, anti-bronchitis, and vomiting inducer (for poisoning treatment) (Patel & Patel, 2021). Alkaloids are responsible for biological activity. Two major alkaloids, emetine and cephaeline, account for 84% of the total alkaloid content of its roots (Smajlović & Dučić, 2021; Uzor, 2016). Additionally, emetine and/or cephaeline have been tested as potential treatments for tumors and viruses (against dengue and SARS-CoV-2) (Bleasel & Peterson, 2020b). Although there is still ongoing debate regarding the use of Ipecac for antiviral purposes, some in vivo evidence supports its bioactivity against certain viruses (Mukhopadhyay et al., 2016). Other compounds from Ipecac have been associated with bioactivities as well, e.g., some circular peptides have immunomodulatory effects (Rosales-López et al., 2020), and the polyphenols are antioxidants (Ben Hlel et al., 2019).
Several commercial formulas contain Ipecac syrup, which have been commercialized in America, India, and Europe since 1762 (García et al., 2005). The pharmaceutical industry utilized both syrup and alkaloids, and Ipecac became a major drug during the 1940s in the USA and Europe (Ocampo-Sánchez, 2004). Syrups are prepared from the dried roots of the Ipecac plant. Plants are produced in three central regions: from Nicaragua to northern Colombia (from southern Central America to northern South America), the southwestern Brazilian Amazon, and the rainforest near the Brazilian Atlantic Coast (García et al., 2005). Nicaragua and Costa Rica account for 20% and 35% of the global Ipecac market, respectively (Ocampo, 2007). Ipecac root production is a crucial economic activity in the northern Costa Rican and southern Nicaraguan regions. Although C. ipecacuanha has been introduced for cultivation in India and various areas in Asia, Costa Rican roots have demonstrated superior quality, with reported alkaloid yields 1.5 to 4.6 times higher than those of Indian and Brazilian counterparts (Han et al., 2013; Ocampo-Sánchez, 2004). These facts underscore the importance of preserving the high phytochemical quality of locally cultivated plants through the use of appropriate agronomic and post-harvest practices.
ThefinalsalepriceofIpecacrootsdepends on the concentration of emetine, cephaeline, and total alkaloids (used as quality parameters). Nonetheless, there is no systematic study of the relationship between the maturity of the plants and the harvest timetooptimizethesellingpriceofthefinal product. Previously, Alvez-García et al. (2005) found some dependence of alkaloid content on the morphological characteristics of the root. In addition, there are variations in alkaloid occurrence rates; for example, the emetine-to-cephaeline ratio has been reported to range from 0.6 to 0.7, according to Ocampo (2007), whereas Rosales-López et al. (2020) reported a lower value of 0.47. Some of the morphological characteristics could be related to plant maturity and the general production of metabolites.
Costa Rican C. ipecacuanha is preferred over other varieties because of its high alkaloid content. The hypothesis in this study is that a specific harvest can be found with optimal alkaloid concentration. This information is relevant to the agricultural production of Ipecac, providing a better understanding of metabolite production and, ultimately, informing the future evaluation of potential related species. This study aims to determine the optimal maturity and harvest time for alkaloid production in dry Ipecac roots.
Sample collection and processing
Plants were harvested from two production fields in La Guaria, San Carlos (Province of Alajuela, Costa Rica). Six samples were taken from each field, approximately 45 days apart, starting one year after sowing. Random samples of Carapichea ipecacuana’s roots were taken from each field. Roots were collected, cleaned,and divided into two groups for two different drying procedures: oven-dried (40°C, four days) and sun-dried. The sun-drying procedure is the method used by Ipecac producers, which involves allowing the sample to dry in the sun for several hours until it becomes brittle. After that, samples were ground to 1 mm in a Wiley cutting mill grinder (Thomas Scientific,NJ).Finally,grounded samples were stored under refrigeration at 4°C and used for further analysis.
Total alkaloid determination
Total alkaloids were determined using a previously reported volumetric method (Pharmacopeia, 2014).For this purpose, 3.75 g of dried and powdered Ipecac root samples were added to a flask with 50mL of ethyl ether and mixed in an orbital shaker at 400 rpm for 5 min. Subsequently, 2.5 mL of 6N ammonium hydroxide was added, and the mixture was shaken at the same speed for an additional hour. A total of 2.5 mL of water was added after agitation, and the flasks were mixed by hand.Flasks were then allowed to settle for a few minutes. The mixture was later filtered using cotton,collecting the organic layer and dumping the aqueousphase.The flask and cotton were washed with 2×15 mL of ethyl ether, and all ether extractions were combined. Ethyl ether was evaporated in a warm water bath (using a hood to extract vapors) until it dried up. The residue was redissolved in 2.5 mL of warm ethanol, allowed to cool, and subsequently mixed with 7.5 mL of a 0.1 N standard solution of sulfuric acid. Alkaloids were back-titrated with 0.1 N sodium hydroxide and a mixture of indicators (1 mg/ mL methyl red and 0.5 mg/mL methylene blue, using ethanol as solvent).
Evaluation of extraction conditions
The optimal extraction conditions were assessed. A total of 0.1 g of dry and ground samples was mixed with 0.5 mL of 6N ammonia. Then, each sample from a separate experiment was extracted with 1 to 5 extraction steps. Each extraction step consists of 2 mL of ethy lether and 10 min in an ultrasonic bath. After each extraction, the mixture was centrifuged for 10 min at 840 x g. Then, the other fractions were mixed in a 10 mL volumetric flask and filled with ethy lether.Finally,1mLwas transfer red to avial.Thesolvent was evaporated and then redissolved in 1 mL of methanol for further HPLC analysis. The procedure was repeated three times.
Emetine and cephaeline quantification using HPLC
Main alkaloids were quantified using the procedure reported by Han et al. (2013) with minor modifications.Methanolic extracts were analyzed in a UHPLC chromatograph Dionex UltiMate3000 (from Thermo Scientific,MA,USA)equippedwithanAcclaim™ 120 C18 column, a column oven (kept at 40°C), and a diode array detector. Amobile phase consisting o f0.08% trifluoro acetic acid (TFA,aqueous) and acetonitrile was utilized, following the gradient described in Table 1. The injection volume was 5 µL. Emetine and cephaeline were analyzed at 285 nm, against pure commercial standards (obtained from Sigma-Aldrich).
Table 1 Solvent gradient utilized for HPLC separation of Ipecac alkaloids
| Run time(min) | Flux (mL/min) | Solvent components (%) | |
|---|---|---|---|
| TFA 0.08% | Acetonitrile | ||
| 0.00 | 0.6 | 100 | 0 |
| 0.50 | 0.6 | 90 | 10 |
| 1.25 | 0.6 | 80 | 20 |
| 2.50 | 0.6 | 50 | 50 |
| 3.75 | 0.6 | 30 | 70 |
| 4.00 | 0.3 | 20 | 80 |
| 4.50 | 0.3 | 100 | 0 |
| 6.00 | 0.3 | 100 | 0 |
| 6.50 | 0.6 | 100 | 0 |
| 7.00 | 0.6 | 100 | 0 |
Note: derived from research.
Antibiotic activity of Ipecac extracts
The antibiotic activity of Ipecac extracts was evaluated against the ESKAPE pathogen group: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 9027, and Enterococcus faecium ATCC 6056.
Modified Kirby-Bauerforagarwelldiffusion method was utilized (Balouiri et al., 2016; Biemer, 1973). Forthispurpose, 100 µL of E. coli suspension (0.5 McFarland scale)insterilesalinesolution(0.85% NaCl) was plated into 90 x 15mm Muller-Hinton agar plates (25mL of agar). Then, four 7 mm wells were perforated into each agar plate using sterile Pasteur pipettes (upside down),andthewellswerefilledwith 50 µL of 60 mg/mL extract solution (in methanol). The other two wells were filled with methanol as a negative control and penicillin/streptomycin (1000 µg/mL each) as a positive control. The plates were incubated at 37°C for 18 h. The percentage of relative inhibition (%RI) was calculated according to equation (1).
where DIZ stands for “diameter of inhibition zone”.
Statistical analysis
Extraction optimization was evaluated using Minitab®,withα=0.05.Repeated ANOVA measurements for total alkaloids were performed using R-studio. Assumptions of no extreme outliers and normality by the Shapiro-Wilktest were verified by utilizing parametric statistics. Pair wise comparisons were performed using the Bonferroni method to adjust the p-values, α=0.05.Three repetitions were used for drying conditions and total alkaloids. Standard deviations were calculated for emetine and cephaeline concentrations from two fields and six repetitions.
Analysis and results
Evaluation of the sun-drying vs. The oven-drying procedure
Local Ipecac producers utilize a sun-drying method for root drying. Root tissue is relatively easy to dry. The traditional method is inexpensive, requires no technological equipment or trained personnel, and is logistically efficient (Belwal et al., 2022). However, an unsuccessful drying step will result in lower alkaloid yield and higher transportation costs. Also, long drying times can lead to a decline in the alkaloid concentration because other organisms can contaminate and degrade it (Kamel et al., 2013).
The regular oven-drying process and the traditional sun-drying protocol were compared.
Results are presented in Fig. 1. Data.
Note: Boxes represent the t-test for pairwise comparison between sun-drying and oven-drying (Two-way ANOVA). The p-value above each graph represents the pairwise comparisonbetween treatment groups. Significance codes for pairwise boxes are: 0.001 '**' and 0.01'*'. The p-adjust utilized is Bonferroni.
Figure 1 Boxplot graph of the effect of drying treatment on final alkaloid concentration. (a) field #1, (b) field #2, (c) both fields combined.
for plants younger than 13 months were excluded, as preliminary findings revealed high variability and uncertainty, likely due to immature root development. Repeated ANOVA analysis for the average of both fields (Fig. 1 (c)) showed no significant difference between the oven-dried samples and the sun-dried samples. Additionally, 16-month-old plants showed no significant differences. These plants are relevant because they yield the highest titer of total alkaloids. However, some of the samples from the individual fields (Fig. 1(a) and 1(b)) exhibited significant differences during certain months of the test period. For example, some oven-dried samples from Field #2, harvested at 17.5, 19, and 20.5 months, exhibited a total alkaloid content (w/w) approximately 0.1 to 0.5 percentage points higher than their sun-dried counterparts. This difference can be attributed to the fact that older plants have developed stronger structural tissue, making them more challenging to dry. Then, the residual moisture lowers the alkaloid content (Ekeoma et al., 2021).
Effect of the plant maturity on total alkaloid production
Fig. 1(c) shows the average concentration of alkaloid production in the combined samples of both fields included in this study.
Clearly, the best results are obtained when plants are between 16 and 19 months old, with the maximum at 16 months. Previously, Yonjan (1988) found that C. ipecacuanha increases its alkaloid concentration during the warmer months of the Indian summer, with the maximum increment occurring at the beginning of the summer. The increment in the total alkaloid concentration is claimed to be related to the post-reproductive phases. Samples from the 16th-old Ipecac roots included in this study were taken during December, the beginning of the dry season (December to March).
After the 16th month, the alkaloid concentration begins to decrease. Therefore, climate conditions can impact alkaloid production.
In the Northern zone, average temperatures range from 21 to 30 °C (Solano, 2023). Seasonal temperature variations between the dry and rainy periods (April to November) are minimal (±3°C) and are unlikely to significantly affect alkaloid production.
Consequently, they were not considered in this study. However, previous studies (Hüther et al., 2024) have shown that larger temperature differences can influence alkaloid levels, although their impact appears to be less significant than that of shading. Precipitation may have some influence over alkaloid concentration, although it was not considered a factor in this study.
Other variables, such as explant hardening and lower altitude, are reported to increase the total alkaloid concentration (Chatterjee et al., 1986).
Cephaeline and emetine production
Chromatographic determination was performed using standards for the main alkaloids, as shown in Fig. 2. Cephaeline has a retention time of approximately 5.3, while emetine has a retention time of around 5.8. Chromatographic separation was successful.
Note: derived from research.
Figure 2 UPLC chromatographs for alkaloids determination (A) a mixture of emetine and cephaeline standards. (B) Ipecac roots sample.
Fig. 3(A) shows the emetine and cephaeline concentrations of Ipecac roots during maturity. Results are consistent with total alkaloid production, showing a maximum cephaeline concentration between 16 and 19 months old (3.2-3.7%) and an emetine maximum titer between 14.5 and 19 months (1.4-1.7%). Additionally, the cephaeline/emetine (C/E) ratio is approximately 2 in plants ranging from 13 to 17.5 months old, and then it increases to approximately three at 20.5 months (Fig. 3(B)). According to the US Pharmacopeia, oral formulations containing Ipecac extracts should not exceed a 2.5 C/E ratio (Bleasel & Peterson, 2020a). Emetine’s highest titer is reached earlier (around 6 months), while cephaeline’s top concentration is reached at 19 months. The trend of C/E ratios increased at 19 months, despite a decrease in the concentration of both alkaloids. All this data suggests the same conclusion: optimal harvest time is around 16 months. Cephaeline and emetine are monoterpenoid- isoquinoline alkaloids. The biosynthetic pathway comprises the transformation of loganin → deacetylisoipicoside→ protoemetine → cephaeline → emetine (Jha et al., 1991). Many of the present alkaloids only differ in the O-methylation of some of the phenolic residues regulated by O-methyltransferases (OMT). The conversion of cephaeline to emetine is catalyzed by ipeOMT1 (Nomura & Kutchan, 2010).
Note: derived from research
Figure 3 Main alkaloids in C. ipecacuanha roots. (A) Cephaeline and emetine concentration in dried roots vs. maturity time. Error bars represent standard deviation. (B) Cephaeline/emetine ratio during maturity. The dotted line represents the trend of the ratio.
Therefore, it is reasonable to hypothesize that the C/E ratio is influenced by changes in ipeOMT1 expression at different growth stages; however, further experimentation is required to validate this hypothesis.
Antibiotic activity of Ipecac extracts
Given that Alkaloids are also known for their antimicrobial properties (Cushnie et al., 2014), the properties of the Ipecac extracts and how they change with the harvest time were explored. Fig.4 summarizes antibio gram results for the antimicrobial activity of C. ipecacuanha against six bacterial strains. All the extracts showed antimicrobial properties, with at least half the activity of penicillin/streptomycin (54-94%). Interestingly, the antimicrobial activity remains unchanged in the presence of Gram-positive bacteria. Moreover, the activity of the extracts against E. faecium is almost as vast as the positive control (penicillin-streptomycin), which is remarkable since this bacterium is one of the most problematic intra-hospital microbes due to its high capacity to generate resistance to antibiotics (Miller et al., 2014).
Note: derived from research.
Figure 4 Antibiotic activity of Ipecac extracts vs. harvest time. Error bars represent the standard deviation. (A) Percent of relative inhibition using penicillinstreptomycin 1000 μg/mL as positive control. A. baumannii was excluded because it was not inhibited by the positive control. (B) Diameter of inhibition zones in antibiograms.
Antibiotic activity is probably related to alkaloid concentration. Then, the extracts from plants harvested at 16 months old are the most active, as expected, due to the high alkaloid concentration (Cushnie et al., 2014).
Antimicrobial properties against A. baumannii, one of the most common multidrug-resistant pathogens (Shi et al., 2024), demonstratedsignificantinhibition,even when our positive control (penicillin-streptomycin 1000 µg/mL) failed to inhibit bacterial growth. The primary mechanisms of action against eukaryotic cells (e.g., antiparasitic activity) and viruses involve the inhibition of protein synthesis and DNA replication. However, the exact antimicrobial mode of action remains unclear (Abookleesh et al., 2022).
Conclusions
The concentrations of emetine, cephaeline, and total alkaloids in Carapichea ipecacuanhaareinfluencedbyharvesttime. Roots harvested at 16 months of age are considered optimal due to their elevated levels of total alkaloids, including emetine and cephaeline, as well as a favorable emetine-to-cephaeline (E/C) ratio. Notably, thesesamplesexhibitsignificantantibioticactivity, in some cases comparable to that of conventional antibiotics. This antimicrobial effect is likely associated with the high alkaloid content.
