Validated spectrophotometric method for quantification of total triterpenes in plant matrices

Abstract

Background

Triterpenes are ubiquitous secondary metabolites present in plants. They can be found in both forms, as genins or conjugated as glycosides. Although distinct analytical methods to quantify these compounds in vegetal tissues are available in the literature, limitations like high cost, complexity on sample preparation, and selectivity are often challenging issues. This study aimed to develop and to validate a simple and rapid spectrophotometric method to detect and quantify total triterpenes in plant matrices.

Methods

The assay was conducted directly into glass tubes using vanillin, acetic acid, and sulphuric acid as reagents, and β-sitosterol as reference standard. The samples were analyzed at 548 nm assessing the quality parameters of selectivity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness.

Results

The method was selective, with precision and accuracy varying from 0.56% to 4.98% and 96.63% to 113.87%, respectively. The values of the limit of detection and quantification were 0.042 μg.mL⁻¹ and 0.14 μg.mL⁻¹, correspondingly. The correlation coefficient (r) at the concentration range of 3.08 μg.mL⁻¹to 24.61 μg.mL⁻¹ was 0.9998. The total of triterpenes found in of B. holophylla and M. ilicifolia leaves were 132.36 ± 20.36 mg EβS.g⁻¹ of dry extract and 53.91 ± 2.6 mg EβS.g⁻¹ of dry extract, respectively.

Conclusion

The method was reliable to quantify total triterpenes extracted from Maytenus ilicifolia and Bauhinia holophylla.

Graphical abstract

Electronic supplementary material

The online version of this article (10.1007/s40199-020-00342-z) contains supplementary material, which is available to authorized users.

Introduction

Triterpenes are ubiquitous secondary metabolites from plants. This class of compounds is derived from polycyclic isoprenic units with 27 to 30 carbons [1]. They can be found in distinct species of plants as genins or conjugated with sugar moieties (saponins). The triterpenes are distributed in different tissues like roots, stalks, leaves, and fruits functioning as structural and defensive compounds [2, 3]. These compounds are of great commercial interest especially for agricultural, pharmaceutical, and cosmetic companies [4]. Previous studies demonstrated that some triterpenes like the ganoderic acids have anti-inflammatory and antitumoral properties [5, 6].

Maytenus ilicifolia Mart. ex. Reiss (Celastraceae) is traditionally used in Brazil due its anti-inflammatory and anti-ulcerogenic activities [7, 8], which are, in part attributed to the triterpenes like pristimerin, maytenin, friedelin, friedelan-3-one, and friedelan-3-ol [9, 10]. The genus Bauhinia is commonly known in Brazil as cow’s-paw, also has been shown apoptotic, anti-inflammatory, antioxidant and anti-ulcerogenic activities [11, 12]. These biological properties are associated to its chemical constituents flavonoids, phenolics acids, alkaloids, and mainly triterpenes. Particularly, the triterpene caffeate [3-β-trans-(3,4-dihydroxy cinnamoyl oxy) olean-12-en-28 oic acid] is the most studied [11].

To better correlate the biological activities of triterpenes with their amount in distinct plants is crucial to have an analytical method able to selectively detect and quantify such compounds in the plant matrices. The methods available to quantify triterpenes in plant matrices include gas and liquid chromatography [13–16]. However, they require derivatization or specific and high cost detectors like evaporative light scattering detector (ELSD) or mass spectrometry (MS) [17, 18]. On the order hand, for practical and economic reasons the spectrophotometric assays of triterpenes using vanillin and mineral acids as oxidant agents are commonly employed [19–22]. Nevertheless, these methods diverge regarding the choice of reference standards, reagents, wavelengths, and reaction conditions to develop the chromophore moiety. Furthermore, some of them were not validated or are specific for phytosterols or saponins [15, 23]. These inconsistencies make the comparison of previous studies and the selection of a suitable method to quantify triterpenes a challenging task. Therefore, this study aimed to validate a spectrophotometric assay able to quantify total triterpenes in plant matrices at 548 nm. The chromophore was obtained upon reaction of the triterpenes (β-sitosterol and ursolic acid, for example, Fig. 1) with vanillin in the presence of sulfuric acid.

Fig. 1.

Chemical structure of β-sitosterol (a) and ursolic acid (b)

Materials and methods

Plant material

The leaves of Maytenus ilicifolia Mart. ex. Reiss (Celastraceae) were collected at the Medicinal Plant Garden from the Federal University of Grande Dourados (UFGD), located in Dourados, Mato Grosso do Sul, Brazil. The samples were collected in April 2014 and the vouchers were identified by Prof. Maria C. Vieira of UFGD Herbarium (DDMS 4882). Bauhinia holophylla (Bong.) Steud. (Fabaceae) leaves were collected in Cerrado (Brazilian savanna) at Ijaci, Southern Minas Gerais State, Brazil (SISBIO no. 24542–3, IBAMA Registration: 5042260). Fertile samples were collected in June 2013 and the vouchers were identified by Andréia Fonseca Silva of PAMG Herbarium (PAMG 57021) at the Agricultural Research Company of Minas Gerais (EPAMIG).

Standard and reagents

The analytical grade reagents acetic acid (99,7%), vanillin, methanol were obtained from Vetec® (Rio de Janeiro, Brazil), ethanol, chloroform and sulfuric acid (99,5%) were acquired from Alphatec® (Macaé, Brazil), Synth® (Diadema, Brazil), and Impex® (Rio de Janeiro, Brazil), respectively. The standards ursolic acid (purity >90.0%), β-sitosterol (purity >70.0%), boldin (purity >98.0%), and rutin (>95.0%), were purchased from Sigma–Aldrich® (MO, USA), whereas the saponin Weiss Rein (Art.7695, Erg. B. 6. E.) was bought from Merck® (Darmstadt, Germany).

Plant extracts

The milled dried leaves of M. ilicifolia and B. holophylla were submitted to extraction with methanol (5 g in 50 mL) in water bath (45 ± 2 °C) during 15 min. The extracts were filtered, dried and stored in refrigerator. For further analysis, the samples were resuspended in ethanol at concentrations of 1 mg.mL⁻¹ (w:v) and 0.05 mg.mL⁻¹ for M. ilicifolia and B. holophylla, respectively.

Standard and sample solutions

The standards of flavonoid (rutin) and saponin were diluted in ethanol 80%, whereas the alkaloid (boldin), phytosterol (β-sitosterol), and triterpene (ursolic acid) were diluted in chloroform. In all cases stock solutions of each standard at 100 μg.mL⁻¹ were prepared in triplicate. The vanillin solution (50 mg.mL⁻¹) was prepared in acetic acid.

Assay procedure

The standard curve of β-sitosterol (n = 3) was obtained independently at three consecutive days by transferring 100, 200, 400, 600, and 800 μL from the stock solution (100 μg.mL⁻¹) to test tubes. The tubes were kept in water bath (85 °C) until dryness followed by the addition of 250 μL of vanillin solution (50 mg.mL⁻¹) and 500 μL of sulfuric acid (99,5%) in that order. The tubes were heated in water bath (60 ± 1 °C) for 30 min and then transferred into an ice bath followed by the addition of 2500 μL acetic acid (99,7%). The resulting solutions were maintained under cooling for 20 min and for additional 20 min at room temperature. The same procedure was conducted after adding 75 μL of plant extracts to the test tubes. A blank solution was similarly prepared and the absorbances were determined at 548 nm using a dual beam UV-Vis Genesys 10 spectrophotometer (Thermo Scientific, USA) equipped with 10 mm matched quartz cells. The content of total triterpenes was expressed as mg equivalent to β-sitosterol per gram of dried extract (mg E βS.g⁻¹).

Method validation

The method was validated assessing the quality parameters of selectivity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness [24, 25].

Selectivity

The selectivity was demonstrated by overlapping the spectrum (450–600 nm) obtained from the stock solutions (100 μg.mL⁻¹) containing the flavonoid (rutin), alkaloid (boldin), saponin, phytosterol (β-sitosterol), and triterpene (ursolic acid). The spectrum corresponding to the extract of M. ilicifolia and B. holophylla were also investigated (Fig. 2).

Fig. 2.

Scanning spectrum of different groups of secondary metabolites. Selectivity of protocol in the dosage of triterpenes and phytosterols

Linearity

The stock solution of β-sitosterol (100 μg.mL⁻¹) was used to prepare independently the calibration solutions at 3.08, 6.15, 12.31, 18.46, and 24.61 μg.mL⁻¹. Each level was prepared in triplicate at three consecutive days and the absorbance was determined randomly.

Precision and accuracy

Furthermore, according to ANOVA analysis the regression was satisfactorily significant (F_calc = 9.33 > F_crit = 4.7), whereas the deviation from linearity was not (F_calc = 0.077 < F_crit = 3.7), suggesting an adequate adjustment of the data to the regression line.²⁹ The linear equation obtained from the OLSM was: absorbance = 0.072 [β-sitosterol] + 0.008. The LOD and LOQ values calculated based on the standard error of the intercept (σ = 0.001) and slope (S = 0.072) were 0.042 μg.mL⁻¹ and 0.14 μg.mL⁻¹, respectively.

The precision and accuracy of the method are presented in Table 1. Accordingly, the recovery of the triterpene ranged from 95.17 to 102.85% and the CV (%) values were ≤ 5.0%. Overall, these data demonstrate that the developed method has adequate precision and accuracy.

Table 1.

Precision and accuracy of the analytical curves 1 (3.08 to 24.61 μg mL⁻¹) – mean ± standard deviation (n = 3)

Day 1
Theoretical (μg mL−1)	Observed (μg mL−1)	CVa (%)	Relative Error (%)
9.23	9.68 ± 0.46	4.77	−4.83
15.38	15.66 ± 0.23	1.44	1.83
21.54	21.95 ± 0.98	4.45	1.90
Day 2
Theoretical (μg mL−1)	Observed (μg mL−1)	CVa (%)	Relative Error (%)
9.23	9.12 ± 0.46	5.00	−1.14
15.38	15.18 ± 0.31	2.02	−1.30
21.54	21.03 ± 0.12	0.56	−2.38
Day 3
Theoretical (μg mL−1)	Observed (μg mL−1)	CVa (%)	Relative Error (%)
9.23	9.07 ± 0.28	3.12	−1.69
15.38	15.82 ± 0.43	2.74	2.85
21.54	21.47 ± 0.46	2.13	−0.31
Inter Day
Theoretical (μg mL−1)	Observed (μg mL−1)	CVa (%)	Relative Error (%)
9.23	9.29 ± 0.46	4.9	−0.67
15.38	15.55 ± 0.41	2.62	1.13
21.54	21.48 ± 0.67	3.13	−0.26

^aCV(%) = coefficient of variation

Limit of detection (LOD) and limit of quantification (LOQ)

The limits were calculated from eqs. (1) and (2), where σ is the standard deviation of the linear coefficient obtained from the three analytical curves and S is the slope obtained from the three analytical curves prepared on different days [26, 27]:

$$\mathit{LOD}=3\sigma /S$$ 1

$$\mathit{LOQ}=10\sigma /S$$ 2

Robustness

The robustness of the method was evaluated by varying the following parameters: temperature of water bath before vanillin addition (75 °C or 95 °C), the brand of sulfuric acid and the period that the samples remained under ice bath (15 min or 30 min). These parameters were previously determined as critical variables of the method.

Statistical analysis

The parameters were estimated by ordinary least squares method (OLSM) and ANOVA analysis were conducted in EXCEL® followed by Bonferroni’s test considering p < 0.05 as the significance level. Results were expressed as mean ± standard deviation.

Results and discussion

Selectivity

The selectivity was assessed by comparing the spectrum corresponding to different classes of natural compounds present in plant matrices such as flavonoids, saponins, alkaloids, phytosterols and triterpenes (Fig. 1). The method was selective for triterpenes at 548 nm since only ursolic acid and β-sitosterol showed maximum absorption at this wavelength, whereas the absorbance of the other compounds remained below the LOQ. Regarding the M. ilicifolia and B. holophylla extracts, one can observe a hypsochromic shift to 520 nm for the maximum of absorption, probably because the triterpenes present in such matrix contain distinct substituents or unsaturation from those of β-sitosterol. The wavelength at 548 nm was chosen to proceed the validation since it represents the intersection with the spectrum of M. ilicifolia and B. holophylla extracts.

Linearity, limit of detection and limit of quantification

The analytical curve (Fig. S1) presented a coefficient correlation of 0.9986 for a concentration range from 3.08 μg.mL⁻¹ to 24.61 μg.mL⁻¹.The range of our analytical curve was in a good agreement with that obtained from a recent study that reported the validation of an HPLC method to quantify isolated triterpenes in Bauhinia variegata, Cecropia palmate Willd. and C. obtuse [28]. According to the authors the standard curve was linear from 0.7 μg.mL⁻¹ to 35 μg.mL⁻¹.

Furthermore, according to ANOVA analysis the regression was satisfactorily significant (F_calc = 9.33 > F_crit = 4.7), whereas the deviation from linearity was not (F_calc = 0.077 < F_crit = 3.7), suggesting an adequate adjustment of the data to the regression line [29]. The linear equation obtained from the OLSM was: absorbance = 0.072 [β-sitosterol] + 0.008. The LOD and LOQ values calculated based on the standard error of the intercept (σ = 0.001) and slope (S = 0.072) were 0.042 μg.mL⁻¹and 0.14 μg.mL⁻¹, respectively.

Precision and accuracy

The precision and accuracy of the method are presented in Table 1. Accordingly, the recovery of the triterpene ranged from 95.17 to 102.85% and the CV (%) values were ≤ 5.0%. Overall, these data demonstrate that the developed method has adequate precision and accuracy.

Robustness

As presented in Table 2, the absorbance values of β-sitosterol were not significantly affected by varying the temperature of water bath before vanillin addition (75 or 95 °C), the brand of sulfuric acid and the period that the samples remained under ice bath (15 and 30 min). The results suggest that the suitability of the method may be easily adjusted when analyzing triterpenes from plant matrices. In order to demonstrate the applicability of the present method, the total triterpenes from leaves of M. ilicifolia and B. holophylla were quantified.

Table 2.

Ratio between the absorbance obtained under distinct conditions with that obtained under the standard condition) – mean ± standard deviation (n = 3)

Parameter	Level	Absa Ratio (%) ± SDb
Temperature	75 °C	98.75 ± 0.04
	95 °C	96.48 ± 0.09
H2SO4	Brand 1	100.94 ± 0.02
	Brand 2	101.49 ± 0.03
Time in ice bath	15 min	101.28 ± 0.02
	30 min	101.56 ± 0.02

^aAbsorbance

^bStandard Deviation

Determination of total triterpenes in leaves of M. ilicifolia

The leaves of M. ilicifolia are commonly used to treat gastroduodenal ulcer and part of its pharmacological properties is attributed in part to the triterpenes [7–9, 30]. However, not many information about the content of total triterpenes in this species is available [31, 32]. Mossi et al. [32] previously described that the content of triterpenes in M. ilicifolia populations in distinct regions of Brazil. The analyses were conducted using gas chromatograph coupled to mass spectrometer detector and total content of triterpenes varied from 8.67 mg.g⁻¹ to 22.1 mg.g⁻¹. The results however, were based on the summation of only three triterpenes (friedelan-3-ol, friedelan-3-one and friedelin) which may lead to an underestimation since other compounds of this class were already described [10, 30, 31]. Similar outcomes could be expected from studies based on extraction and purification steps [31]. On the other hand, the content of total triterpenes found in dried leaves of M. ilicifolia determined for the first time by spectrophotometry was about 2.4-fold higher than those from previous work corresponding to 53.91 ± 2.6 mg EβS.g⁻¹ of dry extract [31, 32]. Despite our method is less selective to estimate a specific triterpene it may has the advantage to be accurate and less expensive and can be successfully applied to estimate the total amount of triterpenes in plant matrices.

Determination of total triterpenes in leaves of B. holophylla

The Bauhinia genus is a source of terpenoid compounds. The total triterpenes of the Bauhinia are not described in the literature. However, Schmidt et al. [28] validated an HPLC method for the quantification of the specific triterpenes (maslinic acid, stigmasterol, lupeol, β-amirin, β-sitosterol and alpha-amirin) present in Bauhinia variegata extracts. In our work, we have been able to express the total triterpenes values of B. holophylla, 132.36 ± 20.36 mg EβS.g⁻¹ of dry extract. This is the first time that the total triterpene contents of B. holophylla leaves are described.

Therefore, the spectrophotometric assay could be an important preliminary analytical tool to screen for triterpenes in plant matrices. Once total triterpenes are determined one could use a more specific analytical method such LC-MS-MS or GC-MS-MS to discriminate and quantify them individually.

Conclusions

A suitable and accessible spectrophotometric method based on the reaction of triterpenes with vanillin in the presence of sulfuric acid was validated. The detection was set at 548 nm and the method has been shown to be linear over 3.08 to 24.61 μg.mL⁻¹, precise, accurate, robust, sensitive e selective for triterpenes, phytosterols and saponins even in the presence of distinct classes of natural compounds. Furthermore, the method was successfully applied to quantify the total triterpenes present in leaves extract of Maytenus ilicifolia and Bauhinia holophylla.

Since the total triterpenes contents in complex matrices can be easily quantified by this methodology, other plants could be evaluated and their biological activities compared to the amount of these compounds.

Authors’ contributions

AMP (PhD student) contributed preparing the extract and executing the method development and validation, analysis of the data and drafted the paper. WVC contributed to design the validation procedure, to the critical discussion of the manuscript and data analysis. AHFC contributed in plant collection, herbarium confection and data analysis. JMDA designed the study, supervised the laboratory work, contributed to critical reading of the manuscript and data analysis. All the authors have read the final manuscript and approved the submission.

Funding information

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

Compliance with ethical standards

This project was registred in Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SISBIO) of the Brazilian Government (A0C2B0F).

Conflict of interest

The authors declare no conflict of interest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.