# Quantification of Barbatusin and 3β-Hydroxy-3-deoxybarbatusin inPlectranthus Species by HPLC-DAD

Authors: Maria Goretti de Vasconcelos Silva; Leandro Bezerra Lima; Maria da Conceição Ferreira de Oliveira; Marcos Carlos de Mattos; Jair Mafezoli
Journal: International Journal of Analytical Chemistry
 (2017)

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## Abstract

The concentration of diterpenes barbatusin (1) and 3β-hydroxy-3-deoxybarbatusin (2) in the extracts from leaves ofPlectranthus grandis, P. barbatus, P. ornatus,andP. amboinicus was evaluated by HPLC-DAD analysis on a Luna C-18 column, using isocratic mixtures of water and acetonitrile as eluents. The regression equations were obtained with good linearity (r2 > 0.99) and limit of quantifications was higher than 0.1 µg/mL. The precision (lower than 3.5%, within day) and accuracy (higher than 81.7% and lower than 107.6%) of the methods were adequate. Barbatusin (1) was detected inP. grandis (15.432 ± 2.28 mg/g) andP. barbatus (5.198 ± 3.45 mg/g) extracts, while compound2was detected inP. grandis(4.068 ± 3.34 mg/g),P. barbatus(0.654 ± 5.86 mg/g),P. amboinicus (0.160 ± 7.25 mg/g), andP. ornatus (0.763 ± 5.10 mg/g). The evaluated validation parameters were satisfactorily achieved, and the developed methodology represents a suitable tool for application in the quantification of barbatusin (1) and 3β-hydroxy-3-deoxybarbatusin (2) inPlectranthusspecies.


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## Body

## 1. Introduction

ThePlectranthus genus (Labiatae) is represented byca. 300 species, which occur essentially in Africa, Asia, Australia, and some Pacific islands. Several species of this genus are cultivated for their edible tubes or as essential oil crops. In addition, some species are used for medicinal purposes such as treating vomiting, nausea, and ear infections; relieving toothache, headache, sores, and burns; or as antiseptic [1]. Species ofPlectranthus show biosynthetic ability to produce bioactive diterpenes like the labdane diterpenoid forskolin, fromP. forskohlii, that shows significant blood pressure lowering properties [2] and the abietane diterpenoids coleon U and horminone that show antimicrobial activity against methicillin-resistantStaphylococcus aureus and vancomycin-resistantEnterococcus faecalis [3]. In Brazil,Plectranthus species are popularly known as “boldo” and widely used as medicinal plants.P. grandis is used in the treatment of gastric and hepatic disturbs, and the gastric hyposecretory activity of this plant was already proven [4]. Several diterpenes present in this plant have shown gastroprotective properties [5–7]. These results validate the popular use ofP. grandis in the treatment of digestive problems. The abietane diterpenes barbatusin (1) and 3β-hydroxy-deoxybarbatusin (2) (Figure 1) were isolated fromP. grandisandP. barbatus [4, 8] and showed good gastric protection activity in rats by oral administration (10 mg/kg). The reduction of the gastric lesions was about 76% for1 and 96% for2 [8].Figure 1
Chemical structures of barbatusin (1) and 3β-hydroxy-3-deoxybarbatusin (2).


## 2. Experimental

### 2.1. General

Acetonitrile and methanol (HPLC grade) were purchased from Tedia®. Deionized water was obtained from a Millipore Milli-Q system. All other solvents and reagents were purchased from Synth®. Chromatography columns were developed on silica (φ mm 0,063–0,200) purchased from Vetec®. Analytical TLC was performed on precoated 0.25 mm thick plates of silica gel 60 F254 from Macherey-Nagel®, and the spots were visualized under UV lamp (254 nm) and by spraying with a solution of perchloric acid-vanillin in EtOH, followed by heating.


### 2.2. Plant Material

Leaves ofP. grandis(#28377), P. barbatus(#24408), P. ornatus(#31929), andP. amboinicus (#28601), grown under environmental conditions at the Horto de Plantas Medicinais Francisco José de Abreu Matos (official medicinal plant garden from Ceará state, Brazil), were collected in January 2013, and their voucher specimens are deposited in Prisco Bezerra Herbarium-UFC.


### 2.3. LC System and Chromatographic Method

A Shimadzu UFLC chromatographic system (model LC-20A), with DAD detector (Diode Assay Detector) SPD-M20A, and LC solutions software (version 1.25) were used for data processing. The analyses were conducted on a Phenomenex® Luna 5u C18 (2) 100A (250 × 4.60 mm, 5µm) analytical column in combination with a Phenomenex guard column.The methodology was based on the analysis of diterpenes in plants by HPLC-DAD [9]. Isocratic elution with 70% acetonitrile in water was used for compound1(method 1) and 45% acetonitrile in water for compound2(method 2). Both methods were carried out at flow rate of 0.8 mL/min. Temperature of the column was 40°C, and detection wavelength was 254 nm. Analyses were carried out in triplicate.


### 2.4. Sample Preparation and Validation

#### 2.4.1. Isolation and Identification of Compounds1 and2

The dried leaves (3.5 Kg) ofP. grandis were extracted with ethanol (5 × 8 L) and yielded 295.0 g of extract after solvent evaporation under reduced pressure. The crude extract was dissolved in 3 L of water/methanol (4 : 6 v/v) and submitted to liquid-liquid extraction with dichloromethane (6 × 100 mL) to yield 60.0 g of dichloromethane fraction. Successive chromatographic treatments in column, using SiO2 as stationary phase and dichloromethane as mobile phase, gave 3.4 g of1 and 140.0 mg of2. The structures of both diterpenes were confirmed by high-resolution mass spectra,1H-NMR (500 MHz, CDCl3), 13C-NMR (125 MHz, CDCl3), and comparison with literature data [10].


#### 2.4.2. Preparation of Samples

Leaves from fourPlectranthus species (P. grandis, P. barbatus, P. ornatus,andP. amboinicus) were dried at 40°C, pulverized to powder, and stored in a desiccator. An accurately weighted amount of 1.0 g of each sample was extracted with 100 mL of ethanol in ultrasonic bath during 1 hour at 25°C. Extracts were filtered and the solvent was completely removed by vacuum distillation at 40°C. Each plant extract was dissolved in methanol (HPLC grade) and transferred to a 25 mL volumetric flask for a final concentration 0.5 mg/mL to be analyzed by HPLC-DAD.


#### 2.4.3. Preparation of Standard Solutions

Barbatusin (1) and 3β-3-hydroxy-3-deoxybarbatusin (2) isolated from leaves ofP. grandis were used as standards for the analytical curves and method validation. Each diterpene was separately dissolved in methanol (HPLC grade), and the final concentration was adjusted to 1.0 mg/mL. The purities of standard compounds1 and2 were determined as ≥99% by HPLC-DAD analyses.


#### 2.4.4. Linearity and Selectivity

Solutions of the standard compounds1 and2 were prepared in methanol at 25, 50, 100, 250, and 500 µg/mL. Then, 20 µL aliquots of these solutions were injected into HPLC equipment, in triplicate. For each analyte, the average peak area values were plotted against the respective concentrations, expressed in µg/mL. Both the correlation coefficient (r) and general standard curve equation were obtained from linear least-square regression analysis (Graphpad Prism® 5.03 software). The method selectivity was determined through the analyses of the standard compounds1 and2 and extracts of the fourPlectranthus species. In all extracts, the peaks corresponding to1 and2 were identified by comparison of their retention times with those from the standard compounds.


#### 2.4.5. Limits of Detection and Quantification

The limits of detection (LODs, S/N≥ 3) of the two diterpenes1 and2were 0.015 and 0.22 µg/mL, and the limits of quantification (LOQ, S/N ≥ 10) were 0.1 and 0.6 µg/mL, respectively.


#### 2.4.6. Precision and Repeatability

The precision of the method was evaluated in two levels, intraday and interdays. Intraday precision was evaluated by nine determinations (triplicate) in three different concentrations. Interdays precision was evaluated using the same three concentrations, during two consecutive days, and by changing operators. The analyses were performed separately for each standard compound. The relative standard deviations (RSD) were calculated for each determination and taken as a measure of precision and repeatability.


#### 2.4.7. Accuracy

The accuracy was determined by recovery tests, which is an estimate of the accuracy of the methods. Extracts containing known amounts of1 (166.5, 249.0, and 374.0 µg/mL) and2 (37.8, 47.8, and 62.8 µg/mL), in quintuplicate, were analyzed, besides the extracts with no addition of standard compounds. The recoveries of both standard compounds were calculated from the corresponding calibration curve according to the following equation: (1)%recovery=amountfoundamountadded×100.


## 2.1. General

Acetonitrile and methanol (HPLC grade) were purchased from Tedia®. Deionized water was obtained from a Millipore Milli-Q system. All other solvents and reagents were purchased from Synth®. Chromatography columns were developed on silica (φ mm 0,063–0,200) purchased from Vetec®. Analytical TLC was performed on precoated 0.25 mm thick plates of silica gel 60 F254 from Macherey-Nagel®, and the spots were visualized under UV lamp (254 nm) and by spraying with a solution of perchloric acid-vanillin in EtOH, followed by heating.


## 2.2. Plant Material

Leaves ofP. grandis(#28377), P. barbatus(#24408), P. ornatus(#31929), andP. amboinicus (#28601), grown under environmental conditions at the Horto de Plantas Medicinais Francisco José de Abreu Matos (official medicinal plant garden from Ceará state, Brazil), were collected in January 2013, and their voucher specimens are deposited in Prisco Bezerra Herbarium-UFC.


## 2.3. LC System and Chromatographic Method

A Shimadzu UFLC chromatographic system (model LC-20A), with DAD detector (Diode Assay Detector) SPD-M20A, and LC solutions software (version 1.25) were used for data processing. The analyses were conducted on a Phenomenex® Luna 5u C18 (2) 100A (250 × 4.60 mm, 5µm) analytical column in combination with a Phenomenex guard column.The methodology was based on the analysis of diterpenes in plants by HPLC-DAD [9]. Isocratic elution with 70% acetonitrile in water was used for compound1(method 1) and 45% acetonitrile in water for compound2(method 2). Both methods were carried out at flow rate of 0.8 mL/min. Temperature of the column was 40°C, and detection wavelength was 254 nm. Analyses were carried out in triplicate.


## 2.4. Sample Preparation and Validation

### 2.4.1. Isolation and Identification of Compounds1 and2

The dried leaves (3.5 Kg) ofP. grandis were extracted with ethanol (5 × 8 L) and yielded 295.0 g of extract after solvent evaporation under reduced pressure. The crude extract was dissolved in 3 L of water/methanol (4 : 6 v/v) and submitted to liquid-liquid extraction with dichloromethane (6 × 100 mL) to yield 60.0 g of dichloromethane fraction. Successive chromatographic treatments in column, using SiO2 as stationary phase and dichloromethane as mobile phase, gave 3.4 g of1 and 140.0 mg of2. The structures of both diterpenes were confirmed by high-resolution mass spectra,1H-NMR (500 MHz, CDCl3), 13C-NMR (125 MHz, CDCl3), and comparison with literature data [10].


### 2.4.2. Preparation of Samples

Leaves from fourPlectranthus species (P. grandis, P. barbatus, P. ornatus,andP. amboinicus) were dried at 40°C, pulverized to powder, and stored in a desiccator. An accurately weighted amount of 1.0 g of each sample was extracted with 100 mL of ethanol in ultrasonic bath during 1 hour at 25°C. Extracts were filtered and the solvent was completely removed by vacuum distillation at 40°C. Each plant extract was dissolved in methanol (HPLC grade) and transferred to a 25 mL volumetric flask for a final concentration 0.5 mg/mL to be analyzed by HPLC-DAD.


### 2.4.3. Preparation of Standard Solutions

Barbatusin (1) and 3β-3-hydroxy-3-deoxybarbatusin (2) isolated from leaves ofP. grandis were used as standards for the analytical curves and method validation. Each diterpene was separately dissolved in methanol (HPLC grade), and the final concentration was adjusted to 1.0 mg/mL. The purities of standard compounds1 and2 were determined as ≥99% by HPLC-DAD analyses.


### 2.4.4. Linearity and Selectivity

Solutions of the standard compounds1 and2 were prepared in methanol at 25, 50, 100, 250, and 500 µg/mL. Then, 20 µL aliquots of these solutions were injected into HPLC equipment, in triplicate. For each analyte, the average peak area values were plotted against the respective concentrations, expressed in µg/mL. Both the correlation coefficient (r) and general standard curve equation were obtained from linear least-square regression analysis (Graphpad Prism® 5.03 software). The method selectivity was determined through the analyses of the standard compounds1 and2 and extracts of the fourPlectranthus species. In all extracts, the peaks corresponding to1 and2 were identified by comparison of their retention times with those from the standard compounds.


### 2.4.5. Limits of Detection and Quantification

The limits of detection (LODs, S/N≥ 3) of the two diterpenes1 and2were 0.015 and 0.22 µg/mL, and the limits of quantification (LOQ, S/N ≥ 10) were 0.1 and 0.6 µg/mL, respectively.


### 2.4.6. Precision and Repeatability

The precision of the method was evaluated in two levels, intraday and interdays. Intraday precision was evaluated by nine determinations (triplicate) in three different concentrations. Interdays precision was evaluated using the same three concentrations, during two consecutive days, and by changing operators. The analyses were performed separately for each standard compound. The relative standard deviations (RSD) were calculated for each determination and taken as a measure of precision and repeatability.


### 2.4.7. Accuracy

The accuracy was determined by recovery tests, which is an estimate of the accuracy of the methods. Extracts containing known amounts of1 (166.5, 249.0, and 374.0 µg/mL) and2 (37.8, 47.8, and 62.8 µg/mL), in quintuplicate, were analyzed, besides the extracts with no addition of standard compounds. The recoveries of both standard compounds were calculated from the corresponding calibration curve according to the following equation: (1)%recovery=amountfoundamountadded×100.


## 2.4.1. Isolation and Identification of Compounds1 and2

The dried leaves (3.5 Kg) ofP. grandis were extracted with ethanol (5 × 8 L) and yielded 295.0 g of extract after solvent evaporation under reduced pressure. The crude extract was dissolved in 3 L of water/methanol (4 : 6 v/v) and submitted to liquid-liquid extraction with dichloromethane (6 × 100 mL) to yield 60.0 g of dichloromethane fraction. Successive chromatographic treatments in column, using SiO2 as stationary phase and dichloromethane as mobile phase, gave 3.4 g of1 and 140.0 mg of2. The structures of both diterpenes were confirmed by high-resolution mass spectra,1H-NMR (500 MHz, CDCl3), 13C-NMR (125 MHz, CDCl3), and comparison with literature data [10].


## 2.4.2. Preparation of Samples

Leaves from fourPlectranthus species (P. grandis, P. barbatus, P. ornatus,andP. amboinicus) were dried at 40°C, pulverized to powder, and stored in a desiccator. An accurately weighted amount of 1.0 g of each sample was extracted with 100 mL of ethanol in ultrasonic bath during 1 hour at 25°C. Extracts were filtered and the solvent was completely removed by vacuum distillation at 40°C. Each plant extract was dissolved in methanol (HPLC grade) and transferred to a 25 mL volumetric flask for a final concentration 0.5 mg/mL to be analyzed by HPLC-DAD.


## 2.4.3. Preparation of Standard Solutions

Barbatusin (1) and 3β-3-hydroxy-3-deoxybarbatusin (2) isolated from leaves ofP. grandis were used as standards for the analytical curves and method validation. Each diterpene was separately dissolved in methanol (HPLC grade), and the final concentration was adjusted to 1.0 mg/mL. The purities of standard compounds1 and2 were determined as ≥99% by HPLC-DAD analyses.


## 2.4.4. Linearity and Selectivity

Solutions of the standard compounds1 and2 were prepared in methanol at 25, 50, 100, 250, and 500 µg/mL. Then, 20 µL aliquots of these solutions were injected into HPLC equipment, in triplicate. For each analyte, the average peak area values were plotted against the respective concentrations, expressed in µg/mL. Both the correlation coefficient (r) and general standard curve equation were obtained from linear least-square regression analysis (Graphpad Prism® 5.03 software). The method selectivity was determined through the analyses of the standard compounds1 and2 and extracts of the fourPlectranthus species. In all extracts, the peaks corresponding to1 and2 were identified by comparison of their retention times with those from the standard compounds.


## 2.4.5. Limits of Detection and Quantification

The limits of detection (LODs, S/N≥ 3) of the two diterpenes1 and2were 0.015 and 0.22 µg/mL, and the limits of quantification (LOQ, S/N ≥ 10) were 0.1 and 0.6 µg/mL, respectively.


## 2.4.6. Precision and Repeatability

The precision of the method was evaluated in two levels, intraday and interdays. Intraday precision was evaluated by nine determinations (triplicate) in three different concentrations. Interdays precision was evaluated using the same three concentrations, during two consecutive days, and by changing operators. The analyses were performed separately for each standard compound. The relative standard deviations (RSD) were calculated for each determination and taken as a measure of precision and repeatability.


## 2.4.7. Accuracy

The accuracy was determined by recovery tests, which is an estimate of the accuracy of the methods. Extracts containing known amounts of1 (166.5, 249.0, and 374.0 µg/mL) and2 (37.8, 47.8, and 62.8 µg/mL), in quintuplicate, were analyzed, besides the extracts with no addition of standard compounds. The recoveries of both standard compounds were calculated from the corresponding calibration curve according to the following equation: (1)%recovery=amountfoundamountadded×100.


## 3. Results and Discussion

Two analytical curves were obtained, one for each standard compound1 (method 1) and compound2 (method 2). The linearity of the concentrations versus peak area plot for both methods was determined, and good correlation coefficients were achieved (r2 = 0.9931 and r2 = 0.9966). Table 1 shows the linear regression equation and values for the LOD and LOQ which indicates that the developed methods are very sensitive and adequate for the investigated samples. The chromatograms of1 and2 andP. grandis extract are shown in Figure 2.Table 1
Analytical parameters of linearity and limits of detection and quantification obtained for1and 2.
Analyte
1
2
Regression equation
y
=
21496
x
-
20448
y
=
14145
x
+
239256
r
2
0.9931
0.9966
LOD (µg/mL)
0.015
0.22
LOQ (µg/mL)
0.1
0.6Figure 2
HPLC chromatograms (254 nm) of standard compounds1 (a) and2 (b) and the ethanol extract ofP. grandis (c and d).
(a)
(b)
(c)
(d)The intraday precision was evaluated by triplicate injection at low, medium, and high concentrations levels. Relative standard deviations were calculated based on observed concentrations. Naturally, interdays precision shows relative standard deviation greater than intraday. The accuracy was performed as recommended, by collecting data from a minimum of nine determinations over a minimum of three concentration levels [11]. Table 2 shows the precision of the methods for1 and2, which were accurate with recovery rates ranging from 81.7 to 104.0% for1 and from 102.1 to 107.6% for2. The found recovery rates (Table 2) were acceptable since the objective of this study is mainly estimating the amount of diterpenes1 and2.Table 2
Precision and accuracy for1 and 2 in Plectranthussamples.
Precision
Accuracy
Analyte
Amountµg/mL
IntradayRSD (%)
InterdayRSD (%)
Amount added (µg/mL)
Amount found (µg/mL)
Recovery(mean ± RSD, %, n=5)
1
85.0
3.42
5.70
186.5
152.3
81.7 ± 1.74
170.0
2.10
3.31
249.0
259.1
104.0 ± 2.10
680.0
1.92
6.01
374.0
384.0
102.7 ± 3.39
2
65.0
3.44
2.68
37.8
40.3
107.6 ± 3.60
130.0
3.34
6.21
47.8
50.4
105.4 ± 2.94
325.0
1.36
10.95
62.8
64.1
102.1 ± 2.73Table3 shows the concentration (mg/g) of diterpenes1 and2 in leaves of the investigatedPlectranthus species. Both compounds were detected inP. grandis andP. barbatus whileP. ornatusandP. amboinicus showed only the presence of2. The highest concentrations of1 and2 were found in the leaves ofP. grandis. Except in this species, diterpene2 was found in low concentration in the leaves of the studied species. No amount of1 inP. ornatusandP. amboinicus was observed by the employed methodology.Table 3
Concentration of diterpenes1 and 2 in leaves of Plectranthusspecies.
Species
1 (mean ± RSD, mg/g, n=3)
2 (mean ± RSD, mg/g, n=3)
P. grandis
15.432 ± 2.28
4.068 ± 3.34
P. barbatus
5.197 ± 3.45
0.654 ± 5.86
P. ornatus
Not detected
0.763 ± 5.10
P. amboinicus
Not detected
0.160 ± 7.25The lack of clear-cut morphological criteria to discriminate some species ofPlectranthus has resulted in numerous taxonomic problems for identification of the specimens [1]. Therefore, this work may represent a new tool for discriminatingPlectranthus species, by using compounds1and2as chemomarkers.


## 4. Conclusions

In summary, the analytical methods were adequate for quantification of diterpenes1 and2 in the leaves of fourPlectranthus species. Barbatusin (1) was identified only in the extracts fromP. grandisandP. barbatus, while compound2 was present in allPlectranthus species. The highest amount of both compounds1 and2was found inP. grandis. This is the first report on the identification of 3β-hydroxy-3-deoxybarbatusin (2) in the extracts fromP. amboinicus andP. ornatus.



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*Source: 2397131-2017-07-03.xml*