Isolation and Identification Carpaine in Carica papaya L. Leaf by HPLC-UV Method

Abstract

The carpaine was isolated from Carica papaya L. leaf, was identified by means of Fourier transform infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, and was determined by HPLC. The powder of dried leaves was macerated and extracted with ethanol/water/HCl (89:10:1 V/V/V) to produce crude alkaloid. The extract was fractionated on a silica gel column chromatography using methanol/chloroform (5:95 v/v) as the eluate. The major fraction was isolated according to R_f values of thin-layer chromatography (TLC) (silica gel, methanol/chloroform [15:85 v/v]) exposed by Dragendroff’s reagent. The extraction procedure, sample preparation, and HPLC conditions were evaluated and optimized. The method was fully validated in terms of accuracy, precision, specificity, and calibration model. Results showed that the developed HPLC method was suitable for the determination of carpaine using a single-point calibration. The calibration model was linear in the concentration range from 0.20 to 1.8 mg/mL. Analysis of different days showed that the method was precise with an average concentration of 0.93 g/kg and relative standard deviation of 0.062%. A recovery of between 97.6 and 100.1% was obtained in a 95% confidence interval indicating that the method was accurate and the content of the carpaine in the powdered Carica papaya leaves was 0.93 g/kg.

Keywords:

• Carica papaya L. leaf
• Carpaine
• Isolation
• Quantification
• Validation
• Quality control

INTRODUCTION

Carica papaya L., belonging to the family of Caricaceae, is a perennial plant. It is cultivated almost all over tropical and subtropical countries of the world particularly in Srilanka, India, Philippines, East Equatorial Africa, South America, and West India.^[1,^] Several species of Caricaceae have been used as remedy against a variety of diseases.^[2−4] Almost all parts of the plant can be utilized by humans for food or for medicinal purposes.^[5−8] The leaves of Carica papaya have been shown to contain many active components that can increase the total antioxidant ability in the blood and reduce lipid peroxidation level. They have been used as an aboriginal remedy for various disorders, including cancer and infectious diseases.^[9,10] Crushed Carica papaya leaves have been used for anthelmintic purpose and fever.^[11] In Ibolnd and Ghaha, the yellow red parts of the dried leaves have been used to treat gastric problems.^[12] Despite its common use, the preparations with known content or standardized products are not commercially available.

The first step in the development of products from medicinal plants, is the characterization of the starting material, i.e., the powdered Carica papaya leaves. The main active constituent in Carica papaya leaves is the macrocyclic lactone carpaine (Fig. 1).^[13−15] This compound can reduce blood pressure and heart rate, movement of the intestinal strips, and can also cause the uterus marked relaxation and the bronchioles dilatation.^[16] It has been reported to have antitumor activity and antihelmintic activity.^[17] Furthermore, it has been shown to be peripheral vasodilator and can decrease blood pressure in intact animals. HPLC-MS and UPLC-Triple TOF-ESI-MS were used for the identification of carpaine in Papaya leaf extract which have already been described in the work of some research groups.^[18,19] However, these methods which were designed for research purposes, are not suitable for routine quality control due to their complex and laborious sample preparation. Furthermore, the data of calibration model, repeatability, and accuracy of the methods were not provided.

FIGURE 1 The structure of carpaine.

The objective of this research project was to develop and validate a method suitable for detecting the content of carpaine in the powdered Carica papaya leaves, and for controlling the quality of Carica papaya leaves used in health food and pharmaceutical preparations.

MATERIALS AND METHOD

Reagents, Solvents, and Instruments

HPLC grade acetonitrile and methanol were purchased from the Oceanpak Alexative chemical Co., Ltd. Mobile phase of ammonium acetate was filtered before usage. The chromatographic separations were carried out on a Waters 2695 HPLC with PAD detector, using a XBridge^TM C¹⁸ 5 μm column (250 mm × 4.6 mm) from Waters.

Plant Material

The plant material of Carica papaya leaves (Suizhonghong) was collected at the Papaya Professional Cooperatives Farm (Zengcheng Guangzhou) between September and December of 2012. The plant material was dried in an electric blast drying oven 101A-1 at 50°C for 48 h until constant mass. Then the dried leaves were milled to fine powder with particle size of 0.38 mm (40 meshes).

Isolation of Carpaine

Six kilograms of powdered plant material was macerated with 18 L mixed solution of ethanol/water/HCl(89:10:1 v/v/v) during 24 h at room temperature. The extract was then removed, stored, and desiccated at room temperature protected from light. The extraction was repeated five times or until no trace of alkaloid was detected using Dragendroff’s reagent. The whole extracts were pooled, and evaporated at 50°C under reduced pressure in a rotary evaporator (Shanghai Shen Shun Biotechnology Co. Ltd.). Then the extract was dissolved in 2500 mL of water/HCl (98:2 v/v) solution, filtered, and extracted with 5000 mL of petroleum ether to remove fat materials. The acid fraction was adjusted to pH 8.0˜9.0 using concentrated NH₄OH solution and further extracted with excess chloroform. The chloroform fractions were then pooled and evaporated at 50°C under reduced pressure until dry; the operation was repeated again. The crude alkaloid 21.34 g (3.56 g/kg) was obtained. TLC was used as an initial method for determining purity of extracts. Samples were spotted onto silica gel 60 F254, developed in methanol: chloroform (15:85 v/v), and plates were then exposed to Dragendroff’s reagent to visualize organic compounds.

Prior to use, the column was initially filled with stationary phase (silica gel 60, 400 g), then equilibrated with mobile phase. The column was considered in equilibrium when only chloroform eluted from the tail end. At this point, a crude extract of Carica papaya leaves (20 g) was dissolved in 10–15 mL of chloroform and loaded into the column, which was chromatographed with methanol/chloroform (5:95 v/v) as eluant, collecting 50 mL each fraction. The fractions were monitored by TLC (silica gel, same solvent) and plates were then exposed to Dragendroff’s reagent to visualize the spots. The fractions containing carpaine, the eluent of fractions 30 to 43, recognized by the violet zone with an R_f value of about 0.78, were collected and evaporated at 50°C under reduced pressure in a rotary evaporator. The residue was dissolved in 100 mL of water/HCl (98:2 v/v) solution, and filtered with a 0.45 μm filter membrane, and then concentrated NH₄OH solution was used to adjust the pH of the filtrate to 8.0–9.0 until there was no precipitation. The precipitate was collected and dried at room temperature, the light yellow powder was obtained.

The identity of the isolated compound was confirmed by means of nuclear magnetic resonance (NMR) spectroscopy and by comparison to literature data.^{[13,15] 1}HNMR, ¹³CNMR, DEPT-135, and DEPT-90 spectra were recorded in deuterated chloroform on a Bruker Avance 500 instrument, operating at 500.15 and using standard software packages. A library search based on the 13C NMR data using the NMR Predict software (MestRe Nova 8.0) confirmed the structure as carpaine (Fig. 1). In addition, the structure assignment was confirmed by mass spectroscopy (MS) using a linear ion trap 4000Q TrapTM of Applied Biosystems. Ionization was performed with electron spray ionization in positive mode. [M H]⁺, [M NH₃]⁺, and [M Na]⁺ ions at m/z 479, 495, and 501 were found, respectively, in the positive mode, indicating a relative molecular mass of 478. The infrared absorption spectrum was confirmed by Fourier infrared spectrometer EQUINOX55 of Germany Bruker spectral instruments company. The infrared characteristic absorption wavenumbers were at 3320, 1716, and 1232 cm⁻¹, respectively. The purity of this reference material was determined by HPLC analysis according to the normalization method, and was calculated to be 95%.

HPLC Analysis

Reference solution

About 200 mg of carpaine reference material was accurately weighed in a measuring flask of 10 mL and dissolved in ethanol. One hundred milliliters of this stock solution was transferred into a measuring flask of 10 mL and diluted with ethanol.

Sample preparation

Ten grams of the powdered Carica papaya leaves were weighed in a flask, the solution of 40 mL ethanol/water/HCl(89:10:1 v/v/v) mixed solution was added and the solution was placed in an ultrasonic bath for 25 min. The resulting extract was filtered into a round bottomed flask and the filter was rinsed with about 50 ml of ethanol/water (90:10 v/v). This extraction procedure was then repeated twice, the extract was collected in the round bottomed flask, and was dried using a rotary evaporator at a temperature not exceeding 50℃. The residue was dissolved in 50 mL water/HCl (98:2 v/v) solution and then filtered. The acid fraction was adjusted to pH 8.0–9.0 using concentrated NH₄OH solution and extracted with excess chloroform three times. The residue was dissolved in ethanol and transferred into a measuring flask of 10 mL. This solution was then filtered through a 0.45 μm membrane filter before injection into the HPLC.

Chromatographic conditions

Ten milliliters of the samples and standard solutions were injected in triplicate and eluted using the following linear gradient at a flow of 0.8 mL/min with A (acetonitrile) and B (ammonium acetate, 0.05 mol/L,pH 8.5): 0 min, 25% A and 75% B; 5 min, 25% A and 75% B; 15 min, 40% A and 60% B; 30 min, 40% A and 60% B; 35 min, 100% A and 0% B; 40 min, 100% A and 0% B; 45 min, 25% A and 75% B; 55 min, 25% A and 75% B. The peaks were detected at 304 nm.

Validation of the Method

Calibration model

A series of carpaine reference solutions were prepared in a concentration ranging from 0.20 to 1.8 mg/mL. By transferring different amounts from the stock solution into measuring flasks of 10 mL and diluting with absolute ethanol, five concentration levels of 0.20, 0.60, 1.00, 1.40, and 1.80 mg/mL were obtained, respectively. These solutions were analyzed in triplicate using the conditions described in HPLC analysis.

A calibration line was made and the least square line and correlation coefficient were calculated. Using student’s t-test, the intercept and the regression coefficient were investigated. The calibration line and the residuals also were inspected and evaluated.

Precision

Six independent crude alkaloid samples were analyzed on three different days by the same analyst and using the same equipment. For each day, and for every set of six samples, a fresh reference solution was prepared. From the results, the mean, the standard deviation, and the relative standard deviation (RSD) were calculated for each day. A Cochran’s test and ANOVA single factor were performed. Within and between-days RSD were calculated.

Accuracy

The accuracy was investigated by performing a recovery experiment. Samples were prepared at three concentration levels in triplicate by adding 2.0, 4.0, and 6.0 mL of the carpaine reference stock solution to 0.5 g of the powdered Carica papaya leaves. Extraction and analysis were then performed according to the described procedure. The recovery was calculated by comparing what was actually found by analysis and what was theoretically calculated, and was expressed in percentage. Student’s t-test was performed in order to investigate the results.

Specificity

The specificity of the method was investigated by checking the peak purity of carpaine. This was performed by comparing the UV spectrum of the peak of the reference material and that of Carica papaya leaves extract.

RESULTS AND DISCUSSION

Since carpaine is not only the active compound in Carica papaya leaves, but also the main constituent, the determination of the amount of carpaine in Carica papaya leaves are indispensable for the quality control of any preparation containing this plant extract as active ingredient. Herein, an HPLC method was developed for its determination. A certain amount of reference material of carpaine was required. Therefore, carpaine was first isolated from the Carica papaya leaves as described in the isolation of carpaine. Overall, this procedure can be considered faster, less complex, and less expensive without decreasing the purity of the final compound, i.e., 95%. The structure of carpaine was confirmed by NMR, mass spectrometry (MS) and Fourier transform infrared spectroscopy (FTIR). The NMR data, ¹HNMR (500 MHz, CDCl3): δ 4.82 (d, J = 26.1 Hz, 2H), 2.89 (dt, J = 12.1, 6.1 Hz, 2H), 2.61 (d, J = 7.2 Hz, 2H), 2.48–2.39 (m, 2H), 2.38–2.27 (m, 2H), 2.10–1.96 (m, 2H), 1.73–1.15 (m, 30H), 1.09 (t, J = 18.3 Hz, 6H). ¹³C NMR (126 MHz, CDCl3): δ 173.51 (s), 70.36 (s), 56.04 (s), 53.62 (s), 37.43 (s), 34.61 (s), 29.78 (s), 29.14 (s), 28.76 (s), 28.72 (s), 26.42 (s), 25.51 (s), 25.39 (s), 18.71 (s), were in good agreement with the literature.^[13,15]

The results on the evaluation of the calibration model were shown in Table 1. The least square line was determined by following equation: y = 134417x – 8526 and the correlation coefficient was at least 0.99. The intercept was not statistically different from (0.0) and its 95% confidence interval included zero. On the contrary, the regression coefficient was statistically different from zero and therefore, it could be concluded that a single-point calibration was allowed. Also graphical inspection showed a linear correlation and the highest residual of 4.5% was still lower than the limit of 5%. It could be concluded that a linear model was applicable in the concentration range from 0.20 to 1.8 mg/mL. The resulting chromatograms of the reference solution and sample solution were shown in Fig. 2a and 2b, respectively. The peak of carpaine had a retention time of 19.92 min and showed an acceptable symmetry. In the sample solution a good separation between carpaine and the other constituents was observed.

TABLE 1 Overview of the results about the calibration model of carpaine

	Carpaine
Range (mg/ml)	0.20–1.8
Number of standards	5
Correlation coefficient	0.995
Intercept ± standard error	−8526 ± 21
Confidence interval (95%)	−28844 to 11792
Slope ± standard error	134417 ± 23

FIGURE 2(a) HPLC chromatogram of the carpaine reference material.

FIGURE 2(b) HPLC chromatogram of the sample solution.

The validation of the method was required to assure the suitability for its intended use. Different characteristics were investigated according to reported guidelines^[20] including calibration model, precision, accuracy, and specificity. The precision experiment which was performed included the investigation of the repeatability and the intermediate precision. The data were shown in Table 2. The mean values of each different day were 0.93, 0.93, and 0.92 g/kg with a RSD of 0.057, 0.071, and 0.067%, respectively. The Cochran’s test was performed in order to check whether it was allowed to compare the results obtained on the different days. Since the calculated value of 0.41 was lower than the critical value of 0.71, it was allowed to perform an ANOVA. This resulted in a calculated F of 0.040, which was lower than the critical value of 3.68, it meant that the results obtained on different days were not statistically different. Since the within-days RSD (0.065%) and the between-days RSD (0.061%) were in the same order of magnitude, the method could be considered to be precise. In addition, the between-days RSD was smaller than the limit (3.81%), calculated by the Horwitz formula: RSD_max = 2/3RSD_Horwitz in which RSD _Horwitz = 2(1-0.5logC).^[21] The overall average amount of carpaine found in Carica papaya leaves was 0.93g/kg with an RSD of 0.062%.

TABLE 2 Data on the precision of the method

	Day 1	Day 2	Day 3
Repeatability
Number of replicates	6	6	6
Mean content (g/kg)	0.93	0.93	0.92
Standard deviation	0.0053	0.0066	0.0060
RSD (%)	0.057	0.071	0.067
Intermediate precision
Number of groups	3
Overall mean (g/kg)	0.93
RSD within (%)	0.065
RSD between (RSDmax) (%)	0.061	(3.81)
Ccalc (Ccrit)	0.41	(0.71)
Fcalc (Fcrit)	0.040	(3.68)

The results of the recovery experiment were shown in Table 3. The average of the recovery was 99.0% with an RSD of 1.66%. The 95% confidence interval was situated between 97.6 and 100.1%. Student’s t-test resulted in a calculated value of 2.08 while the theoretical value was 2.31. Thus, it showed that the recovery was not statistically different from 100%, meaning that the method was considered to be accurate.

TABLE 3 Results of the recovery experiment

Solution	Determined amount	Theoretical amount	Recovery (%)
1a	3.91	4	97.8
1b	3.87	4	96.8
1c	3.88	4	97.0
2a	8.02	8	100.3
2b	8.10	8	101.3
2c	8.07	8	100.9
3a	11.87	12	98.9
3b	11.79	12	98.3
3c	11.83	12	98.5

Regarding the specificity, no deviations were observed during the comparison of the UV spectra of the peaks of carpaine in the reference material and in the plant extracts. In addition, the peaks showed equal symmetry factors and width at half height, indicating that no interference induced by other constituents in sample solution.

CONCLUSION

In this study, Carpaine was isolated by a silica gel column chromatography using methanol/chloroform (5:95 v/v) as the eluate, and identified by means of IR, MS, NMR spectroscopy. An HPLC-UV method was established to detect the content of carpaine which was fully validated in terms of accuracy, precision, specificity, and calibration model. Using that method, the content of carpaine in the powdered Carica papaya leaves was determined to be 0.93 g/kg. This information will help us to discover ecological resources of carpaine and will also contribute to the future application of Carica papaya leaves as a raw material in the pharmaceutical industries and of Carica papaya in the health food.

FUNDING

This research was financially supported by the Natural Science Foundation of Guangdong Province (China) “Dynamic main composition and modeling of respiration rate of postharvest fruit in modified atmospheric environment” (10151063201000021) and the Key Technology R&D Program of Zhuhai (Guangdong China) “Papaya storage and preservation” (PC20061044).