Journal of Medicinal Plants Research Vol. 6(29), pp.4526-4534, 1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1253 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Quantification of α-, β- and γ-mangostin in Garcinia mangostana fruit rind extracts by a reverse phase high

performance liquid chromatography

Abdalrahim F. A. Aisha1, Khalid M. Abu-Salah2, Mohammad J. Siddiqui3, Zhari Ismail1 and Amin Malik Shah Abdul Majid1*

1School of Pharmaceutical Sciences, University of Science Malaysia (USM), Minden 11800, Pulau Pinang, Malaysia.

2Biochemistry Department and King Abdulla Institute for Nanotechnology, King Saud University (KSU), Riyadh 11451,

Saudi Arabia. 3International Medical University (IMU), Kuala Lumpur 57000, Malaysia.

Accepted 1 February, 2012

Garcinia mangostana fruit rinds contain high concentration of xanthones such as α-, and γ-mangostin. The α-mangostin-rich extracts have been used widely in nutritional supplements, herbal cosmetics and pharmaceutical preparations. This study aims to develop a reverse phase HPLC method for the quantification of α-, β- and γ-mangostin in G. mangostana fruit rind extracts. The method was validated at 244, 254, 316 and 320 nm. Selectivity was determined by comparing the retention time and the UV-Vis spectra of α-, β- and γ-mangostin in G. mangostana extracts with those of the reference compounds. Linearity was in the range 0.2 to 200 µg/ml at R

2 > 0.9999.

The intraday and interday precision was

determined as a relative standard deviation, and was found to be (0.4 ± 0.4) and (0.3 ± 0.3)%, respectively. The percentage recovery was in the range (96.3 ± 2.5) to (100.5 ± 3.4)%. The limits of detection and quantification were in the range 0.06 to 0.12 and 0.14 to 0.37 µg/ml, respectively. The reported method was applied for the determination of α-, β- and γ-mangostin concentration in 7 extracts of G. mangostana fruit rinds, and it could be considered as an important analytical tool for quality control, stability studies, pharmacokinetics, and standardization purposes. Key words: Alpha-, beta-, and gamma-mangostin, Garcinia mangostana, mangosteen.

INTRODUCTION Garcinia mangostana L., (Guttiferae) or mangosteen is a tropical tree cultivated for centuries in the tropical rainforests of Southeast Asia and many other countries (Ji et al., 2007). The pericarps of the fruit have been used in folk medicine by Southeast Asians in the treatment of several human diseases such as skin and wound infections (Harborne et al., 1999). Mangosteen is also used worldwide as an ingredient of several commercial products including nutritional supplements, herbal cosmetics and pharmaceutical preparations (Ji et al., 2007). Previous phytochemical studies on G. mangostana reported this plant as one of the richest *Corresponding author. E-mail: aminmalikshah@gmail.com. Tel: +6046534582. Fax: +6046534582.

sources of xanthones, where more than 50 xanthones have been isolated such as α-, β- and γ-mangostin (Figure 1) (Ee et al., 2006; Peres et al., 2000; Pedraza-Chaverri et al., 2008; Zhang et al., 2010).

G. mangostana is gaining more and more interest due to the remarkable pharmacological effects of the fruit rind xanthones such as analgesic (Cui et al., 2010), anti-oxidant (Jung et al., 2006), anti-tumor (Doi et al., 2009; Akao et al., 2008), anti-inflammatory (Chen et al., 2008; Tewtrakul et al., 2009), anti-allergy (Nakatani et al., 2002), anti-bacterial (Sakagami et al., 2005; Chomnawang et al., 2009), anti-tuberculosis (Suksamrarn et al., 2003), anti-fungal (Kaomongkolgit et al., 2009), anti-viral activities (Chen et al., 1996) and enhancement of the immune system (Tang et al., 2009).

Due to the increasing interest in G. mangostana, reliable procedures are needed for the quantitative

Figure 1. Chemical structure of α-, β- and γ-mangostin.

analysis of its bioactive ingredients. Few analytical methods have been reported for the standardization of G. mangostana extracts (Walker, 2007; Yodhnu et al., 2009; Pothitirat and Gritsanapan, 2009; Li et al., 2011). Though these methods are validated and reliable, they are either time consuming or have been developed for analysis of α-mangostin only.

This study was conducted in order to develop and validate a new HPLC method for the quantification of the major xanthone components of G. mangostana including α-, β and γ-mangostin. The effect of the wavelength on the validation parameters was also investigated.

MATERIALS AND METHODS

Preparation of raw material

Ripened G. mangostana fruit was obtained from a local fruit farm at the Island of Penang, Malaysia, on June 2009. Taxonomic authentication was performed by Taxonomist, USM. A voucher specimen (No: 11155) was deposited at the Herbarium at School of

Biological Sciences, USM, Malaysia. The fruit rinds were separated from the edible part, chopped using an electric grinder, and dried at 45 to 50°C for 24 h.

Abdalrahim et al. 4527 Chemicals and reagents The reference compounds were obtained ChromaDex, USA. The solvents were of HPLC or analytical grades, and were acquired from Merck. The reverse phase Nucleosil C18 column (5 µm, 4.6 × 250 mm) was purchased from Macherey-Nagel, USA. Extraction

Three extracts of the G. mangostana fruit rind powder were prepared including methanolic, 75% ethanolic and toluene extracts. The methanolic extract was prepared at room temperature at 5:1

(solvent: solid) ratio for 48 h, and the 75% ethanolic and the toluene extracts were prepared at 60°C for 48 h. Methanol was evaporated to dryness at 50°C using a rotary evaporator, and the crude extract was extracted sequentially (3 × 100 ml, 10 min each) with petroleum ether, chloroform, ethyl acetate and methanol. The ethanolic and toluene extracts were concentrated, at 55 to 60°C, until a yellow precipitate starts to form. Subsequently, the concentrated solutions were kept at 2 to 8°C for 24 h. A yellow mass was formed in both extracts and was collected and dried at

50°C. This material was considered for the 75% ethanolic and toluene extracts. Instrumentation and HPLC conditions The instrument consisting of Dionex-Ultimate® 3000 Rapid Separation LC (RSLC) system, was equipped with a auto sampler, quaternary pump, degasser, column oven, and a DAD detector.

The chromatographic analysis was carried out using a reverse phase Nucleosil C18 column (5 µm, 4.6 × 250 mm). The column temperature was set at 30°C, the mobile phase was consisting of A (acetonitrile) and B (0.1% H3PO4 in water), the elution program was isocratic at 95% (A) and 5% (B) for 10 min, the flow rate was maintained at 1 ml/min, and the injection volume was 10 µl. The spectral data from the DAD-3000RS Diode Array Detector was collected at 244, 254, 316 and 320 nm, and data acquisition was

performed by Chromeleon software version 6.8. Preparation of the standard mixture

Stock solution of the standard mixture consisting of α-, β- and γ-mangostin reference compounds was prepared at 2 mg/ml in HPLC grade methanol. The solution was further diluted to obtain 200, 100, 25, 10, 5, 1 and 0.2 µg/ml. The sample extracts were prepared at 1 mg/ml in the same solvent, and were further diluted to obtain 200 µg/ml. The stock solutions were filtered through 0.45 µm syringe filters. Method validation

The described method was validated according to the ICH guidelines (ICH, 1997). The following validation characteristics were evaluated: selectivity, linearity, precision, accuracy and the limits of detection and quantification (LOD and LOQ).

Linearity

Linearity was determined by injecting 10 µl of the standard mixture in a concentration range of 0.2 to 200 µg/ml. The calibration curves

were obtained for each individual compound by plotting the peak area versus the concentration. Regression analysis was performed in order to determine the linearity, in terms of R

2, of the calibration

4528 J. Med. Plants Res.

Figure 2. UV-Vis spectra of the reference compounds. α-Mangostin (A), β-mangostin (B), and γ-mangostin (C). The spectra were

collected by scanning the peaks at their start, apex and end.

graphs.

Selectivity

The selectivity of the method was determined by comparing the retention time and the ultraviolet-visible (UV-Vis) spectra of α-, β- and γ-mangostin obtained in the sample extracts with those of the reference compounds. Precision

The peak area and the retention time were considered as the parameters for the precision analysis. The standard mixture was analyzed at 7 concentration points in the range 0.2 to 200 µg/ml. The intraday and interday precision was determined in terms of the relative standard deviation (%RSD), (n = 5).

Accuracy Accuracy was determined as a percentage recovery of α-, β- and γ-mangostin added to the toluene extract at 10 µg/ml. The recovery was studied at a concentration of 1, 5, 10 and 25 µg/ml of each individual reference compound. The peak area of the compounds in the toluene extract (B), the individual reference compounds (C) and

their combinations (A) was recorded. The percentage recovery was calculated as the following: Percentage recovery = ((A – B) / C) × 100 The results are presented average ± SD (n = 3). Limits of detection and quantification

The LOD and LOQ were calculated through the slope and standard

deviation method (ICH, 1997), using the following formula:

LOD = (3.3 × δ) / S, and LOQ = (10 × δ) / S, Where: δ: is the standard deviation of the Y intercept of the linear regression equations. S: is the slope of the linear regression equations. Determination of α-, β- and γ-mangostin concentration in G. mangostana extracts

Ten microliters of G. mangostana extracts were injected at 200 µg/ml, and the peak area corresponding to α-, β- and γ-mangostin

was recorded. The concentration of the marker compounds in the samples was calculated by applying the linear regression equations of the standard calibration curves. The results are presented as a

wt/wt percentage using the formula: Wt/wt percentage = (the found concentration / 200 µg/ml) × 100 (n = 3). Statistical analysis Statistical calculations were carried out using the SPSS 16.0

software package. The student’s t-test or One-way ANOVA analysis was applied, and differences were considered significant at P < 0.01.

RESULTS The UV-Vis spectra of α-, β- and γ-mangostin reference compounds showed peaks at 244 and 316 nm, in addition to 259 nm in γ-mangostin (Figure 2). In this

study, peak detection was carried out at 244 and 316 nm, and at two commonly used wavelengths 254 and 320 nm. Selectivity The selectivity of the method was determined firstly by comparing the retention time of α-, β- and γ-mangostin obtained in the sample extracts with those of the reference compounds. The retention time of α-, β- and γ-mangostin reference compounds were 4.72 ± 0.001, 6.97 ± 0.001 and 3.97 ± 0.002 min, respectively. The retention time of the same compounds in the G. mangostana extracts was 4.71 ± 0.001, 6.93 ± 0.01 and 3.97 ± 0.001 min, respectively. The UV-Vis spectra of the target compounds were recorded in same manner of the reference compounds. These spectra were also used, as a second parameter, for the selectivity evaluation. Eight compounds were detected, and showed distinct UV-Vis spectra. The spectra of α-, β-, and γ-mangostin detected in the fruit rind extracts were found to match those of the reference compounds (Figure 3). Taken together, these findings confirm the selectivity of the developed method. Linearity Linearity of the developed method was presented in terms of regression coefficient (R

2) and was > 0.9999 in

the 3 reference compounds at the 4 studied wavelengths. Precision The precision was presented in terms of the %RSD of the retention time and the peak area (n = 5) of the standard mixture. α-, β- and γ-mangostin reference compounds were eluted at 4.72, 6.97 and 3.98 min, respectively. The %RSD was less than 0.003% which indicated good reproducibility of the retention time. The %RSD of the peak area, as a second parameter, was also calculated in the range 0.2 to 200 µg/ml. Table 1 depicts the average %RSD values of the 3 reference compounds. Statistical analysis by One-way ANOVA indicates no significant effect of the wavelength on the method’s precision, P > 0.01. Likewise, analysis by Student’s t-test indicates no significant difference between the intraday and interday precision, P > 0.01. Accuracy and recovery The accuracy of the method was evaluated by performing a recovery study at 4 concentrations: 1, 5, 10 and 25 µg/ml. The results are presented as average percentage recovery ± SD (Table 2). Statistical analysis was performed by One-way ANOVA and indicates no

Abdalrahim et al. 4529 significant effect of the wavelength on the percentage recovery of the reference compounds, and hence on the method’s accuracy, P values > 0.01. LOD and LOQ The linear regression equations of the reference compounds along with the LOD and LOQ values are presented in Table 3. Statistical analysis by One-way ANOVA indicates no significant effect of the wavelength on the LOD and LOQ, P > 0.01. Concentration of α-, β- and γ-mangostin in G. mangostana extracts Seven extracts from G. mangostana fruit rinds were analyzed (Figure 4). The concentration of α-, β- and γ-mangostin in the extracts was calculated by applying the linear regression equations of the reference compounds, and the results are presented as average (wt/wt)% ± SD. No significant difference in the concentration of the marker compounds was found when the same samples were analyzed at 244, 254, 316 and 320 nm, P values > 0.01. The results showed that α- and γ-mangostin are the main xanthone components of the extracts, with much less amount of β-mangostin. The highest concentration of α-, and γ-mangostin was obtained in the toluene extract, whereas the highest β-mangostin content was achieved in the petroleum ether sub-extract of the methanolic extract (Table 4). Analysis of an enriched mixture of xanthones A fraction obtained by column chromatography from the toluene extract was enriched with α-, β-, and γ-mangostin, and was analyzed for separation of the compounds. The HPLC chromatogram is shown in Figure 5, which shows the presence of 10 peaks. DISCUSSION AND CONCLUSION α- and γ-mangostin were selected as reference compounds because they constitute the major xanthone components in G. mangostana extracts. β-mangostin is one of the most hydrophobic xanthones in G. mangostana, and hence the compound has been selected as a marker for the end of elution. The use of acetonitrile at 95% and the 0.1% H3PO4 at 5% resulted in short elution time (<10 min), while maintaining high resolution. The wide concentration range (0.2 to 200 µg/ml) gives flexibility to the developed method. Consequently, it can be applied in analysis of samples containing high concentration of the target compounds

4530 J. Med. Plants Res.

Figure 3. UV-Vis spectra of 8 xanthones from G. mangostana extracts. The compounds are labeled with the name and the

retention time. The spectra were collected by scanning the peaks at their start, apex and end.

Abdalrahim et al. 4531

Table 1. Precision analysis of the HPLC method. The relative standard deviation of the peak area was calculated in the intraday and interday data, and the results are presented as average ± SD (n = 5). P value was calculated by two tests: One-way ANOVA to study the effect of the wavelength on the precision, and Student’s t-test to study the difference between the intraday and interday data. Differences are considered significant at P < 0.01.

Compounds Wavelength (nm)

P value (One-way ANOVA)

244 254 316 320

Intraday data

α-mangostin 0.23 ± 0.14 0.27 ± 0.20 0.59 ± 0.78 0.40 ± 0.63 0.54

β-mangostin 0.21 ± 0.22 0.31 ± 0.30 0.67 ± 0.53 0.61 ± 0.71 0.55

γ-mangostin 0.17 ± 0.08 0.19 ± 0.09 0.46 ± 0.38 0.51 ± 0.48 0.20

Interday data

α-mangostin 0.17 ± 0.13 0.20 ± 0.15 0.26 ± 0.16 0.33 ± 0.28 0.03

β-mangostin 0.27 ± 0.33 0.20 ± 0.08 0.27 ± 0.11 0.57 ± 0.64 0.04

γ-mangostin 0.18 ± 0.10 0.23 ± 0.18 0.54 ± 0.42 0.34 ± 0.14 0.04

P value (student t-test)

α-mangostin 0.41 0.47 0.31 0.79

β-mangostin 0.73 0.37 0.10 0.91

γ-mangostin 0.93 0.95 0.69 0.40

Table 2. Accuracy of the HPLC method in the concentration range 1 to 25 µg/ml. The study

was performed at 4 wavelengths and the results are shown as average percentage recovery of the reference compounds ± SD, (n = 3). P value was calculated by One-way ANOVA.

Compounds Concentration (µg/ml)

244 nm 254 nm 316 nm 320 nm P value

α-mangostin 99.0 ± 2.6 98.3 ± 1.7 97.8 ± 1.3 97.8 ± 1.3 0.41

β-mangostin 97.8 ± 1.7 97.0 ± 1.2 97.3 ± 1.5 96.3 ± 2.5 0.38

γ-mangostin 100 ± 2.9 100.0 ± 2.9 99.8 ± 2.5 100.5 ± 3.4 0.53

Table 3. Summary of the calibration data of the reference compounds. The regression equation is (y = ax + b), where (a) is the slope and (b) is the y intercept. The data are presented as average ± SD (n = 5).

Wavelength (nm) a b LOD (µg/ml) LOQ (µg/ml) R2

α-mangostin

244 0.90 ± 0.004 0.33 ± 0.021 0.07 ± 0.01 0.21 ± 0.02 1.00

254 0.78 ± 0.003 0.33 ± 0.020 0.09 ± 0.01 0.26 ± 0.04 0.99

316 0.64 ± 0.003 0.24 ± 0.015 0.08 ± 0.03 0.24 ± 0.08 1.00

320 0.59 ± 0.002 0.22 ± 0.014 0.08 ± 0.02 0.23 ± 0.05 1.00

β-mangostin

244 0.87 ± 0.003 0.40 ± 0.022 0.08 ± 0.02 0.25 ± 0.06 1.00

254 0.78 ± 0.004 0.37 ± 0.017 0.07 ± 0.01 0.21 ± 0.04 1.00

316 0.62 ± 0.003 0.28 ± 0.015 0.08 ± 0.02 0.24 ± 0.05 1.00

320 0.57 ± 0.002 0.24 ± 0.012 0.07 ± 0.03 0.21 ± 0.07 1.00

γ-mangostin

244 0.75 ± 0.003 0.31 ± 0.019 0.08 ± 0.01 0.25 ± 0.03 1.00

254 0.75 ± 0.003 0.30 ± 0.018 0.08 ± 0.03 0.24 ± 0.08 1.00

316 0.55 ± 0.002 0.20 ± 0.015 0.09 ± 0.03 0.27 ± 0.10 1.00

320 0.52 ± 0.002 0.18 ± 0.012 0.08 ± 0.01 0.23 ± 0.03 1.00

4532 J. Med. Plants Res.

Figure 4. HPLC chromatograms of G. mangostana extracts at 244 nm. Standard mixture of α-, β-, and γ-mangostin (A),

toluene extracts (B), methanolic extract (C), and the sub-extracts of the methanolic extract; petroleum ether (D), chloroform (E), ethyl acetate (F), methanol (G). The 75% ethanolic extract (H).

Abdalrahim et al. 4533

Table 4. α-, β- and γ-mangostin content in G. mangostana fruit rind extracts. Results are depicted as average (wt/wt)% ± SD (n = 3).

Extracts α-mangostin β-mangostin γ-mangostin

Toluene 72.0 ± 0.2 0.9 ± 0.01 16.9 ± 0.04

Methanol 33.0 ± 0.1 1.3 ± 0.01 7.3 ± 0.02

Petroleum ethera

51.0 ± 0.1 5.0 ± 0.01 0.7 ± 0.02

Chloroformb

46.0 ± 0.8 1.9 ± 0.02 10.1 ± 0.20

Ethyl acetatec

42.0 ± 0.7 1.7 ± 0.02 9.3 ± 0.20

Methanold

7.0 ± 0.1 0.4 ± 0.01 1.8 ± 0.02

75% Ethanol 53.0 ± 0.3 1.4 ± 0.01 11.0 ± 0.07 a–d

refer to sub-extracts of the G. mangostana methanolic extract.

Figure 5. HPLC chromatogram of the enriched mixture of known and unknown xanthones. γ-, α-, and β-mangostin were eluted at 3.97, 4.71 and 6.93 min, respectively. The other peaks refer to unknown xanthones.

such as G. mangostana extracts and its commercial products. The method can also be used in pharmacokinetics, where low concentration of the marker compounds in the blood or animal tissues is expected. The use of different wavelengths also gives flexibility to the developed method especially when used in cosmetics, pharmaceutical preparations or in pharmacokinetic studies where interference from the additives or blood components is expected, so the wavelength without or with the minimal noise can be selected.

Analysis of the enriched mixture of different xanthones showed the presence of 10 isolated peaks. Consequently, and providing the availability of the reference compounds, it can be concluded that our method can be used for quantification of 10 xanthones in G. mangostana extracts. It is noteworthy to mention that the reported method was applied in the drug release

studies of 2 nanoparticle formulations prepared from α-mangostin and the toluene extract of G. mangostana (Aisha et al., 2012). The method was also used to study the accumulation of α-mangostin in the gastrointestinal tract wall of mice after long term treatment with the formulated and non-formulated toluene extract (unpublished data). Overall, we report a novel reverse phase HPLC method for the quantification of α-, β- and γ-mangostin in G. mangostana extracts. The reported method was found to be rapid, selective, precise, accurate and with high sensitivity. This method provides several advantages over the previously published methods as it required less elution time and hence less solvent, more flexibility of the wavelength, and the number of marker compounds. We believe that this method may have an important contribution to the research on G. mangostana, and could be considered as an analytical tool for quality control assurance in

4534 J. Med. Plants Res. cosmetics and pharmaceutical industry of G. mangostana. ACKNOWLEDGEMENTS This work was supported by the Malaysian Ministry of Science and Technology (MOSTI) Science fund (305/PFARMASI/613219) and by the Malaysian Ministry of Higher Education (FRGS-MOHE 203/PFARMASI/61154). This work was also funded by the research chair of "Drug Targeting and Treatment of Cancer use Nanoparticles "at King Saud University, Riyadh, Saudi Arabia. The first author would like to acknowledge with thanks the University of Science Malaysia for providing fellowship during the academic year 2010/2011. Finally, we would like to thank Mr. Shanmugan A/C Vellosamy, School of Biological Sciences, for authentication of the plant samples. REFERENCES Aisha AF, Ismail Z, Abu-Salah KM, Majid AM (2012). Solid dispersions

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