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Research Paper
Journal of Pharmacy And Pharmacology
Stability studies of antiandrogenic compounds in Curcuma aeruginosa Roxb. extract Nungruthai Suphroma, Jukkarin Srivilaia, Ganniga Pumthongb, Nantaka Khoranac, Neti Waranuchb, Nanteetip Limpeanchobd and Kornkanok Ingkaninana a Bioscreening Unit, Department of Pharmaceutical Chemistry and Pharmacognosy, bDepartment of Pharmaceutical Technology, cDepartment of Pharmaceutical Chemistry and Pharmacognosy, dDepartment of Pharmacy Practice, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok, Thailand
Keywords antiandrogen; Curcuma aeruginosa; degradation; germacrone; stability Correspondence Kornkanok Ingkaninan, Bioscreening Unit, Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand. E-mail: [email protected] Received June 28, 2013 Accepted December 7, 2013 doi: 10.1111/jphp.12216
Abstract Objectives Curcuma aeruginosa Roxb. extract is a 5α-reductase antagonist that can be used to treat hair loss. We aimed to study the stability of antiandrogenic constituents, germacrone and other sesquiterpene components in the extract. Methods Germacrone and the extract were analyzed as solid forms or solublized with polyethylene glycol-40 (PEG-40) or methanol using high-performance liquid chromatography and gas chromatography with flame ionization detector. The effects of pH, temperature and light on their stability were studied. Key findings Degradation of antiandrogenic compounds in C. aeruginosa was highly sensitive to temperature especially pure anhydrous germacrone, which was completely lost within 3 days at 45°C. Curiously, degradation was slower than as a dried extract. Paradoxically, when solubilized with PEG-40, it was largely intact even after 90 days at 45°C. The MS spectrum of a major degradation product suggested that it was elemenone probably produced by Cope rearrangement. Two other putative degradation products were germacrone-1,10-epoxide and germacrone-4,5-epoxide suggesting that oxidation of double bonds was an important mechanism. Germacrone stability was unaffected by pH (2.0–9.0) but only as dried extract it was slightly degraded by light. Conclusion Antiandrogenic constituents of C. aeruginosa were instable at high temperature and in solid form. Thus, the extract would be optimately stored as a solution or otherwise as solid form at low temperature.
Introduction Curcuma aeruginosa Roxb. (Zingiberaceae) is a native plant of tropical areas and is commonly known as Wanmahamek in Thailand. It is a perennial with oblong tuberous roots, and the fresh rhizome emits a ginger-like aroma. Leafy shoots are 45–60 cm high and the plant blooms during the rainy season.[1–3] In Thai traditional medicine, a tincture of the rhizome is used to treat uterine pain, uterine inflammation, postpartum uterine and perimenopausal bleeding.[4] Various pharmacological effects of this plant have been delineated such as postcoital contraception, anti-HIV actions, hepatoprotection, antimicrobial effects, antioxidation, reduce platelet activation and antinociception.[1,5–7] Recently, our group found that a hexane extract of C. aeruginosa rhizomes showed high antiandrogenic activity both in vitro and in vivo. It was shown to be an effective ingredient in a hair tonic for androgenic alopecia.[8] Six 1282
sesquiterpenes, that is, germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol and isocurcumenol were isolated from C. aeruginosa extract (Figure 1), and these were shown to inhibit the conversion of testosterone to dihydrotestosterone. Of the six components, germacrone was the most active. Our studies demonstrated that germacrone also showed an antiandrogenic action on testosterone-induced growth suppression of human prostate cancer cells [Lymph Node Carcinoma of the prostate (LNCaP)] and on the hamster flank gland model. A possible mechanisms was blockade of 5α-reductase activity whereas no binding of germacrone to androgen receptors could be detected.[9] Thus, germacrone has potential applications in the treatment of androgen-dependent disorders. But its success as a cosmetic or health product depends on its chemical stability. Therefore, the present study was
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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Stability studies of Curcuma aeruginosa
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Chemical structures of germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol, and isocurcumenol.
undertaken to assess its stability as a pure compound or in an extract of C. aeruginosa along with other active ingredients. In particular, we assessed their chemical stability to pH, temperature, oxygen and light.
Materials and Methods General experimental procedures Thin layer chromatography (TLC) analysis was performed on silica gel 60 F254 TLC on an aluminum sheet (20 × 20 cm; Merck, Darmstadt, Germany). A silica gel
column (0.040–0.063 mm granule size; Merck) was used for the chromatographic isolation of degradation products. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV400 (Bruker, Billerica, MA, USA) spectrometer at 400 MHz for proton and 100 MHz for carbon with tetramethylsilane as the internal standard. Mass spectrometry (MS) spectra were obtained using electron impact ionization mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Fourier transform infrared spectroscopy spectra were recorded on a Spectrum One spectrometer (Perkin Elmer, Shelton, CT, USA).
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Chemicals Methanol, acetonitrile [high-performance liquid chromatography (HPLC) grade], hexane and ethyl acetate were bought from RCI Labscan Ltd (Bangkok, Thailand). Polyethylene glycol-40 (PEG-40) hydrogenated castor oil was purchased from Sigma-Aldrich (St. Louis, MO, USA). Orthophosphoric acid and disodium hydrogen phosphate were purchased from BDH (Poole, UK). Hydrochloric acid, potassium chloride, potassium hydroxide, acetic acid and sodium acetate were purchased from Merck. The reference compounds, germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol and isocurcumenol were also isolated from C. aeruginosa extract in our lab, and the structures were confirmed by MS and NMR data.
Plant material Fresh rhizomes of C. aeruginosa were collected from Khaokhor District, Phetchabun Province, Thailand. The specimen (collection number: ganniga001) was stored at the Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, and at the PBM herbarium, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. The plant materials were identified by Professor Wongsatit Chuakul, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.
The 5 mg/ml of C. aeruginosa extract or 0.1 mg/ml of germacrone were prepared in the mixture of PEG-40 and buffer solution (15 : 85 v/v). The resultant solutions were transferred to microcentrifuge tubes and stored at room temperature (25°C) for 14 days. The samples were analyzed at various time intervals. All of samples were kept at −80°C before analyses. Effect of temperature C. aeruginosa extract and germacrone were tested both in solid and solubilized forms. For solid form test, 5 mg of C. aeruginosa extract or 0.1 mg of germacrone were placed into microcentrifuge tubes. For solubilized forms, 5 mg/ml of C. aeruginosa extract or 0.1 mg/ml of germacrone were mixed with PEG-40 and pH 5.5 acetate buffer solution (15 : 85 v/v) and maintained at 4, 25 or 45°C for 0–180 days. The content of sesquiterpene was then measured. Effect of oxygen The solid form of germacrone (5.0 mg) was placed into amber glass vials and prepared in a normal atmosphere and a nitrogen atmosphere; after which the vials were capped and kept at 45°C for 14 days. The chemical degradations of germacrone in both conditions were monitored at days 0, 3, 7 and 14.
Preparation of Curcuma aeruginosa extract
Effect of light
The powdered rhizomes of C. aeruginosa (5 kg) were extracted with hexane (2.5 l) for 3 days at room temperature and filtered. The maceration procedure was repeated three times. The solvent was evaporated under reduced pressure to produce the dried extract (30 g).
A 0.1 mg of either germacrone or 5 mg C. aeruginosa extract in anhydrous or solubilized in 1 ml of PEG-40, and pH 5.5 acetate buffer (15 : 85 v/v) were placed in microcentrifuge tubes. The samples were exposed to direct visible light (400–700 nm) from fluorescence lamp (36W, 220V), 8 hr/day and kept at 25°C for 0, 3 or 7 days and then the amount remaining was then measured.[9]
Stability studies C. aeruginosa extract or germacrone (as a pure compound) were prepared and transferred to microcentrifuge tubes and then maintained under the various conditions (see below). A relative humidity of 75% was achieved by equilibration of the samples with a saturated solution of sodium chloride.[10] Three sample tubes were randomly collected at the various intervals and the amount of remaining of compound(s) of interest were measured. Effect of pH Acid-based degradation studies were carried out to determine the optimal pH stability of C. aeruginosa sesquiterpenes. The samples were dissolved in PEG-40 and diluted with buffer solution to (either pH; 2.0, 5.5, 7.4 and 9.0). The four buffer solutions were composed of hydrochloric/potassium chloride buffer (pH 2.0), acetate buffer (pH 5.5) and phosphate buffers (pH 7.4 and 9.0). 1284
High-Performance Liquid Chromatography and Gas Chromatography with Flame Ionization Detector for quantitative analysis of Curcuma aeruginosa extract Quantitative measurement of chemical constituents of C. aeruginosa extract used both HPLC and gas chromatography (GC) systems. The HPLC (Shimadzu; Kyoto, Japan) was equipped with a SPDM10AVP photodiode array detector, an LC-10ATVP pump, CTO-10AS VP column oven and an injector with a 20 μl loop. A phenomenex Luna 5u C-18column (150 × 4.6 mm) was used as a stationary phase. The flow was isocratic at 1.0 ml/min with acetonitrile and 0.5 mM phosphate buffer pH 3.0 (73 : 27 v/v). The detector was set at 214 nm. GC analysis was performed on a Hewlett Packard (Agilent Technologies, Palo Alto, CA, USA) model6890 gas chromatograph equipped with a flame ionization detector
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(GC-FID). A fused silica capillary Hewlett Packard HP-5 (5% phenyl methyl siloxane) column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was used for the GC separation. High purity helium was used as carrier gas with flow rate 1.0 ml/min. The injector was set at 150°C in splitless mode (1.0 μl). The FID detection was set at 250°C. The identification of the components was based on comparing their retention times with those for reference compounds under the same condition. The following GC temperature programs were applied. GC-FID system 1: the initial temperature was set at 50°C and held for 2 min, then the temperature increased at a rate of 10°C/min up to150°C. Then, the rate was decreased to 2°C/min until 190°C followed by15°C/min to 250°C and held for 7 min. GC-FID system 2: the column temperature program was set at 100°C and held for 2 min, then programmed at 15°C/ min to 160°, then at 3°C/min to180°C and finally held for 4 min at 180°C.
Statistical analyses The Kruskal–Wallis test was used for comparing more than two samples. If it indicated significant effect, Nemenyi’s test was performed to compare the difference of each pair. In addition, the responses at various times were compared with those at day 0 using Mann–Whitney U-test. A significance level of P ≤ 0.05 denoted significance in all cases.
Isolation and identification of degradation products The degradation product (1) of germacrone after storage at 25°C for 6 months was characterized using GC-MS and
Figure 2
Stability studies of Curcuma aeruginosa
comparison the data with the literatures. The column, flow rate and injection volume were as described above. The column temperature was set at 100°C and held for 2 min, then programmed at 15°C/min to 160°C, after that, increased for 3°C/min to 180°C at which it was held for 10 min and finally increased at 10°C/min to 250°C and held for 5 min. The temperature of the ion source was set at 250°C with ionization mode electron impact ionization (EI). The scanning mass range was 50–800 amu. Two degradation products of germacrone appeared after 7 and 14 days at 45°C under nitrogen were investigated. The samples (15.0 mg) were pooled and further fractionated on a silica gel column using gradient elution of hexane and ethyl acetate to yield 2 (3.4 mg) and 3 (6.1 mg). The structures of these degradation products were elucidated using spectroscopic methods. Elemenone (1): oil; EI-MS: m/z = 218 [M]+; 203(6), 186(8), 175(20), 161(5), 147(11), 135(75), 121(30), 107(100), 91(38), 79(24), 67(58), 53(22). Germacrone-1,10-epoxide (2): colorless crystals; infrared spectroscopy (IR) (KBr) ν (cm−1): 1658 (C = O stretching), EI-MS: m/z 234 [M]+, 219(7), 149(47), 135(60), 121(57), 107(100), 91(41), 67(59). 1H NMR (CDCl3, 400 MHz) δ ppm: 5.02 (1H, br d, J = 8.4 Hz, H-5), 3.06 (1H, br t, J = 12.4 Hz, H-6a), 2.97 (1H, d, J = 10.9 Hz, H-9a), 2.85 (1H, br d, J = 11.2 Hz, H-6b), 2.71 (1H, d, J = 10.6 Hz, H-1), 2.48 (1H, d, J = 10.9 Hz, H-9b), 2.20 (1H, overlapping, H-3a), 1.99 (1H, br d, J = 9.6 Hz, H-3b), 1.76 (3H, s, H-15), 1.66 (3H, s, H-13), 1.54 (3H, s, H-12), 1.41 (2H, m, H-2) and 1.26 (3H, s, H-14). Germacrone-4,5-epoxide (3): colorless crystals; IR (KBr) ν (cm−1): 1677 (C = O stretching), EI-MS: m/z 234 [M]+,
The high-performance liquid chromatography fingerprint of 1.0 mg/ml C. aeruginosa extract. The peak of germacrone is detected at 9.1 min.
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167(61), 121(39), 67 (64), 53(22). 1H NMR (CDCl3, 400 MHz) δ ppm: 5.18 (1H, br d, J = 8.6 Hz, H-1), 3.44 (1H, br s, H-9b), 3.04 (1H, br s, H-9a), 2.87 (1H, dd, J = 13.7, 2.1 Hz, H-6a), 2.42 (1H, dd, J = 11.0, 2.1 Hz, H-5), 2.30 (1H, m, H-2a), 2.23 (1H, m, H-2b), 2.13 (1H, dd, J = 12.9, 6.6 Hz, H-3a), 2.05 (1H, dd, J = 13.7, 11.0 Hz, H-6b), 1.82 (3H, s, H-12), 1.81 (3H, s, H-13), 1.72 (3H, s, H-15),1.14 (1H, m, H-3b), 1.03 (3H, s, H-14); 13C NMR (CDCl3, 100 MHz): δ 204.9 (C, C-8), 134.5 (C, C-11), 133.0 (C, C-7), 129.9 (CH, C-1), 126.7 (C, C-10), 65.0 (CH, C-5), 61.0 (C, C-4), 55.7 (CH2, C-9), 37.8 (CH2, C-3), 29.8 (CH2, C-6), 24.8 (CH2, C-2), 22.9 (CH3, C-12), 20.6 (CH3, C-13), 17.2 (CH3, C-15) and 16.0 (CH3, C-14).
Results and Discussion The HPLC and GC-FID methods were used to assess chemical stability. A HPLC fingerprint of a C. aeruginosa extract showed a well separated and prominent peak for germacrone (Figure 2). However, other components of the extract were not well separated so we developed a GC-FID detection method (named ‘GC-FID system 1’). It was able to resolve the sesquiterpenes in C. aeruginosa. Notwithstanding this, only germacrone, zederone, curcumenol and isocurcumenol could be detected because dehydrocurdione and zedoarondiol were degraded at the higher temperatures used in this protocol (Figure 3a). When germacrone was
Figure 3 The gas chromatography chromatogram of (a) 3.0 mg/ml C. aeruginosa extract analyzed using gas chromatography with flame ionization detector system 1 and (b) 0.5 mg/ml germacrone analyzed using gas chromatography with flame ionization detector system 2 (i = isocurcumenol, g = germacrone, c = curcumenol, z = zederone).
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studied as a pure compound, a more rapid detection method was developed as the ‘GC-FID system 2’ where the peak appeared at 13 min instead of 22 min (Figure 3b).
Effect of pH The extract and germacrone were solubilized and then incubated in various pH buffer solutions (pH 2.0, 5.5, 7.4 and 9.0) at 25 ± 2°C. The remaining germacrone contents were measured by HPLC after various incubation times (Figure 2). In general, there was little evidence of any degradation of germacrone at any pH up to 7 days whether in an extract (Figure 4a) or the pure compound (Figure 4b). After 14 days, there was some suggestion of degradation but there
Stability studies of Curcuma aeruginosa
was no clear pH dependency. The data also showed no significant difference between germacrone in an extract and pure germacrone. As cosmetics are commonly formulated to have a pH of around 5.5, this value was used in subsequent determinations.
Temperature dependent degradation Germacrone was stored either as a dried C. aeruginosa extract (Figure 5a) or as a pure solid compound (Figure 5b) for various periods up to 180 days at various temperatures (4, 25 and 45°C). As might be expected, degradation was most rapid at 45°C. It was completely lost after only 3 days when in the solid pure form (Figure 5b), but the deg-
Figure 4 The effect of various pH condition on the stability of germacrone in (a) C. aeruginosa extract and (b) as pure compound analyzed by high-performance liquid chromatography. (#P ≤ 0.05 against data of pH 7.4 at the same day).
Figure 5 The effect of temperature on the stability of germacrone in solid form of (a) C. aeruginosa extract and (b) as pure compound analyzed by gas chromatography with flame ionization detector (*P < 0.05 against data of day 0,# and ##P ≤ 0.05 against data of 4°C and 25°C at the same day, respectively). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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Figure 6 The (a) gas chromatography chromatogram of the pure germacrone sample after degradation at 25°C for 180 days and (b) electron impact ionization-mass spectrometry spectrum of the 11.67 min gas chromatography peak.
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The proposed degradation products of germacrone.
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radation was slower when present in the dried extract (Figure 5a). This difference between the pure and extract forms was also seen at the higher temperatures (25 and 45°C). This suggests that other components of the extract might provide protection against germacrone degradation. Correspondingly, additional chromatogram peaks appeared in both pure and extract forms after storage and these were studied further to identify possible degradation products.
Identification of temperature degradation products The peaks of unknown compounds were observed in the GC chromatograms after keeping germacrone for the period of time at 4, 25 and 45°C. These germacrone degra-
Figure 8 Thin layer chromatography fingerprints of purified germacrone which prepared in nitrogen atmosphere following maintenance at 45°C for 0, 3, 7 and 14 days. The compounds were visualized by either (a) 254 nm light, or (b) colorizing with reaction with anisaldehyde reagent. Hexane : EtOAc (95 : 5 v/v) was the mobile phase. The dashed line shows the fractions which were recombined into a single sample for further study.
Stability studies of Curcuma aeruginosa
dation products were determined by GC-MS and an example record is shown in Figure 6. The peak of degradation compound 1, was observed at the retention time of 11.67 min. EI-MS spectrum of 1 shows m/z 218 [M]+, which equals the molecular weight of germacrone but the fragmentation pattern was different[9] (see experimental section). This result suggests that germacrone might be rearranged possibly through a Cope rearrangement (Figure 7a). An idea supported by reports of Reichardt et al. (1989)[11] and Yang et al. (2007).[12] Even though Cope rearrangement is normally activated by heat, it can also occur at room temperature.[13] Even though the amount of
Figure 10 The stability of germacrone (as a pure compound) in solution with either 15% PEG-40/buffer or 100% MeOH at 45°C. Germacrone was measured by gas chromatography with flame ionization detector. There were no statistical differences between both solution conditions.
Figure 9 Degradation of solubilized germacrone at 4, 25 and 45°C either (a) in the crude extract of C. aeruginosa or (b) as the pure compound. Remaining germacrone was measured by gas chromatography with flame ionization detector. (*P < 0.05 against data of day 0 and #P ≤ 0.05 against data of 4°C). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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spectrum 1 was not enough for the full identification by NMR, spectrum 1 was identified as elemenone by comparison of MS spectrum with the report of Zhou et al. (2007).[14] Oxygen may mediate the germacrone degradation and to test this, purified germacrone was maintained at 45°C under nitrogen gas. Germacrone remained stable over 3 days, whereas its complete degradation was observed when germacrone was kept under air. However, further heating under nitrogen for another 4 days converted the germacrone into a yellow oily substance. At least three degradation products at days 7 and 14 were detected on TLC
analysis (Figure 8). These products were subsequently isolated using silica column chromatography. Two degradation compounds were identified as germacrone-1,10-epoxide (2)[15] and germacrone-4,5-epoxide (3, Figure 7)[16] by using 1D- and 2D-NMR. Two double bonds at positions C1–10 and C4–5 in the 10-membered ring had been oxidized to epoxide rings presumably by traces of molecular oxygen attacking sensitive endocyclic double bonds (Figure 7). Since both of these compounds are also crystalline at room temperature, the formation of an oily intermediate after 3 days suggests that the formation of these epoxides was via intermediates.
Figure 11 Degradation of zederone, curcumenol and isocurcumenol in solid form (left) and solubilized form (right) of C. aeruginosa extract analyzed 4°C 25°C 45°C, *P < 0.05 against data of day 0 and #P ≤ 0.05 against data of 4°C). by gas chromatography with flame ionization detector (
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When solubilized in 15% PEG-40/buffer solution, germacrone degradation was much slower (Figure 9) than in solid forms (Figure 5). Furthermore, the degradation rates were rather similar whether germacrone was pure or not at all temperatures. The improvement of stability of germacrone in solution form might be due to the fact that germacrone in solution form was exposed to O2 less than that in solid form or PEG-40 hydrogenated castor oil could protect germacrone from the degradation reaction by forming micelles. To test this, germacrone was also prepared in methanol and kept at 45°C for 14 days. The results showed that the degradation rate was similar to that in PEG-40 (Figure 10). Therefore, the micelle formation should not be the cause to retard the degradation of gemacrone in solution form.
Degradation of other sesquiterpene components in Curcuma aeruginosa extract Temperature stability C. aeruginosa extract,
of other zederone,
sesquiterpenes in curcumenol and
isocurcumenol was evaluated using the same GC-FID condition as that for germacrone (Figure 11). It is noted that in solid form, germacrane type sesquiterpenes (germacrone and zederone) significantly degraded more quickly than that of guaiane type sesquiterpenes (curcumenol and isocurcumenol). The endocyclic double bonds in cyclodecadiene system and epoxide ring of germacrane molecules might play an important role in the stability of the compounds. The flexible cyclodeca-1(10) and −4(5)diene ring systems are believed to be important intermediates in biosynthesis of several classes of sesquiterpenes including guaiane type.[17–19] The thermally isomerization of molecules might also be the reason for the instability of these compounds. Moreover, the exocyclic double bond at C7, which found in all molecules studied should not be ignored. It might also play the pivotal role for the instability of these sesquiterpenes. However, other factors might involve in the degradation of these compounds which need further investigation. When comparing the degradation profiles of these sesquiterpenes in solid and solubilized extracts, zederone
Figure 12 The effect of light on the stability of germacrone in solid form(left) and solubilized form (right) of (a) C. aeruginosa extract and (b) pure compound stored at 25°C. The remaining of germacrone were analyzed by gas chromatography with flame ionization detector ( dark light, #P ≤ 0.05 against data of dark at the same day). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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Figure 13 The effect of light on the stability of zederone, curcumenol and isocurcumenol in solid form (left) and solubilized form (right) of C. aeruginosa extract stored at 25°C. The remaining of marker compounds was analyzed by gas chromatography with flame ionization detector. dark light). There were no statistical differences observed between light and dark conditions. (
was more stable in solubilized form, which was similar to those we observed in germacrone (Figures 5a and 9a) while no difference between both forms was found in curcumenol and isocurcumenol (Figure 11).
The light sensitivity of zederone, curcumenol or isocurcumenol in C. aeruginosa extract were also studied (Figure 13a–13c). Light did not seem to affect the stability of these compounds in solid and solubilized form of C. aeruginosa extract during 7 days of exposure.
Effect of light Exposure to 7 days of diurnal room lighting degraded germacrone in dried C. aeruginosa extract by ∼40% while that maintained in the dark was unaffected. However, solubilized C. aeruginosa extract was apparently unaffected by light (Figure 12a). For pure germacrone, light did not change the stability profile both in solid and solubilized form (Figure 12b). 1292
Conclusion The antiandrogenic compound, germacrone, as the solubilized pure compound or as a constituent in an extract of C. aeruginosa was stable at pH 2.0–9.0 for at least 14 days. Germacrone in solid form degraded by >40% after 1 month of storage at 25 or 45°C. The major temperature degradation products were germacrone-1,10-epoxide and
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germacrone-4,5-expoxide. It is noted that the degradation was greatly slowed as PEG-40 suspension or in methanol or when stored under a N2 atmosphere. In contrast, the less potent antiandrogenic sesquiterpenes, zederone, curcumenol and isocurcumenol, were far more stable. The further investigation on the antiandrogenic profile of degradation products might be interesting.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
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Acknowledgements and Funding This work was supported by the grant under the programme Strategic Scholarships for Frontier Research Network for the PhD Program Thai Doctoral degree, the Center of Excellence for Innovation in Chemistry (PERCHCIC), Office of the Higher Education Commission, Ministry of Education, the National Research Council of Thailand (NRCT) and Naresuan University, Thailand. We thank Dr C. Norman Scholfield, Faculty of Pharmaceutical Sciences and Dr Katechan Jampachaisri, Faculty of Science, Naresuan University for helping with the manuscript preparation.
8. Pumthong G et al. Curcuma aeruginosa, a novel botanically derived 5alpha-reductase inhibitor in the treatment of male-pattern baldness: a multicenter, randomized, doubleblind, placebo-controlled study. J Dermatol Treat 2011; 23: 385–392. 9. Suphrom N et al. Anti-androgenic effect of sesquiterpenes isolated from the rhizomes of Curcuma aeruginosa Roxb. Fitoterapia 2012; 83: 864–871. 10. Jin P et al. The solution and solid state stability and excipient compatibility of parthenolide in feverfew. AAPS PharmSciTech 2007; 8: 200–205. 11. Reichardt PB et al. Thermal instability of germacrone: implications for gas chromatographic analysis of thermally unstable analytes. Can J Chem 1989; 67: 1174–1177. 12. Yang FQ et al. Optimization of GC–MS conditions based on resolution and stability of analytes for simultaneous determination of nine sesquiterpenoids in three species of Curcuma rhizomes. J Pharm Biomed Anal 2007; 43: 73–82. 13. Adio AM. (−)-trans-β-Elemene and related compounds: occurrence, synthesis, and anticancer activity. Tetrahedron 2009; 65: 5145–5159.
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14. Zhou X et al. Analysis of volatile components of Curcuma sichuanensis X. X. Chen by gas chromatography-mass spectrometry. J Pharm Biomed Anal 2007; 43: 440–444. 15. Pomkeua S. Chemicals Constituents from the Rhizomes of Curcuma Zedoaria (Christm.) Rosc. and the Stems of Citrus Medica Linn. Songkla, Thailand: Prince of Songkla University, 2010. 16. Yoshihara M et al. The absolute stereostructure of (4S,5S)-(+)germacrone 4,5-epoxide from zedoariae rhizoma cultivated in Yukushima island. Chem Pharm Bull 1984; 32: 2059–2062. 17. de Kraker J-W et al. (+)-Germacrene A biosynthesis. Plant Physiol 1998; 117: 1381–1392. 18. Adio AM. Germacrenes A–E and related compounds: thermal, photochemical and acid induced transannular cyclizations. Tetrahedron 2009; 65: 1533–1552. 19. Faraldos JA et al. Conformational analysis of (+)-germacrene A by variable-temperature NMR and NOE spectroscopy. Tetrahedron 2007; 63: 7733–7742.
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Research Paper
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Stability studies of antiandrogenic compounds in Curcuma aeruginosa Roxb. extract Nungruthai Suphroma, Jukkarin Srivilaia, Ganniga Pumthongb, Nantaka Khoranac, Neti Waranuchb, Nanteetip Limpeanchobd and Kornkanok Ingkaninana a Bioscreening Unit, Department of Pharmaceutical Chemistry and Pharmacognosy, bDepartment of Pharmaceutical Technology, cDepartment of Pharmaceutical Chemistry and Pharmacognosy, dDepartment of Pharmacy Practice, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok, Thailand
Keywords antiandrogen; Curcuma aeruginosa; degradation; germacrone; stability Correspondence Kornkanok Ingkaninan, Bioscreening Unit, Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand. E-mail: [email protected] Received June 28, 2013 Accepted December 7, 2013 doi: 10.1111/jphp.12216
Abstract Objectives Curcuma aeruginosa Roxb. extract is a 5α-reductase antagonist that can be used to treat hair loss. We aimed to study the stability of antiandrogenic constituents, germacrone and other sesquiterpene components in the extract. Methods Germacrone and the extract were analyzed as solid forms or solublized with polyethylene glycol-40 (PEG-40) or methanol using high-performance liquid chromatography and gas chromatography with flame ionization detector. The effects of pH, temperature and light on their stability were studied. Key findings Degradation of antiandrogenic compounds in C. aeruginosa was highly sensitive to temperature especially pure anhydrous germacrone, which was completely lost within 3 days at 45°C. Curiously, degradation was slower than as a dried extract. Paradoxically, when solubilized with PEG-40, it was largely intact even after 90 days at 45°C. The MS spectrum of a major degradation product suggested that it was elemenone probably produced by Cope rearrangement. Two other putative degradation products were germacrone-1,10-epoxide and germacrone-4,5-epoxide suggesting that oxidation of double bonds was an important mechanism. Germacrone stability was unaffected by pH (2.0–9.0) but only as dried extract it was slightly degraded by light. Conclusion Antiandrogenic constituents of C. aeruginosa were instable at high temperature and in solid form. Thus, the extract would be optimately stored as a solution or otherwise as solid form at low temperature.
Introduction Curcuma aeruginosa Roxb. (Zingiberaceae) is a native plant of tropical areas and is commonly known as Wanmahamek in Thailand. It is a perennial with oblong tuberous roots, and the fresh rhizome emits a ginger-like aroma. Leafy shoots are 45–60 cm high and the plant blooms during the rainy season.[1–3] In Thai traditional medicine, a tincture of the rhizome is used to treat uterine pain, uterine inflammation, postpartum uterine and perimenopausal bleeding.[4] Various pharmacological effects of this plant have been delineated such as postcoital contraception, anti-HIV actions, hepatoprotection, antimicrobial effects, antioxidation, reduce platelet activation and antinociception.[1,5–7] Recently, our group found that a hexane extract of C. aeruginosa rhizomes showed high antiandrogenic activity both in vitro and in vivo. It was shown to be an effective ingredient in a hair tonic for androgenic alopecia.[8] Six 1282
sesquiterpenes, that is, germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol and isocurcumenol were isolated from C. aeruginosa extract (Figure 1), and these were shown to inhibit the conversion of testosterone to dihydrotestosterone. Of the six components, germacrone was the most active. Our studies demonstrated that germacrone also showed an antiandrogenic action on testosterone-induced growth suppression of human prostate cancer cells [Lymph Node Carcinoma of the prostate (LNCaP)] and on the hamster flank gland model. A possible mechanisms was blockade of 5α-reductase activity whereas no binding of germacrone to androgen receptors could be detected.[9] Thus, germacrone has potential applications in the treatment of androgen-dependent disorders. But its success as a cosmetic or health product depends on its chemical stability. Therefore, the present study was
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Chemical structures of germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol, and isocurcumenol.
undertaken to assess its stability as a pure compound or in an extract of C. aeruginosa along with other active ingredients. In particular, we assessed their chemical stability to pH, temperature, oxygen and light.
Materials and Methods General experimental procedures Thin layer chromatography (TLC) analysis was performed on silica gel 60 F254 TLC on an aluminum sheet (20 × 20 cm; Merck, Darmstadt, Germany). A silica gel
column (0.040–0.063 mm granule size; Merck) was used for the chromatographic isolation of degradation products. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV400 (Bruker, Billerica, MA, USA) spectrometer at 400 MHz for proton and 100 MHz for carbon with tetramethylsilane as the internal standard. Mass spectrometry (MS) spectra were obtained using electron impact ionization mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Fourier transform infrared spectroscopy spectra were recorded on a Spectrum One spectrometer (Perkin Elmer, Shelton, CT, USA).
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Chemicals Methanol, acetonitrile [high-performance liquid chromatography (HPLC) grade], hexane and ethyl acetate were bought from RCI Labscan Ltd (Bangkok, Thailand). Polyethylene glycol-40 (PEG-40) hydrogenated castor oil was purchased from Sigma-Aldrich (St. Louis, MO, USA). Orthophosphoric acid and disodium hydrogen phosphate were purchased from BDH (Poole, UK). Hydrochloric acid, potassium chloride, potassium hydroxide, acetic acid and sodium acetate were purchased from Merck. The reference compounds, germacrone, zederone, dehydrocurdione, curcumenol, zedoarondiol and isocurcumenol were also isolated from C. aeruginosa extract in our lab, and the structures were confirmed by MS and NMR data.
Plant material Fresh rhizomes of C. aeruginosa were collected from Khaokhor District, Phetchabun Province, Thailand. The specimen (collection number: ganniga001) was stored at the Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, and at the PBM herbarium, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. The plant materials were identified by Professor Wongsatit Chuakul, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.
The 5 mg/ml of C. aeruginosa extract or 0.1 mg/ml of germacrone were prepared in the mixture of PEG-40 and buffer solution (15 : 85 v/v). The resultant solutions were transferred to microcentrifuge tubes and stored at room temperature (25°C) for 14 days. The samples were analyzed at various time intervals. All of samples were kept at −80°C before analyses. Effect of temperature C. aeruginosa extract and germacrone were tested both in solid and solubilized forms. For solid form test, 5 mg of C. aeruginosa extract or 0.1 mg of germacrone were placed into microcentrifuge tubes. For solubilized forms, 5 mg/ml of C. aeruginosa extract or 0.1 mg/ml of germacrone were mixed with PEG-40 and pH 5.5 acetate buffer solution (15 : 85 v/v) and maintained at 4, 25 or 45°C for 0–180 days. The content of sesquiterpene was then measured. Effect of oxygen The solid form of germacrone (5.0 mg) was placed into amber glass vials and prepared in a normal atmosphere and a nitrogen atmosphere; after which the vials were capped and kept at 45°C for 14 days. The chemical degradations of germacrone in both conditions were monitored at days 0, 3, 7 and 14.
Preparation of Curcuma aeruginosa extract
Effect of light
The powdered rhizomes of C. aeruginosa (5 kg) were extracted with hexane (2.5 l) for 3 days at room temperature and filtered. The maceration procedure was repeated three times. The solvent was evaporated under reduced pressure to produce the dried extract (30 g).
A 0.1 mg of either germacrone or 5 mg C. aeruginosa extract in anhydrous or solubilized in 1 ml of PEG-40, and pH 5.5 acetate buffer (15 : 85 v/v) were placed in microcentrifuge tubes. The samples were exposed to direct visible light (400–700 nm) from fluorescence lamp (36W, 220V), 8 hr/day and kept at 25°C for 0, 3 or 7 days and then the amount remaining was then measured.[9]
Stability studies C. aeruginosa extract or germacrone (as a pure compound) were prepared and transferred to microcentrifuge tubes and then maintained under the various conditions (see below). A relative humidity of 75% was achieved by equilibration of the samples with a saturated solution of sodium chloride.[10] Three sample tubes were randomly collected at the various intervals and the amount of remaining of compound(s) of interest were measured. Effect of pH Acid-based degradation studies were carried out to determine the optimal pH stability of C. aeruginosa sesquiterpenes. The samples were dissolved in PEG-40 and diluted with buffer solution to (either pH; 2.0, 5.5, 7.4 and 9.0). The four buffer solutions were composed of hydrochloric/potassium chloride buffer (pH 2.0), acetate buffer (pH 5.5) and phosphate buffers (pH 7.4 and 9.0). 1284
High-Performance Liquid Chromatography and Gas Chromatography with Flame Ionization Detector for quantitative analysis of Curcuma aeruginosa extract Quantitative measurement of chemical constituents of C. aeruginosa extract used both HPLC and gas chromatography (GC) systems. The HPLC (Shimadzu; Kyoto, Japan) was equipped with a SPDM10AVP photodiode array detector, an LC-10ATVP pump, CTO-10AS VP column oven and an injector with a 20 μl loop. A phenomenex Luna 5u C-18column (150 × 4.6 mm) was used as a stationary phase. The flow was isocratic at 1.0 ml/min with acetonitrile and 0.5 mM phosphate buffer pH 3.0 (73 : 27 v/v). The detector was set at 214 nm. GC analysis was performed on a Hewlett Packard (Agilent Technologies, Palo Alto, CA, USA) model6890 gas chromatograph equipped with a flame ionization detector
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(GC-FID). A fused silica capillary Hewlett Packard HP-5 (5% phenyl methyl siloxane) column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was used for the GC separation. High purity helium was used as carrier gas with flow rate 1.0 ml/min. The injector was set at 150°C in splitless mode (1.0 μl). The FID detection was set at 250°C. The identification of the components was based on comparing their retention times with those for reference compounds under the same condition. The following GC temperature programs were applied. GC-FID system 1: the initial temperature was set at 50°C and held for 2 min, then the temperature increased at a rate of 10°C/min up to150°C. Then, the rate was decreased to 2°C/min until 190°C followed by15°C/min to 250°C and held for 7 min. GC-FID system 2: the column temperature program was set at 100°C and held for 2 min, then programmed at 15°C/ min to 160°, then at 3°C/min to180°C and finally held for 4 min at 180°C.
Statistical analyses The Kruskal–Wallis test was used for comparing more than two samples. If it indicated significant effect, Nemenyi’s test was performed to compare the difference of each pair. In addition, the responses at various times were compared with those at day 0 using Mann–Whitney U-test. A significance level of P ≤ 0.05 denoted significance in all cases.
Isolation and identification of degradation products The degradation product (1) of germacrone after storage at 25°C for 6 months was characterized using GC-MS and
Figure 2
Stability studies of Curcuma aeruginosa
comparison the data with the literatures. The column, flow rate and injection volume were as described above. The column temperature was set at 100°C and held for 2 min, then programmed at 15°C/min to 160°C, after that, increased for 3°C/min to 180°C at which it was held for 10 min and finally increased at 10°C/min to 250°C and held for 5 min. The temperature of the ion source was set at 250°C with ionization mode electron impact ionization (EI). The scanning mass range was 50–800 amu. Two degradation products of germacrone appeared after 7 and 14 days at 45°C under nitrogen were investigated. The samples (15.0 mg) were pooled and further fractionated on a silica gel column using gradient elution of hexane and ethyl acetate to yield 2 (3.4 mg) and 3 (6.1 mg). The structures of these degradation products were elucidated using spectroscopic methods. Elemenone (1): oil; EI-MS: m/z = 218 [M]+; 203(6), 186(8), 175(20), 161(5), 147(11), 135(75), 121(30), 107(100), 91(38), 79(24), 67(58), 53(22). Germacrone-1,10-epoxide (2): colorless crystals; infrared spectroscopy (IR) (KBr) ν (cm−1): 1658 (C = O stretching), EI-MS: m/z 234 [M]+, 219(7), 149(47), 135(60), 121(57), 107(100), 91(41), 67(59). 1H NMR (CDCl3, 400 MHz) δ ppm: 5.02 (1H, br d, J = 8.4 Hz, H-5), 3.06 (1H, br t, J = 12.4 Hz, H-6a), 2.97 (1H, d, J = 10.9 Hz, H-9a), 2.85 (1H, br d, J = 11.2 Hz, H-6b), 2.71 (1H, d, J = 10.6 Hz, H-1), 2.48 (1H, d, J = 10.9 Hz, H-9b), 2.20 (1H, overlapping, H-3a), 1.99 (1H, br d, J = 9.6 Hz, H-3b), 1.76 (3H, s, H-15), 1.66 (3H, s, H-13), 1.54 (3H, s, H-12), 1.41 (2H, m, H-2) and 1.26 (3H, s, H-14). Germacrone-4,5-epoxide (3): colorless crystals; IR (KBr) ν (cm−1): 1677 (C = O stretching), EI-MS: m/z 234 [M]+,
The high-performance liquid chromatography fingerprint of 1.0 mg/ml C. aeruginosa extract. The peak of germacrone is detected at 9.1 min.
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167(61), 121(39), 67 (64), 53(22). 1H NMR (CDCl3, 400 MHz) δ ppm: 5.18 (1H, br d, J = 8.6 Hz, H-1), 3.44 (1H, br s, H-9b), 3.04 (1H, br s, H-9a), 2.87 (1H, dd, J = 13.7, 2.1 Hz, H-6a), 2.42 (1H, dd, J = 11.0, 2.1 Hz, H-5), 2.30 (1H, m, H-2a), 2.23 (1H, m, H-2b), 2.13 (1H, dd, J = 12.9, 6.6 Hz, H-3a), 2.05 (1H, dd, J = 13.7, 11.0 Hz, H-6b), 1.82 (3H, s, H-12), 1.81 (3H, s, H-13), 1.72 (3H, s, H-15),1.14 (1H, m, H-3b), 1.03 (3H, s, H-14); 13C NMR (CDCl3, 100 MHz): δ 204.9 (C, C-8), 134.5 (C, C-11), 133.0 (C, C-7), 129.9 (CH, C-1), 126.7 (C, C-10), 65.0 (CH, C-5), 61.0 (C, C-4), 55.7 (CH2, C-9), 37.8 (CH2, C-3), 29.8 (CH2, C-6), 24.8 (CH2, C-2), 22.9 (CH3, C-12), 20.6 (CH3, C-13), 17.2 (CH3, C-15) and 16.0 (CH3, C-14).
Results and Discussion The HPLC and GC-FID methods were used to assess chemical stability. A HPLC fingerprint of a C. aeruginosa extract showed a well separated and prominent peak for germacrone (Figure 2). However, other components of the extract were not well separated so we developed a GC-FID detection method (named ‘GC-FID system 1’). It was able to resolve the sesquiterpenes in C. aeruginosa. Notwithstanding this, only germacrone, zederone, curcumenol and isocurcumenol could be detected because dehydrocurdione and zedoarondiol were degraded at the higher temperatures used in this protocol (Figure 3a). When germacrone was
Figure 3 The gas chromatography chromatogram of (a) 3.0 mg/ml C. aeruginosa extract analyzed using gas chromatography with flame ionization detector system 1 and (b) 0.5 mg/ml germacrone analyzed using gas chromatography with flame ionization detector system 2 (i = isocurcumenol, g = germacrone, c = curcumenol, z = zederone).
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studied as a pure compound, a more rapid detection method was developed as the ‘GC-FID system 2’ where the peak appeared at 13 min instead of 22 min (Figure 3b).
Effect of pH The extract and germacrone were solubilized and then incubated in various pH buffer solutions (pH 2.0, 5.5, 7.4 and 9.0) at 25 ± 2°C. The remaining germacrone contents were measured by HPLC after various incubation times (Figure 2). In general, there was little evidence of any degradation of germacrone at any pH up to 7 days whether in an extract (Figure 4a) or the pure compound (Figure 4b). After 14 days, there was some suggestion of degradation but there
Stability studies of Curcuma aeruginosa
was no clear pH dependency. The data also showed no significant difference between germacrone in an extract and pure germacrone. As cosmetics are commonly formulated to have a pH of around 5.5, this value was used in subsequent determinations.
Temperature dependent degradation Germacrone was stored either as a dried C. aeruginosa extract (Figure 5a) or as a pure solid compound (Figure 5b) for various periods up to 180 days at various temperatures (4, 25 and 45°C). As might be expected, degradation was most rapid at 45°C. It was completely lost after only 3 days when in the solid pure form (Figure 5b), but the deg-
Figure 4 The effect of various pH condition on the stability of germacrone in (a) C. aeruginosa extract and (b) as pure compound analyzed by high-performance liquid chromatography. (#P ≤ 0.05 against data of pH 7.4 at the same day).
Figure 5 The effect of temperature on the stability of germacrone in solid form of (a) C. aeruginosa extract and (b) as pure compound analyzed by gas chromatography with flame ionization detector (*P < 0.05 against data of day 0,# and ##P ≤ 0.05 against data of 4°C and 25°C at the same day, respectively). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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Figure 6 The (a) gas chromatography chromatogram of the pure germacrone sample after degradation at 25°C for 180 days and (b) electron impact ionization-mass spectrometry spectrum of the 11.67 min gas chromatography peak.
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The proposed degradation products of germacrone.
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radation was slower when present in the dried extract (Figure 5a). This difference between the pure and extract forms was also seen at the higher temperatures (25 and 45°C). This suggests that other components of the extract might provide protection against germacrone degradation. Correspondingly, additional chromatogram peaks appeared in both pure and extract forms after storage and these were studied further to identify possible degradation products.
Identification of temperature degradation products The peaks of unknown compounds were observed in the GC chromatograms after keeping germacrone for the period of time at 4, 25 and 45°C. These germacrone degra-
Figure 8 Thin layer chromatography fingerprints of purified germacrone which prepared in nitrogen atmosphere following maintenance at 45°C for 0, 3, 7 and 14 days. The compounds were visualized by either (a) 254 nm light, or (b) colorizing with reaction with anisaldehyde reagent. Hexane : EtOAc (95 : 5 v/v) was the mobile phase. The dashed line shows the fractions which were recombined into a single sample for further study.
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dation products were determined by GC-MS and an example record is shown in Figure 6. The peak of degradation compound 1, was observed at the retention time of 11.67 min. EI-MS spectrum of 1 shows m/z 218 [M]+, which equals the molecular weight of germacrone but the fragmentation pattern was different[9] (see experimental section). This result suggests that germacrone might be rearranged possibly through a Cope rearrangement (Figure 7a). An idea supported by reports of Reichardt et al. (1989)[11] and Yang et al. (2007).[12] Even though Cope rearrangement is normally activated by heat, it can also occur at room temperature.[13] Even though the amount of
Figure 10 The stability of germacrone (as a pure compound) in solution with either 15% PEG-40/buffer or 100% MeOH at 45°C. Germacrone was measured by gas chromatography with flame ionization detector. There were no statistical differences between both solution conditions.
Figure 9 Degradation of solubilized germacrone at 4, 25 and 45°C either (a) in the crude extract of C. aeruginosa or (b) as the pure compound. Remaining germacrone was measured by gas chromatography with flame ionization detector. (*P < 0.05 against data of day 0 and #P ≤ 0.05 against data of 4°C). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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spectrum 1 was not enough for the full identification by NMR, spectrum 1 was identified as elemenone by comparison of MS spectrum with the report of Zhou et al. (2007).[14] Oxygen may mediate the germacrone degradation and to test this, purified germacrone was maintained at 45°C under nitrogen gas. Germacrone remained stable over 3 days, whereas its complete degradation was observed when germacrone was kept under air. However, further heating under nitrogen for another 4 days converted the germacrone into a yellow oily substance. At least three degradation products at days 7 and 14 were detected on TLC
analysis (Figure 8). These products were subsequently isolated using silica column chromatography. Two degradation compounds were identified as germacrone-1,10-epoxide (2)[15] and germacrone-4,5-epoxide (3, Figure 7)[16] by using 1D- and 2D-NMR. Two double bonds at positions C1–10 and C4–5 in the 10-membered ring had been oxidized to epoxide rings presumably by traces of molecular oxygen attacking sensitive endocyclic double bonds (Figure 7). Since both of these compounds are also crystalline at room temperature, the formation of an oily intermediate after 3 days suggests that the formation of these epoxides was via intermediates.
Figure 11 Degradation of zederone, curcumenol and isocurcumenol in solid form (left) and solubilized form (right) of C. aeruginosa extract analyzed 4°C 25°C 45°C, *P < 0.05 against data of day 0 and #P ≤ 0.05 against data of 4°C). by gas chromatography with flame ionization detector (
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When solubilized in 15% PEG-40/buffer solution, germacrone degradation was much slower (Figure 9) than in solid forms (Figure 5). Furthermore, the degradation rates were rather similar whether germacrone was pure or not at all temperatures. The improvement of stability of germacrone in solution form might be due to the fact that germacrone in solution form was exposed to O2 less than that in solid form or PEG-40 hydrogenated castor oil could protect germacrone from the degradation reaction by forming micelles. To test this, germacrone was also prepared in methanol and kept at 45°C for 14 days. The results showed that the degradation rate was similar to that in PEG-40 (Figure 10). Therefore, the micelle formation should not be the cause to retard the degradation of gemacrone in solution form.
Degradation of other sesquiterpene components in Curcuma aeruginosa extract Temperature stability C. aeruginosa extract,
of other zederone,
sesquiterpenes in curcumenol and
isocurcumenol was evaluated using the same GC-FID condition as that for germacrone (Figure 11). It is noted that in solid form, germacrane type sesquiterpenes (germacrone and zederone) significantly degraded more quickly than that of guaiane type sesquiterpenes (curcumenol and isocurcumenol). The endocyclic double bonds in cyclodecadiene system and epoxide ring of germacrane molecules might play an important role in the stability of the compounds. The flexible cyclodeca-1(10) and −4(5)diene ring systems are believed to be important intermediates in biosynthesis of several classes of sesquiterpenes including guaiane type.[17–19] The thermally isomerization of molecules might also be the reason for the instability of these compounds. Moreover, the exocyclic double bond at C7, which found in all molecules studied should not be ignored. It might also play the pivotal role for the instability of these sesquiterpenes. However, other factors might involve in the degradation of these compounds which need further investigation. When comparing the degradation profiles of these sesquiterpenes in solid and solubilized extracts, zederone
Figure 12 The effect of light on the stability of germacrone in solid form(left) and solubilized form (right) of (a) C. aeruginosa extract and (b) pure compound stored at 25°C. The remaining of germacrone were analyzed by gas chromatography with flame ionization detector ( dark light, #P ≤ 0.05 against data of dark at the same day). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1282–1293
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Figure 13 The effect of light on the stability of zederone, curcumenol and isocurcumenol in solid form (left) and solubilized form (right) of C. aeruginosa extract stored at 25°C. The remaining of marker compounds was analyzed by gas chromatography with flame ionization detector. dark light). There were no statistical differences observed between light and dark conditions. (
was more stable in solubilized form, which was similar to those we observed in germacrone (Figures 5a and 9a) while no difference between both forms was found in curcumenol and isocurcumenol (Figure 11).
The light sensitivity of zederone, curcumenol or isocurcumenol in C. aeruginosa extract were also studied (Figure 13a–13c). Light did not seem to affect the stability of these compounds in solid and solubilized form of C. aeruginosa extract during 7 days of exposure.
Effect of light Exposure to 7 days of diurnal room lighting degraded germacrone in dried C. aeruginosa extract by ∼40% while that maintained in the dark was unaffected. However, solubilized C. aeruginosa extract was apparently unaffected by light (Figure 12a). For pure germacrone, light did not change the stability profile both in solid and solubilized form (Figure 12b). 1292
Conclusion The antiandrogenic compound, germacrone, as the solubilized pure compound or as a constituent in an extract of C. aeruginosa was stable at pH 2.0–9.0 for at least 14 days. Germacrone in solid form degraded by >40% after 1 month of storage at 25 or 45°C. The major temperature degradation products were germacrone-1,10-epoxide and
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germacrone-4,5-expoxide. It is noted that the degradation was greatly slowed as PEG-40 suspension or in methanol or when stored under a N2 atmosphere. In contrast, the less potent antiandrogenic sesquiterpenes, zederone, curcumenol and isocurcumenol, were far more stable. The further investigation on the antiandrogenic profile of degradation products might be interesting.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
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Acknowledgements and Funding This work was supported by the grant under the programme Strategic Scholarships for Frontier Research Network for the PhD Program Thai Doctoral degree, the Center of Excellence for Innovation in Chemistry (PERCHCIC), Office of the Higher Education Commission, Ministry of Education, the National Research Council of Thailand (NRCT) and Naresuan University, Thailand. We thank Dr C. Norman Scholfield, Faculty of Pharmaceutical Sciences and Dr Katechan Jampachaisri, Faculty of Science, Naresuan University for helping with the manuscript preparation.
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