Food & Nutrition Research

 The complete Freund’s adjuvant (CFA) was purchased from Sigma (F5881, Sigma-Aldrich, USA). Diclofenac was purchased from Novartis, Beijing. The nutmeg oil and Gas chromatographymass spectrometry (GCMS) results of nutmeg oil were kindly provided by the Functional Oil Laboratory Associated by Oil Crops Research Institute, Chinese Academy of Agricultural Sciences and Infinite Co., LTD., Guangzhou 510000, China. 2% tween20 was used for emulsification of nutmeg oils and diluted before the application. All other reagents were purchased from Sigma-Aldrich, USA. 

Nutmeg oil used in the study was distilled from dried seeds of Myristica fragrans Houtt (produced in Guangdong, China). Briefly, dried seeds (purchased from Chinese medicine Yinpian factory, Guangzhou medicine company, Guangzhou, China) were pulverized and mixed with water in a ratio of 1 g to 10 mL. Subsequently, the nutmeg oil was obtained by distilling the mixture in steam distillation apparatus for 8 h until no oil drop was seen in distilled fraction. 

Nutmeg oil (0.103 g) was placed in a 10-mL volumetric flask with n-Hexane (chromatographically pure) as a solvent. The solution of nutmeg volatile oil (0.5 mL) was inserted into the GC injector. GCMS analyses were performed using an Agilent 7890 (II) GC coupled to an Agilent 5975 series mass selective detector operating in the electron impact ionization mode at 70 eV with a mass range of 50450 m/z. Volatile compounds were separated using an HP-5MS capillary column (polydimethylsiloxane 5% diphenyl, 30 m0.32 mm0.25 mm, Hewlett Packard). 

The temperature was programmed from 408C (1 min) to 2208C at 58C/min, with a final 1 min hold. 

Helium was used as the carrier gas, with a volumetric flow rate of 1.0 mL/min. For the analyses of volatile oil of nutmeg, split mode was used with a ratio of 1:60 and an injector temperature of 2408C. All samples were analyzed in triplicate. Retention indices (RI) were calculated using an n-alkane series. 

The components were identified by the comparison of their RI relative to C4-C30 n-alkanes (Sigma Chemical Co., St. Louis, MO) that were obtained from the HP-5MS column and compared to the data provided by the NIST mass spectral libraries. Positive identification was assumed when a good match of the mass spectrum and RI was achieved. 

 Young male Wistar rats (190250 g) were acclimatized for 7 days under specific pathogen-free conditions before the initiation of experiments. The animals were kept in a temperature-controlled laboratory (22258C) with a 12-h lightdark cycle. The care and use of animals for this study was performed according to the Guide for Animal Experimentation, South-Central University for Nationalities and the Committee of Research Facilities for Laboratory Animal Sciences, South-Central University for Nationalities, China. The protocols were approved by the Committee on the Ethics of Animal Experiments of the South-Central University for Nationalities, China (Permit Number: 2013-SCUEC-AEC-002). All efforts were made to minimize suffering.

Rats were randomly divided into control group (no CFA treatment) and CFA-induced inflammatory pain group (CFA treatment). The detailed sample sizes of different groups are included in the Results section. Inflammatory pain was induced by a subcutaneous injection of 150 ml CFA into the bottom of the left hind paws of rats. The control rats were injected with the same volume of saline. After the CFA injection, rats in control group were randomly divided into three groups (control) and sacrificed in the end of the first, second, or third week, respectively. The CFA group were randomly divided into CFA-treated only (CFA), CFA-treated with continuous oral administration of diclofenac sodium (CFAdiclo, 30 mg/kg/day, Novartis, Beijing) afterward, and CFAtreated with continuous oral administration of high-dose nutmeg oil (CFANOhigh, 20 mg/kg/day) and low-dose nutmeg oil (CFANOlow, 10 mg/kg/day). These rats were sacrificed in the end of the first, second, or third week as well. The detailed schedule was shown in Fig. 1A. 

The extent of inflammation was assessed by paw swelling. The paw volume was monitored with a water displacement plethysmometer (PV-200; Chengdu Taimeng Software Co. Ltd., Chengdu, China), in triplicate. Then, the paw swelling was expressed as the ratio of the volume of the right paw for each rat to the volume of its left paw. Data acquired in the end of the first, second, and third week after the CFA injection were presented in percentage for the control group and treated groups. 

All behavioral assessments were performed under the ethical guidelines by the International Association for the Study of Pain (IASP). All of the behavioral tests were conducted blindly. All behavioral tests were performed at the following time points: 1, 2, or 3 weeks after the CFA injection. To eliminate diurnal rhythm, these tests were performed at the same time of the day. The pain score test was performed as follows: both the hind paws of the rat were uniformly marked with red ink. In a self-made semienclosed rat-walking trail, marked rats were allowed to walk for a distance of 0.5 m, and photos of both hind paws were taken. Subsequently, five persons were asked to grade the pain scores independently and doubleblinded with the following criteria: compared with the left paw, a deeper color in any of the four areas (a, toes; b, anterior sole; c, posterior sole; d, heel) of the right paw scores 1, and hence, the rat in Fig. 1B got a score of 4. 

Animals were placed on a metal mesh grid under a plastic chamber, and the tactile threshold was measured by applying a von Frey filament (North Coast Medical, Gilroy, CA, USA) to the mid-plantar surface of the hind paw until a positive response for withdrawal behavior was elicited. Different calibrated fine von Frey filaments (0.40, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, 15.1, and 21.0 g) were presented serially to the hind paw in ascending order of strength with sufficient force to evoke slight bending. 

A brisk paw withdrawal response was considered as a positive response.

If there was no response, the next filament with greater force would be tested. When no response was observed at 21 g of pressure, the animal was recognized as being at the cut-off value. The 50% withdrawal response threshold was determined using the updown method (19).    

To determine nociceptive responses to heat stimuli, a previously described paw withdrawal response latency  (WRL) measurement was used (20). In brief, animals were placed in a plastic chamber with a glass floor and allowed to acclimatize for 10 min. A radiant heat source was placed under the glass floor beneath each hind paw, and paw WRL was measured to the nearest 0.1 sec using a plantar tester (Ugo Basile Srl, Varese, Italy). The intensity of the light source was calibrated to produce a paw withdrawal response within a range of 1625 sec in naive animals. The test was examined in triplicate on both the ipsilateral and contralateral hind paws, and the mean WRL in each hind paw was calculated. The cut-off time was set at 60 sec.       

Rats with different treatments were sacrificed 1, 2, or 3 weeks after the CFA-injection, respectively. The blood samples were collected and pipetted into a disposable test tube with EDTA and PI (peptidase inhibitors; 1 mM phosphoramidon, 4 mg/mL bacitracin and 1 mM captopril; Sigma Chemical Co., St Louis, MO, USA). After 1 h, the blood plasma was collected and the substance P content in the blood was measured using a highly sensitive radio-immuno-assay as previously described (21).    

The skin specimens with diameter of 3 mm around then CFA-injected regions were collected and fixed immediately in 10% phosphate-buffered formalin solution for 1624 h, embedded in paraffin and cut into 4-mm-thick
sections and followed by hematoxylin and eosin (H&E) or Masson’s trichrome staining by standard techniques.
Histopathologic examinations of the liver sections were conducted using a Nikon 50i light microscope (Nikon Inc,Tokyo, Japan).
 

Briefly, skin sections from different treatments were incubated with mouse anti-COX-2 primary antibody
(dilution of 1:300, CX229, Cayman chemical, Michigan, USA) overnight at 48C. After washing, the sections were
incubated with appropriate biotin-conjugated secondary antibody (dilution of 1:100; Santa Cruz Biotechnology, CA, USA) for 30 min at room temperature. 

The color development (brown) was performed using a DAB substrate kit (Nichirei, Japan), and the sections were counterstained with hematoxylin (blue). Then, multispectral imaging analysis of sections was performed by using a
Nikon 50i light microscope (Nikon Inc, Tokyo, Japan) with a Nuance Multispectral Imaging System (Cambridge Research and Instrumentation Inc., Woburn, MA) according to the method instructions (22).
 

All results of behavior tests (except otherwise instructed) in the paper were presented as percentages between the  CFA-injected paws and control paws in the form of mean9SD. Statistical analysis was carried out by a one-way analysis of variance followed by Bonferroni posttests in Origin8.0 (Originlab Corp., MA, USA). 

All tests were two-tailed, P<0.05 were considered to be statistically significant, and in all graphs, *, **, or *** represents
P,0.05, 0.01, or 0.001, respectively. The results of immunostaining and substance P detection were presented as mean+-SEM.
 
 

The major compounds of the nutmeg oil used in this study were sabinene (25.00%), 4-terpineol (11.54%), a-pinene (12.79%), limonene (6.87%), and g-terpinene (6.52%). The detailed data describing all the compounds are presented in Fig. 2 and Table 1 in comparison of a recent study (23).    

To evaluate the anti-inflammatory and analgesic effects of nutmeg oil, CFA-injected rats model was used in our study as instructed in the Materials and Methods section. As shown in Fig. 3A, before the CFA-injection, no swelling was observed in left hind paws of each group. However, after the CFA-injection, the injected hind paws of rats displayed a certain level of swelling compared with the control group (Fig. 3B, in percentage: 163.05+-12.69 of CFA-injected group, n=18 vs. 99.24+-2.47 of control group, n=24), and the extent of swelling decreased in a time-dependent manner. With the application of different treatment (diclo, sodium diclofenac as positive control, 30 mg/kg/day, n=22; NOlow, low dose of nutmeg oil, 10 mg/Kg/day equivalent to 100 mg/day for a 60-Kg weight human, n=12; NOhigh, high dose of nutmeg oil, 20 mg/Kg/day, n=17), the extent of swelling decreased  more rapidly compared with the CFA group. More importantly, in the presence of high dose nutmeg oil, the decrease was much more significant than diclofenactreated group (week 1, 27.43% of NOhigh vs. 24.17% of diclo; week 2, 39.51% of NOhigh vs. 33.29% of diclo; week 3, 41.90% of NOhigh versus 35.52% of diclo).    

To better characterize the inflammation-associated pain states of rats under different treatment, pain score test was introduced in the current study. The detailed procedure of pain score test was in the Materials and Methods section. As summarized in Fig. 4A, the CFA injection increased the pain score for 7-folds from 0.3790.30 at day 0 to 2.45+-0.52 at day 1 and remained in a relatively high level (1.9590.46 at week 3) for as long as 3 weeks, suggesting that the CFA injection successfully induced a significant and sustainable pain to hind paws of rats. With the application of diclofenac, the positive control drug, pain score dropped to 1.89+-0.32 at week 1 and further decreased to 1.625+-0.25 at week 3. 

However, a high dose of nutmeg oil treatment reduced the pain score to 1.90+-0.51 at week 1 and 1.46+-0.19 at week 3, which surpass the effect of diclofenac application, suggesting that high dose application of nutmeg oil showed a better effect of alleviating the inflammatory pain response induced by the CFA injection. The pain score test was conducted by five persons independently and double-blindly. However, there could still be some bias and subjectivity during the experiments. In addition, the score demonstrated in this test was an overall and complex readout of pain behavior. 

Hence, a traditional touch pain test and heat pain test was also used to test the anti-inflammatory and analgesic effects of nutmeg oil. As shown in Fig. 4B, we observed a significant sensitization of both touch and heat-induced pain response after the CFA injection. In accordance with our previous results, both the low- and high-dose treatment of nutmeg oil alleviated the hypersensitivity of touch pain behavior of CFA-injected rats. 

The touch pain threshold enlarged about 3-folds from (in percentage) 24.72+-12.63 at day 1 to 71.67+-32.72 at week 3 for low dose application of nutmeg oil and from 16.19910.29 at day 1 to 55.71+-31.78 at week 3 for high dose. In the   contrary, the threshold for CFA group merely increased from 10.09+-3.73 at day 1 to 18.33+-4.27 at week 3 suggesting a poor self-recovery on the allodynia. It is interesting to note that the positive control drug, sodium diclofenac, showed less potency in raising touch pain threshold compared with nutmeg oil (from 12.5094.71 at day 1 to 30.83+-10.73 at week 3). Consistent with the touch pain test result, heat pain thresholds were also greatly reduced by the CFA injection and restored by high-dose treatment of nutmeg oil (from 28.49+-7.37 at day 1 to 80.22+-16.67 at week 3). 

However, in our experiments, the effects of low-dose treatment of nutmeg oil or diclofenac on alleviating the heat hyperanalgesia were merely observable (Fig. 4C)       

It has been demonstrated that CFA-injection could induce COX-2 expression in rats (2426). To test whether pain alleviation of nutmeg oil application was mediated by inhibition of COX-2, skin samples around the CFA injected regions were collected and conducted to immunohistochemistry staining. As shown in Fig. 5A and B, the expression pattern of COX-2 varied after diclofenac or high dose of nutmeg oil treatment. Both treatments decreased COX-2 expression induced by the CFA injection, especially at week 2. Moreover, substance P release has been demonstrated to be correlated with pain response in rats (22). Hence, the blood substance P levels were investigated in the current study as summarized in Fig. 5C. Compared with CFA-treated rats, diclofenac application could greatly diminish CFA-injection induced increase of blood substance P level, while application of high-dose nutmeg oil nearly abolished the rise, suggesting a better analgesic effect compared with diclofenac       

We have demonstrated that nutmeg oil could potentially alleviate the CFA-injection-induced joint swelling, allodynia, and heat hyperanalgesia of rats through inhibition of COX-2 expression and blood substance P level, which made it possible for nutmeg oil to be a potential chronic inflammation and pain reliever.
 

SST and TTL carried out the cellular and animal experiments; RHC, FLM, and CJW carried out the analysis of composition of nutmeg oil; WKZ, FLM, and HBT proposed and designed the research; WKZ and HBT performed the data analysis and wrote the paper; YSL and XJL assisted in the data analysis and interpretation.
 

This study was supported by the National Natural Science Foundation of China (8137384231400705), and the Natural Science
Foundation of China Hubei (2013CFB451) and the Modernization Engineering Technology Research Center of Ethnic Minority
Medicine of Hubei province (2015ZD005).
 

The authors declare no conflict of interest