Extraction optimization of Eucommia ulmoides Oliver and its effect on bone quality in OVX rats

Purpose: To maximize the yield of extract from Eucommia ulmoides Oliver and its effect on bone quality. Methods: Different extraction indices were optimized with response surface methodology (RSM) for maximization of extract yield from Eucommia ulmoides Oliver. Box–Behnken design (BBD) was used to identify the effects of temperature, time, and liquid to solid ratio on extract yield from Eucommia ulmoides Oliver. After 4-week acclimatization, thiry-two rats were randomly assigned to 4 groups (n = 8): group 1 (sham) given vehicle only; group 2 (OVX rats given Eucommia ulmoides Oliver extract at a dose of 4 g/kg; group 3 (OVX + vehicle); group 4 (OVX + EUOE), i.e., OVX rats given Eucommia ulmoides Oliver extract (4 g/kg). Sham rats had intact ovaries. After surgery, the rats received gentamicin intramuscularly for 3 successive days. Two months after surgery, blood and trabecular bones was taken for analysis. Results: Temperature and liquid-to-solid ratio had marked impact on extract yield from Eucommia ulmoides Oliver, with the best conditions being temperature of 88 °C, time of 137 min, and liquid to solid ratio 16:1. Using these optimized conditions, the maximum yield of extract obtained experimentally (2.53 %) was very close to the predicted value of 2.49 %. There was a good fit between the mathematical model evolved and the data on extract yield. The extract significantly (p < 0.01) increased the Ca and P and Cr levels in OVX + EUOE group compared to those in OVX control. Moreover, the extract significantly (p < 0.01) increased macro-mechanical indices of trabecular bone in OVX+EUOE group, relative to those in OVX control. Conclusion: The yield of Eucommia ulmoides Oliver extract has been successfully optimized using RSM. The extract exhibited strong effects on bone quality.


INTRODUCTION
Eucommia ulmoides Oliver (Eucommiaceae) is a medicinal herb in Traditional Chinese medicine TCM) [1]. The medicinal parts of E. ulmoides are its stem bark and leaves. Several bioactive compounds with pharmacological effects have been isolated from E.
The effects of variable indices and their interactions are determined using RSM [5]. It is a very popular and reliable method for the optimization of operations involving plant products [6][7][8]. The technique minimizes the number of trials needed to ascertain the relationships among multiple factors, and it facilitates simultaneous investigations of numerous indices [9][10][11].
This study was carried out to develop effective response surface models to obtain optimal extract conditions from Eucommia ulmoides Oliver, based on temperature, time, and solid: liquid, for efficient extraction of Eucommia ulmoides. The effect of Eucommia ulmoides Oliver extract on bone quality in OVX rats was also investigated.

EXPERIMENTAL Preparation of extract from Eucommia ulmoides Oliver
The Eucommia ulmoides Oliver was first refluxed with 90 % ethanol for 5 h. Then, the resultant residue was extracted with water at different temperatures for various tested times to obtain the aqueous extract. The supernatant fraction was concentrated and dried. Three major extraction parameters: temperature, time, and the liquid: solid ratio were considered in this study.

Study design
Experiments were conducted to optimize extract yield from E. ulmoides Oliver with RSM, using a 3-tier, 3-parameter BBD. The BBD permits investigation of factors to at least one at the center point (0) [12]. Variables such as temperature (A), time (B), and liquid: solid ratio (C) were used.
In all, 17 runs were used to measure extract yield, while maintaining the independent variables at levels −1, 0, and +1 (Table 1). Optimum extraction conditions were chosen and validated on the basis of BBD results. Regression coefficients (β) for assessment of the degree of influence of each factor on response were deduced from 3-D response surface plots. For individual responses, model F-value, absence of fit and R 2 were determined.

Induction of osteoporosis in rats
A total of thiry-two rats (mean bwt = 240 ± 20 g) were assigned to 4 groups (8 rats per group): vehicle-treated (sham, group 1); OVX rats treated with Eucommia ulmoides Oliver extract (4 g/kg, group 2); OVX + vehicle (group 3), and OVX rats given extract at a dose of 4 g/kg b.w. Each aniaml was fasted for 6 h prior to excision of bilateral ovary under pentobarbitone anaesthesia (50 mg/kg). The ovaries of rats in the sham group were not removed. Post-surgery, gentamicin was administered intramuscularly to the rats for 3 successive days. Two months after surgery, blood and trabecular bones were taken for analysis.

Tests on bone biomechanics
Indices of biomechanics of L5 vertebrae were determined as described earlier [13].

Statistical analysis
Results are presented as mean±SD. The reliability of RSM calculations and equations were determined. Estimation of dissolution was done with 3 formulations chosen at random, using the experimentally-derived models.

Optimized extraction temperature
The influence of temperature on extraction yield of Eucommia ulmoides Oliver was studied ( Figure 1). The results indicated a sharp rise in the extract yield with temperature up to 90 o C, and a slow decrease subsequently ( Figure 1). The highest extract yield (2.42 %) was obtained at 90 o C. Therefore, extraction temperature of 90 o C was chosen for optimization of other parameters.

Optimized extraction time
The extraction yield from Eucommia ulmoides Oliver increased rapidly with time for about 140 min, with no increases thereafter ( Figure 2). Maximum extract yield (2.39%) was attained after 160 min. Thus, 140 min was selected for optimizing other indices.

Optimized liquid: solid ratio
The optimum liquid: solid ratio was determined using a wide range of 11:1 to -18:1. The optimum liquid-to-solid for maximum extract yield from Eucommia ulmoides Oliver extraction yield at solid-to-liquid ratio of 1:16 was 2.51 % (Figure 3).
Further increases in the liquid-to-solid ratio had no marked effect on extract level. Thus, the optimized solid-to-liquid ratio of 1:16 was used in subsequent experiments.
The quadratic model was chosen as optimized model for EUOE extraction since the linear term was significant (p < 0.0001), as inferred from results of regression analysis in Table 3.
Using optimized conditions i.e. temperature of 88 °C, time of 137 min and solid: liquid ratio of 1:16 to test the model, the actual extract yield from Eucommia ulmoides Oliver was 2.53 % (n = 3), demonstrating that the experimental and predicted values were comparable (p > 0.05).
Thus, the response model is adequate for use in optimizing extract yield.

Effect of EUOE on Ca and P and Cr levels in OVX rats
The experimental analyses revealed that the Ca and P and Cr levels in sham were markedly higher than those in OVX control ( Figure 5 and Figure 6). However, there were greater decreases in the Ca and P and Cr levels in OVX + EUOE rats than in OVX control ( Figure 5 and Figure 6). There were no marked differences in any of the parameters between the sham, and sham + EUOE groups.   Table 3 indicates that macro-mechanical indices were markedly higher in sham rats than in OVX rats. Ultimate load was markedly greater in OVX + EUOE group, relative to OVX groups. The stiffness was significantly greater in the OVX + EUOE group when compared to the OVX groups. The energy to failure was significantly greater in OVX + EUOE group than in OVX groups. The other indices were comparable among the sham, and sham + EUOE groups.

DISCUSSION
In this optimization, yields in range of parameters are shown in Table 1. Some specimens were prepared and tested based on these optimized values, and their mean values were obtained. Using linear regression and statistical analysis [14,15], the results demonstrated that all the input parameters had significant impacts on yield of extract from EUOE. Temperature and solid: liquid ratio had the most effect on extract yield. The interaction plot indicated significant interaction between the temperature and liquid: solid ratio with respect to extract yield which is also evident in the output mathematical model of yield response.
The response points are assessed based on 3D response surface and 2D contour plots [16]. There were marked interactions between temperature and liquid: solid ratio. These two factors had the highest influence on extract levels.
In Japan, Eucommia ulmoides Oliver is called Tuchong, while Chinese call it Du-zhong Chinese) [17]. It is a deciduous tree which belongs to the family of Eucommiaceae [18]. The present study showed that EUOE treatment improves Ca and P and Cr levels and trabecular bone biomechanical properties in OVX rats

CONCLUSION
The extraction conditions for Eucommia ulmoides Oliver have been successfully optimized using RSM. The optimized conditions obtained are temperature of 88 °C, time of 137 min and solidto-solid ratio of 1:16. The results suggest that EUOE has potential for application in the suppression of bone degeneration in humans.

DECLARATIONS
License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.