Comparative Studies on Chemical Contents and Effect in Kidney-Yang Deficiency Rats of Salt-Processed Product and Wine-Processed Product of Cuscutae Semen

Cuscutae Semen mainly includes salt-processed product (SPP) and wine-processed product (WPP), which are most commonly used in traditional Chinese medicine. However, the differences between SPP and WPP have not been reported. In this paper, comparative studies between SPP and WPP on chemical contents and effect in kidney-yang deficiency rats have been investigated. UPLC-MS/MS was used to study the differences in chemical components. Kidney-yang deficiency was induced by hydrocortisone in rats. Rats were orally administrated with different doses of Jinkui Shenqi Pills, SPP, and WPP for 28 days. The average organ coefficients, renal function indexes, sex hormone levels, and testicular morphology were detected. As a result, the contents of flavonoids and chlorogenic acids were higher in SPP than in WPP, which may be caused by different processing methods. The improvement on reproduction of SPP was reflected in organ coefficients, renal function indexes, and biochemical properties of seminal plasma; furthermore, WPP was in sex hormone levels and morphology of testis. As a conclusion, these results indicated that both SPP and WPP can improve the reproductive function of kidney-yang deficiency rats with different mechanisms, which may be due to the differences in chemical contents between WPP and SPP as well as different processing methods. It is the first time that the differences between SPP and WPP have been studied in reproductive function in rats with kidney-yang deficiency.


Introduction
Cuscutae Semen (the dry mature seed of Cuscuta australis R. Br. or Cuscuta chinensis Lam., belonging to the family Convolvulaceae) is first recorded in the "Shen Nong's Herbal" as an upper-grade drug about 2000 years ago [1]. It is widely applied in Chinese medicine practice to nourish the liver and kidney, treat impotence and seminal emission, prevent miscarriage, and improve eyesight [2][3][4].
In order to ensure the safety and effect in clinical application, most of the herbal drugs are always processed (known as Pao Zhi in Chinese) before prescription [5]. It is believed that herbal drugs after processing can change the therapeutic range, enhance the effect, or reduce toxicity in traditional Chinese medicine (TCM). e processing of Cuscutae Semen, mainly including salt-processed product (SPP) and wine-processed product (WPP), has been applied for a long history [1,6]. WPP is the initial processed product of Cuscutae Semen, which is first recorded in "A Handbook of Formulas for Emergencies" (Zhou Hou Bei Ji Fang) in the Jin Dynasty of China [7]. SPP is first documented in "Comprehensive Recording of Sage-Like Benefit" (Sheng Ji Zong Lu) in the North Song Dynasty [8]. ere is different understanding on the effect of SPP and WPP by the theory of TCM. e salt processing is supposed to lead drugs into channel tropism of kidney and enhance functions of the kidney, such as nourishing kidney and improving reproductive function. Wine processing is presumed to change the property of medicine and promote yang-qi, such as kidney-yang [5]. SPP is more adept at nourishing kidney and astringent while WPP can enhance the role of warming spleen and kidney [5]. However, to the best of our knowledge, no study on differences between SPP and WPP has been described. Based on the works we had done before, this paper compared chemical compositions and reproductive function in rats with kidney-yang deficiency between SPP and WPP for the first time. We expect to find the theoretical basis for the processing of traditional Chinese medicine.
Previous phytochemical investigations on Cuscutae Semen have led to the isolation of a series of natural products, including flavonoids, chlorogenic acids, and polysaccharides [9,10]. e flavonoids and chlorogenic acids are reported to be biologically active constituents in Cuscutae Semen [1,3,11]. Cuscutae Semen flavonoids can improve reproductive injury induced by glycosides of Tripterygium wilfordii Hook.f. [9], regulate endocrine dysfunction (sex hormone receptors) [12], ameliorate kidneyyang deficiency symptoms [13], and improve the immune function of kidney-yang deficiency rats [14]. Modern pharmacological experiments have indicated that chlorogenic acids have antiviral effects and protective effects on the liver injury and gallbladder [11]. Our previous study reveals that the contents and biological activities of total flavonoids between the crude products (CP) and SPP of Cuscutae Semen are different [1]. Flavonoids and chlorogenic acids are also identified by UPLCs in our previous study [15]. However, up to now, the comparative studies on chemical contents and effect between SPP and WPP have not been described. In this study, the contents of bioactive chemicals are determined to differentiate chemical components between SPP and WPP.
As the symptom of kidney-yang deficiency rats is similar to the kidney-yang deficiency in TCM, hydrocortisone-induced kidney-yang deficiency rats are used for comparative studies on reproductive function between SPP and WPP [16].
Acetonitrile (MS grade) and formic acid (MS grade) were supplied from ermo Fisher Scientific Inc. All the other chemicals of analytical grade were commercially available from Beijing Chemical Works (Beijing, China). Other reagents were of analytical grade. Deionized water for preparing samples was purified by MilliQ50 SP Reagent Water System (Bedford, MA, USA). Distillated water for mobile phase was purchased from A. S. Watson Group (Hong Kong) Ltd.

Collection of Samples.
We followed the methods of Zhang et al. [15]. e crude products of Cuscutae Semen (lot number: 160161211) were obtained from Beijing Kangmei Pharmaceutical Co., Ltd. (Beijing, China) and authenticated as Cuscutae Semen by Yang Yaojun, the professor of Pharmacognosy Department in Beijing University of Chinese Medicine. Voucher specimens were deposited in the School of Chinese Materia, Beijing University of Chinese Medicine. SPP (lot number: 160715) was prepared in the laboratory, which meant that the CP was mixed with salt solution and then stir-heated at 80°C in a metallic pan and dried in the air (2 kg salt per 100 kg of Cuscutae Semen). WPP (Lot number: 160716) samples were mixed with wine and then stir-heated at 100°C and dried in the air (20 kg wine per 100 kg of Cuscutae Semen).

Preparation of Samples.
SPP and WPP were selected from the samples we collected and prepared in our lab (as described in Section 2.2). e samples were accurately weighed as 0.8 kg and soaked 8 times in 60% ethanol for one night. And then, they were extracted under reflux twice for 1 h each time. e filtrate was combined, and the ethanol was removed. e ethanol extracts were dried in vacuum at 60°C. en, the ethanol extracts were obtained. e w/w extraction yield of SPP and WPP was 9.16% and 9.10%, respectively. A part of the ethanol extracts of SPP were used for content determination. Another part of the extracts were diluted in distilled water when the rats were treated orally. Sample preparation in WPP was the same.  Evidence-Based Complementary and Alternative Medicine was considered as the peak area three times greater than the noise level. LOQ was the peak area ten times greater than the noise level.

Effect of Reversing the Kidney-Yang Deficiency
2.5.1. Animals and Housing. Male Wistar rats (160-180 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. Before the experiment, all rats were allowed to acclimatize in environmentally controlled quarters (temperature of 21-23°C, relative humidity of 45-65%, and 12:12 h light/dark cycle) with food and water freely available for one week. e experiment complied with the ethical guidelines suggested by the Institutional Animal Ethics Committee and Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Health, and Government of China.

Treatment of Animals.
Sixty rats were randomly partitioned into five groups. Four groups were given an intramuscular injection of 20 mg/kg hydrocortisone sodium succinate (purchased from Tianjin Biochem Pharmaceutical Co., Ltd., Tianjin, China) for 14 days to establish the kidneyyang deficiency model except for normal control group (NC) which was injected with an equal volume of physiological saline. After this treatment, the kidney-yang deficiency model was made.
On the 15th day, five groups were treated as follows: Jinkui Shenqi Pills group (JK) was treated orally at dose of 105 mg/kg/d Jinkui Shenqi Pills (purchased from Beijing Tongrentang Group Co., Ltd., Beijing, China); SPP group was treated orally with 0.18 g/kg ethanol extracts of SPP; WPP group was treated orally with 0.18 g/kg ethanol extracts of WPP; model group (M) and normal control group (NC) were treated with an equal volume of distilled water. All the samples were administered by gastric perfusion.
Twenty-four hours after the last treatment (after 4 weeks' treatment), all rats were anesthetized and sacrificed. Blood samples were collected from abdominal aorta into Eppendorf tubes and then were centrifuged at 3000 rpm for 15 min to collect the serum and then stored at − 80°C until the determination of renal function indexes and hormone levels. One kidney and one testicle were rapidly removed and weighed as well as epididymis, prostate gland, and seminal vesicle. e rest of the testicles were fixed in Bouin's fixative solution for 24 h at 4°C, which were subsequently embedded in paraffin using standard procedures and stained with hematoxylin-eosin (HE).

Morphological Observation of Testis.
e left testicles were washed twice with phosphate-buffered saline (PBS). After fixed 1 h in Bouin's fixative, they were stained with hematoxylin-eosin (HE), followed by examination for histopathological changes under the microscope.

Statistical Analysis.
e UPLC-MS/MS data for quantitative analysis were acquired and processed using the Agilent Mass Hunter Workstation Software (version B.07.00).
Data for animal experiments were expressed as mean-± standard deviations and analyzed with one-way analysis of variance (ANOVA) by SAS software 9.3. P < 0.05 was considered statistically significant.

Quantitative Analysis by UPLC-MS/MS
e regression equations, together with LOD and LOQ values for the quantitation method, are shown in Table 1. e calibration curves were in good linearity with correlation coefficients over 0.9900. e RSD for peak area was within 5.79%, which suggested that the investigated compounds were stable within 24 h. e RSD of repeatability and precision was within 3.86% and 1.96% (Table 1), respectively, which suggested that the method was reproducible and precise. e recovery test (Table 2; recovery ranged from 101.69% to 104.80%) suggested good accuracy of the method. e results were validated by the reliability of the method.

Contents Analysis in SPP and WPP.
e contents of fifteen major compounds in SPP and WPP of Cuscutae Semen samples are shown in Table 3. e results showed a significant difference between the two processed products. Except for quercetin, SPP had a higher content of other 14 components than WPP. 3-CQA and hyperoside were considered as the major constituents in SPP and WPP.

Kidney-Yang Deficiency Model Was Successfully Made.
After intramuscular injection of hydrocortisone sodium succinate for 14 days, the rats showed withered clothing hair, decreases in body weight, slowed reaction, aversion cold, weakness, tendency to cluster, and decreased activity as in kidney-yang deficiency. Based on these clinical parameters, the model was regarded as successful.

Average Organ Coefficients.
All rats and their organ were weighed, and the average organ coefficients were calculated. Compared with the normal control group, the coefficients of kidney, testicle, epididymis, seminal vesicle, and prostate gland were decreased in the model group (P < 0.05 or P < 0.01). Compared with the model group, the organ coefficients of the JK, SPP, and WPP groups were increased. e SPP group was better in the epididymis and prostate gland than the WPP group (P < 0.05 or P < 0.01).
e results are shown in Figure 2 and Table 4.

Analysis of Renal Function Indexes.
In traditional Chinese medicine, the kidney is thought to be related to reproduction. Based on the theory, reproductive ability, growth, and development were determined by the essence and vitality of the kidney [17]. e detection of renal function can be used to evaluate the reproductive function in kidney-yang deficiency rats. As shown in Table 5 and Figure 3, the levels of CRE, BUN, and UA in the serum were determined. Compared with the NC group, the levels of CRE, BUN, and UA were increased in the model group (P < 0.05 or P < 0.01). Compared with the M group, the CRE, BUN, and UA levels of all treated groups were lower (P < 0.05 or P < 0.01). e contents of CRE were significantly different between SPP and WPP. e SPP group had better renal injury improvement than the WPP group.

Hormone Analyses.
Compared with the normal control group, testosterone (T) and luteinizing hormone (LH) levels in the serum of the model group were lower. Estradiol (E 2 ) and follicle-stimulating hormone (FSH) levels were higher in the model group (P < 0.01). Compared with the M group, T and LH levels increased while E 2 and FSH levels decreased in the SPP and WPP groups as shown in Figure 4 and Table 6. In the regression equation, x is the peak area, y the concentration of each analyte (ng/mL), and r the correlation coefficient. LOD, limit of detection (S/N � 3); LOQ, limit of quantification (S/N � 10). Precision, repeatability, and stability are shown in RSD (%). 6 Evidence-Based Complementary and Alternative Medicine   Figure 2: Average organ coefficients (100%) in each group. Significant differences with the NC group were designated as Δ P < 0.05, ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with the WPP group # P < 0.05, ## P < 0.01.

Analysis of Biochemical Properties of Seminal Plasma.
As shown in Figure 5 and Table 7, compared with the NC group, the levels of fructose, acid phosphatase (ACP), α-glucosidase, and zinc in the seminal vesicle of the model group were decreased (P < 0.05 or P < 0.01). Compared with the M group, biochemical indexes of seminal plasma in all treated groups were increased (P < 0.05 or P < 0.01). e contents of fructose, ACP, and Zn were significantly increased in the SPP group compared with the WPP group. e SPP group had improved this condition better than the WPP group.

Morphological Observation of Testis.
Testicular histology was regular in the NC group ( Figure 6 A-1, A-2).
ere was no evidence of disruption or injury among the seminiferous tubules. e seminiferous tubules were arranged compactly. e spermatogenic cells were well developed. e spermatogonia (SG), primary spermatocytes (PS), round spermatids (RTS), and elongated spermatids (EST) were observed. e number of cells was at normal levels and the cell layer was clear. ere were visible mature sperms within the lumen, rich mesenchymal cells, and clear and complete structures.
Of the model group (Figure 6 B-1, B-2), the seminiferous cells were significantly lower and the cells were loose. e spermatogenic cells were arranged disorderly and were found shortened. Cell nuclei were seriously sparse and vacuoles were existed. e sperm counts had significantly dropped. e stromal cells were atrophy with the number decreased. Based on these pathologies, the kidney-yang model was successfully made.
In the JK group ( Figure 6 C-1, C-2), compared with the model group, the seminiferous tubules were arranged more compactly.
e number of spermatogenic cells increased. e cells were arranged in a neat and orderly manner. Leydig cells were abundant; meanwhile, the degeneration degree was improved.
In the SPP group ( Figure 6 2 D-1, D-2), the seminiferous tubules were arranged compactly. Compared with the model group, the number of spermatogenic cells increased and the cells were arranged in order. With improved degradation degree and increased number of Leydig cells, vacuoles were still existed.
In the WPP group ( Figure 6 E-1, E-2), compared to the model group, the seminiferous tubules were arranged compactly and the number of spermatogenic cells increased.
ere were visible mature sperms within the lumen. With improved degradation degree and a rich number of Leydig cells, the vacuoles were improved obviously.

Discussion
Based on the TCM theory, kidney-yang is essential for human growth and development as well as reproductive function. e kidney-yang deficiency syndromes are used in TCM to elucidate a status in which the patients show a group of symptoms mainly including soreness and weakness of waist and knees, fatigue, cold feeling of the whole body, impairment of hearing, looseness of teeth, and even reproductive dysfunction [14]. In this study, hydrocortisoneinduced kidney-yang deficiency rats were used for a comparative study on reproductive function between SPP and WPP, in which rats will show similar symptoms with those have been described in TCM kidney-yang deficiency [16]. 0.3170 ± 0.0227 * * 0.4290 ± 0.0216 * * 0.1315 ± 0.0061 * * 0.1284 ± 0.0127 * * 0.1395 ± 0.0098 * * SPP 0.2991 ± 0.0193 * 0.4478 ± 0.0294 * * 0.1397 ± 0.0103 * * # 0.1111 ± 0.0096 * 0.1327 ± 0.0220 * ## WPP 0.2935 ± 0.0253 # 0.4234 ± 0.0334 * * 0.1262 ± 0.0085 * 0.1318 ± 0.0177 * * 0.1277 ± 0.0127 * e values are presented as mean ± S.D. Significant differences with the NC group were designated as Δ P < 0.05, ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with the WPP group # P < 0.05, ## P < 0.01. e values are presented as mean ± S.D. Significant differences with the NC group were designated as Δ P < 0.05, ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with the WPP group # P < 0.05.  Figure 4: Sex hormone levels in each group. Significant differences with the NC group were designated as ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with the WPP group # P < 0.05. e values are presented as mean ± S.D. Significant differences with the NC group were designated as ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with WPP group # P < 0.05.  Figure 5: Biochemical indexes of seminal plasma in rats. Significant differences with the NC group were designated as Δ P < 0.05, ΔΔ P < 0.01, with M group * P < 0.05, * * P < 0.01, with WPP group # P < 0.05, ## P < 0.01. e values are presented as mean ± S.D. Significant differences with the NC group were designated as Δ P < 0.05, ΔΔ P < 0.01, with the M group * P < 0.05, * * P < 0.01, with the WPP group # P < 0.05, ## P < 0.01. 10 Evidence-Based Complementary and Alternative Medicine e hydrocortisone-induced kidney-yang deficiency symptoms rats are commonly used to evaluate the pharmacological activity of reinforcing kidney-yang herbs [18].
Cuscutae Semen is a traditional Chinese medicine commonly used in reinforcing kidney-yang and alleviating or even reversing the symptoms of kidney-yang deficiency. e processing of Cuscutae Semen, mainly including saltprocessed product (SPP) and wine-processed product (WPP), has been applied for a long history. However, no study has investigated the differences between SPP and WPP. Our previous study showed that phenolic compounds are the major constituents of Cuscutae Semen. Flavonoids and chlorogenic acids were identified [15]. In this study, we determined fifteen major compounds in SPP and WPP including seven flavonoids and eight chlorogenic acids. Compared with WPP, most of the contents of fifteen major compounds were higher in SPP. e content distinction may be caused by different processing methods. We speculate that the content differences of chemical composition between SPP and WPP may be related to the effect differences. e value of organ coefficients is equal to the percentage of the body weight divided by the absolute organ weight. In this study, the organ coefficients of the SPP and WPP groups were increased, which indicated that SPP and WPP could reverse organ coefficients decline. In general, the effect of SPP was better than that of WPP.
Clinically, serum CRE, BUN, and UA are important indexes to evaluate renal function [19,20]. When the renal function is disrupted, CRE, BUN, and UA increase. In this study, the SPP and WPP groups could decrease the contents of CRE, BUN, and UA. It indicated that the processed products of Cuscutae Semen were able to improve the renal injury induced by hydrocortisone. e experimental results showed that the SPP group mainly reduced the CRE content. Overall, SPP is better to alleviate kidney damage than WPP.
Experimental studies [21] indicate that kidneys are closely correlated with the reproductive endocrine system. FSH, LH, T, and E 2 are important indexes to evaluate the reproductive function. e results showed that hydrocortisone caused gonadal and reproductive function injury. SPP and WPP regulated the levels of sex hormones (increase E 2 and FSH and decrease T and LH) and improved testis injury in kidney-yang deficiency rats, so as to regulate the disturbance of reproductive function. In general, the effect of WPP on the regulation of sex hormone levels was stronger than that of SPP.
Seminal vesicle which plays an important role in male reproduction is an important accessory gland in the male reproductive system. With the development of biochemical technology, the evaluation of seminal plasma biochemical indexes and the function of male accessory gonad on male reproductive ability have been paid more and more attention. α-Glucosidase, ACP, fructose, and Zn are major indexes to evaluate the function of male reproductive ability [22][23][24]. e results showed that processed products of Cuscutae Semen improved the reproductive function in rats by enhancing the accessory gonad function of kidney-yang deficiency rats. SPP was more adept at enhancing seminal plasma biochemical indexes than WPP. e testis is the major organ of the male reproductive system. Experimental studies have shown that the kidneyyang deficiency can affect the morphology of testicular tissue [25]. In the model group, the seminiferous tubules showed obvious damage, indicating that the model of kidney-yang deficiency was successfully established. e testes of the Cuscutae Semen processed products group rats were improved. Based on the results, the effect of the WPP group was better than that of the SPP group.
Total flavonoids, efficacious components of Cuscutae Semen, have been reported to show regulatory effects on reproductive and endocrine function [12,26], such as regulating LH, T, and E 2 levels. Chlorogenic acids, other active components, exhibited protective effects on sperm plasma [27,28]. Previous studies have also shown that quercetin, astragalin, kaempferol, feruloylquinic acid, and caffeic acid have beneficial and/or protective effects on improving the reproductive function, preventing sperm damage, ameliorating sperm function, and improving testicular function [27][28][29][30][31][32]. In this study, SPP and WPP showed protective effects on reproductive, endocrine, and testicular function. e protective effects may be related to these components which need further investigation.

Conclusions
e contents of flavonoids and chlorogenic acids were higher in SPP than in WPP. e content distinction may be caused by different processing methods. Pharmacological experimental results demonstrated that both SPP and WPP could improve the reproductive function in kidney-yang deficiency rats. e improvement on reproduction of SPP was reflected in organ coefficients, renal function indexes, and biochemical properties of seminal plasma; furthermore, WPP was in sex hormone levels and morphology of testis. e different improvement mechanism may be due to the differences in chemical contents between WPP and SPP as well as different processing methods. e pharmacological experiment results are in accordance with the theory of Cuscutae Semen processing, which provides the theoretical basis for the clinical application of Cuscutae Semen and the further development and utilization. However, the action mechanism of the salt-processed product and wine-processed product of Cuscutae Semen needs further study. Relative standard deviation CRE: Creatinine E 2 : Estradiol FSH: Follicle-stimulating hormone LH: Luteinizing hormone 3,5-DiCQA: 3,5-Dicaffeoylquinic acid 4,5-DiCQA: 4,5-Dicaffeoylquinic acid CA: Caffeic acid MRM: Multiple reaction monitoring BUN: Blood urea nitrogen ESI: Electron spray ionization T: Testosterone UA: Uric acid.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Ying Zhang, Shuya Xu, and Xiangri Li conceived and designed the experiment. Ying Zhang and Tianying Jia performed the experiment and data analysis. Ying Zhang, Wenjia Qu, Zhe Jia, and Xiangri Li drafted the paper. Xinfang Xu, Ting Han, and Mengnan Liu revised the manuscript. All authors have contributed to the final version and approved the publication of the final manuscript.