Study on the Effects of Chinese Materia Medica Processing on the Hypoglycemic Activity and Chemical Composition of Anemarrhenae Rhizoma

Purpose To compare the hypoglycemic effects of different extracts of Anemarrhenae Rhizoma (AR) before and after being stir-baked with salt water on the diabetic mice and to detect the contents of 8 components in the corresponding active parts simultaneously using the UPLC-MS method, in order to screen the better extracts for diabetes and to clear the material basis for enhancing hypoglycemic activity of Anemarrhenae Rhizoma stir-baked with salt water (SAR). Methods Taking spontaneous type II diabetic db/db mice as models and fasting blood glucose (FBG), oral glucose tolerance test (OGTT), glycated hemoglobin or glycosylated hemoglobin (HbAlc), serum resistin (RESISTEIN), fasting insulin (FINS), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) as indicators, the hypoglycemic effects of different active parts of Anemarrhenae Rhizoma were evaluated. The chromatographic separation was performed on a Waters BEH C18 (2.1 mm × 50 mm, 1.7 μm) column using acetonitrile (B) and 0.1% formic acid in water (A) as mobile phases, and the flow rate was 0.3 ml/min. The column temperature was set as 28°C, and the injection volume was 10 μL. A mass spectrometer was connected to the UPLC system via an electrospray ionization (ESI) interface. Full-scan data acquisition was performed in the negative ion mode. Result In the study of pharmacodynamics, the hypoglycemic effect of Anemarrhenae Rhizoma stir-baked with salt water is better than that of Anemarrhenae Rhizoma and the hypoglycemic effect of ethanol extract of Anemarrhenae Rhizoma is more remarkable than that of the decoction. The measured components all have a good linear relationship within their respective linear ranges (r ≥ 0.9990); the average recovery rates are 98.86%–100.69%, RSD <2.90%. Compared with the raw Anemarrhenae Rhizoma, the contents of Timosaponin AIII, Timosaponin BII, Timosaponin BIII, Anemarrhenasaponin I, Anemarrhenasaponin Ia, and Mangiferin of Anemarrhenae Rhizoma stir-baked with salt water are all higher, the changes of Timosaponin AI and Anemarrhenasaponin AII are not obvious, and all the contents of chemical composition in the ethanol extract of Anemarrhenae Rhizoma and Anemarrhenae Rhizoma stir-baked with salt water were obviously higher compared with the water decoction. Conclusion The processing method, stir-baking with salt water, can increase the contents of active compositions in Anemarrhenae Rhizoma and strengthen the hypoglycemic effect. The ethanol extract of Anemarrhenae Rhizoma stir-baked with salt water is the better active site.


Introduction
Traditional Chinese medicine (TCM) has its unique theoretical system and application form. Chinese material medica processing can reduce the toxicity of TCM and increase its efficiency, which is an indispensable guarantee for effectiveness and safety of traditional Chinese medicine clinical practice [1]. TCM processing is a unique and traditional pharmaceutical technology adopted according to the theory of TCM, the nature of Chinese crude drug, and the needs of dispensing preparation and clinical application. TCM processing makes TCM different from natural medicine [2]. TCM processing was listed in the first batch of national intangible cultural heritage in 2006. TCM processing is a major feature on the clinical prescription of TCM. Raw decoction pieces and processed decoction pieces are used for different symptoms. ere are many records in ancient books to guide Chinese medicine practitioners to use processed products; meantime, the researchers of traditional Chinese medicine processing have been struggling to explain different treatments of raw and cooked by analyzing the changes of pharmacodynamic substances and the regular pattern of the body's action caused by the processing of TCM [3].
Anemarrhenae Rhizoma refers to the dry rhizome of the plant Anemarrhena asphodeloides Bge. of the family Liliaceae. It is bitter and sweet with cold nature and attributive to lung, stomach, and kidney meridians, taking on effects of heat-clearing, fire-purging, nourishing Yin, and moisturizing dryness [4]. In clinical Chinese medicine, in addition to the use of AR, there is also the application of SAR, which has a history of nearly a thousand years. e traditional theory of TCM believes that the processing method, stir-baking with salt water, can introduce the medicines into the kidney meridian and enhance the effect of nourishing Yin for lowering fire. Our research has found that the stir-baking with salt water can enhance the hypoglycemic effect of Anemarrhenae Rhizoma (AR) significantly, which is just consistent with the traditional theory of the processing of AR because TCM believes that the main pathogenesis of diabetes is the loss of Yin and Jin and the excessive dryness and heat. e deficiency of Yin is the root cause, and the dryness and heat are the superficies [5]. Anemarrhenae Rhizoma stirbaked with salt water (SAR) can improve this symptom properly. In order to explain the processing effect of AR from the chemical composition and medicinal effect, domestic researchers of traditional Chinese medicine have done a lot of research on the hypoglycemic effect of traditional Chinese medicine [6], but there are few studies on the synergistic effect of hypoglycemic effect after processing. In order to explain the processing principle of the hypoglycemic effect of SAR, we have carried out many experimental studies. e research studies on the hypoglycemic effect of SAR have been reported mainly by our research group until now. We found the contents of three components, Timosaponin AIII, Timosaponin BIII, and Mangiferin, increased in the SAR and confirmed the hypoglycemic activity of these three components [7].
On the basis of previous research, this thesis used spontaneous type 2 diabetic db/db mice as experimental animals and used fasting blood glucose (FBG), oral glucose tolerance test (OGTT), glycated hemoglobin or glycosylated hemoglobin (HbAlc), serum resistin (RESISTEIN), fasting insulin (FINS), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) as indicators to evaluate the hypoglycemic effects of water decoction from AR (WAR) and SAR (WSAR) and ethanol extract from AR (EAR) and SAR (ESAR), and ultraperformance liquid chromatography coupled with a triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) was used to analyze the changes in the contents of Timosaponin AIII, Timosaponin BII, Timosaponin BIII, Mangiferin, Anemarrhenasaponin I, Anemarrhenasaponin Ia, Timosaponin AI, and Anemarrhenasaponin AII before and after being stir-baked with salt water. Combining the results of the efficacy and the component analysis, the principle of Chinese materia medica processing can be illustrated, that is, to explain why the hypoglycemic effect of SAR can be enhanced.

Animals.
ere were 30 healthy SPF 7-9-week-old male db/db mice and 6 db/m mice, weighing 50 g-60 g, provided by Changzhou Card Vince Laboratory Animal Co., Ltd. All experiments involving animals were approved by the Animal Care and Welfare Committee of Liaoning University of Traditional Chinese Medicine (use license no. SYXK(Liao) 2019-0004).

Effects of Anemarrhenae Rhizoma on Hypoglycemic
Activity in db/db Mice 3.1.1. Sample Preparation. Add 30 mL salt water (containing 3 g salt) to every 100 g AR decoction pieces, mix well and stir thoroughly, and stir-bake at 150°C∼160°C for 8 min [8].
Preparation of water decoction: weigh AR/SAR decoction pieces, add 15 times the amount of water to a group and decoct 3 times, each time for 1 h, filter the combined water decoction, and concentrate to make the concentration of 0.25 g·mL −1 of AR and SAR water decoction, respectively.
Preparation of ethanol extract: AR/SAR was extracted 3 times with 15 times the amount of 75% ethanol under reflux, 1 h each time, filtered, combined the filtrate, and concentrated until no alcohol smell to obtain the extract, which was dispersed in water to prepare ethanol extract of AR and SAR with a concentration of 0.25 g·mL −1 , respectively.

Grouping and Administration.
After adaptive feeding for 7 days and fasting for 12 hours, blood was collected from the tail vein to determine the fasting blood glucose level. e mice whose average blood glucose level was ≥11.0 mmol L −1 [9] can be used for experiments. Number the db/db mice with qualified blood glucose, and randomly group them using the Syntax window in SPSS. Numbers 1-6 are the db/ db control group, numbers 7-12 and 13-18 are the raw and salt Anemarrhenae water decoction groups, respectively, and numbers 19-24 and 25-30 are the raw and salt Anemarrhenae ethanol extract groups, respectively; db/m is the normal control group. Except for the db/m normal control group and the db/db control group, each group was given the corresponding liquid medicine for 2 weeks (5 g kg −1 d −1 ).

3.1.3.
e Effect on Fasting Blood Glucose (FBG) [10] in Spontaneous Type 2 Diabetic db/db Mice. After 7 days and 14 days of administration and fasting for 12 hours, blood was collected from the tail vein, and a blood glucose meter was used to determine the fasting blood glucose of mice (FBG).

3.1.4.
e Effect of Glucose Tolerance (OGTT) [11] in Spontaneous Type 2 Diabetic db/db Mice. After the last blood glucose measurement, a glucose solution (2 g·kg −1 ) was administered by gavage. e blood glucose value and the area under the curve (AUC) were measured for 0 h, 0.5 h, 1 h, and 2 h, and the glucose tolerance (OGTT) was calculated.

e Influence of Processing on the Compounds in
Anemarrhenae Rhizoma

Preparation of Sample Solution. Weigh raw and salt
Anemarrhena powder separately (each 10 g), and prepare the extract as described in Section 3.1.1 and then weigh, dissolve with 100 ml methanol, filter, and pass through a 0.22 μm microporous filter membrane. Take additional filtrate and set aside.

Mass Spectrometric
e results show that the 8 components are within their respective linear ranges, r > 0.9990, and the linear relationship is good.

Effects of AR and SAR on Glucose Tolerance (OGTT) in
Type 2 Diabetic db/db Mice. Compared with the db/db control group, after glucose load, the ethanol extract of SAR group significantly decreased blood sugar and glucose tolerance (AUC means glucose tolerance) at 0.5, 1, and 2 h (p < 0.01), while after 1 h and 2 h, the blood sugar and glucose tolerance of the water decoction group of AR and SAR and the ethanol extract group of SAR were significantly reduced (p < 0.01), indicating that Anemarrhenae Rhizoma has a good effect on improving glucose tolerance. e ethanol extract of SAR has the best effect. e results are shown in Table 4 and Figure 3.

Effects of AR and SAR on HbAlc, RESISTEIN, and FINS of Type 2 Diabetic db/db Mice.
Compared with the db/db control group, the water decoction and ethanol extract of AR and SAR had significant improvement on HbAlc, FINS, HOMA-IR, ISI, and RESISTEIN (p < 0.01 or p < 0.05), and the ethanol extract of SAR was superior to the water decoction. It indicated that the effect of improving insulin resistance of ESAR is the best. e results are shown in Table 5 and  Evidence-Based Complementary and Alternative Medicine oxide (NO) in the pancreas of type 2 diabetic mice (p < 0.01). e water decoction of AR and SAR and the ethanol extract of AR could also increase the content of nitric oxide (NO) in the pancreas of diabetic mice (p < 0.05). e ethanol extract of SAR has the best curative effect. e results are shown in Table 6 and Figure 5.

Measurement Results of 8 Components of the Sample.
According to calculations, the contents of these 8 components in the ethanol extract of AR and SAR are higher than those in the water decoction, and the contents of the ethanol extract of SAR are the highest, which proves that the hypoglycemic effect of SAR is better than that of AR. e results are shown in Table 7 and Figure 6.

Discussion and Conclusions
is paper focuses on the hypoglycemic effects of AR, which is enhanced after being stir-baked with salt water, as reported in our previous research.
In view of the fact that oxidative stress [19] is the main cause of aggravated impairment and dysfunction of pancreatic β-cells [20] in recent studies of type 2 diabetes mellitus, in addition to the basic indicators, such as fasting blood sugar, serum insulin, and glucose tolerance, SOD, NO, and MDA were also detected, which can reflect oxidative stress. Especially in the pathogenesis of type 2 diabetes mellitus, oxidative stress as the central link of pancreatic β-cell damage is more obvious. Oxidative stress can block insulin action pathways leading to insulin resistance (IR), reduce the insulin gene expression, and promote β-cell apoptosis [21]. In scientific research, the levels of oxidative stress are mainly determined by reactive oxygen species (ROS) [22], reactive nitrogen species (RNS) [23], and lipid peroxides. ROS induces oxidative stress in vivo, but it is difficult to determine directly because of its instability. RNS mainly includes nitric oxide and peroxynitrite anion. Lipid peroxide is the product of unsaturated fatty acid by ROS, which can indirectly reflect the level of oxidative stress in vivo. MDA is commonly used as product index, and SOD plays an increasingly important role in the body's antioxidant mechanism as antioxidant enzyme, which cannot be ignored.
Anemarrhenae Rhizoma stir-baked with salt water can significantly improve the activity of pancreatic islet β-cells, reduce the content of NO in cells, and can significantly increase the activity of SOD in pancreatic β-cells, as well as reduce MDA activity [7]. e mechanism of its damage to pancreatic β-cells may be related to the enhancement of cells' ability to scavenge oxygen free radicals, resistance to exogenous NO, inhibition of MDA factor, and effective protection of pancreatic β-cells.
e research results have shown that compared with the water decoction, the contents of Timosaponin AI, Anemarrhenasaponin AII, Timosaponin AIII, Timosaponin BII, Timosaponin BIII, Anemarrhenasaponin I, Anemarrhenasaponin Ia, and Mangiferin in the ethanol extract of AR and SAR were increased, and the 8 components both in the water decoction and in the ethanol extract of SAR were higher than those of AR. e research results also suggest that SAR has an improved therapeutic effect on carbohydrate metabolism and islet damage in diabetic mice. SAR is better than AR, and the ethanol extract of SAR has the most significant effect. Its hypoglycemic mechanism is related to reducing fasting blood glucose, improving glucose tolerance and insulin resistance, and protecting and repairing pancreatic islet tissue. It also suggests that these saponins are the material basis for the hypoglycemic effect of SAR. e hypoglycemic effect of the ethanol extract of SAR is better than that of the water decoction. e reason maybe the increase of the content of Timosaponin AIII, Timosaponin BIII, Mangiferin, and other components in the ethanol extract of SAR, which strengthens the hypoglycemic effect of SAR.
Anemarrhenae Rhizoma stir-baked with salt water has a long history of application, but the research on how processing enhances its hypoglycemic effect is very limited. e hypoglycemic effect experiment was combined with the analysis of chemical components to explain the influence of processing on the hypoglycemic effect of Anemarrhenae Rhizoma, which explains the traditional theory of processing in enhancing the effect of Anemarrhenae Rhizoma to nourish Yin for lowering fire from a modern point of view. It not only proves that the use of SAR in the treatment of diabetes conforms to the traditional Chinese medicine theory but also uses modern scientific knowledge to explain the traditional Chinese medicine theories, which can provide a useful modern research basis for the clinical use of SAR.

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 there are no conflicts of interest. 10 Evidence-Based Complementary and Alternative Medicine