Effects of Different Processed Products of Radix Codonopsis on Intestinal Flora of Rats with Spleen Deficiency

Jiabao MA, 2, Zhengteng YANG, Jiangcun WEI3*, Jian CUI, Song CHEN3, Dongmei HUANG3, Lifu WEI

1. First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 2. Guangxi Zhuang-Yao Pharmaceutical Engineering Technology Research Center, Nanning 530200, China; 3. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China

Abstract [Objectives] This study aimed to observe the effects of different processed products of Radix Codonopsis on intestinal flora in rats with spleen deficiency. [Methods] Rat models with spleen deficiency were established by bitter-cold purgation method with Radix et Rhizoma Rhei. Normal group, model group, Radix Codonopsis group, fried Radix Codonopsis group, rice-fried Radix Codonopsis group, honey-fried Radix Codonopsis group and bran-fired Radix Codonopsis group were designed. After subjecting to corresponding treatments, the changes in the quantity of intestinal microorganisms of the rats were detected. [Results] There was no significant change in the quantity of intestinal microorganisms of the rats in the normal group and model group. The rats in the rice-fried Radix Codonopsis group were administered after successful modeling, and the abundance of Bifidobacterium and Lactobacillus in the intestines of the rats increased, and the quantity of Escherichia coli and Staphylococci reduced. The rats in the Radix Codonopsis group, fried Radix Codonopsis groups, honey-fried Radix Codonopsis group and bran-fried Radix Codonopsis group were administered after successful modeling, and the abundance of Bifidobacterium and Lactobacillus in the intestines of the rats increased (the increases were smaller than those in the rice-fried Radix Codonopsis group), and the abundance of E. coli and Staphylococci reduced, close to normal levels. [Conclusions] Different processed products of Radix Codonopsis have obvious regulation effect on intestinal flora of rats with spleen deficiency, and the regulation effect of rice-fried Radix Codonopsis on rats with spleen deficiency is better than that of Radix Codonopsis, fried Radix Codonopsis, honey-fried Radix Codonopsis and bran-fried Radix Codonopsis.

Key words Radix Codonopsis, Processed product, Intestinal flora, Spleen deficiency rat model

1 Introduction

Radix Codonopsis (Dangshen in Chinese) is a commonly used traditional Chinese medicine, also known as Huangshen, Fangfeng Dangshen and Fangdangshen, and it is a perennial herbaceous plant. According to the 2015 edition of theChinesePharmacopoeia, Radix Codonopsis refers to dried roots ofCodonopsispilosula(Franch.) Nannf.,C.pilosulaNannf. Var. Modesta (Nannf.) LT Shen orC.tangshenOliv. Radix Codonopsis is sweet in taste, neutral in nature, metabolized through the spleen and lung channels, with functions of strengthening spleen, tonifying lung, invigorating spleen-stomach, replenishing qi, nourishing blood and engendering fluid. It is often used clinically for qi deficiency in spleen and lung, lack of appetite, burnout syndrome, deficiency of blood, shallow complexion and thirst[1-2]. Using the quantity ofBifidobacterium,Lactobacillus,EscherichiacoliandEnterococcusin mouse feces as the detection indices, the changes in the intestinal flora of the mice after administration were observed[3]. The results show that Radix Codonopsis has a good regulation effect on the intestinal flora of the mice. In this study, the effects of different processed products of Radix Codonopsis on the intestinal flora of mice with spleen deficiency were investigated to screen out which processed product of Radix Codonopsis is most conducive to the growth and reproduction ofBifidobacteriumandLactobacillus, to provide experimental basis for research on the interaction of active ingredients of Radix Codonopsis in the body and their mechanism of actioninvivo.

Intestinal flora is also closely related to spleen deficiency syndrome of traditional Chinese medicine. Related microecological study[4]show that the quantity of beneficial physiological bacteria such asBifidobacteriumandLactobacillusin the intestine of rats is negatively correlated with the severity of the symptoms of spleen deficiency in rats, that is, the more severe the symptom of spleen deficiency in rats, the smaller the quantity ofBifidobacteriumandLactobacillusin the intestine of rats, leading to disorder of intestinal flora. Spleen is one of the internal organs of humans and vertebrates, and has a role in regulating metabolism. It is one of the "five internal organs" in traditional Chinese medicine, and is called "the foundation of acquired constitution". Spleen is closely related to the various immune functions, physiological conditions and pathological conditions in the body[5].

2 Materials and methods

2.1Materials

2.1.1Apparatus and instruments. D1810C electronic analytical balance (Shanghai Shensheng Technology Co., Ltd.); HWS-26 electric-heated thermostatic water bath (Shanghai Qixin Scientific Instrument Co., Ltd.); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

2.1.2Drugs and reagents. The culture media used were purchased from Beijing Solarbio Science & Technology Co., Ltd. The pure water used was self-made.

2.1.3Medicinal materials. Radix Codonopsis (Shanxi, batch No. 180101762) and Radix et Rhizoma Rhei (Sichuan, batch No. 180307021) were purchased from Guangdong Kangmei Pharmaceutical Co., Ltd. Radix Codonopsis was identified as the dried roots ofCodonopsispilosula(Franch.) Nannf.,C.pilosulaNannf. var. modesta (Nannf.) L. T. Shen orC.tangshenOliv. by Professor Li Bin from Laboratory of Medicinal Plants, Guangxi University of Chinese Medicine. Radix et Rhizoma Rhei was identified as the dried roots and rhizome ofRhizomaofficinaleBaill. All the processed products were prepared by using the above-mentioned Radix Codonopsis in accordance with theNationalStandardforthePreparationofChineseHerbalPiecesand the 2015 edition ofChinesePharmacopoeia.

2.1.4Animal. Male SD rats of SPF grade, weighing (220 ± 20) g, were provided by the Experimental Animal Center of Guangxi Medical University (production license SCXK Gui 2014-0002; quality certificate 0001757). The rats were housed in air-conditioned cages[humidity (70±5)%, temperature (25±1) ℃]. They were subjected to routine feeding and provided with free access to feed and drinking water. Natural lighting was adopted.

2.2Methods

2.2.1Sample preparation. A certain amount (200 g) of each of Radix codonopsis, fried Radix Codonopsis, rice-fried Radix Codonopsis, honey-fried Radix Codonopsis and bran-fried Radix Codonopsis was pulverized and added with 10 times the amount of distilled water to prepare into suspension (0.38 g/mL), respectively. The rat models established were administered by gavage.

A certain amount (500 g) of Radix et Rhizoma Rhei was pulverized, soaked in 10 times the amount of water for 1 h, decocted at 85 ℃ for 15 min, filtered and stood to collect the supernatant. Water in the supernatant was recovered using a rotary evaporator at 60 ℃. Thus, the decoction of Radix et Rhizoma Rhei was obtained[6].

2.2.2Establishment of rat models of spleen deficiency. Rat models of spleen deficiency were established using bitter-cold purgation method with Radix et Rhizoma Rhei[7]. The specific procedure was as follows. After weighing the rats on the 1st d, each rat was intragastrically administered with 3 mL of 100% Radix et Rhizoma Rhei preparation. Administration was carried out twice a day, one at 8:00 and the other at 18:00, lasting for 9 d. The performance of rat models successfully established was as follows: showing blurred eyes, diarrhea, declined weight, yellow and dry hair, lack of energy, significantly reduced activity, hand and foot paralysis, weakness and squinting, and clumping together or curling up for fear of cold. The above performance could prove the success of modeling.

2.2.3Animal grouping and treatment. Seventy rats were randomly and evenly divided into seven groups: normal group, model group, Radix Codonopsis group, fried Radix Codonopsis group, rice-fried Radix Codonopsis group, honey-fried Radix Codonopsis group and bran-fried Radix Codonopsis group. The rats in the Radix Codonopsis group and fried Radix Codonopsis groups were administered intragastrically with corresponding processed products of Radix Codonopsis at a dose of 1 mL/100 g. The rats in the normal group and model group were given with equivalent amount of distilled water. The administration was carried out once a day and lasted for 14 d.

2.2.4Observation of changes in body weight of rats. The body weight of the rats in each group before and after the administration was measured and recorded. They were all weighed before intragastric administration.

2.2.5Observation of intestinal flora. The feces of each rat before and after administration were collected. A part of the feces of each rat were used for slide review using Gram staining, and the changes in the intestinal flora were observed under a microscope and recorded.

On the next day after the administration ended, each rat was anesthetized and dissected strictly according to aseptic operation. They were sacrificed after collecting blood from the veins. Then, six rats were randomly selected from each group, and the contents of their ceca were sampled with disposable inoculating loops, followed by removal of each organ. The content of cecum was inoculated into 4 kinds of media:Bifidobacteriummedium (BS),Lactobacillusmedium (LBS),E.colimedium[8]and Staphylococcus medium[9]. The culture was conducted at 37 ℃ for 48 h. According to different characteristics of the colonies, they were classified, and subsequently, each type of bacteria was subjected to pure culture, Gram staining, isolation and identification.

2.2.6Judgment basis for experimental results. According toTechnicalSpecificationforInspectionandEvaluationofHealthFood(2016 edition), the microorganisms were identified. The quantity ofBifidobacterium,Lactobacillus,StaphylococcusandE.colibefore and after the experiment and among the groups was compared.

2.2.7Statistical analysis. Data were analyzed statistically using SPSS 21.0 statistical software. Measurement data are expressed as mean±SD. For data of normal distribution, one-way ANOVA was used; for comparisons between any two groups,LSD-ttest was used; and for data with uneven variances, Games-Howell test was used. The significance level was set at α=0.05.

3 Results and analysis

3.1SignsobservationThe model rats began to show symptoms of spleen deficiency on the next day, such as diarrhea, thin stools, loss of appetite, abdominal distension, clumping together and fear of cold. During the subsequent modeling, the aforementioned symptoms of spleen deficiency were gradually aggravated. After the modeling was completed, the rats were subjected to administration treatment. The rats in the normal group acted normally. The rats in the model group still showed symptoms of spleen deficiency, such as laziness and low food intake. After administration of rice-fried Radix Codonopsis, the symptoms of spleen deficiency in the rats were eased to a certain extent, manifested by increased food intake, increased weight gain and more vigor. Compared with other administration groups of Radix Codonopsis, the recovery of spleen deficiency symptoms in the rice-fried Radix Codonopsis group was significantly better.

3.2ChangesinbodyweightofratsbeforeandafteradministrationThe experimental results show that before administration, compared with the normal group, the body weight of rats in the other groups significantly declined (P<0.05), indicating the success of modeling of spleen deficiency.

After administration, compared with the model group, the body weight of rats in the Radix Codonopsis administration groups increased significantly (P<0.05), indicating that different processed products of Radix Codonopsis can improve the symptoms of spleen deficiency in rats (Table 1).

Table1Changesinbodweightofratsafteradministration

n=10)

Note: 1. normal group; 2. model group; 3. Radix Codonopsis; 4. fried Radix Codonopsis group; 5. rice-fried Radix Codonopsis group; 6. honey-fried Radix Codonopsis group; 7. bran-fried Radix Codonopsis group. Compared with normal group,*P<0.05,**P<0.01; compared with model group,△P<0.05,△△P<0.01. The same as below.

3.3EffectsofdifferentprocessedproductsofRadixCodonopsisonBifidobacteriumandLactobacillusintheintestineofratsBefore administration, compared with the normal group, the abundance ofBifidobacteriumandLactobacillusin the rats of the other groups significantly decreased (P<0.05), indicating that the modeling of spleen deficiency was successful.

After administration, compared with the model group, the abundance ofBifidobacteriumandLactobacillusin the rats of the Radix Codonopsis groups increased significantly (P<0.05), indicating that different processed products of Radix Codonopsis can improve the symptoms of spleen deficiency in rats (Table 2).

3.4EffectsofdifferentprocessedproductsofRadixCodonopsisonStaphylococcusandE.coliintheintestineofrats

Before administration, compared with the normal group, the abundance ofStaphylococcusandE.coliin the rats of the other groups significantly increased (P<0.05), indicating that the modeling of spleen deficiency was successful.

Table2EffectsofdifferentprocessedproductsofRadixCodonopsisonBifidobacteriumandLactobacillusintheintestineofrats

Log10 (cfu/g)

After administration, compared with the model group, the quantity ofStaphylococcusandE.coliin the rats of the Radix Codonopsis administration groups decreased significantly (P<0.05), indicating that different processed products of Radix Codonopsis can improve the symptoms of spleen deficiency in rats (Table 3).

Table3EffectsofdifferentprocessedproductsofRadixCodonopsisonStaphylococcusandEscherichiacoliintheintestineofrats

Log10 (cfu/g)

4 Conclusions and discussion

Radix et Rhizoma Rhei is a traditional Chinese medicine for purgation[10-11]. It was first recorded inDivineFarmer’sClassicofMateriaMedica. Radix et Rhizoma Rhei is mainly metabolized through spleen, stomach, large intestine, heart and liver channels. Long-term or excessive administration of Radix et Rhizoma Rhei will damage the spleen and stomach and consume vital energy, leading to spleen deficiency. According to the theory of traditional Chinese medicine and the experience of establishing spleen deficiency animal models of many domestic scholars, if rat is administered with a large amount of Radix et Rhizoma Rhei decoction, spleen deficiency will appear, and as a result, the bacterial flora in the intestine of the rat is disturbed[12].

Through animal experiment, Song Keyuetal.[3]studied the effects of Radix Codonopsis and Poria on intestinal flora of mice. Using the quantity ofBifidobacterium,Lactobacillus,E.coliandEnterococcusin mouse feces as the detection index, the changes in mouse intestinal flora after administration were observed. The results show that Radix Codonopsis and Poria have better regulating effect on the intestinal flora of the mice. It suggests that Radix Codonopsis has a certain promotion effect on the reproduction ofBifidobacteriumandLactobacillus. Based on the conclusion that Radix Codonopsis has the effect of promoting the growth and reproduction of beneficial bacteria in the intestinal flora, decoctions of different processed products of Radix Codonopsis were used to treat rats with spleen deficiency, and it was found that the abundance ofBifidobacteriumandLactobacillusin the intestine of the rats increased, and the abundance ofE.coliandStaphylococcusreduced, returning to the normal levels or dropping below the normal levels. It suggests that suspensions of different processed products of Radix Codonopsis do have therapeutic effect on spleen deficiency syndrome. Based on the changes in the quantity of beneficial bacteria in the intestine, it could be concluded that rice-fried Radix Codonopsis has better curative effect on spleen deficiency syndrome.

Studies[13-14]have shown that codonolactones are an important material basis for spleen strengthening, volatile oils were dry ingredients, and polysaccharides from Radix Codonopsis show a certain immune regulation effect. However, in the animal body where Radix Codonopsis acted as a whole, the interactions between the three components mentioned above were not discussed, and the target and mechanism need to be explored and are also the focus of future research.