Insight into the secret of how indigo naturalis works: in vitro and in vivo validation of the critical role of bile

Background: Indigo Naturalis (IN) has positive therapeutic effects on cancer, leukemia, colitis, psoriasis, fever and other diseases that seriously endanger human health. It contains 10% organic matter and 90% inorganic matter with extremely hydrophobic. However, the clinical usage of IN is mainly by oral administration. And how IN exerts its effect after oral administration is still a secret remained to be discovered. Methods: In this study, in vitro and in vivo experiments were performed. The vitro experiments simulate the in vivo process of IN by different digestive uid and the effects of a small amount and normal amount of bile on the organic and inorganic matter release of IN were compared. And the surface morphology and elements of IN in different solutions were also observed. In addition, 2, 4-dinitrophenol-induced fever rat model was also used to investigate the intervention effect of IN on the fever of normal rats and bile duct ligation rats. Results: It was found that bile plays an important role in promoting the carrier dissolution and release of inorganic and organic matters from IN. And IN had better antipyretic effect on normal rats than on bile duct ligation rats, which further proved that bile is important to the curative effect of IN. Conclusions: The results indicate that the calcium carbonate carrier translates into a slightly soluble state in the acidic simulated gastric uid. And a small amount of organic matter was released from the calcium carbonate carrier. When IN entering the simulated intestinal uid which mixed with bile, a large amount of carrier dissolved, and organic substances such as indirubin dissolved out in large quantities, thereby being absorbed into the blood and exerting therapeutic effect. The above ndings have certain guiding signicance for the clinical application of IN. For patients with insucient bile secretion such as cholangiotomy, oral IN may have poor curative effect and even doesn't work.

Background IN is a Traditional Chinese Medicine with a long history of application. It is widely used in internal medicine, surgery, pediatrics, gynecology, infectious and other diseases [1]. The earliest medicinal e cacy of IN is recorded in Theory of Medicine which published in the Tang Dynasty. It can be taken internally or externally and used alone or in combination with gypsum and bupleurum [2]. IN is a dry powder, blocks or granules derived from dried leaves and stems of Baphicacanthus cusia (Nees) Bremek., Polygonum tinctorium Ait., or Isatis indigotica Fort [3]. The traditional manufacturing method is to harvest the stems and leaves in autumn, place them in the soaking pool and introduce the river water till they are submerged completely. Then immerse and ferment for several days. When the pool liquid turn green [4] remove the residue, add lime, stir fully, stand for a 1 to 2 days to make Ca (OH) 2 react with CO 3 2in the immersion liquid to generate CaCO 3 crystal nucleus, providing a carrier for sedimentation and attachment for indigo and indirubin. Then discard the supernatant and collect the sediment at the bottom and move it to the small pool, let it stand, and then discard the supernatant to get crude indigo. The crude indigo is puri ed through water y removal and other impurities, and after drying, the nished product is obtained [5].
Due to its manufacturing process, the material composition of IN is about 10% organic and about 90% inorganic matter [6]. Its organic components are mainly indole alkaloids, such as indigo, indirubin [7], etc. Preparation of simulated gastric uid (SGF) [12] 16.4 mL of dilute hydrochloric acid was added to 800 mL of distilled water, shaken and diluted to 1000 mL.
The volume ow rate is 1 mL·min -1 . Column temperature was 25°C. The injection amount is 10 μL.

Preparation ofdissolution samples from IN
Collect the ltrates obtained in Figure 1 under item 3.1.1 respectively, accurately measure 10 mL, extract with 30 mL ethyl acetate for 30 min each time, and extract 5 times. The upper liquid was placed in an evaporation dish and evaporated in a water bath, 10 mL DMF was added to dissolve. After cooling and passing 0.22 microporous membrane, take the continuous ltrate. The rats were subjected to adaptive measurement of anal temperature every morning and evening (at 9:00 a.m. and 17:00 p.m.) for 3d before the experiment [13]. Before each measurement, apply a small amount of glycerin on the probe of the electronic thermometer, insert the rat's rectum 3 cm, (make sure that the depth of each insertion is consistent), record the temperature after the reading is stable. And rectal temperatures with uctuations beyond 0.5°C between the morning and evening measurements were excluded. A total of 60 rats were randomly divided into six groups (n = 10 per group) according to weight, including normal control group, model group, IN group, bile duct ligation normal (BLN) group, bile duct ligation model group (BLM), bile duct ligation IN (BLIN) group.

Bile duct ligation
Rats of BLN group, BLM and BLIN group were anesthetized with 20% urethane (1g·kg -1 ). The belly of the rat is shaved, and the abdomen was disinfected 3 times with 75% alcohol. The abdominal cavity was opened along the midline of the abdomen, the common bile duct was ligated, sutured, and the wound was disinfected with iodophor. Incubate until awake, and feed in a single cage for 3 days [14].

Model preparation and drug intervention
Prior to the modeling, the rats were fasted for 12 h (free access to water). The rectal temperature of all rats was measured twice prior to modeling (original temperature). Subsequently, 2,4-dinitrophenol dissolved in physiological saline (3 mg·mL -1 ) was injected in the back of rats (10 mL·kg -1 ) to induce fever in rats except normal group and the BLN group. Fever in rats (both body temperature increased by 0.3°C), accompanied by tremors, shortness of breath and atrophy, indicate that the model was successfully created. Immediately after the successful modeling, the IN group and the BLIN group were intragastric administrated with the IN solution (0.9 g·kg -1 ), and others groups were gavaged distilled water according to body weight.
After drug intervention treatment, the anal temperature was measured once every 30 min for each group and recorded continuously for 5 h. And HT-19 Infrared thermal imager is used to take pictures of different groups of rats to observe the body temperature status. The change curve of anal temperature was plotted and processed by SPSS 21.0 statistical software.

Statistical analysis
All data were reported as x(_) ± s. Signi cance of each group was analyzed with one-way analysis of variance. The values of various groups were evaluated by one-way ANOVA and difference test. *P < 0.05 and **P < 0.01, calculated using SPSS software (version 21), were considered statistically signi cant.

Inorganic matter dissolve from IN
The concentration of calcium ion in different solutions was calculated respectively. It was discovered that the SSBH group has higher concentration than other groups. And it was bile rather than stir that play an important role in the solubility of calcium ion. It was showed in gure 2. In summary, the slight dissolution of the carrier does not release a large amount of active ingredients, and bile rather than stirring plays an important role in the process of releasing the active ingredients and acting.
Bile on antipyretic effect of IN It can be observed that the temperature of the model group is signi cantly higher than that of the other groups in Figure 6. In the fever rats, the tails are red, and the temperature of the control group does not change much, the perianal is redder, while the tail is bluish compared to other groups with lower temperature. Of course, the perianal temperature measured by the infrared instrument is far less accurate than the rectal temperature measured by the electronic thermometer, but you can visually see the fever rats in different group.
The rectal temperatures of the rats at different time points were recorded to assess the pyrexia rat model versus control rats. The temperature rise value of each group of rats at the temperature monitoring point  [15][16][17]. It also indicates that the normality of bile secretion has a signi cant in uence on the effectiveness of some drugs when used in clinically. For patients with insu cient bile secretion, oral IN may not be effective as expected or even has no effect. Because bile is secreted into the intestine, it can be inferred that the main absorption site of IN after oral administration is in the intestine.
The lipopolysaccharide model requires intraperitoneal injection, and it is a bacterial fever model. The use of lipopolysaccharide model may cause abdominal sutures in bile duct ligation rats, such as dehiscence and low immunity. Moreover, the model of endotoxin may lead to lower survival rate of surgery rats, so it should not be used. The dry yeast fever model is due to local skin ulceration after injection, which is more harmful to rats after surgery. However, 2,4-dinitrophenol is a strong metabolic stimulant which can stimulate aseptic in ammation in animals by injecting subcutaneously and is a non-infectious fever model. It induced fever quickly and the harm is small. Thus, 2,4-dinitrophenol rats fever model was used to induce fever. The results showed that the normal rats and the bile duct ligation rat model group had stable fever, and there was no signi cant difference which means the selected model is reasonable. And normal group has better antipyretic effect than bile duct ligation group at the same dose of IN. It is also proved that bile is the key to the antipyretic effect of IN. Of course, according to literature reports, bile duct ligation will affect the change of intestinal ora, such as Bi dobacteria, etc., which may also be one of the in uencing factors of IN's antipyretic effect [21,22].
In this experiment, a simple method was used to explore the oral absorption mechanism of IN.

Declarations
Ming Yang, Dingkun Zhang, Li Han and Xiaorong Xu conceived and designed the paper; Xiaorong Xu, Fei Ran and Huamei Gou performed the experiment. Yanan He and Fang Wang gave some advice for improving the paper; other people gave some advice for improving the picture; Xiaorong Xu wrote the paper.

Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was conducted in strict accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocol and experimental designs were approved by the Ethical Committee of Chengdu University of Traditional Chinese Medicine (Approval ID: 2017BL-003). All possible steps were taken to avoid the animals' suffering at any stage of the experiments. At the end of the study, the animals were sacri ced following anesthesia with excessive urethane.