Traditional Uses, Origins, Chemistry and Pharmacology of Bombyx batryticatus: A Review

Bombyx batryticatus (B. batryticatus), a well-known traditional animal Chinese medicine, has been commonly used in China for thousands of years. The present paper reviewed advances in traditional uses, origin, chemical constituents, pharmacology and toxicity studies of B. batryticatus. The aim of the paper is to provide more comprehensive references for modern B. batryticatus study and application. In Traditional Chinese Medicine (TCM) culture, drugs containing B. batryticatus have been used to treat convulsions, headaches, skin prurigo, scrofula, tonsillitis and fever. Many studies indicate B. batryticatus contains various compounds, including protein and peptides, fatty acids, flavonoids, nucleosides, steroids, coumarin, polysaccharide and others. Numerous investigations also have shown that extracts and compounds from B. batryticatus exert a wide spectrum of pharmacological effects both in vivo and in vitro, including effects on the nervous system, anticoagulant effects, antitumor effects, antibacterial and antifungal effects, antioxidant effects, hypoglycemic effects, as well as other effects. However, further studies should be undertaken to investigate bioactive compounds (especially proteins and peptides), toxic constituents, using forms and the quality evaluation and control of B. batryticatus. Furthermore, it will be interesting to study the mechanism of biological activities and structure-function relationships of bioactive constituents in B. batryticatus.


Introduction
Bombyx batryticatus (B. batryticatus) is the dried larva of Bombyx mori L. (silkworm of 4-5 instars) infected by Beauveria bassiana (Bals.) Vuill [1]. It is one of the most popular traditional Chinese medicines, called "Jiangcan" in Chinese vernacular and has been used in China for thousands of years. In addition, it is also widely used in Korea and Japan [2]. B. batryticatus is derived from silkworm spontaneously infected by Beauveria bassiana originally [3]. Currently, it is mainly produced through artificial breeding techniques by artificial inoculation of Beauveria bassiana [4].

Origin
B. batryticatus (Figure 1), derived from silkworm spontaneously infected by Beauveria bassiana, is the by-product of sericulture, which was described in "Sheng Nong's herbal classic" (Han Dynasty), "Xin Xiu Ben Cao" (Tang Dynasty), "Zheng Lei Ben Cao" (Song dynasty), "Tang Ye Ben Cao" (Yuan dynasty) and "Ben Cao Pin Hui Jing Yao" (Ming dynasty). Dictionary of Chinese Pharmacy by Chen (2010) revealed the formation of B. batryticatus that before silkworm became moth, it was infected by Beauveria bassiana and eventually died [32]. In addition, the lethal mechanism is that when spore of Beauveria bassiana infected silkworm, it can secrete chitinase, then dissolve the epidermis and body wall of silkworm and invade into its body, continuously reproduce and eventually cause the death of silkworm. After silkworm is infected by Beauveria bassiana, it becomes stiff and its surface covered with white conidias of Beauveria bassiana [33].

Origin
B. batryticatus (Figure 1), derived from silkworm spontaneously infected by Beauveria bassiana, is the by-product of sericulture, which was described in "Sheng Nong's herbal classic" (Han Dynasty), "Xin Xiu Ben Cao" (Tang Dynasty), "Zheng Lei Ben Cao" (Song dynasty), "Tang Ye Ben Cao" (Yuan dynasty) and "Ben Cao Pin Hui Jing Yao" (Ming dynasty). Dictionary of Chinese Pharmacy by Chen (2010) revealed the formation of B. batryticatus that before silkworm became moth, it was infected by Beauveria bassiana and eventually died [32]. In addition, the lethal mechanism is that when spore of Beauveria bassiana infected silkworm, it can secrete chitinase, then dissolve the epidermis and body wall of silkworm and invade into its body, continuously reproduce and eventually cause the death of silkworm. After silkworm is infected by Beauveria bassiana, it becomes stiff and its surface covered with white conidias of Beauveria bassiana [33]. With development of prevention technology of silkworm diseases, the source of B. batryticatus was significantly deficient. Thus, for meeting the market demands, its artificial breeding techniques, namely artificial inoculation of Beauveria bassiana, have received more attention and obtained certain development in recent years [4]. The detailed procedure of artificial breeding of B. batryticatus is as follows: Beauveria bassiana is mixed with warm water and sprayed on silkworms of 4-5 instars; after inoculation for 15-20 min, silkworms are fed with mulberry leaves, and fed every 5.0-6.0 h until they become stiff and white; finally, stiff silkworms are mixed with lime and dried in a ventilated place. The temperature and humidity of the feeding room should be set at 24.0-26.0 °C and 90.0%, respectively [14].
It was recorded that B. batryticatus firstly appeared in Yu county, Henan province in Qin and Han Dynasties [3]. During Tang and Song Dynasties, Henan and Shandong were main producing regions of B. batryticatus recorded in "Ben Cao Tu Jing and Zheng Lei Ben Cao". Later, during Ming and Qing Dynasties, its main regions moved to south area, such as Jiangsu and Zhejiang, which was recorded in "Ben Cao Chong Yuan". Subsequently, Sichuan and Guangdong became the main With development of prevention technology of silkworm diseases, the source of B. batryticatus was significantly deficient. Thus, for meeting the market demands, its artificial breeding techniques, namely artificial inoculation of Beauveria bassiana, have received more attention and obtained certain development in recent years [4]. The detailed procedure of artificial breeding of B. batryticatus is as follows: Beauveria bassiana is mixed with warm water and sprayed on silkworms of 4-5 instars; after inoculation for 15-20 min, silkworms are fed with mulberry leaves, and fed every 5.0-6.0 h until they become stiff and white; finally, stiff silkworms are mixed with lime and dried in a ventilated place. The temperature and humidity of the feeding room should be set at 24.0-26.0 • C and 90.0%, respectively [14].
It was recorded that B. batryticatus firstly appeared in Yu county, Henan province in Qin and Han Dynasties [3]. During Tang and Song Dynasties, Henan and Shandong were main producing regions of B. batryticatus recorded in "Ben Cao Tu Jing and Zheng Lei Ben Cao". Later, during Ming and Qing Dynasties, its main regions moved to south area, such as Jiangsu and Zhejiang, which was recorded in "Ben Cao Chong Yuan". Subsequently, Sichuan and Guangdong became the main producing regions of B. batryticatus. Currently, the main regions of B. batryticatus bred artificially are Sichuan, Jiangsu, Zhejiang, Guangdong, Shandong and Guangxi in China, and the quality of B. batryticatus in Sichuan is considered to be the best [34].

Chemistry
There are various chemical constituents in B. batryticatus, including protein and peptides, fatty acids, flavonoids, nucleosides, steroids, coumarin, polysaccharide and others. In this section, the major chemical constituents and structures of B. batryticatus are presented (Table 2 and Figure 2).

Proteins and Peptides
As a traditional animal medicine, the main chemical constituents in B. batryticatus are proteins. It is reported that the content of proteins in B. batryticatus varies in the range within 43.9-74.3% [45,55]. Currently, some research on peptides in B. batryticatus have been reported. BB octapeptide is a novel platelet aggregation inhibitory peptide isolated from B. batryticatus, and its molecular mass and the Cyclo-(Ala-Pro) ACIBB -The molecular mass is 1200.0 Da, and consisting of 7 kinds of amino acids [40] Enzymolysis polypeptides by pepsin - The molecular mass were 500.0-1000.0 Da, and the number of amino acid was less than 10 [41] Homoarginine (2)

Polysaccharide
One study of Ying et al. (2015) showed that polysaccharide yield of B. batryticatus was about 4.4% and it possessed good antioxidant activity [56]. In addition, BBPW-2 was isolated from B. batryticatus

Trace Elements
18 trace elements have been found in B. batryticatus, including Al, Fe, Ca, Mg, P, B, Ba, Cu, Cr, La, Mn, Ni, Pb, Sr, Ti, U, Y and Zn. Among them, the contents of Al, Fe, Zn, La and Mn were relatively high [57].

Effects on Nervous System
The characteristic pharmacological activity of B. batryticatus is the effects on nervous system, including anticonvulsant and antiepileptic effects, hypnotic effects, neurotrophic effects and others. The beauvericin can significantly prolong latent period of nikethamide-induced and isoniazid-induced convulsion in mice (125.0 and 250.0 mg/kg, s.c.) [58,59]. In addition, β-sitosterol and ergost-6,22-dien-3,5,8-triol were demonstrated to obviously prolong latent period of isoniazid-induced convulsion in mice (125.0 mg/kg, s.c.) [59]. Chloroform fraction of ethanol extract of B. batryticatus at dose of 20.0 g/kg showed significant effect on nikethamide-induced convulsion in mice [60] [61]. Later, another interesting study reported that ammonium oxalate (30.0 and 60.0 mg/kg) also can inhibit epileptic discharge frequency, amplitude, time and pyramidal cell necrosis in hippocampus region of epileptic rats induced by penicillin [62].
In 2003, it was reported that ethanol extracts of B. batryticatus had a significant hypnotic effect on mice (25.0 g/kg, p.o. or 12.5 g/kg, s.c.) and rabbits [63]. The extracts (extracted by water and precipitated by ethanol) of B. batryticatus (20.0 g/kg, p.o.) were found to exhibit sedation effect on mice through inhibiting its spontaneous activity [64].

Anticoagulant Effect
Anticoagulant effect is another characteristic pharmacological activity of B. batryticatus. In 2014, it was reported that BB octapeptide, a novel peptide, can inhibit rabbit platelet aggregation induced by collagen and epinephrine in vitro, with the IC 50 values of 91.1 and 104.5 µM, respectively [35]. In addition, BB octapeptide also significantly prevented paralysis and death in pulmonary thromboembolism model at doses of 10.0, 30.0 and 50.0 mg/kg, and significantly reduced ferric chloride-induced thrombus formation in rats (5.0, 10.0 and 20.0 mg/kg) [35]. One investigation by Wang et al. (1989) revealed that water extracts of B. batryticatus (20.0 mg/mL) could inhibit blood coagulation [67]. Zhao et al. (2005) demonstrated that increasing total concentration of ammonium oxalate in water extracts of B. batryticatus (33.7-42.3 mg/mL) can prolong TT (thrombase time) [68]. ACIBB (9.0, 18.0 and 36.0 mg/kg, i.v.), belonging to peptide, can significantly inhibit venous thrombosis in rats dose-dependently, by decreasing the contents of Fbg (fibrinogen) and PLg (plasminogen), increasing the activities of tPA (tissue plasminogen activator) and AT-III (antithrombin-III), as well as prolonging APTT (activated partial thromboplastin time), PT (prothrombin time) and TT [69]. Similarly to ACIBB, water extracts of B. batryticatus (350.0 mg/kg, i.v.) also possessed fibrinolytic activity and inhibited venous thrombosis [70]. Injection of B. batryticatus (150.0 mg/L) was reported that can also inhibit venous thrombosis through increasing tPA activity and decreasing PAI-1 activity [71].

Antitumor Effect
Numerous studies have been conducted on antitumor effects of B. batryticatus in recent years. B. batryticatus possesses significant anti-proliferative effects on human cancer cell lines, such as cervical cancer, liver cancer and gastric cancer [8]. In 2011, it was reported that ethanol extracts of B. batryticatus possessed significant anti-cervical cancer effect against HeLa cells at concentrations of 3.0-11.0 mg/mL, and anticancer mechanisms may be associated with induction of apoptosis by down-regulating the expression of Bcl-2 [72]. Another study reported that flavonoids isolated from B. batryticatus (50.0-500.0 µg/mL) also showed strong anti-cervical cancer activities through suppressing proliferation of HeLa cells in a concentration-dependent manner [73]. Later, an oligosaccharide BBPW-2 in B. batryticatus was demonstrated to have notable anti-cervical cancer (HeLa), anti-liver cancer (HepG2) and anti-breast cancer (MCF-7) activities above the dose of 1.0 mg/mL, and the action mechanism was that BBPW-2-induced cellcycle disruption in the G0/G1 and G2/M phases of early and late apoptotic as well as necrotic cells [52]. In addition, ethanol extract of B. batryticatus also had significant anti-cervical cancer activity against HeLa cells with IC 50 value of 1.7 mg/mL by inducing apoptosis via the regulation of the Bcl-2 and Bax [74]. Recently, it has been reported that ethanol extract of B. batryticatus can induce apoptosis of human gastric cancer cells SGC-7901 through upregulating expressions of Bax and P21 and downregulating Bc1-2 expressions with IC 50 value of 3.2 mg/mL [75]. Another investigation demonstrated that ergosterol, β-Sitosterol and palmitic acid isolated from B. batryticatus exerted significant anti-melanoma activities at the lowest concentrations of 0.1, 0.1 and 0.3 mmol/L, respectively [76].

Effects on Viruses
In 2016, one study demonstrated that the supernatant (after ethanol extraction and water precipitation) of B. batryticatus possessed antiviral effects against RSV viruses, and the EC 50 value was 2.7 × 10 −2 g/mL [79]. Interestingly, the research of Zhang et al. (2014) indicated that ethanol extracts of B. batryticatus can significantly increase the virulence of HearNPV via inhibition of the ALP (alkaline phosphatase) activity at concentrations of 40.0-80.0 µg/mL [80].

Antioxidant Effect
Reactive oxygen species (ROS) is one of main causes of various types of diseases. In addition, recently, increasing studies have been performed on the antioxidant effect of B. batryticatus. In 2013, the study of Jiang et al. (2013) demonstrated that flavonoids isolated from B. batryticatus had strong abilities to scavenge DPPH radicals and hydroxyl radicals at concentrations of 5.0 × 10 −3 -0.1 mg/mL and 0.1-0.4 mg/mL, respectively [73]. Another investigation reported that methanol extract of B. batryticatus possessed notable DPPH radical scavenging, ferric ion-scavenging and lipoxygenase-scavenging activities at the lowest concentrations of 2.0, 8.0 and 4.0 mg/mL, respectively [81]. Later, polysaccharides isolated from B. batryticatus possessed a powerful hydroxyl radical-scavenging effect and reducing power at concentrations of 2.5 × 10 −2 -0.3 mg/mL [56]. In addition, water extracts of B. batryticatus (1.0 × 10 −7 -1.0 × 10 −6 ) were reported to possess notable antioxidant effects through inhibiting lipid peroxidation and enhancing SOD activity [66].

Other Pharmacological Effects
Increasing investigations suggest that B. batryticatus possesses a wide range of other biological activities, such as hypoglycemic effects, anti-fertility effects, improving immune function effects and others. It was reported that flavonoids isolated from B. batryticatus can significantly promote proliferation of HEK293 normal human embryo kidney cell lines at concentrations of 50.0-500.0 µg/mL [73].  [81]. Another investigation revealed that powder of B. batryticatus presented notable hypoglycemic effects in clinical use at the dose of 15.0 g/day for 2 months (p.o.) [82,83]. Additionally, powder of B. batryticatus was also reported to relieve headache caused by disturbing-up of liver Yang at a dose of 18.0 g/day for 3 days (p.o.) in clinic [84]. In 2002, one interesting study indicated that water extracts of B. batryticatus exerted significant anti-fertility effect on mice, and the results showed that water extracts can significantly reduce the weight of ovary, uterus and pregnancy rate in female mice, and increase the weight of testes and seminal vesicles in male mice [85]. Furthermore, another study reported that polysaccharide isolated from B. batryticatus can significantly improve immune function via increasing the immune organ weights, improving phagocyte phagocytosis and lymphocyte transformation rate [86].

Summary of Pharmacological Effects
B. batryticatus possesses a wide spectrum of pharmacological effects, including effects on the nervous system, anticoagulant effects, antitumor effects, antibacterial and antifungal effects, effects on viruses and antioxidant effects, etc. (Table 4). These pharmacological effects show that the extracts and the compounds from B. batryticatus can used to prevent or treat certain diseases, in particular convulsions, epilepsy, thrombus and cancer. However, there is not enough systematic data on chemical compounds of B. batryticatus and their pharmacological effects. Reducing ferric chloride-induced thrombus formation BB octapeptide 5.0 mg/kg (i.v.) in vivo [35] Inhibiting blood coagulation fibrinolytic Water extracts 20.0 mg/mL in vitro [67] Prolonging TT Increasing whole the concentration of ammonium oxalate in water extracts 33. 7 mg/mL in vitro [68] Inhibiting venous thrombosis and prolonging APTT, PT, TT ACIBB 9.0 mg/kg (i.v.) in vivo [69] Water extracts 350.0 mg/kg (i.v.) in vivo [70] Increasing tPA activity and decreasing PAI-1 activity Injection 150.0 mg/L in vitro [71] Antitumor effect

Toxicity
Throughout its long history, B. batryticatus has been generally considered to be a safe TCM in China [5,14]. However, recent poisoning accidents of B. batryticatus were reported by numerous investigations, which is not consistent with traditional understanding of B. batryticatus safety.  [87][88][89]. Based on the literature, it can be found that occurrences of poisoning accidents for B. batryticatus mainly result from the following reasons: overdose and misuse of B. batryticatus, and quality problems caused by non-standard procedure of production and processing [87][88][89][90][91][92][93][94][95]. Furthermore, as a traditional animal medicine, B. batryticatus is easily contaminated by aflatoxin, which is regarded as carcinogenic or a teratogenic toxic substance in the procedure of processing, storage and transportation [96]. Therefore, it is urgent and important to standardize methods of production and processing and select the proper doses according to the using form of B. batryticatus to avoid adverse reactions and even poisoning.
It was reported that metabolism of ammonium oxalate in the body can produce ammonia easily, and high content of ammonium oxalate may cause blood ammonia poisoning [97]. The content of ammonium oxalate in B. batryticatus is in the range of 5.0-13.0% [53,54]. Thus, overdosing B. batryticatus can possibly cause poisoning. Additionally, toxins secreted by Beauveria bassiana when using infected silkworm, such as beauvericin, chitosan, chitinase and cellulase, can induce cell death procedurally [9]. Currently, the recognized cause of adverse reactions of B. batryticatus is an allergic reaction. Some allogeneic proteins in B. batryticatus, can cause sensitization, immune response and even cause metabolic disorder and dysfunction of central nervous system [97]. One investigation demonstrated that proteins secreted by Beauveria bassiana can cause adverse effects on mice [97]. However, to date the specific constituents causing adverse reactions or poisoning have not been clarified in B. batryticatus. Thus, further studies should be carried out to confirm which constituents are causing side effects or poisoning in B. batryticatus and explore corresponding content ranges.

Future Perspectives and Conclusions
B. batryticatus is one of the most important and frequently used traditional animal medicines, which has been used to treat convulsions, cough, asthma, headaches, skin prurigo, scrofula, tonsillitis and other diseases in China. Recently, B. batryticatus has received increasing attention. However, certain aspects still need to be further studied and explored.
There is limited research on bioactive compounds and the mechanism of biological activities of B. batryticatus. Thus, it is essential to strengthen research on bioactive compounds, action mechanisms of the bioactive compounds and their structure-function relationships in B. batryticatus. Current investigations of B. batryticatus mainly focus on its small molecule compounds, but rarely investigate its macromolecular compounds. In addition, as an animal Chinese medicine, the main chemical constituents in B. batryticatus are proteins. Therefore, future investigations of B. batryticatus could be concentrated on its macromolecular compounds, particularly its proteins and peptides. In addition, mechanisms of biological activities of B. batryticatus should be further explored with techniques of modern molecular biology and pharmacology.
Many monographs of TCM record that powder of B. batryticatus is used directly in a total of 65 prescriptions where B. batryticatus is as the main drug [5,14]. However, in the Pharmacopoeia of the People's Republic of China of all editions except 1963 edition, the only using form of B. batryticatus is decoction. Therefore, further studies should be done to explore which using form (decoction or powder) of B. batryticatus is more reasonable and scientific. Furthermore, based on scientific using form of B. batryticatus, further studies should be done to analyze reasons of adverse reaction or poisoning caused by B. batryticatus and then to establish its safety evaluation system.
Lack of standardized methods of production and processing is another issue of B. batryticatus. In the process of production, lime is often used to dry silkworm infected by Beauveria bassiana to avoid contamination by miscellaneous bacteria, but lime lacks quality standard and contains a high content of heavy metal and other toxic substances, which seriously affects the quality and safety of B. batryticatus [4]. When B. batryticatus is processed by stir-frying with bran to a yellowish color, processing degree is mainly evaluated by experience of pharmaceutical worker, which lacks quantifiable indices and is not objective. Thus, it is crucial to standardize the procedure of production and processing using modern technologies for ensuring quality of B. batryticatus.
Additionally, as an animal medicine containing complicated compounds, quality evaluation and control of B. batryticatus remains challenging for modern researchers. Currently, quality criteria of B. batryticatus in the Pharmacopoeia of the People's Republic of China only includes a description, microscopic identification, check (impurity, contents of water, total ash, acid insoluble ash and aflatoxin) and extract [1], which is inadequate to reflect the holistic quality of B. batryticatus. Therefore, it is urgent and important to establish suitable quality evaluation and control systems that can reflect the holistic quality of B. batryticatus, such as the fingerprint of the protein or peptide.
In conclusion, this paper provides a comprehensive overview on the traditional uses, chemistry, pharmacology and toxicity of B. batryticatus. In addition, this review also provides some trends and perspectives for the future development of B. batryticatus. Jianghua Li sorted out the references; He Xiao and Yongchuan Li drawn the structures of chemical constituents, Wei Peng designed Pharmacology section; Chunjie Wu conceived and designed the whole structure of the review.

Conflicts of Interest:
The authors declare that there is no conflict of interest.