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Isatis indigotica: a review of phytochemistry, pharmacological activities and clinical applications

Abstract Objectives Isatis indigotica Fort. (I. indigotica) is an herbaceous plant belonging to Cruciferae family. Its leaf (IIL) and root (IIR) are commonly used in traditional Chinese medicines (TCMs) with good clinical efficacies such as clearing away heat and detoxification, cooling blood and reducing swelling. This review aimed to provide a systematic summary on the phytochemistry, pharmacology and clinical applications of I. indigotica. Key findings This plant contains alkaloids, organic acids, flavonoids, lignans, nucleosides, amino acids, and steroids. Previous pharmacological researches indicated that I. indigotica possesses promising antivirus, antibacterial, immunoregulatory, anti-inflammation, and cholagogic effects. Importantly, it can inhibit various viruses, such as influenza, hepatitis B, mumps, herpes simplex, cytomegalovirus, and coxsachievirus. Clinically, it is frequently used to treat various viral diseases like viral influenza, parotitis and viral hepatitis. Consequently, I. indigotica may be beneficial for the prevention and treatment of coronavirus disease 2019 (COVID-19). Summary This paper reviewed the chemical constituents, pharmacological effects and clinical applications of I. indigotica which may guide further research and application of this plant.


Antiviral activity
Epigoitrin, an alkaloid from I. indigotica, can reduce the susceptibility to H1N1 virus and the production of pro-inflammatory cytokines to alleviate pneumonia in restraint-stressed mice. [41] Plant-derived compounds such as indigotin, sinigrin, aloe-emodin and hesperetin display anti-SARS coronavirus effects, effectively blocking the cleavage processing of the 3C-like protease. [40,41] The injection of IIL extracts can inhibit the infection and proliferation of influenza A, encephalitis B, mumps viruses, etc. [42] The result from the hemagglutination titer test showed a direct inhibitory effect of IIL against influenza A virus. [43] However, there are few studies on its antiviral mechanism of action. 4(3H)-quinazolinone, a compound isolated from the leaves, has the capacity to inhibit influenza and coxsackie virus. [44] In the early stage of viral myocarditis (VMC), the leaves may improve and protect the myocardial cells by inhibiting the synthesis of the virus, enhancing the phagocytosis of leukocytes and reducing the permeability of capillaries. [45] The root aqueous extract can inhibit human H7N9 avian influenza virus in vitro possibly by blocking the absorption of H7N9 avian influenza virus to host cells by inhibiting the hemaglutinin of H7N9 avian influenza virus, so as to prevent the virus invading the host cells. [46] It has a good curative effect on virus-caused pharyngitis, acute upper respiratory tract infection and pneumonia, especially catarrhal inflammation such as cough, nasal obstruction, runny nose and sneeze. [47] Polysaccharides from I. indigotica can inhibit hepatitis B virus (HBV) in vitro, reduce extracellular and intracellular DNA level of HBsAg, HBeAg and HBV in HepG2.2.15 cells in a time and dose-dependent manner. [48,49] Peptides reduces the mortality of mice infected with influenza virus and inhibits the proliferation of the virus. [50] Aqueous extract of leaves can antivirus such as HSV-II, Dengue virus II and Cytomegalovirus. [51,53] Aqueous extract of roots can anti HSV-I, inhibits virus replication and proliferation in cells. [52] Antibacterial activity The aqueous, ethanol and n-butanol extracts of the leaves have antibacterial effects on Staphylococcus aureus and Escherichia coli. [53,54] The leaf decoction showed an antibacterial effect in vitro on Staphylococcus aureus, Staphylococcus albus, Streptococcus A and Streptococcus B by use of disk diffusion test. [55] Tryptanthrin, a component isolated from the leaves, has strong inhibitory effects on Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, and Microsporum canis, which can cause tinea pedis. [56,57] The roots have a broad-spectrum antibacterial effect, in which tryptanthrin is the main antibacterial active ingredient. The root aqueous extract can inhibit Escherichia coli, Staphylococcus epidermidis, Pneumococcus, Himophilus influenzae, and Streptococcus. [58] The total organic acids from roots also show strong antibacterial activity on Escherichiacoli by cylinder-plate test. [59,60] Salicylic acid can inhibit excessive release of TNF-α and NO in serum of mice, [61] and the roots decoction can decrease the levels of TNF-α and IL-6 in peritoneal macrophages of mice. [62] Anti-endotoxin Bacterial endotoxin is the lipopolysaccharide component existing in the extracellular of gram-negative bacteria, which can stimulate the body's defence system to release inflammatory factors, such as tumour necrosis factor and nitric oxide, causing fever, disseminated intravascular coagulation, multiple organ failure, and even death. [63,64] The leaf extract can directly neutralize and degrade endotoxin to reduce the thermophilic and lethality of endotoxin in actinomycin D sensitized mice with endotoxin lethal attack. [65] The chloroform extract of the leaves has the anti-endotoxin effect on Escherichia coli O 111 B 4 with dilution in vitro to 64 times still destroying the endotoxin, and the endotoxin dripped into the vein of rabbits is also destroyed, suggesting that the leaves contain anti-endotoxin active substances. [66,67] IIR can significantly reduce the level of serum lipid peroxide and improve the activity of superoxide dismutase, suggesting its functions of anti-lipid peroxidation, scavenging free radicals and antagonizing endotoxin. [68] The result of bacterial endotoxin destruction test showed that the different pH value significantly affected the action intensity of the root aqueous extract against bacterial endotoxin, the reason being that the active ingredients contained in the roots against bacterial endotoxin are extracted more easily in an acid environment. [69]

Immunopotentiation
The leaf decoction can promote IL-2 secretion of spleen lymphocytes induced by concanavalin A in normal mice to enhance immunity but has no effect on TNF-α secretion of peritoneal macrophages and the activity of leukocytes, pathological damage and dysfunction. [70,71] Polysaccharide of the roots has immunopotentiation effects, which can promote specific immune, non-specific immune, humoral immune or cellular immune effects. [72] Intraperitoneal injection of polysaccharide 50mg/ kg significantly enhanced the immune function of normal mice with increasing the spleen weight and a total number of leukocytes and lymphocytes. [73,74] However, it also markedly reduced spleen index and the total number of leukocytes and lymphocytes in the immunosuppressed mice induced by hydrocortisone, and inhibited the delayed anaphylaxis in immunosuppressed mice induced by dinitrochlorobenzene and cyclophosphamide. [75] Further study showed that lectin from the roots could bind to glycoprotein on the cell surface to promote the development of thymus and the proliferation of thymocytes, indirectly maintaining the microenvironment of the thymus, promoting the secretion of thymosin and cytokines by T-lymphocytes and thymic epithelial cells, and improving the immunity of the body. [76] Anti-inflammation The leaf decoction has a significant inhibitory effect on methanal induced arthritis in mice and suppresses the local inflammatory reaction and capillary permeability of rabbit skin caused by xylene. [77,78] Total alkaloids and amino acids from the leaves also alleviate mouse ear oedema, suggesting the anti-inflammatory effects. [79] 70% ethanol extract of the roots can inhibit ear swelling of mice caused by xylene and foot swelling of rats caused by egg white to a certain extent. [80] Anti-tumour Indirubin, an alkaloid from I. indigotica, possesses an anti-tumour activity, which strongly inhibits transplanted tumour growth of animals and alleviates chronic myeloid leukaemia. [81,82] Owing to poor water-soluble and liposoluble properties, the indirubin's derivatives named derivative III were designed and synthesized to increase solubility with an inhibitory rate of 58% against leukaemia cells. [83] Indirubin is likely to participate in regulating the metabolism of lung cancer cells by inducing the activity of cytochrome P4501A1 and 1B1mRNA enzyme in MCF-7 lung cancer cells. [84,85] Curdione isolated from the roots can inhibit the proliferation of hepatocarcinoma BEL-7402 cells and ovarian cancer A2780 cells, induce differentiation, reduce the telomerase activity and boost the conversion of tumour cells into normal cells. [86] Indirubin displays significant cytotoxicity in HL-60 cells, eliciting cell pyknosis, condensation and even lyses. [87] Others IIL also has a cholagogic effect, which can promote bile excretion and relieve pain. [88,89] It can depress adenosine diphosphateelicited platelet aggregation in rabbits due to the efficacy of promoting blood circulation and removing stasis. [90] Indigotin has a significant protective effect against liver injury caused by carbon tetrachloride [91,92] and the leaves can detoxify the effects of lead poisoning mice. [93] All the pharmacological effects of this plant are summarized in Table 2.

Toxicity
I. indigotica is generally considered nontoxic, however, the adverse reactions of its leaves occur from time to time as reported in the literature. [94,95] The extracts of roots of I. indigotica, also called Banlangen, can induce the micronucleus rate of polychromatic erythrocytes in mouse bone marrow and increase the sperm deformity rate of mice, suggesting certain genotoxicity in mammalian somatic cells and germ cells. [96,97]

Hepatitis
The leaves of I. indigotica show significantly improvement effects on acute common infectious hepatitis. 32 cases of icterohepatitis were treated with the leaves of I. indigotica in combination with roots of Salviae miltiorrhizae, roots of Curcumae longae, roots of Dryopteridis crassirhizomatis and fruits of Ziziphus jujuba, and the effective rate was 94%. [98,99] Yigan-Jiedu decoction composed of the leaves and roots of I. indigotica, roots of Salviae miltiorrhiza, roots of Astragalus membranaceus, and the whole herb of Lysimachia christinae apparently improved the symptoms and signs of 86 cases with chronic hepatitis B when compared with the control group. [100] Another injection named Shu-gan-ning, composed of roots of I. indigotica, Ganoderma lucidum, fruits of Kochia scoparia, fruits of Gardenia jasminoides, and roots of Scutellaria baicalensis, quickly alleviated jaundice symptoms of 45 cases with acute icteric hepatitis, and the clinical effective rate was 91%. [101,102] Qinggan-Lidan decoction, consisted by the roots of I. indigotica, whole herb of Artemisia carvifolia, fruits of Gardenia jasminoides, barks of Phellodendri chinensis, the whole herb of Bupleurum chinense, Poria cocos, roots of atractylodis macrocephalae, and semens of Coix lacryma-jobi, treated 100 cases with acute icteric hepatitis and the effective rate was 100%. The compound decoction is simple, easy to use, economical and cheap, and has few reported side effects. [103] Parotitis Total 92 cases of children mumps were treated with the formula containing the leaves combined with ganciclovir. The time of fever abatement, parotid swelling abatement and parotid pain abatement was significantly shortened in the treatment group when compared with the control group, and their effective rates were 97.83% and 80.43%, respectively. [104,105] The formula comprised of the roots of I. indigotica, borneolum syntheticum and cactus cured all 45 cases of epidemic parotitis, with 15 cases cured in two days, accounting for 33%, 21 cases in three days accounting for 47%, 9 cases in four days accounting for 20%. [106] The external application of jinhuang ointment combined with the oral administration of the root granules has an effective rate of 100% when treating 60 cases of children mumps and no adverse reactions and complications were reported in any of the patients. [107]

Upper respiratory tract infection
Total 56 cases of upper respiratory tract infection were treated with the root granules, and the effective rate was 98.21%, which is higher than that of 80.36% observed in the control group treated with ribavirin only. [108,109] A similar result for the root granules was observed in another 60 cases of upper respiratory tract infection, with the effective rate of 100% versus 87% in the control group treated with ribavirin only. [110] Oseltamivir phosphate combined with the  KM mice Reduces the production of pro-inflammatory cytokines to alleviate pneumonia.

Escherichia coli
Rabbit Reduces high body temperature in rabbits caused by endotoxin. 5 mL/kg (ip) [44] Alkaloid Newcastle disease virus Chicken embryo fibroblasts Blocks the absorption of virus, protects cells and reduces virus infection. 7.8-31.3 μg/mL [45] Root aqueous extract H7N9 avian influenza virus Chicken embryos Inhibit human H7N9 avian influenza virus in vitro by blocking the absorption of H7N9 avian influenza virus to host cells.

Respiratory syncytial virus
Hep-2 cells Inhibits the proliferation of respiratory syncytial virus after invading Hep-2 cells.
10-120 μg/mL [47] Polysaccharide HSV-II BALB/C mice Reduces the incidence rate, mortality and prolongs the average survival time in mice.

50, 100 and 200
μg/mL [49] Peptides H1N1 KM mice Reduces the mortality of mice infected with influenza virus and inhibits the proliferation of virus.
50, 100 and 200 mg/kg (ig) [50] Leaf aqueous extract HSV-II Vero cells Inhibits the replication and Inhibits proliferation of HSV-II in cells.

Cytomegalovirus
Guinea pig embryo lung cells Antiguinea pig cytomegalovirus activity.

Streptococcus pneumoniae Staphylococcus aureus
Organic acid Alkaloid

Escherichia coli
Oxford Cup Components have strong antibacterial activity.

Salicylic acid
Lipopolysaccharide Balb/c mice Inhibits excessive release of TNF-α and NO in serum of mice.
2 mg/mL [61] Balb/c mice Promotes the humoral immune response of the body and produces immune effect.

K562 cells
The drug containing serum inhibits the proliferation of cells.
1 g/mL [68] root granules showed significant clinical efficacy in the treatment of influenza A (H1N1) when compared the control group of patients received oseltamivir phosphate alone, and the total effective rate was 97.14%. [111] Others The decoction comprised of the leaves and roots of I. indigotica, herba lysimachiae and radix et rhizoma rhei displayed significant improvement effects in the treatment of pointed condyloma 28 cases, among whom, 14 cases were cured, 12 improved and 2 ineffective, having an effective rate of 92.8% when oral decoction was combined with fumigation and washing. [112] 35 cases of palmoplantar pustulosis were treated topically with the formula consisting of the leaves, herba violae, flos lonicerae, radix sophorae flavescentis, fructus kochiae, fructus cnidii, semen plantaginis, rhizoma atractylodis, and alum, and the total effective rate was 68.57%. [113] 136 cases of epidemic kerato-conjunctivitis were treated with the root granules in combination with herba houttuyniae injection, 110 cases recovered, and the cure time was 2-15 days, averaging 5.6 days. [114] The compound granule could treat viralmyocarditis, which consists of the leaves and roots of I. indigotica, fructus forsythiae, and rhizoma bistortae, and the effective rate was 85.5%, among whom, 23 cases were excellent, 77 fine, 17 ineffective for ventricular premature beats symptom. [115]

Conclusions and Perspectives
Natural agents which are commonly derived from plants or herbs could not only give us essential foods for living, including sugars, lipids, proteins and vitamins, but also supply us some precious medicinal secondary metabolites for preventing various diseases, such as berberine, artemisinin, emodin, and taxol. [116][117][118] As a natural plant, I. indigotica contains alkaloids, organic acids, flavonoids, lignans, nucleosides, amino acids, and steroids. Previous pharmacological researches indicated that I. indigotica possesses promising antivirus, antibacterial, immunoregulatory, antiinflammation, and cholagogic effects. Importantly, it can inhibit various viruses, such as influenza, hepatitis B, mumps, herpes simplex, cytomegalovirus, and coxsachievirus. Clinically, it is frequently used to treat various viral diseases like viral influenza, parotitis and viral hepatitis. Consequently, I. indigotica may be beneficial for the prevention and treatment of coronavirus disease 2019 (COVID-19). I. indigotica has the function of immune regulation, which reinforces its anti-virus effects in turn. Therefore, I. indigotica may be effective for the prevention and treatment of COVID-19, however, this need to be investigated further. Although numerous chemical constituents have been isolated and identified from I. indigotica, the active components, mechanisms of action and their target remain unknown. As the clinic application of Chinese medicines is characterized by compatibility, the therapeutic mechanism of I. indigotica combined with other medicines should be investigated further. However, it is rather difficult to clarify the mechanism at the molecular level based on the compatibility of the crude extracts or components. The compound-based Chinese medicine formula (CCMF) may be promising for clarification of the mechanism and target due to its clear composition of compounds derived from Chinese medicines. The action targets of compounds can be investigated through such techniques as CETSA, DARTS, and MST. When the mechanism of compatibility for CCMF is defined, the scientific connotation for the TCM compatibility theory will probably be clarified.

Author Contribution
QC and HYL reviewed the literature and wrote the manuscript, WP and KR revised the manuscript, QCL, XL and HZ conceived and designed the study and revised the manuscript.

Conflicts of Interest
The authors confirm that this article content has no conflicts of interest.