The Progress of Anti-HBV Constituents from Medicinal Plants in China

Abstract Hepatitis B virus (HBV) infection causing acute and chronic hepatitis is a serious problem worldwide, whereas the current treatment methods are unsatisfactory. Traditional Chinese herbs that have long been used for medicinal purposes are fascinating sources for novel anti-HBV candidates. This paper summarizes the progress of anti-HBV constituents from diverse medicinal plants in China to provide information for searching new anti-HBV drugs from natural sources. Graphical Abstract


Introduction
Hepatitis B infection caused by the hepatitis B virus (HBV) can become either acute or chronic diseases. HBV infections are among the top ten causes of death, and lead to 0.89 million deaths every year. It is estimated that 257 million people are living with chronic HBV infection. China is one of the high prevalence areas accounting for one-third of the infections. The major complications of chronic hepatitis B (CHB) are cirrhosis and hepatocellular carcinoma (HCC) [1,2]. Hepatitis B vaccination is effective in preventing HBV infections; however, it is invalid to those who have already been infected with HBV. Interferons and nucleoside analogs are the most common drugs for CHB in clinic. Interferons (INF-a and PegIFN-a) take effects by enhancing the immune system of patients, but their application is limited by the low curing rate and serious side effects. Nucleoside analogues (mainly lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoprox and clevudine) can inhibit HBV replication by targeting on viral DNA polymerase; however, the drugresistance and relapse are challenging after long-term treatment [3][4][5]. Currently, there is no cure for CHB, and new treatment methods with unprecedented targets are urgently needed. Traditional Chinese herbs that have long been used for medicinal purpose in China are fascinating sources for anti-HBV candidates [6,7]. This paper summarized the progress of anti-HBV constituents from medicinal plants in China.

Active Constituents from Medicinal Plants
According to the traditional Chinese medicine (TCM) theory, CHB is characteristic with the syndrome of liver stagnation and spleen deficiency. Thus, traditional Chinese herbs with the properties of ''clearing heat removing dampness'' and ''promoting blood circulation and removing blood stasis'' are preferable in searching anti-HBV candidates.
Compound 19 displayed inhibition on HBV DNA replication with an IC 50 value of 0.07 mM (SI = 23.6), and compound 20 showed slightly decreased activity with an IC 50 value of 0.12 mM (SI = 17.1) [12].
Artemisia annua (Huanghuahao) is famous for the production of artemisinin that forms the basis of the most important treatments of malaria. Romero et al. reported that artemisinin (21) showed inhibition on HBsAg secretion with an IC 50 value of 55 lM [13]. 5,4 0 -Dihydroxyl-6,7,3 0trimethoxyflavone (22) from Artemisiae argyi (Ai) could suppress the secretions of HBsAg and HBeAg with IC 50 values of 8.1 and \ 3.5 mg/L [14] (Fig. 1). Swertia mileensis (Qingyedan) belonging to the Swertia genus of the family Gentianaceae is an endemic Chinese herb to Yunnan Province (China). This herb has long been used for acute and chronic hepatitis in the Yi and Hani minority areas of China. S. mileensis is the only plant in Swertia genus, which is documented in Chinese Pharmacopoeia . Previous investigation revealed the liver protective effects of S. mileensis and the active constituents, e.g. sweroside, swertisin, and oleanolic acid, whereas the anti-HBV active constituent is still unclear. The in vitro anti-HBV screening manifested that the ethanol extract of S. mileensis was more effective than the water part. The following bioassay-guided isolation gave rise to 47 compounds, involving 29 new and 13 active ones. Swerilactones A and B (23,24) with an unprecedented 6/6/6/6/6 pentacyclic ring system are the first cases of C 18 -skeleton secoiridoid dimers. Their structures were confirmed by detailed spectroscopic data and X-ray crystallographic analyses. Swerilactone A showed anti-HBV property against HBsAg secretion with an IC 50 value of 3.66 mM, and against HBeAg secretion with an IC 50 value of 3.58 mM. However, swerilactone B exhibited no activity at the highest tested concentration of 4.64 mM. Swerilactones C and D (25, 26) with a 6/6/6/6/6 pentacyclic ring system are another type of secoiridoid dimers with C 20 skeleton, and their structures were determined by X-ray crystallographic analyses. Swerilactones C and D both showed inhibitory activities against the secretions of HBsAg with IC 50 values of 1.24 and 2.96 mM, and HBeAg with IC 50 values of 0.77 and 1.47 mM, respectively. Swerilactones E and F (27,28) are two unusual C 16skeleton lactones with a naphthyl or dihydronaphthyl ring,   (Fig. 2). Swerilactosides A-C (38-40) are three unusual secoiridoid glycoside dimers, which further enriched the skeleton types of secoiridoid glycosides. In addition to the above mentioned compounds, 11 new secoiridoid aglycones, including six C 9 -skeletons (swerimilegenins A-F, 41-46), one bis-C 9 -skeleton (swerimilegenin G, 47), and four C 10skeletons (swerimilegenins H-K, 48-51) were further obtained from the low-polarity part of S. mileensis, as well as six known ones. Two known compounds, erythrocentaurin (52) and gentiogenal (53) [20,21] (Fig. 3).

Swertia Plants
Swertia leducii (Mengzi Zhangyacai) is an annual herbaceous plant mainly distributed in Mengzi County of the Yunnan Province. Many traits of S. leducii are similar to those of S. mileensis, except for petioles and flowers. Due to their close morphology, S. leducii was always used as the alternative for S. mileensis in producing ''Qing-Ye-Dan'' medicines. In order to clarify the anti-HBV constituents, a pair of novel enantiomeric lactones, (±)-sweriledugenin A (54, 55), were isolated from S. leducii under the guidance of LCMS investigation. Both (?)-and (-)sweriledugenin A showed activities inhibiting HBV DNA replication with IC 50 values of 36.86 (SI = 10.5) and 26.55 (SI = 31.6) lM on the HepG 2.2.15 cell line in vitro [22]. This investigation is a valuable attempt for guided isolation from a completed natural complex.
Swertia punicea (Zihong Zhangyacai), the congener species of S. mileensis, are used for the treatment of hepatitis in the folk of Yunnan Province. The anti-HBV screening manifested that the EtOAc extract of S. punicea showed inhibitory activities on HBsAg and HBeAg with IC 50 values of 0.69 and 0.15 mg/mL, respectively. In order to clarify the active components, swerpunilactone A (56) and its plausible precursors, (±)-gentiolactone (58) and bellidifolin (59) were isolated from the whole plants of S. punicea. Simultaneously, swerpunilactone B (57), (±)gentiolactone, and norbellidifolin (60)  3) mM, respectively [23]. This is the first report of xanthone and secoiridoid heterodimers connected with C-C bond.
Swertia macrosperma (Dazi Zhangyacai), the congeneric species of S. mileensis, is widely used for curing hepatitis in Yunnan Province, China. Previous phytochemical studies demonstrated that xanthones, triterpenoids, and secoiridoid glycosides were the main constituents, whereas their anti-HBV effects remain unclear. The bioassay on HepG2.2.15 cell line in vitro revealed that the water extraction of S. macrosperma showed activity inhibiting HBsAg and HBeAg with IC 50 values of 0.22 (SI = 19.0) and 0.14 (SI [ 30.0) mg/mL. Subsequent separation on this part provided eight new and 46 known compounds, seven of which showed anti-HBV potency. Gentiocrucines A-E (61-65) as five unusual lactonic enamino ketones (LEKs) are the only cases after the first report of gentiocrucine (66). Compared to gentiocrucine, gentiocrucines A-E maintain prolific substituents on N-7, and their isolation further enriches the structure types of LEKs. In addition, gentiocrucines A-E and gentiocrucine were also obtained from the congener plant, S.  Swertia kouitchensis (Guizhou Zhangyacai), a perennial herb mainly distributed in Southern China, is widely used as a fork medicine to treat jaundice, indigestion and sore throat. Detailed investigation on the ethanol part of S. kouitchensis yielded three new secoiridoids, swertiakoulactone (71) and swertiakosides A and B (72, 73), as well as 28 known compounds. Swertiakoside B is a new secoiridoid glycoside in which the glycosyl is attached to the aglycon by the OH-1 0 and OH-2 0 simultaneously. Swertiakoulactone showed moderate activities inhibiting the HBsAg secretion (IC 50 = 1.10 mM, SI = 4.4) and HBV DNA replication (IC 50 = 1.16 mM, SI = 4.1) [27,28].
Swertia cincta (Xinan Zhangyacai), the congener plant of S. mileensis, is generally used to treat hepatitis in the folk region of Yunnan Province ( [29,30]. Swertia angustifolia (Xiaye Zhangyacai), the congener plant of S. mileensis, is used for treating hepatitis and cholecystitis in the folk region of Yunnan Province (China). The phytochemical investigation on this plant gave rise to seven new secoiridoids, swertianglide (81) and swertianosides A-F (82-87), together with 29 known compounds. Swertianoside A, an unusual secoiridoid glycoside dimer with two molecules of secoiridoids connected with a glucosyl group, showed significant activities inhibiting the secretions of HBsAg (IC 50 = 0.18 mM, SI = 3.1) and HBeAg (IC 50 = 0.12 mM, SI = 4.7), and HBV DNA replication (IC 50 = 0.22 mM, SI = 2.5). As a comparison, other secoiridoid glycosides displayed no activity or cytotoxicity at the tested concentration. This result suggests the importance of two secoiridoid aglycones in the structure [31,32].
Swertia yunnanensis (Yunnan Zhangyacai) as the congener plant of S. mileensis is always used as the alternative of Qingyedan for treating jaundice, hepatitis, and cholecystitis in Yunnan, Sichuan, and Guizhou Provinces of China. The in vitro anti-HBV bioassay manifested that the ethanol extracts of S. yunnanensis showed significant inhibition on the secretions of HBsAg and HBeAg with IC 50 values of 0.79 mg/mL (SI = 2.4) and 0.34 mg/mL (SI = 5.4), respectively, and on HBV DNA replication with an IC 50 value of 0.08 mg/mL (SI = 18.9). Detailed investigation on the ethanol part yielded 24 compounds, involving five new ones (88-92). Sweriyunnanlactone A (88) with a phenyl ring is the first example of C 28 -skeleton secoiridoid trimer which was obtained under the guidance of LC-MS analysis. Its structure was determined by extensive HRESIMS, 1D and 2D NMR spectroscopic data, and GIAO 13  Swertia patens (Xiejing Zhangyacai), mainly distributed in Yunnan and Sichuan provinces of China, is used as a folk medicine to treat hepatitis in folk. However, its chemical constituents and bioactivities were seldom reported. In continuing efforts to discover anti-HBV active compounds from Swertia, phytochemical investigation on S. patens led to the isolation of two new secoiridoids, swerpatic acid (104) and swerpalactone (105), together with 28 known compounds. Swerpatic acid (106) [43,44] (Fig. 6).
Cyperus rotundus Linneus (purple nutsedge), belonging to the family Cyperaceae, is a perennial herb indigenous to China and distributed in tropical and subtropical regions throughout the world. The rhizomes of C. rotundus (Xiangfu) recorded in every edition of Chinese Pharmacopoeia have been widely used as a traditional Chinese     [50][51][52].
Morus alba (Sang) belonging to the genus of Morus in Moraceae family is widely cultivated in China, whose leaves are indispensable food for silkworms. Its root bark (Mori cortex, Sang-Bai-Pi) is a traditional Chinese herb recorded in Chinese Pharmacopoeia (2000 edition) for anti-inflammation, antihypertension, hypoglycemic and diuretic purposes. In order to reveal the anti-HBV active constituents, phytochemical investigation on the root bark of M. alba provided two polyphenols, mulberrofuran G (160) and isomulberrofuran G (161), one new flavonoid, sanggenol P (162) and nine known compounds. Mulberrofuran G (160) exhibited moderate activity against HBV DNA replication, with an IC 50 value of 3.99 lM (SI = 2.0), but weak activity against HBsAg and HBeAg with SI values less than 1. Isomulberrofuran G (161) as the isomer of mulberrofuran G was inactive at the tested concentration. The MS/MS fragmentation pathway for mulberrofuran G and isomulberrofuran G was investigated for the first time, and he characteristic ions at m/z 451 and 439 in MS 2 could be considered as their respective diagnostic ions [53,54] (Fig. 10).
Pericampylus glaucus (Xiyuanteng), a climbing shrub, is widely distributed in southwest China, whose roots are Primary SARs study suggested that a long carbon chain (n [ 8) in the amide alkaloids is helpful for the anti-HBV ability [56][57][58] (Fig. 11).
Scutellaria baicalensis (Huangqin) is a widely used Chinese herbal medicine in antiinflammatory and anticancer therapy. The main constituent, wogonin (191), from this plant showed potent anti-HBV activity both in vitro and in vivo. Wogonin could inhibit HBsAg and HBeAg with an IC 50 value of 4 lg/mL, and DHBV DNA polymerase with an IC 50 value of 0.57 lg/mL [65].
Sophora tonkinensis (Yuenanhuai) is mainly present in Guangxi, Guizhou and Yunnan Provinces of China, whose roots and rhizomes are commonly used as the traditional  (Fig. 13).

Conclusion and Perspective
This paper summarized the progress of anti-HBV constituents from medicinal plants in China. In total, 198 compounds involving monoterpenes, sesquiterpenes, triterpenes secoiridoids and their polymers, alkaloids, xanthones, lignans, enynes, amides and polyphenols were reviewed. Especially, four secoiridoid trimers, swerilactones H-K can obviously suppress HBV DNA replication with IC 50 values ranging from 1.53 to 5.34 lM, comparable to the positive drug lamivudine (3TC). With the consideration of the novel structures, they should have different action mechanism with nucleosides. Oxymatrine has been used to treat hepatitis B in clinic with confirmed efficiency and safety, and its target is host Hsc70 instead of viral polymerase. Thus, natural products from traditional Chinese herbs will provide prolific candidates for searching new anti-HBV drugs with diverse mechanisms.
Hepatitis B as a type of chronic hepatitis is accompanied with high levels of alanine transaminase (ALT) and aspartate transaminase (AST). In addition to anti-HBV efficiency, TCMs can reduce the ALT and AST levels by improving the hepatic function of patients, and relieve HBV-associated clinical symptoms, e.g. anorexia, hypodynamia, nausea and vomiting, to improve the life quality of patients. Thus, TCMs maybe have advantages in treating HBV infection with multi-component and multi-target properties. Although, TCMs are fascinating sources for anti-HBV candidates, the complicated constituent and time-consuming isolation restrict the following drug development. Chemical modification on active natural products is an effective method in searching for anti-HBV agents, which can rapidly yield plenty of candidates from the starting substrates, and rationally increase the bioactivity, decrease the toxicity, or improve their physicochemical properties under the guidance of SARs.