Journal Pre-proof

Thirteen bisbenzylisoquinoline alkaloids in five Chinese medicinal plants: Botany,

traditional uses, phytochemistry, pharmacokinetic and toxicity studies

Han Zhang, Xiaoming Wang, Yaqing Guo, Xiaomei Liu, Xizi Zhao, Tekleab Teka,
Chunxiao Lv, Lifeng Han, Yuhong Huang, Guixiang Pan
PII: S0378-8741(20)33454-1
DOI: https://doi.org/10.1016/j.jep.2020.113566
Reference: JEP 113566

To appear in: Journal of Ethnopharmacology

Received Date: 23 June 2020

Revised Date: 13 October 2020
Accepted Date: 5 November 2020

Please cite this article as: Zhang, H., Wang, X., Guo, Y., Liu, X., Zhao, X., Teka, T., Lv, C., Han, L.,
Huang, Y., Pan, G., Thirteen bisbenzylisoquinoline alkaloids in five Chinese medicinal plants: Botany,
traditional uses, phytochemistry, pharmacokinetic and toxicity studies, Journal of Ethnopharmacology,
https://doi.org/10.1016/j.jep.2020.113566.

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 Botany OCH3 H3CO
OCH3 H3CO

N N N
N
H3C OCH3 O
H
CH3 P450s 3A4/5 H 3C OCH3 O CH3
H H H DNA

OH O O O
OCH3 H3CO

Traditional uses S S SH SH H 3C
N
H
OCH3 O
N
H
CH3

DNA
Protein
OH O

Demethylation
P450s Free transcription factor Repressor protein and its
bound transcription factor

Phytochemistry Translocation to nucleus, gene expression

N
OCH3 H3CO

N
H 3C OCH3 O CH3
H H

OCH3 H3CO OCH3 H3CO Protein

N OH O
N N N
H3C OCH3 O CH3 H3C OCH3 O CH3
H H H H
Toxicity
GS

Toxicology
OCH3 O OH O

Tetrandrine

Bisbenzylisoquinoline
Pulmonary toxicity
OCH3 H3CO

Pharmacokinetics H3C
N
H
OCH3 O
N
H
CH3
Liver toxicity
OCH3 O
Genotoxic

Nephrotoxicity

Mitochondrial dysfunction

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 1 Thirteen bisbenzylisoquinoline alkaloids in five Chinese medicinal plants:
2 Botany, traditional uses, phytochemistry, pharmacokinetic and toxicity studies
3

4 Han Zhang 1#, Xiaoming Wang 1#, Yaqing Guo 1#, Xiaomei Liu 1, Xizi Zhao 1, Tekleab
5 Teka 1, Chunxiao Lv2, Lifeng Han1,*, Yuhong Huang 2, Guixiang Pan 2,*
6

7 1 Tianjin University of Traditional Chinese Medicine, Tianjin-301617, China.

8 2 Second Affiliated hospital of Tianjin University of Traditional Chinese Medicine,
9 Tianjin-300250, China.

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10 * Corresponding author: hanlifeng_1@sohu.com (L.-F.H.); guixiangp@163.com

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11 (G.-X.P.); Tel./Fax: +86-22-60637919 (G.-X.P)
12
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These authors contributed equally to this work.
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13

14 Abstract:
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15 Relevance: Bisbenzylisoquinoline (BBIQ) alkaloids are generally present in plants of

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16 Berberidaceae, Monimiaceae and Ranunculaceae families in tropical and subtropical

17 regions. Some species of these families are used in traditional Chinese medicine, with
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18 the effects of clearing away heat and detoxification, promoting dampness and
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19 defecation, and eliminating sores and swelling. This article offers essential data
20 focusing on 13 representative BBIQ compounds, which are mainly extracted from five
21 plants. The respective botany, traditional uses, phytochemistry, pharmacokinetics, and
22 toxicity are summarized comprehensively. In addition, the ADME prediction of the 13
23 BBIQ alkaloids is compared and analyzed with the data obtained.
24 Materials and methods: We have conducted a systematic review of the botanical
25 characteristics, traditional uses, phytochemistry, pharmacokinetics and toxicity of
26 BBIQ alkaloids based on literatures collected from PubMed, Web of Science and
27 Elsevier during 1999-2020. ACD/Percepta software was utilized to predict the
28 pharmacokinetic parameters of BBIQ alkaloids and their affinity with enzymes and
29 transporters.
30 Results: Botany, traditional uses, phytochemistry, pharmacokinetic and toxicity of 13
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31 alkaloids, namely, tetrandrine, dauricine, curine, trilobine, isotrilobine, cepharanthine,
32 daurisoline, thalicarpine, thalidasine, isotetrandrine, liensinine, neferine and
33 isoliensinine, have been summarized in this paper. It can't be denied that these
34 alkaloids are important material basis of pharmacological effects of family
35 Menispermaceae and others, and for traditional and local uses which has been
36 basically reproduced in the current studies. The 13 BBIQ alkaloids predicted in this
37 paper showed strong affinity and inhibitory effect on P-glycoprotein (P-gp), with poor
38 oral absorption and potent binding ability with plasma protein. BBIQ alkaloids
39 represented by tetrandrine play a key role in regulating P-gp or reversing multidrug

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40 resistance (MDR) in a variety of tumors. The irrationality of their usage could pose a

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41 risk of poisoning in vivo, including renal and liver toxicity, which are related to the
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formation of quinone methide during metabolism.
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43 Conclusion: Although there is no further clinical evaluation of BBIQ alkaloids as
44 MDR reversal agents, their effects on P-gp should not be ignored. Considering their
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45 diverse distribution, pharmacokinetic characteristics and toxicity reported during

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46 clinical therapy, the quality standards in different plant species and the drug dosage
47 remain unresolved problems.
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48 Keywords: Tetrandrine, bisbenzylisoquinoline, botany, traditional uses,

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49 phytochemistry， pharmacokinetics, toxicology.

50 1. Introduction
51 Bisbenzylisoquinoline (BBIQ) alkaloids are formed of two benzyl isoquinoline
52 units by oxygen bridges. In recent years, interest and attention for BBIQ alkaloids
53 have raised subtly because of a wide range of pharmacological activities, such as
54 anti-inflammatory, antibacterial, anti-cancer and anti-hypertensive effects. BBIQ
55 alkaloids are mainly present in the species of Berberidaceae, Menispermaceae,
56 Monimiaceae and Ranunculaceae, which are commonly cultivated in China, India,
57 Japan, Chile, and other countries (Jin et al., 2010). There are 78 species of 19 genera
58 of Menispermaceae in China, which are mainly distributed in tropical and subtropical
59 regions. This family, a famous medicinal plant group, is dioecious and the plants are
60 mostly vines (Wang, 2003). The plants of the family Stephaniaceae grow in the south
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61 and southwest of China, and are used in folk medicine for antipyretic, analgesic,
62 antibacterial and anti-inflammatory, hemostasis, blood stasis, detumescence and
63 diuresis. Some of them, such as Stephania rotunda Lour., have the effects of clearing
64 away heat and detoxification, promoting dampness and defecation, and eliminating
65 sores and swelling. Its decoction is commonly used in folk for diuresis and
66 detumescence. The plants of another family Stephaniaceae are rich in BBIQ alkaloids,
67 including Tetrandrine (Tet), dauricine (Dau), curine, trilobine, cepharanthine (Cep),
68 isotetrandrine and isotrilobine, as well as flavonoids, anthraquinones and other
69 components. Tetrahydropalmatine (rotundine), dicentrine and Cep extracted from the

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70 root tubers of the plants of Menispermaceae are all important active ingredients (Shao

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71 et al., 2019). Moreover, Thalictrum dasycarpum Fisch., C.A.Mey. & Avé-Lall.
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(Ranunculaceae) and Nelumbo nucifera Gaertn. (Nymphaceae) are also two plants
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73 with a large number of BBIQ alkaloids.
74 The understanding of these five kinds of medicinal plants in traditional Chinese
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75 medicine is consistent with the relevant research results of modern pharmacology. In

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76 recent years, researchers have found that BBIQ alkaloids are the material basis of the
77 pharmacological effects. Details of selected alkaloids concerning the pharmacology
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78 are provided with Table 1.

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79 According to the literatures, BBIQ alkaloids displayed poor bioavailability.

80 Previous studies have pointed out the poor absorption of Dau, with absolute
81 bioavailability of 16.6% in rat (Chen et al., 2001). Neferine (Nef) exists in the form of
82 ions in an acidic (pH 1-2) environment, and usually can't penetrate lipid membranes.
83 Considering the diversity of BBIQ alkaloids and limited data, we have predicted the
84 physicochemical and biochemical properties of 13 bio-active alkaloids by
85 ACD/percepta software. These basic characteristics, such as Log P, molecular weight,
86 binding rate of plasma protein, solubility and affinity with Cytochrome P450 (CYP)
87 and P-glycoprotein (P-gp), could reflect their pharmacokinetic property. The results of
88 predicted parameters are shown in Table 2.
89 It has been certified that BBIQ alkaloids have a great potential as a substrate and
90 inhibitor of P-gp. P-gp is the most widely studied transporter and considered to be the
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 91 main factor related to multidrug resistance (MDR). Recently, looking for tumor MDR
92 reversal agents from plant medicine has become a novel direction for anti-cancer
93 researchers. Many studies have shown that BBIQ alkaloids can reverse MDR and
94 have the clinical potential effect for chemosensitizer. Tian et al. pointed out that Dau
95 and daurisoline were capable of reversing MDR significantly in naturally resistant
96 human hepatoma cell line BEL-7402, acquired drug-resistant MCF-7/ADR and
97 KBv200 cells (Tian et al., 1996). Tet is a natural compound that can inhibit P-gp
98 non-competitively (Fu et al., 2002) and potentiates the effectiveness of the primary
99 drug (antineoplastic, antimalarial, tuberculostatic) against a drug-resistant cell, called

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100 as chemosensitizers (Ford, 1995). However, this appears to pose a risk of serious toxic

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101 effects with the improper use of Tet. Renal and liver toxicity are the main poisoning
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related to Tet. Typical symptoms of toxic effects are hypotension, ataxia, glomerular
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103 necrosis and acute renal failure. In addition, high-dose of Tet may cause respiratory
104 depression and death. Up to now, the pharmacokinetics and potential toxicity of Tet
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105 and other BBIQ alkaloids have not been systematically reviewed yet.
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106 In this review, we compared and analyzed the physicochemical properties and
107 ADME properties of 13 BBIQ alkaloids with the data obtained from ACD/percepta
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108 software. This article mainly focused on five plants that contain such alkaloids, and
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109 summarized the botany, traditional useage, phytochemistry, pharmacokinetics, and

110 toxicity of BBIQ alkaloids comprehensively. The five traditionally used medicinal
111 plants are Stephania cephalantha Hayata, Cocculus orbiculatus (L.) DC., Nelumbo
112 nucifera Gaertn., Thalictrum alpinum L. and Menispermum canadense L.. Our review
113 aims to help motivate more discovery of new medications and pharmacological
114 activities.

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115 Table 1. Summary of partial BBIQ alkaloids.
NO. Name Molecular Plant source Chemical Structure Pharmacological Activity Ref
Formula
1 Tetrandrine C38H42N2O6 Cyclea barbata (Wall.) Miers Rheumatoid (Shen et al., 2010; Wang et
(Menispermaceae) Arthritis al., 2015)
Cyclea peltata (Lam.) Hook.f. Hypertension
& Thomson (Menispermaceae) Inflammation
Cocculus orbiculatus (L.) DC. Sepsis
(Menispermaceae) Silicosis

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Menispermum canadense L. Neuralgia
(Menispermaceae)

pr
2 Dauricine C38H44N2O6 Cardiopetalum calophyllum Protection of cerebral injury (Dong et al., 2014; Wang et

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Schlecht. (Annonaceae) Induction of cell apoptosis al., 2012)

Pr
Menispermum canadense L. Suppression of cancer cell
(Menispermaceae) growth and angiogenesis

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3 Curine C36H38N2O6 Cissampelos pareira L. Vasodilatory (Ribeiro-Filho et al., 2013;

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(Menispermaceae) Anti-inflammatory Ribeiro-Filho et al., 2019;
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Analgesic Ribeiro-Filho et al., 2015)

4 Trilobine C35H34N2O5 Anisocycla cymosa Troupin Anti-inflammatory (Tan et al., 1991; Xu et al.,
(Menispermaceae) Inhibited ADP-induced 1986)
Anisocycla grandidieri Baill. platelet aggregation
(Menispermaceae) Analgesic
Cocculus orbiculatus (L.) DC. Antipyretic
(Menispermaceae)
Stephania cephalantha Hayata

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 (Menispermaceae)
5 Isotrilobine C36H36N2O5 Albertisia papuana Becc. Antiplasmodial (Chen et al., 1991; Marshall
(Menispermaceae) et al., 1994)
Anisocycla jollyana (Pierre)
Diels (Menispermaceae)
6 Cepharanthine C37H38N2O6 Stephania cephalantha Hayata Antimalarial (Huang et al., 2014; Zhou et
(Menispermaceae) Antioxidant al., 2012)
Stephania epigaea H.S. Lo Anti-allergic
(Menispermaceae) Anti-inflammatory

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Antitumor
Anti-viral

pr
7 Daurisoline C37H42N2O6 Menispermum dauricum DC. Focal ischemia/reperfusion (Liu et al., 1998; Q. N. Liu et

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(Menispermaceae) injury al., 2010)

Pr
Polyalthia nitidissima Benth. Arrhythmia
(Annonaceae) Platelet aggregation

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Antagonizing potassium and

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calcium channel
8 Thalicarpine C41H48N2O8 u
Thalictrum dasycarpum Fisch., Antitumor (Wu et al., 1977; Wu et al.,
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C.A.Mey. & Avé-Lall. Hypotensive 2002)
(Ranunculaceae) Antimicrobial
Thalictrum minus L.
(Ranunculaceae)
9 Thalidasine C39H44N2O7 Thalictrum alpinum L. Antitumor (Kupchan et al., 1969; Wu et
(Ranunculaceae) al., 1991)
Thalictrum cultratum Wall.
(Ranunculaceae)

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10 Isotetrandrine C38H42N2O6 Atherosperma moschatum Anticancer (Dong et al., 1991; Wang et
Labill. (Monimiaceae) Anti-inflammatory al., 2016)
Berberis boliviana Lechl. Antibacterial activities
(Berberidaceae) Antioxidant
Stephania cephalantha Hayata Anti-hypertensive
(Menispermaceae)
Thalictrum alpinum L.
(Ranunculaceae)
11 Liensinine C37H42N2O6 Nelumbo nucifera Gaertn. OCH3 H3CO Inhibit sodium and calcium (Hu et al., 2015; Zhou et al.,

f
N N

oo
(Nymphaceae) H3C OCH3 O CH3 channel 2015)
H H
Block hERG channel

pr
OH HO Inhibit autophagy

e-
Anti-arrhythmia

Pr
Anti-oxidant
Anti-HIV

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12 Neferine C38H44N2O6 Nelumbo nucifera Gaertn. Anti-hypertensive (Kadioglu et al., 2017; Zhou

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(Nymphaceae) Anti-thrombotic et al., 2013)

u Anti-inflammatory
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Anti-cancer
Neuro-protective effect
Antidepressant
Antioxidant
13 Isoliensinine C37H42N2O6 Nelumbo nucifera Gaertn. Anti-HIV (Xiao et al., 2005; Xiao et al.,
(Nymphaceae) Anti-cancer 2006)
Cocculus orbiculatus (L.) DC. Inhibit bleomycin-induced
(Menispermaceae) pulmonary fibrosis in mice

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 Antiproliferative
Antioxidant
Anti-inflammatory

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pr
e-
Pr
al
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116 2. Methods
117 The references related to BBIQ alkaloids in this review covered ancient and
118 modern books, dictionaries of traditional Chinese medicine, doctoral and master's
119 theses, English and Chinese literatures and some electronic databases, including
120 PubMed, Web of Science and Elsevier. We searched more than 1000 articles published
121 during 1999-2020 with tetrandrine, dauricine, neferine, curine, trilobine, isotrilobine,
122 cepharanthine, daurisoline, thalicarpine, thalidasine, isotetrandrine, liensinine,
123 isoliensinine and bisbenzylisoquinoline as the key words, mainly in English, followed
124 by botany, traditional uses, phytochemistry, pharmacokinetics, toxicology as the
125 second key words for searching. The retrieval results were classified according to

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126 different fields, namely, botany, traditional uses, phytochemistry, pharmacokinetic and

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127 toxicity. In the selection of literature, the latest research results directly related to the
128 research content were given priority as the first-hand literature. The sources of the
129
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literature were carefully reviewed, and the botanical and Latin names were verified by
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130 the standard database (http://www.theplantlist.org). Results related to the above
131 research contents that did not meet the quality standards were excluded. In addition,
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132 ACD Labs Percepta software (Advanced Chemistry Development, Inc., Toronto,
133 Ontario, Canada) was used to predict the physicochemical, ADME properties of
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134 thirteen BBIQ alkaloids, as well as the affinity with CYPs and P-gp. The prediction
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135 was based on a database, which includes a variety of interaction constants and three
136 predictive algorithms, namely, classic, GALAS and consensus. Classic pattern was
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137 based on the principal of isolating carbons, while GALAS was to make adjustment
138 with data from the most similar compounds. Consensus included both classic and
139 GALAS algorithms.
140 3. Botany
141 Family Menispermaceae has served as the most abundant source for BBIQ
142 alkaloids (Schiff, 1987). At least 112 alkaloids have been identified in 21 genera of
143 Menispermaceae. The content of BBIQ alkaloids, such as Tet and fangchinoline, is the
144 most abundant in the genus Cyclea of Menispermaceae, followed by Thalictrum.
145 There are 78 species of 19 genera of Menispermaceae in China, which are mainly
146 distributed in tropical and subtropical regions (Zhu et al., 1983). The family is, mostly
147 vines and, distributed in the south and southwest of China. According to literatures,
148 they are used historically for antipyretic, analgesic, antibacterial and
149 anti-inflammatory, hemostasis, blood stasis, detumescence and diuresis (Wei et al.,
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150 2019). The representative herbal remedy is Rhizoma Menispermi, the root of
151 Menispermum dauricum DC. It has been detected with a great deal of BBIQ alkaloids,
152 such as Dau, daurinoline, dauricinoline, Tet, sinomenine, menisperine, dauricoline,
153 dauriciline, dauriporphinoline and daurisoline (Shao et al., 2019; Wei et al., 2015). R.
154 Menispermi is a perennial twining vine, reaching more than 10 meters in length with
155 slender rhizomes. They are found on hillsides, forest margins, thickets, and gravelly
156 beaches, or on rocks, especially in north, northeast and east of China. Additionally, the
157 leaves and the stems of this plant are also used medicinally, including heat-clearing
158 and detoxification (Bai et al., 2014).
159 Radix Stephaniae Cepharanthae is originated from the root tuber of Stephania

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160 cephalantha Hayata. It grows in different habitats ranging from fertile and moist grass,

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161 hillside, roadside shade or shrub to calcareous mountains. The base of old stem is
162 slightly lignified, with fine furrow and purplish. The leaf blade is round triangle or
163
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oblate (Ma et al., 2000). Root tubers contain numerous alkaloids, including Cep,
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164 isotetrandrine, cepharanoline, cepharamine, cepharanone, cepharadione and Tet, while
165 the seeds contain dehydrostephanine, stephanine, fenfangjines and fangchinoline.
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166 Trilobine, isotrilobine and magnoflorine widely exist in Cocculus orbiculatus (L.)
167 DC., Stephania tetrandra S.Moore and Cocculus laurifolius DC. Cocculus
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168 orbiculatus (L.) DC. is another woody lianas in Menispermaceae, mainly produced in
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169 Henan, Shanxi, and Jiangxi provinces in China. The young branches are densely
170 pilose, and the old branches are nearly glabrous, with straight lines on the surface. Its
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171 drupe is nearly globose, purplish red or blue black at maturity. The stem of this plant
172 is also used for medicine, which was described in Yao Xing Lun for dispelling wind,
173 removing dampness and detumescence (Li et al., 2020).
174 In addition, the properties and relative contents of alkaloids in different parts of
175 plants are varied. Nelumbo nucifera Gaertn. is a perennial aquatic herb with numerous
176 longitudinally ventilated holes, and adventitious fibrous roots. Its mature seed, Semen
177 Nelumbinis, is a traditional medicine used in Eastern Asia for hundreds of years for
178 anxiety and post menstrual depression (Kang et al., 2005). Semen Nelumbinis mainly
179 contains linoleic acid, oleic acid and palmitic acid, while young leaves and radicle in
180 mature seeds of Plumula Nelumbinis, containing liensinine, isoliensinine, Nef,
181 nuciferine and pronuciferine. Plumula Nelumbinis is mainly produced in Hunan,
182 Hubei, Fujian, Jiangxi and Jiangsu provinces. The product is slightly rod-shaped,
183 yellow white, brittle and easy to break, with several small holes in the section. In
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184 addition to its ornamental use, it is also served as a staple food in Eastern Asia.
185 The plants in family Thalictrum L.and Ranunculaceae are widely used in China
186 because of their officinal values. The main active constituents of Thalictrum L. are
187 isoquinoline alkaloids including simple isoquinolines, benzylisoquinolines, and also a
188 lot of dimeric alkaloids as BBIQ (Schiff, 1991). Radix et Rhizoma thalictri sibirici,
189 roots and rhizomes of Thalictrum aquilegiifolium var. sibiricum Regel & Tiling, is
190 native to Zhejiang, Shandong and Inner Mongolia provinces. It grows on the loose
191 and fertile sandy loam and humus loam. The whole plant is glabrous and the stem is
192 erect and branched and it has corymbose inflorescence, with many branches and
193 dense flowers. The distribution of some alkaloids in these plants is listed in Table 1.

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194
195 Fig. 1. Hand painted medicinal parts of Stephania cephalantha Hayata, Cocculus
196 orbiculatus (L.)DC., Nelumbo nucifera Gaertn., Thalictrum aquilegiifolium var.
197 sibiricum Regel & Tiling and Menispermum dahuricum DC. from Dictionary of
198 Traditional Chinese Medicine (2006 edition)
199 4. Traditional uses
200 At present, many BBIQ alkaloids have been identified in the species of
201 Menispermaceae. Radix Stephaniae Cepharanthae is a perennial vine of the genus
202 Stephania, commonly referred as Stephania cephalantha Hayata. It tastes bitter,
203 pungent, cold in nature, small in toxicity, and enters into hot meridian. Many
204 prescriptions related to it are recorded in Chinese ancient medical books，such as Pan

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205 Zhu Ji and Xuan Ming Lun. It is prepared in the form of cream and powder
206 respectively, and used for gestational typhoid fever, and toxic damage to the body. In
207 Guizhou Province, the Miao ethnicity uses its root tuber as medicine, known as
208 "jiafeilie", treating patients with sore throat, rheumatic arthralgia, abdominal pain,
209 diarrhea, and traumatic bleeding (He et al., 2010). In Japan, the crude extract from
210 Radix Stephaniae Cepharanthae was used in a variety of acute and chronic diseases,
211 including alopecia areata (Morita et al., 2002) and snakebites (Kimoto et al., 1997).
212 Alkaloids are the basic conceivable bio-active ingredients for treating parotiditis,
213 gastric ulcers, and leukopenia (Nakaoji et al., 1997).
214 Radix Cocculi, the root of Cocculus orbiculatus (L.) DC., was first recorded in

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215 Shennong's classic of Materia Medica and listed as inferior. Wang et al. found that

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216 Radix Cocculi has a great pharmacological diversity in combination with different
217 drugs, and has achieved good clinical efficacy. For example, the combination of
218
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Atractylodes macrocephala Koidz. is common for removing rheumatism, and it can
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219 interact with Astragalus propinquus Schischkin for replenishing Qi (Wang, 2003). Li
220 et al. used Fangji Huangqi Decoction to treat 108 cases of cirrhosis ascites, which
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221 could protect liver, improve liver function and increase blood protein (Li et al., 1999).
222 Besides, Radix Cocculi decoction was tested for the heart failure induced by viral
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223 myocarditis in Kyoto University of Japan. The results indicated that Radix Cocculi
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224 decoction had preventive effect on myocardial damage (Morita et al., 2002).
225 The rhizomes of Menispermum canadense L., R. Menispermi, was first found in the
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226 Chinese pharmacopoeia in 1977, with the meaning of Euchresta japonica Regel in the
227 north. According to the Chinese Pharmacopoeia (2015 edition), Rhizoma Menispermi
228 is bitter and cold, with small toxicity. It has the effects of clearing heat and
229 detoxification, dispelling wind and relieving pain, and can be used to treat sore throat,
230 enteritis and dysentery, rheumatic arthralgia disease and other diseases (Bai et al.,
231 2014). Su et al. appraised the anti-inflammatory properties of its rhizome extract. The
232 results showed that M. dauricum could effectively reduce colon inflammation in mice
233 as a protective agent (Su et al., 2016). The main BBIQ alkaloids in R. Menispermi are
234 characterized to exert powerful effects of including antibacterial, anti-inflammatory,
235 anti-tumor, and anti-depression.
236 The health care function of Nelumbo nucifera Gaertn. has been recorded in the
237 Compendium of Materia Medica. Its seed is described as "bitter, cold, clearing heart
238 and tranquilizing mind, communicating heart and kidney, astringent essence and
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239 hemostasis" (Li et al., 2000). The young leaves and radicle named Plumula
240 Nelumbinis is commonly used in traditional Chinese medicine as sedative, antipyretic
241 and hemostatic agent. The research of modern pharmacology mainly focuses on blood
242 pressure lowering, antiarrhythmic, antioxidant and anti-platelet aggregation (Xie et al.,
243 2013). In traditional Korean herbal therapy, Plumula Nelumbinis is widely used in
244 patients with anxiety, insomnia and post-menopausal depression. Japanese scholars
245 reported that central nervous inhibitory effect of methanol extract from Plumula
246 Nelumbinis, and its main component Nef, was comparable to that of diazepam
247 (Sugimoto et al., 2008). Nef, like diazepam, inhibited motor activity and lowered
248 body temperature. The combination of thiopental sodium with them could prolong the

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249 sleep time of mice induced by thiopental sodium.

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250 The genera Thalictrum L. and Ranunculaceae have more than 200 species in the
251 world, which are distributed in Asia, Europe, Africa, North America and South
252
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America (Yu, 1999). The pharmacological effects of Thalictrum alkaloids include
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253 anti-tumor, anti-parasite, anti-platelet aggregation, anti-silicosis and anti-virus (Gao,
254 1999). The Central Hospital of Changning District in Shanghai has used whole herb
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255 preparation of Thalictrum faberi Ulbr. in the treatment of gastric cancer, and achieved
256 certain curative powers. The study of Thalictrum alkaloids began in the early 1960s.
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257 First of all, thalicarpine and thalidasine were isolated from the Thalictrum robustum
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258 Maxim., which was conducted by Professor Kupchan of the University of Wisconsin.
259 In 1973, thalicarpine began to be used in clinical trials to treat leukemia. Thalictrum
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260 alpinum L. is also used in Tibetan and Mongolian medicine. In Tibetan medicine, "E
261 zheng" refers to T. javanicum B1. The whole herb can treat arthritis, and the root is
262 used for fever, falling and beating. "Gong bu e zheng" is the rhizome and root of
263 Thalictrum foliolosum DC. and Thalictrum baicalense Turcz. ex Ledeb., which can
264 cure conjunctivitis, infectious hepatitis, carbuncle, swelling and furuncle.
265 In the 18th and 19th centuries, the United States, the Soviet Union and the United
266 Kingdom used thalicarpine as a laxative, stomach tonic, diuretic, antipyretic and
267 anti-rabies drug (Hooker, 1875). In India, Thalictrum aquilegiifolium var. sibiricum
268 Regel & Tiling is used to treat gastritis, malaria, fever, jaundice, leprosy, rheumatism,
269 conjunctivitis, and tropical sores. It is also used for tonic, sweating, laxative and
270 snakebites in Japan (Chopra, 1956 ). Indian people in Nevada, the United States,
271 make tea with Thalictrum L. to treat gonorrhea and cold. In Canada, the roots of some
272 plants of Thalictrum L. are used as a drug for snakebites (Lloyd, 1884-1885).
13
273 5. Phytochemistry
274 Benzyl carbon is the consistent structural character of these alkaloids, which is
275 substituted at β-position of nitrogen atom, and easy to be oxidized by oxidants (such
276 as potassium permanganate) in the presence of oxygen and ultraviolet light (Weber et
277 al., 2019; Yang et al., 1999). When H2O2 is used as oxidant, nitrogen atom will be
278 protonated, and the product is usually a mixture of N-oxides. The product can be
279 reduced to original alkaloid in the presence of 6% H2SO3 or zinc powder.
280 Separating and purifying pure active components from the limited plant resources
281 has a wide range of economic and social benefits in the consumer society (Gao et al.,
282 1999). A total of 49 alkaloids were isolated from the MeOH extract of Stephania

of
283 cephalantha Hayata with anti-HSV-1 activity (IC50 values ranging from 14.8-43.2

ro
284 μg/mL) (Nawawi et al., 1999). The structure of BBIQs is listed in Fig. 2. Ji et al.
285 studied the separation and preparation of Tet and fangchinoline with Stephania
286
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tetrandra S.Moore as raw material. They were isolated by column chromatography,
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287 and then identified by TLC. The contents of Tet and fangchinoline determined by
288 HPLC in sample were 100.97% and 98.54% respectively (Ji et al., 2015). Wei et al.
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289 established the extraction, isolation and purification methods for extracting Tet from
290 the root of Stephania tetrandra S.Moore, with good yield (0.76 g/100 g) and high
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291 purity ( 90%) (Wei et al., 2016). The process was conducted with four steps: the reflux
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292 extraction, extraction, higher column chromatography and recrystallization. Two

293 compounds, menisporphine and dauriporphine, were separated and purified from
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294 Menispermum dauricum DC. using ultrasonic-assisted solvent extraction technology,

295 normal or reverse phase column chromatography and semi-preparative HPLC (Ti et
296 al., 2017). The alkaloids isolated from Stephania tetrandra S. Moore were studied,
297 and the main alkaloids were Tet, oxofangchirine, fangchinoline, cycleanine,
298 cycleahomine, tetrandrine 2'-N-α-oxide, tetrandrine2'-N-β-oxide,
299 2'-N-methyltetrandrinium chloride, etc.

14
 OCH3
O H3CO
N
H3C OCH3 N N
H3C O O R1
H O H H

O R2O

O H
R1 R2
H3CO CH3
N 6. Cepharanthine CH3 CH3
17. 2-Norcepharanthine H CH3
H3CO
18. Cepharanoline CH3 H
14. 3,4-Dehydrocycleanine 19. 2-Norcepharanoline H H

OCH3

N
H3 C OR OCH3 R3O
H O
N N
H3C O R4O CH3

of
H H

ro
O H OR2 O
H3CO CH3
N

H3CO

15. (-)-Cycleanine R=CH3

-p 20. Obaberine
21. Oxyacanthine
R1
CH3
CH3
R2
CH3
H
R3
CH3
CH3
R4
CH3
CH3
re
16. (-)-Norcycleanine R=H 22. Stephibaberine CH3 CH3 H CH3
23. Homoaromoline CH3 CH3 CH3 H
lP

O H3CO 24. Aromoline CH3 H CH3 H

N N
H3C O O CH3
H
O
na

H3CO OCH3 H3CO

O N N
CHO R1 OR3 O CH3
H H
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25. Secocepharanthine
OR2 O
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OCH3 H3CO
R1 R2 R3
N N
H3C O H3CO 10. Isotetrandrine CH3 CH3 CH3
H
27. Berbamine CH3 H CH3
28. Thalrugosine CH3 CH3 H
OCH3 O 29. Obamegine CH3 H H
30. 2-Norisotetrandrine H CH3 CH3
26. 3',4'-Dihydrostephasubine 31. 2-Norberbamine H H CH3
300
301 Fig. 2. Structures of main BBIQ alkaloids isolated from Stephania cephalantha
302 Hayata (Nawawi et al., 1999).
303 In the 1960s, the chemical structure of liensinine was identified by chemical
304 degradation method, which is in good agreement with the results obtained by modern
305 methods. After 1980s, a series of modern analytical techniques were attempted to be
306 applied in the structural analysis of liensinine (Lou et al., 1995). ESI/QTOF/MS is a
307 commonly used identification technology. According to the chromatographic
308 characteristics, ultraviolet spectrum and mass spectrometry fragments, 57 alkaloids

15
309 have been initially identified in Plumula Nelumbinis. Among them, more than half of
310 compounds belonged to BBIQ alkaloids (Fig. 3). Yang et al. isolated a new skeleton
311 BBIQ from the seed of Nelumbo nucifera Gaertn., named neoliensinine. It was
312 clarified with the relaxing effect on mesenteric vascular smooth muscle, similar to
313 other two alkaloids, Nef and isoliensinine (Yang et al., 2018). Wang et al. invented a
314 preparation process for extracting and purifying isoliensinine and liensinine from
315 Plumula Nelumbinis and compared with the existing method. This method has the
316 advantages of low cost, high yield, excellent quality, and is suitable for industrial
317 production (Wang, 2000). Pan et al. have disclosed a countercurrent chromatography
318 method of separating liensinine, isoliensinine and Nef, in which the purity of the

of
319 separation can be higher than 95% (Pan et al., 2005). Berbamine, isotetrandrine and

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320 Cep have been extracted and isolated from Stephania cephalantha Hayata with a
321 separation system of hexane-ethyl acetate-methanol-water and their purity was 98.7%,
322
-p
97.3% and 99.3%, respectively (Yuan et al., 2013).
re
lP
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16
 of
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-p
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323
324 Fig. 3. Structures of main BBIQ alkaloids isolated from Nelumbo nucifera Gaertn.
325 (Yang et al., 2019).
326 According to current evidence, more than 400 alkaloids have been reported from
327 the plant genus Thalictrum L. from 1963 to now (Naman et al., 2015). Since then, a
328 great deal of new alkaloids are conducted on these species.
17
329 6. Pharmacokinetic behaviors
330 6.1 ADME properties
331 Analyzing the table of prediction results, 11/13 compounds had high oral
332 bioavailability after administration of 50mg, but conclusions are inconsistent with the
333 actual experimental results. The pharmacokinetics of Tet have been fully studied in
334 rats and human. Though Tet has a favorable pharmacokinetic profile (Table 2), it
335 displays poor bioavailability in intestinal tracts (Song et al., 2008). After a single
336 administration dose of 100 mg, the absorption was slow with Tmax of 14.00 ± 10.02 h.
337 The AUC and Tmax of Tet in the Xiaoxuming decoction group were higher than those
338 in the single-dose Tet group. This research indicated that other complex components

of
339 that present in the decoction can improve pharmacokinetic behavior of Tet (Li et al.,

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340 2009). According to current evidence, the bioavailability of Tet analogue,
341 Bromotetrandrine (BrTet), was 56.9% after intramuscular injection (Zheng et al.,
342
-p
2010). Its rapid distribution and slow elimination from the plasma allow it to be
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343 administered once or twice per treatment cycle.
344 After oral ingestion, the pharmacokinetic parameters of Dau and Nef were similar.
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345 Dau was rapidly absorbed and eliminated from rat plasma, achieving Cmax at less than
346 0.43 h. The absolute bioavailability of Dau in rat was low as 16.6%, indicating poor
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347 permeability and absorption (Wei et al., 2015). Yasuda et al. demonstrated that the
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348 mean Cmax after oral administration with 60 mg tablets of Cep was just 3.5±0.3 ng/mL
349 in plasma, and the absolute bioavailability was 6-9% (Desgrouas et al., 2014).
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350 Nevertheless, studies in rats and dogs have shown that the bioavailability of Nef was
351 as high as 65%. These data showed that Nef is easier to absorb, metabolize and
352 excrete than Cep in vivo. Trials are needed in human for science-based medical
353 practice in case of the differences in transporters expression and distribution profiles
354 across species.
355 Therefore, we look specifically at the distinction in relation to the pharmacokinetic
356 study displayed in the previous section. First and foremost, the low in vivo
357 bioavailability of BBIQ was majorly due to its low solubility, such as Tet of 1.1mg/ml.
358 As lipid-soluble drugs, BBIQ alkaloids pass through the cell membrane by simple
359 diffusion. The rate of simple diffusion mainly depends on the oil-water partition
360 coefficient of the drug and the difference in drug concentration on both sides with the
361 membrane. According to recent analysis, the Papp values of Tet in Caco-2 cells were
362 all smaller than 1×10-6 (cm/s), indicating its poor permeability, which was consistent
18
363 with the oral absorption result of Tet (Yang et al., 2017). Moreover, it was noted that
364 the drug must be dissolved in body fluids before reaching the cell membrane, so one
365 of the reasons for the low bioavailability of Tet in vivo may be due to its poor
366 solubility.
367 The other two reasons are supposed to be the elimination of the first pass and the
368 effect of efflux transporters. Although there is no relevant report till date, according to
369 the prediction results in Table 3, the probability of 11/13 compounds as P-gp
370 substrates is more than 50%. Early studies showed that P-gp has a significant effect
371 on isoliensinine, Nef and liensinine (Tian, 2013). Breast cancer resistance protein
372 (BCRP) is now clear to be an efflux transporter that may prevent drugs from

of
373 penetrating tissues, and essential to drug disposition and distribution. It has been

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374 reported that BCRP was responsible for the transportation of liensinine and Dau,
375 while isoliensinine, Nef and Tet are not the substrates of BCRP (Tian, 2013). In
376
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conclusion, as the substrate of efflux transporters, the absorption of these BBIQ
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377 alkaloids can also be reduced, resulting in low bioavailability.
378 Considering the low bioavailability and short half-life of conventional oral
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379 preparations of Tet, some scholars have developed oral nano-preparations. Compared
380 with the solution, the t1/2 and MRT of Tet solid lipid nanoparticles (SLNs) was
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381 prolonged while the CL was reduced (p<0.05) (S. Li et al., 2011). Besides,
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382 nanocapsules can promote the absorption of Tet in intestinal mucosa, improving its
383 Vd and bioavailability. There was a significant difference in AUC between SLNs and
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384 the tablets. This increasing accumulation of Tet in vivo would make it play a better
385 role in clinical practice in the future (Zhao et al., 2013).
386 The prediction results of ADME profiling show that the plasma protein binding
387 ability of the drug is strong. This indicates that such drugs are slowly eliminated from
388 the body and the effect is maintained for a long time. The quantitative and qualitative
389 results supports the notion of the long half-lives and slow elimination of Tet. The
390 excretion pathway was mainly through the kidney, followed by the liver (Li et al.,
391 2016). The drug taken at the current dose (oral, 60-100 mg, 3 times/day) were
392 implicated to be the reason for the accumulation of Tet and its metabolites (i.e.,
393 berbamine) (Yang et al., 2017). Dau could be monitored in urine samples of healthy
394 rats up to 72 h, while its metabolites were no longer detected beyond 24 hours (Han et
395 al., 2007). The long T1/2γ and MRT of total alkaloids in Plumula Nelumbinis suggested
396 slow excretion or metabolism of the total BBIQ alkaloids (TAL) of Plumula
19
397 Nelumbinis, which may be the reason for their long-lasting anti-hypertensive effects
398 (Huang et al., 2006). However, Nef may accumulate in tissues, and its overdose may
399 tend to cause poisoning in patients with liver and kidney inadequacy.
400 On the other hand, because of the difficulty in penetrating the blood-brain barrier
401 (BBB), it is complicated for BBIQ alkaloids to act on the central nervous system.
402 After intravenous injection, Nef was widely distributed in the kidneys and lungs,
403 followed by the liver and heart. Nevertheless, it was barely detected in the brain,
404 indicating its poor permeability of the BBB (Huang et al., 2007). However, Cep could
405 be detected in brain, spleen, lung, liver and serum at 0.5h after oral administration.
406 The content of Cep in brain and liver was higher than heart, lung, kidney, spleen and

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407 serum. The lung was the target organ of Cep entering into the rat body, and the

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408 content exceeded the highest detected value of brain, liver and spleen from 24 to 48h
409 (Huang et al., 1998). There is also evidence that Tet can be used as an adjuvant drug
410
-p
for the treatment of refractory epilepsy (Han et al., 2015), and it plays a
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411 neuroprotective role in Parkinson's disease (Li et al., 2015). Therefore, this lack of
412 uniformity between the prediction and the reality of the penetration rate of BBIQ
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413 alkaloids across BBB makes it indispensable for further experiments to investigate the
414 possibility of using these drugs in central system.
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415 Table 2. Pharmacokinetic parameters of four major BBIQ alkaloids.

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Drugs Species Dose Pharmacokinetic parameters

Tet (1), p.o., Rabbits 10 mg/kg t1/2α (min) 48.698±45.353
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single treatment t1/2β (min) 732.919±847.32

(Jiang et al., Cmax(μg/mL) 92.448±23.795
2011) Tmax(min) 40±0.0
AUC0−24 h (μg/L·min) 15555.578±6465.068
AUC(0−∞) (μg/L·min) 18986.217±7462.308
MRT0−24h min 362.591±45.898
MRT(0−∞) min 742.143±238.013
Tet (1), i.v., 5 mg/kg t1/2α (min) 4.721±3.728
single treatment t1/2β (min) 283.808±162.937
Cmax(μg/mL) 388.81±160.04
Tmax(min) -
AUC0−24 h (μg/L·min) 51522.865±18403.98
AUC(0−∞) (μg/L·min) 5
MRT0−24h min 59861.149±26962.19

20
 MRT(0−∞) min 6
378.177±152.207
569.529±278.123
Tet (1), p.o., Rats 50 mg/kg AUC(0−72h) (μg/L·h) 6279.2±2411.5
single treatment AUC(0−∞) (μg/L·h) 7002.7±2528.0
(Song et al., Cmax (μm/L) 237.1±95.9
2008) Tmax (h) 6.0±1.8
T1/2 (h) 20.6±3.7
Ke (L/h) 0.034±0.006
Tet (1), p.o., Rats 9.3 mg/kg t1/2 (h) 49.562
single treatment Cmax(mg/L) 0.169
(Li et al., 2009) Tmax(h) 4

of
AUC0−t (mg/L·h) 2.255

ro
AUC(0−∞) (mg/L·h) 6.570
MRT0−t h 11.375
-p MRT(0−∞) h 66.638
re
Tet (1), i.v., Rats 16 mg/kg AUC(0−∞) (μg/L·h) 3081.94±728.21
single treatment Cmax (μg/L) 630.18±183.45
lP

(S. Li et al., t1/2 (h) 7.22±1.13

2011) MRT(0−∞) (h) 10.06±1.57
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CL (L/h/kg) 5.41±1.22
Tet (1), p.o., Human 100 mg t1/2 (h) 22.34±21.22
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single treatment Cmax (ng/mL) 67.26±23.22

(Yang et al., Tmax (h) 14.00±10.02
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2017) AUC0−t (ng/mL·h) 1257.70±330.42

AUC0−∞(ng/mL·h) 1554.16±25.07
Dau (2), p.o., Rats 123.36 Tmax1 (h) 0.3270.25
single treatment mg/kg Tmax2 (h) 9.4072.41
(Wei et al., 2015) t1/2 (h) 20.5373.76
Cmax1 (μg/L) 187.5773.2
Cmax2 (μg/L) 170.2772.2
AUC0−t (μg/L·h) 5387.171553.6
AUC0−∞ (μg/L·h) 6462.271880.2
Dau (2), p.o., Human 300 mg Tmax1 (h) 1.14±0.30
single treatment Tmax2 (h) 4.25±0.97
(X. Liu et al., Cmax1 (ng/mL) 33.6±12.5
2010) Cmax2 (ng/mL) 16.9±4.17
t1/2 (h) 2.87±0.86

21
 AUC(0−12 h) (ng/mL·h) 121±28.1
AUC(0–∞) (ng/mL·h) 132±29.9
MRT (h) 4.28 ±0.41
CL/F (L/h) 1428±316
V/F (L) 5891±1946
Cep (6), i.v., Human 50 mg AUC(0−48 h) (ng/mL·h) 252.9±14.8
single treatment AUC(0–∞) (ng/mL·h) 377.8±22.7
(Kohtaro et al., Cmax (ng/mL) 433±25
1989) t1/2 (h) 36.9±3.6
-1)
Ke1(h 0.0196±0.002
Cep (6), i.v., Human 50 mg Cmax (ng/mL) 135.9 ±66.8
single treatment t1/2 (h) 131.9±48.4

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(Hao et al., 2010) AUC(0−192 h) (ng/mL·h) 566.6±216.6

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Nef (12), p.o., Rats 20 mg/kg AUC0−t (mg/mL·h) 2.11
single treatment AUC0−∞ (mg/mL·h) 2.25
(Huang et al.,
-p MRT0−t (h) 11.34
re
2007) MRT0−∞ (h) 13.80
Tmax (h) 0.17
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Cmax (μg/ml) 0.19

CLtot (L/kg·h) 8.8
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t1/2β (h) 22.9

Daurisoline (7), Rabbits 2.5 mg/kg Vc (L/kg) 2.2±0.4
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i.v., single Vd (L/kg) 13.1±27

treatment (Gu et t1/2α (h) 0.024±0.009
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al., 2003) t1/2β (h) 3.0±0.6

CLs (L/kg·h) 3.1±0.6
AUC (μg/mL·h) 0.84±0.13
5 mg/kg Vc (L/kg) 2.6± 0.6
Vd (L/kg) 18± 6
t1/2α (h) 0.030± 0.019
t1/2β (h) 3.4±0.9
CLs (L/kg·h) 3.6±0.4
AUC (μg/mL·h) 1. 41±0.17
10 mg/kg Vc (L/kg) 0.97±0.15
Vd (L/kg) 44±4
t1/2α (h) 0.018±0.004
t1/2β (h) 6.9±0.6
CLs (L/kg·h) 4.4±0.3

22
 AUC (μg/mL·h) 2.30±0.18
Liensinine (11), Rats 5.0 mg/kg t1/2 (h) 9.81±2.39
i.v., single Vd (L/kg) 56.20±24.81
treatment (Hu et MRT (h) 11.70±2.42
al., 2015) CL (L/kg·h) 3.82±0.91
AUC0−t (ng/mL·h) 1164.09±322.26
AUC0−1 (ng/mL·h) 1369.09±319.15
Liensinine (11), Rats 5.0 mg/kg t1/2(h) 9.28 ±3.25
i.v., single Cmax(ng/mL) 570.20±119.70
treatment (Peng CL (L/kg·h) 3.25±1.00
et al., 2015) AUC0−t (ng/mL·h) 1397.20±322.91
AUC0−∞ (ng/mL·h) 1647.80±454.25

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Liensinine (11), Rat 5.0 mg/kg t1/2(h) 8.2±3.3

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i.v., single Cmax(ng/ml) 668.4±156.9
treatment (Lv et CL (L/kg·h) 3.0±0.9
al., 2015)
-p
AUC0−t (ng/mL·h) 1595.7±400.3
re
AUC0−∞ (ng/mL·h) 1802.9±466.4
Liensinine (11), Rats 8.54 Vc (L/kg) 0.766
lP

i.v., single mg/kg t1/2α (min) 2.514

treatment (Xu et t1/2β (min) 49.522
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al., 2001) AUC (μg/mL·min) 114.878

CLs (L/kg·min) 0.0743
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Nef (12), i.v., Rats 25 mg/kg t1/2α(h) 0.221±0.042

single treatment t1/2β (h) 1.440±0.186
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(Feng et al., Vd L/kg 0.647±0.091

2016) AUC0−4.5 (μg/mL·h) 26.963±0.897
AUC0−∞(μg/mL·h) 28.336±1.189
MRT0−t (h) 1.042±0.055
CL (L/kg·h) 0.769±0.024
Nef (12), i.v., Dogs 5 mg/kg AUC0−24h (ng/mL·h) 835.1±227.1
single treatment AUC0−∞ (ng/mL·h) 844.3±228.2
(Zhao et al., t1/2γ(h) 8.95±4.12
2007) Vd (L/kg) 0.079±0.024
CL (L/kg·h) 0.0091±0.0035
Nef (12), p.o., 5 mg/kg AUC0−24h (ng/mL·h) 1090.7±304.0
single treatment AUC0−∞ (ng/mL·h) 1103.6±302.3
(Zhao et al., MRT (h) 4.62±1.18
2007) Tmax (h) 0.33±0.14

23
 Cmax (ng/ml) 856.16±251.49
t1/2 (h) 7.45±3.64
F (%) 65.3
Nef (12), i.v., Rats 5.0 mg/kg t1/2(h) 5.49±1.17
single treatment Vd (L/kg) 11.00±2.84
(Hu et al., 2015) MRT (h) 7.99±1.20
CL (L/kg·h) 1.42±0.37
AUC0−t (ng/mL·h) 3540.90±985.19
AUC0−1 (ng/mL·h) 3755.40±1076.12
Nef (12), i.v., Rats 25.0 t1/2α(h) 0.212±0.030
single treatment mg/kg t1/2β (h) 2.500±0.189
(Feng et al., Vd (L/kg) 0.702±0. 058

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2016) AUC0−4.5 (μg/mL·h) 32.013±0.548

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AUC0−∞ (μg/mL·h) 44.364±0.785
MRT0−t (h) 1.368±0.028
-p CL (L/kg·h) 0.565±0.034
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Isoliensinine Rats 5.0 mg/kg t1/2 (h) 7.88±0.84
(13), i.v., single Vd (L/kg) 32.47±1 1.79
lP

treatment (Hu et MRT (h) 10.82±1.16

al., 2015) CL (L/kg·h) 2.87±1.03
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AUC0−t (ng/mL·h) 1695.52±578.42

AUC0−1 (ng/mL·h) 1925.22±671.78
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Isoliensinine Rats 5.0 mg/kg t1/2 (h) 7.16±1.33

(13), i.v., single Cmax(ng/mL) 446.96 ±101.89
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treatment (Peng CL (L/kg·h) 2.67±1.01

et al., 2015) AUC0−t (ng/mL·h) 1837.22±545.28
AUC0−∞ (ng/mL·h) 2049.57±626.21
416 p.o., oral administration; i.v., intravenous injection
417 6.2 The affinity with P-gp and CYP enzymes
418 According to perception of the affinity with P-gp, the probabilities of inhibiting
419 P-gp of 13/13 alkaloids are nearing or over 90%, suggesting their potent effect of
420 reversing P-gp-mediated MDR. Previous quantitative structure-activity relationship
421 (QSAR) analysis showed that Nef could bind with P-gp drug binding pocket and
422 inhibit its efflux, thus increasing the accumulation of substrate in cells (Kadioglu et al.,
423 2017). The Papp value of Rho-12 from BL to AP was decreased from 7.32±0.82 to
424 2.38±0.24 with 5 µg/ml Tet, while verapamil (VER), the gold standard of P‑gp
425 inhibitor, led to a decrease in the secretion of Rho-123 from 7.32±0.82 to 2.37±0.18
24
426 (Cao et al., 2018). Compared to classical MDR reversal agent verapamil and
427 cyclosporin A (CsA) (Li et al., 2014), Tet exhibited stronger activity to reverse MDR
428 than anticancer drugs in leukemia cells, such as daunorubicin, vinblastine and
429 doxorubicin (DOX) (Xu et al., 2006). These findings demonstrated that Tet was an
430 inhibitor of P‑gp and can significantly inhibited the transportation of the P-gp
431 substrate across Caco-2 cells. With Tet treatment, the protein expression level of P-gp
432 in Caco-2 cells decreased in a dose-dependent manner. After 48 h of exposure, Tet
433 reduced P-gp protein level by 25.8 to 68.3% at a dose of 1.25 µM to 20 µM (p<0.05
434 or p<0.001 vs. solvent) (Shan et al., 2013). Zhou et al. further explored the
435 mechanism of Cep in reversing MDR in Eca109/CDDP cells. Cep could activate JNK

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436 signal and induce the expression of MDR1 mRNA and P-gp by increasing the

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437 expression of c-Jun (Zhou et al., 2017). Moreover, the P-gp expression was inhibited
438 in a time- and concentration-dependent manner in K562/ADR cells (Peng et al., 2012).
439
-p
Nef has been proven to significantly reduce the expression of MDR-1 mRNA and
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440 P-gp proteins in various drug-resistant cells, such as K562/G01 (Qin et al., 2011) and
441 K562/A02 cell lines (Qin et al., 2010). It could increase the sensitivity of
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442 drug-resistant cells by increasing the accumulation of chemotherapeutic drugs.

443 Due to the limited research on enzymes and transporters related to BBIQ alkaloids,
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444 we also predicted their affinity with CYP enzymes, including CYP3A4, CYP2D6,
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445 CYP2C9, CYP2C19 and CYP1A2. According to the results, we can see that CYP3A4
446 shows a greater affinity with BBIQ alkaloids, which indicates that most BBIQ
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447 alkaloids may be metabolized by CYP3A4. BBIQ alkaloids are formed of two benzyl
448 isoquinoline units connected by oxygen bridges, which contain two nitrogen atoms in
449 tertiary amine state in the molecular structure. The benazepine structure of this type of
450 alkaloids makes them stronger in affinity with metabolic enzymes such as CYPs, with
451 more active products yielded in phase I reaction. Li et al. presented in-depth
452 evaluations of 26 phase I metabolites of Tet identified in blood, urine and feces (Li et
453 al., 2016). The main metabolic pathways of Tet were demethylation, hydroxylation,
454 hydrogenation, acetylation and tarine conjugation (Wang et al., 2015). Another
455 research showed that Tet was metabolized into berbamine, which was commonly used
456 for anti-cancer, anti-arrhythmic and anti-thrombosis. Synergistic effect needs to be
457 recognized as investigating the pharmacological effects of Tet and its metabolites. The
458 proposed metabolic pathways of Tet are summarized in Fig. 4. So far, the results have
459 confirmed the substrate specificity of CYP51 and Tet, yet studies rarely focus on the
25
460 mechanism of metabolic enzymes of Tet (He et al., 2017).
461
462
463
464
465
466
467
468
469

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470

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471
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473
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474
475
lP

476
477
na
ur
Jo

26
478 Table 3. ADME properties of BBIQ alkaloids.
NO. Alkaloids PhysChem profiling ADME profiling Oral
Log P Molecular No. of No. of No. of Rings Solubility Caco-2 Binding CNS Bioavailability
weight hydrogen hydrogen rotatable (mg/ml) rate of
bond bond bonds plasma
donors acceptors protein

f
oo
1 Tetrandrine 4.80 622.75 0 8 4 8 1.1 196×10-6 97.40% -3.3 99.1%
-6
2 Dauricine 5.62 624.77 1 8 10 6 0.21 37×10 95.45% -3.9 98.9%

pr
3 Curine 4.25 594.70 2 8 2 8 1.1 143×10-6 87.81% -3.4 99.1%

e-
-6
4 Trilobine 5.24 562.65 1 7 2 9 0.085 82×10 97.01% -3.6 97.8%

Pr
5 Isotrilobine 5.69 576.68 0 7 2 9 0.035 113×10-6 97.77% -3.5 82.1%
-6
6 Cepharanthine 5.27 606.71 0 8 2 9 1.5 156×10 97.03% -3.5 99.1%

al
7 Daurisoline 5.48 610.74 2 8 9 6 2.7 21×10-6 94.28% -4.0 99.2%

rn
-6
8 Thalicarpine 5.45 696.83 0 10 11 7 0.004 122×10 96.17% -3.7 15.8%
u 236×10-6
Jo
9 Thalidasine 4.05 652.78 0 9 5 8 0.69 96.67% -3.0 99.1%
-6
10 Isotetrandrine 4.80 622.75 0 8 4 8 1.1 196×10 97.40% -3.3 99.1%
-6
11 Liensinine 5.01 610.74 2 8 9 6 6.2 50×10 94.40% -3.8 99.2%
-6
12 Neferine 5.57 624.77 1 8 10 6 0.77 43×10 95.97% -3.9 99.2%
-6
13 Isoliensinine 4.75 610.74 2 8 9 6 1.9 69×10 94.11% -3.7 99.1%
479
480 Table 4. Probability prediction as substrate or inhibitor of CYPs and P-gp.
NO. Alkaloids Substrates Inhibitors (IC50 < 50 μM)

27
 CYP3A4 CYP2D6 CYP2C9 CYP2C19 CYP1A2 P-gp CYP3A4 CYP2D6 CYP2C9 CYP2C19 CYP1A2 P-gp
1 Tetrandrine 0.99 0.94 0.42 0.83 0.01 0.75 0.57 0.06 0.06 0.08 0.02 0.98
2 Dauricine 0.98 0.69 0.36 0.68 0.02 0.77 0.83 0.24 0.06 0.14 0.01 0.98
3 Curine 0.96 0.36 0.58 0.92 0.01 0.60 0.50 0.06 0.07 0.07 0.01 0.88
4 Trilobine 0.93 0.93 0.57 0.84 0.01 0.52 0.32 0.12 0.08 0.05 0.06 0.98
5 Isotrilobine 0.99 0.79 0.61 0.93 0.03 0.39 0.29 0.06 0.05 0.06 0.03 0.98

f
oo
6 Cepharanthine 0.99 0.92 0.28 0.78 0.00 0.85 0.78 0.37 0.12 0.16 0.02 0.95
7 Daurisoline 0.96 0.56 0.48 0.73 0.01 0.66 0.81 0.39 0.07 0.19 0.01 0.94

pr
8 Thalicarpine 0.98 0.83 0.05 0.60 0.08 0.24 0.60 0.39 0.04 0.08 0.07 0.99

e-
9 Thalidasine 0.99 0.96 0.21 0.69 0.02 0.61 0.66 0.07 0.08 0.06 0.02 0.99

Pr
10 Isotetrandrine 0.99 0.94 0.42 0.83 0.01 0.75 0.57 0.06 0.06 0.08 0.02 0.98
11 Liensinine 0.97 0.52 0.38 0.63 0.01 0.61 0.82 0.46 0.09 0.18 0.01 0.92

al
12 Neferine 0.98 0.81 0.36 0.65 0.01 0.70 0.88 0.31 0.07 0.19 0.01 0.97

rn
13 Isoliensinine 0.97 0.66 0.46 0.69 0.01 0.65 0.86 0.87 0.09 0.24 0.02 0.91

u
Jo
481

28
 f
oo
pr
e-
Pr
al
u rn
Jo

482
483 Fig. 4. Metabolic pathway of Tet (Wang et al., 2015).
484

29
485 In human liver microsomes, at least 15 major metabolites of Dau were generated by
486 CYPs, which were dehydrogenated, bis-dehydrogenated, demethylated,
487 bis-demethylated, and hydroxylated (Wang et al., 2009), with little changes in benzyl
488 isoquinoline skeletons. The metabolic pathway of Dau is represented in Fig. 5. The
489 target compounds can increase the hydrophilicity through phase II metabolism,
490 making it easier to excrete from bile and/or urine. Wang et al. detected three quinone
491 methide metabolites which could be captured by GSH in human liver microsomes,
492 including Dau, 2-N-demethyl dauricine and 2′-N-demethyl dauricine (Wang et al.,
493 2009). Additionally, CYP3A4, CYP2A1, CYP2D6, CYP1A2, CYP2C9 and CYP2C19
494 were suggested to be involved in phase І metabolism by co-incubation with

of
495 isoenzyme-selective inhibitors. However, quinone methide intermediates could

ro
496 increase the risk of poisoning by reacting with proteins or DNA.
OCH3 H3CO CH2-2H OCH3 H3CO OCH3 H3CO

H3 C
N

H
OCH3

OH
H3CO

O
N

H
CH3 H3C
N

H
OCH3

OGL1
-p H3CO

O
N

H
CH3 H3C
N

H
OCH3

OH
H3CO

O
N

H
CH3
re
O O

M5;N-demethyl--di--dehydrogenated dauricine M8:glucuronide conjugated dauricine M6:hydroxydauricine

lP

-4H

OCH3 H3CO 2H
OCH3 H3CO OCH3 H3CO

N N N N
H3CO CH3 N N
H3C OCH3 H3 C OCH3 H3CO CH3
H H3C OCH3 H3CO CH3
H H H H H
na

OH O O
OH OH O

M2:tetra--dehydroxydauricine MO:dauricine M1:di--dehydrogenated dauricine

ur

-2CH2

OCH3 H3CO OCH3 H3CO OCH3 H3CO

N N N N N N
H3CO CH3 H3 C OCH3 H3CO CH3 H3 C OCH3 H3CO CH3
H3 C OCH3
H H H H H
H
Jo

O OH O OGla O
OH

497 M4:di-demethyldauricine M3:N-demethyldauricine M7:glucuronide conjugated N demethyldauricine

498 Fig. 5. Metabolic pathway of Dau (Han et al., 2007).

499 Nef was metabolized into four major metabolites in human liver microsomes,
500 which were 2,2'-N-didemethyl-neferine (M1), liensinine (M2-1),
501 2'-N-demethyl-neferine (M2-2) and 2-N-demethyl-neferine (M2-3). Similar to Tet, the
502 main metabolic pathway of Nef was demethylation. M2-3 produced by CYP2C8 and
503 CYP3A4 was also involved in the metabolism, while the production of M2-2 was
504 related to CYP2D6 and CYP3A4. The production of M1 and M2-l was much less than
505 that of M2-2 and M2-3, mainly metabolized by CYP3A4 (Shen, 2014). Although
506 CYP3A4 participated in the metabolism of Nef with a slower reaction rate compared
507 with CYP2C8 and CYP2D6, it contributed a lot to the demethylation of Nef. In
508 addition, Nef could enhance the expression of mRNA of CYP3A and CYP2D in rats
509 (Shen et al., 2014), which suggested that Nef induced enzymes to accelerate its own
30
510 metabolism at a higher dose (20 mg/kg). Considering the differences in CYP3A4
511 expression of individuals, metabolic activity polymorphism of CYP3A4 or in
512 combination with drugs that significantly induces CYP3A4 expression, dose of Nef
513 needs to be reduced to the appropriate level to avoid hepatotoxicity (Fig. 6).
514

of
ro
515
516
-p
Fig. 6. Metabolic pathway of Nef (Shen, 2014).
re
517 6.3 P-gp-mediated pharmacokinetic herb-drug interactions and MDR reverse
lP

518 effect
519 At present, cancer MDR modulators are commonly used clinically to inhibit the
na

520 function of transporters and enhance the sensitivity of cancer cells to anti-cancer
521 drugs. Classical reversal agents that reverse MDR by inhibiting ABC transporters
ur

522 have been clinically available, but their performance has been questioned for
Jo

523 dose-dependent side effects. Some natural compounds in traditional Chinese medicine
524 could overcome this shortcoming and be new reversal agents. The network pathways
525 composed of calcium channels and efflux pumps regulate the signal transduction
526 system in cancer cells synergistically, especially T-type Ca2+ channels (Zhao et al.,
527 2020). Previous studies have demonstrated the inhibitory effect of a variety of calcium
528 channel blockers on P-gp. The structures of BBIQ alkaloids are similar to that of
529 calcium channel blockers (Felix et al., 1992), which explains the reason why these
530 compounds generally have P-gp-inhibition effect.
531 Liensinine, isoliensinine and Nef, the quality control components of Plumula
532 Nelumbinis, have anti-tumor, cardiac protection, antidepressant and insulin sensitizing
533 effects (Nishimura et al., 2013). Tang et al. (Tang et al., 2003) studied the effect of
534 Nef on apoptosis resistance of adriamycin (ADR) resistant human breast cancer cells,
535 and the results showed that Nef could reverse the apoptosis resistance of MCF-7/ADR

31
536 cells. The mechanisms of action may be related to the inhibition of P-gp function and
537 expression, and the increase of ADR accumulation in MCF-7/ADR cells. Nef can also
538 enhance the sensitivity of K562/A02 cells to STI 571, down-regulating the
539 transcription of MDR mRNA and the expression of P-gp, thus reversing the MDR of
540 leukemia. These results indicate that Nef not only has a good anti-tumor effect, but
541 also is a highly effective and low toxicity chemosensitizer, which can reverse the
542 MDR of tumor cells and help improve the efficacy of chemotherapic drugs.
543 It has been demonstrated that Tet is a potent inhibitor of P-gp-mediated drug efflux.
544 Compared to classical MDR reversal agents such as daunorubicin, vinblastine and
545 doxorubicin (DOX), Tet possessed a more effective reversal activity against drug

of
546 resistance cancer cells (Xu et al., 2006). By inhibiting mRNA expression, 2% Tet

ro
547 cream could significantly increase the fungal sensitivity of patients with tinea
548 corporis to ketoconazole (Shi et al., 2011).
549
-p
Moreover, P-gp-mediated modulators alter the cytoplasmic distribution of drugs in
re
550 MDR cells. Ikeda et al. showed that the distribution of adriamycin (ADM) in K562
551 cells was changed by the action of Cep. Since the acidification of the cytoplasm was
lP

552 inhibited, the drug was transferred from the vesicle to the nucleoplasm. Cep could
553 perturb the functions of membrane proteins on the lipid bilayers (such as vacuolar
na

554 H+-ATPase) (Ikeda et al., 2005). However, as the researchers did not get sufficient
ur

555 results on the changes of mdrl mRNA, the correlation between intracellular drug
556 redistribution and the role of P-gp still awaits to be further investigated. No one has
Jo

557 yet studied if other BBIQs can also change the ADM subcellular distribution.
558 Cep increased the level of intracellular Rho123 and the accumulation of ADR by
559 inhibiting the efflux function of P-gp, reversing the drug resistance of K562/ADR
560 cells (Peng et al., 2012). The combination of 5-Fu and CDDP with Cep appeared to
561 modulate the inhibition level of P-gp in various treatment cells, normal tissues and
562 solid tumors simultaneously (H. Li et al., 2011). Additionally, Zhou et al. evaluated
563 inhibition effect of Cep on efflux transporter MRP7, and it reversed paclitaxcel
564 resistance by acting in part as a competitive inhibitor (Zhou et al., 2009). Thus, the
565 efflux of anti-cancer drugs mediated by P-gp, MRP1, and MRP7 can enhance the
566 activity of anti-cancer drugs under the effect of Cep.
567 Changing cell processes such as cell cycle, DNA damaged repair and apoptosis will
568 affect the metabolism of anti-cancer drugs and further weaken the capacity of drugs to
569 kill cancer cells. Overexpression of Bcl-2 in the process of cancer is further to
32
570 promote the deterioration and development of tumors. Therefore, this molecule which
571 presents on the surface of mitochondria is often used as a therapeutic target for
572 anti-cancer drugs, by which inhibiting it can restore the activation of apoptosis
573 cascade. The up-regulation of MDR1 mRNA by Cep hydrochloride (CEH) is related
574 to the activation of JNK signaling pathway. Previous research has reported the
575 expression of MDR1 was consistently dependent on JNK and NF-κB, and the
576 mechanisms include up-regulation of p53 and down-regulation of the expression of
577 Bcl-2 without affecting Bax. Bcl-2 can reduce the lethality of anti-cancer drugs on
578 cancer cells, which is conducive to enhance the inhibition of cancer growth. Cep
579 interfered with the cell cycle of ESCC cell lines and inhibited the proliferation by

of
580 up-regulating p53 and p21 (Zhou et al., 2017).

ro
581 Similarly, Nef could significantly reduce the expression of Bcl-2 in
582 HepG2/thermotolerance cells and overcome the MDR of thermostable hepatocellular
583
-p
carcinoma cells (Ai et al., 2007). With the synergistic effect of hyperthermia, Nef
re
584 reduced the expression of P-gp (Huang, Cao, et al., 2011) as well as DNA damage
585 driven by the accumulation of ADM in MCF-7/Adr cells (Huang, Li, et al., 2011). It
lP

586 has been reported that drugs can regulate the expression of P-gp by activating ERK
587 signaling pathway, which is most likely attributable to their inhibition of protein
na

588 synthesis (McCubrey et al., 2007).

ur

589 7. Toxicology
590 There is a need to evaluate the toxicity associated with the curative effect of BBIQ
Jo

591 alkaloids, including renal toxicity (Chen et al., 2011), liver toxicity (Tainlin et al.,
592 1982), genotoxicity (Whong et al., 1989) and cardiotoxicity (Yu et al., 2016). Rats
593 with cardiotoxicity exhibited symptoms of hypotension and slowness of the heart rate,
594 and the toxic dose can give rise to breathing problems, ataxia, glomerular necrosis,
595 acute renal failure and even death from respiratory depression. The LD50 of Tet in rats
596 was 646.00 mg/kg, and the 95% confidence limit was 504.58-788.42 mg/kg, which
597 suggested a moderate toxicity (Chen et al., 2011). The safe dose range in basic
598 research and clinical application, and the possible side effects are appropriate to be
599 monitored.
600 7.1. Pulmonary toxicity
601 The concentration of lactate dehydrogenase in bronchoalveolar lavage fluid
602 increased significantly, followed by damaged membrane integrity of a large number of
603 cells in the lung after intraperitoneally injection of with Dau (150 mg/kg). It was
33
604 indicated that Dau was more toxic than 4-ipomeanol, a model agent of pulmonary
605 toxin. The structure of Dau contains para-methylene phenol moiety, which is
606 metabolized to quinone methylate, an electrophilic active intermediate. Xie et al.
607 speculated that the formation of the quinone methide intermediate may be associated
608 with Dau-induced toxicity. The quinone methylates of Dau was conjugated to GSH
609 with it incubated in human liver microsomes or mice lung microsomes (Jin et al.,
610 2010) (Fig. 7). A time-dependent GSH depletion suggested that the protein
611 modification by the reactive metabolite triggered the cytotoxicity (Xie et al., 2016).
612 Metabolic studies have also showed that CYP3A enzyme was mainly related to the
613 production of reactive metabolites of Dau.

of
614 Similar to Dau, pulmonary toxicity was corroborated in CD mice after

ro
615 administration of Tet (150mg/kg). Demethylated by phase I metabolism, the methoxy
616 group of the benzene ring of Tet was converted into phenolic hydroxyl group, forming
617
-p
p-methylene phenol group. The metabolite could be further metabolized into quinone
re
618 methylates, resulting in toxicity (Fig. 9). Jin et al. detected a GSH conjugates derived
619 from O-demethylated tetrandrine in incubations of Tet with NADPH- and
lP

620 GSH-supplemented human liver and mice lung microsomes. The formation of the
621 conjugate could be inhibited by ketoconazole, indicating that CYP3A was mainly
na

622 responsible for the metabolic activation of Tet. From the above studies, quinone
ur

623 methide intermediate was related to the pulmonary toxicity of Tet (Jin et al., 2011).
624 The structures of liensinine, Nef and isoliensinine are similar to those of Dau and
Jo

625 Tet, and their molecular structures contain para-methylene phenol moiety as well. It
626 has been shown that CYP3A mediates the metabolism of Nef, while liensinine and
627 isoliensinine can also be widely metabolized by liver microsomes. In addition,
628 liensinine and isoliensinine are concentrated in the lung and kidney after intravenous
629 injection, while a little in other tissues. The pharmacokinetic study of Nef in rats
630 showed that the content of Nef was higher in the kidney and lung, followed by the
631 liver and heart. Therefore, liensinine, isoliensinine and Nef may produce pulmonary
632 toxicity at high doses. However, there is scarce information available on pulmonary
633 toxicity of these three alkaloids.

34
634
635 Fig. 7. Proposed metabolic formation of GSH conjugate from Tet.

of
636 7.2. Liver toxicity
637 D-Tetrandrine has been shown to be drug-dependent and tissue-toxic in beagles and

ro
638 monkeys. Hepatotoxicity occurred after intravenous injection of the experimental
639
-p
group and showed severe cases of sudden drop in blood pressure (Gralla et al., 1974).
re
640 Treatment with Tet led to significant ATP depletion, mitochondrial membrane
641 potential depolarization and release of cyt c accompanied by caspase 3 activation in
lP

642 rat primary hepatocytes. Caspase-dependent mitochondrial apoptosis pathway was

643 proved to be a primary mechanism in Tet-induced toxic effect on primary cultured
na

644 hepatocytes (Yan et al., 2006), in which ROS was a critical inducer. ROS associated
ur

645 with CYP450, particularly CYP2D1/2D2, played a key role in mitochondrial

646 permeability transition (Qi, 2007).
Jo

647 7.3. Genotoxic

648 Tet is considered to be a weak mutagen, which promotes the mutagenicity of
649 various mutagenic compounds in cells by affecting DNA repair function. In addition,
650 Tet enhanced the error prone repair in salmonella typhimurium TA98 and was also a
651 genotoxic enhancer (Whong et al., 1989). The gene mutation was induced by Tet in rat
652 liver homogenate by metabolic activation, and the frequency of chromosome
653 replication was inhibited from the increase of Tet content. Thus, it was apparent to be
654 a weak gene poison. Tet could indirectly inhibit DNA synthesis, affecting cell division
655 and leading to cell death (Xing, Shi, et al., 1989). Additionally, the frequencies of
656 sister-chromatid exchange (SCE) induced by mitomycin C (MMC) or cigarette-smoke
657 condensate (CSC) were enhanced by Tet (Xing, Wu, et al., 1989).
658 7.4. Nephrotoxicity
659 Yuan et al. clarified the hepatorenal toxicity of Tet and aristolochic acid (AA).
35
660 Weaker nephrotoxicity of Tet made local renal tissue hydropic degeneration highly
661 unlikely as compared to AA. However, in the subsequent hepatotoxicity experiments,
662 continuous administration of Tet for 2 months caused liver dysfunction and
663 histopathology. The toxicity was reduced in the incubation experiment with rat liver
664 extract, which was majorly due to the detoxification of metabolic enzymes in the liver
665 mixture (Yuan et al., 2011). Significant nephrotoxicity also appeared in monkeys, and
666 higher doses of Tet showed observable signs of toxicity in dogs (Gralla et al., 1974).
667 7.5. Mitochondrial dysfunction
668 The results of mitochondrial function evaluation showed that the metabolism of Tet
669 through CYP2E1 led to the change of mitochondrial permeability and dysfunction. It

of
670 was interesting to note that ROS produced by CYP2E1 could induce oxidative stress,

ro
671 and ROS clearance reduced the toxic effect of Tet (Qi et al., 2013).
672 There is a lack of research undertaken to evaluate the toxicity of Cep till date.
673
-p
Cepharanthin™ (CEP) which consists of several biscoclaurine alkaloids, namely Cep
re
674 (30%), isotetrandrine (36%), berbamine (14%) and cycleamine (13%) has shown no
675 obvious side effects in the treatment of leukopenia, which was safe in volunteer
lP

676 subjects with intravenous injection of CEP (Nakajima et al., 2004).

677 We have summarized the effect of BBIQ alkaloids on transporters, which affect the
na

678 ADME and interaction of drugs in various ways. In addition to acting as an

ur

679 anti-cancer agent to reverse MDR, it may also lead to the accumulation of drugs and
680 the toxicity of various tissues, including normal tissues. Current studies have
Jo

681 limitations towards of reducing the toxicity of drugs to normal tissues and cells, with
682 medications accumulating in the target area.
683 8. Future perspectives and conclusions
684 BBIQ alkaloids have been reported to show a variety of pharmacological activities
685 (Schiff, 1991). Most of them have the effects of anti-inflammation, anti-cancer,
686 anti-hypertensive, anti-arrhythmia and anti-ventricular hypertrophy. Nevertheless,
687 they are a kind of alkaloids with low solubility, which affects the absorption process
688 to a certain extent. In terms of metabolism, the quinone methide are formatted because
689 of their unique structural characteristics, which may be the common cause of the
690 toxicity of these compounds. Furthermore, most metabolic modes produce a variety of
691 isomers, which makes it a difficult task to identify their structures. Therefore, there is
692 still warrant for further pharmacological investigation.
693 Firstly, the variation of percentages of BBIQ alkaloids in different plant species
36
694 may give rise to the different pharmacological effects of these medicinal plants.
695 Traditional Chinese medicine monomers have made great achievements in
696 experimental research, and also occupies a large proportion in clinical use. Modern
697 research methods and science and technology are used to develop monomers with
698 specific pharmacological effects. Then, the pharmacology, toxicology, quality control,
699 dosage specification and mechanism of action could be standardized, shedding new
700 light on the compatibility of traditional Chinese herbal compound or Chinese patent
701 medicine.
702 Secondly, most of the BBIQ alkaloids have significant inhibitory effects on P-gp. It
703 is necessary to further screen out whether these constituents have effects on P-gp and

of
704 to what extent they can inhibit P-gp. The possibility and reasonable dose of BBIQ

ro
705 alkaloids acting on MDR reversal agent could be elaborated through a large number
706 of studies
707
-p
Thirdly, at present, the separation and extraction methods of these alkaloids have
re
708 been mature. However, due to their wide distribution and diversity, their quality
709 standards in different plant species should be improved. The determination of quality
lP

710 evaluation markers could provide a scientific basis for the research of its production
711 technology, with the objective of promoting more BBIQ alkaloids in clinical
na

712 application in the future.

ur

713 In conclusion, this study summarized the main distributions of BBIQ alkaloids in
714 plants, as well as the botanical characteristics, traditional uses, phytochemistry,
Jo

715 pharmacokinetics, toxicity, and MDR reversal effects. In addition to the common
716 alkaloids discussed above, there are a large number of trace alkaloids worthy of
717 research and development. With the deepening of research, they will also be widely
718 used in the clinical treatment of various diseases.

719 Acknowledgments: This work was supported by the National Natural Science
720 Foundation of China (grant no. 81303182, 81173523) and the National Major
721 Scientific and Technological Special Project for "Significant New Drugs
722 Development" (Grants No. 2018ZX09734-002).

723 Conflicts of Interest: The authors declare that there is no conflict of interest in this
724 article.
725 References:

726
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727 Ai, X. H., Tang, X. Q., Liu, Y. P., et al. (2007). [Effect of neferine on
728 adriamycin-resistance of thermotolerant hepatocarcinoma cell line
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761 diltiazem-binding site. Biochemistry, 31(47), 11793-11800.
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772 Gao, G. Y. (1999). Study on chemical constituents of Thalictrum atriplex and its
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778 Gralla, E. J., Coleman, G. L., & Jonas, A. M. (1974). Toxicology studies with
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779 d-tetrandrine (NSC-77037), a plant alkaloid with vascular and lymphotoxic

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780 effects in dogs and monkeys. Cancer Chemother Rep 3, 5(1), 79-85. Retrieved
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782 Gu, S. F., Shi, S. J., & Chen, H. (2003). [Determination of daurisoline in rabbit
783 plasma and study on its pharmacokinetics]. Yao Xue Xue Bao, 38(12), 908-910.
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