Ethnopharmacology,Phytochemistry,and Pharmacological Activities of Spatholobus suberectus Vine Stem

Abstract

Spatholobus suberectus vine stem is an important medicinal material in Chinese, Vietnamese, and Korean traditional medicines. Its decoction has long been used to treat blood disorders, such as anemia and menstrual irregularity, as well as rheumatoid diseases. Previous phytochemical investigations characterized 88 compounds from S suberectus, which mainly belonged to the phenolic class, especially of the flavonoid type. Pharmacological studies showed the beneficial effects of extracts of the plant on the cardiovascular tract, which is ethnopharmacologically relevant to the plant's use in traditional medicine. Besides that, the extracts and isolated compounds also exhibited antimicrobial, anticancer, antiinflammatory, and antioxidant activities. The past study results support the use of S suberectus vine stem in traditional medicine and also revealed new directions of pharmacological effects of this medicinal plant.

Keywords

flavonoids catechin blood disorders hematopoietic study

Introduction

Spatholobus suberectus Dunn (Leguminosae) is a woody climber, which has been widely cultivated for medicinal purposes in China, Vietnam, and Korea. The medicinal plant has different common names depending on the country, for example, “Ji-xue-teng” in China, “Gye-hyeol-deung” in Korea, and “Ke huyet dang” in Vietnam. These mentioned names can be literally translated as “chicken's blood vine” because the plant decoction has a red color, just like chicken's blood. According to the 5 elements theory of traditional Chinese medicine, red is associated with heart and blood conditions. Therefore, S suberectus decoction is traditionally used to activate and nourish blood in blood disorders such as menstrual irregularities, dysmenorrhea, and amenorrhea. In addition, S suberectus vine stem is also used for the treatment of bone-and-joint-related diseases, such as rheumatism, osteoarthritis, and backache.^1,2 Due to the popular use of the herb, characterization of its chemical composition is of great importance for determining bioactive compounds and quality control. Phytochemical studies revealed that the major chemical constituents were flavonoids, lignans, procyanidins, and megastigmane-type sesquiterpenoids. Of these, isoflavonoids were considered as the characteristic compound class of the plant, with genistein, formononetin, sativan, and liquiritigenin as notable representatives. Besides that, the content of catechin derivatives was also considerably high in the alcoholic and aqueous extracts. In terms of pharmacology, biological assays were also performed to explain scientifically the traditional uses of S suberectus vine stem in treating blood-related conditions. Published results indicated that the herbal extracts showed benefits in stimulating hematopoietic processes, preventing platelet aggregation, and improving blood cell counts in biochemical profiling. In addition, the extracts and isolated compounds also showed promising cytotoxic and antiproliferative activities against malignant cancer, especially breast cancer cells, as well as antiinflammatory, antiviral, and antibacterial effects.

The above-mentioned results assert the potential application of S suberectus stem in the healthcare system as a dietary supplement or herbal medicine. This review hopes to provide a clear summary of recent advances in S suberectus research, so that further biological and clinical studies can be carried out to exploit the full potential of this precious medicinal plant.

Search Strategy

The literature search for this review was performed on PubMed and Web of Science core collection databases until the end of September 2022. The search strategy used keywords “Spatholobus suberectus,” “Spatholobi Caulis,” and “Caulis Spatholobi” to cover all possible publications which are suitable for the topic of interest. After that, the title and abstract of each publication were scanned to exclude irrelevant publications, such as plant cultivation, plant classification, and genetic articles on agricultural topics. Vietnamese and Chinese pharmacopeia, as well as other professional books, were also used in this review. The final number of references used for this review was confirmed to be 70 after full-text reading and duplicate removal. The search results of single keyword and keyword combinations are described in Table 1.

Table 1.

Search Strategy in PubMed and Web of Science Databases Using Keyword Combinations.

No.	Search keyword	Number of matched publications
No.	Search keyword	PubMed	Web of Science
1	Spatholobus suberectus	89	96
2	Spatholobi caulis	27	22
3	Caulis spatholobi	15	22
4	(1) or (2) or (3)	125	134

Traditional Uses

S suberectus is a woody climber and shrubby at a young age with papery or subleathery leaves (9–19 × 5–14 cm). The plant can be naturally found in open or dense forests, mountain slopes, or valleys (800–1700 m) and it has also been widely cultivated for medicinal purposes in Asian countries, such as China, Vietnam, and South Korea.³ The dried vine stems, which are the plant part used in traditional medicine, are cut back to elliptical and oblong slices with 0.3 to 1.0 cm of thickness. In the cut surface, it is clear to observe reddish-brown xylem and brown resinous secretion, as well as numerous vessel pores.² In traditional Chinese medicine, red is associated with the fire element. The red and bitter herb is also believed to affect the functions of the heart, blood vessels, and small intestine. Therefore, the traditional indications are for menstrual irregularities, dysmenorrhea, amenorrhea, and blood deficiency since the herb can activate and nourish blood, as well as regulate menstruation.^1,2 In Vietnam, “Ke huyet dang” (literally translated as “chicken's blood vine”) is a mutual name of several medicinal plants since they share physical similarities (woody climber with red resinous secretion). The most mistaken plants are Sargentodoxa cuneata, Millettia sp., Butea superba, and Mucuna birdwoodiana.^4,5 The same phenomenon also happens in China and South Korea as well, which raises an urgent issue of using the correct taxonomic species for medicinal purposes.

Phytochemistry

There have been a number of phytochemical studies on S suberectus in 2004 to 2022, which revealed nearly 90 compounds belonging to different classes, such as simple phenols, euflavonoids, isoflavonoids, procyanidins, coumarins, and lignans (Table 2). The compounds were isolated and identified by conventional chromatographic procedures and spectroscopic analyses (nuclear magnetic resonance and mass spectrometry [MS]).

Table 2.

Phytochemical Constituents of Spatholobus suberectus Vine Stem.

No.	Compound name	Reference
Simple phenolic
1	Protocatechuic acid	^6,7
2	p-Hydroxybenzoic acid	⁶
3	Protocatechuic acid methyl ester	⁶
4	trans-p-Hydroxycinnamic acid	⁶
5	Salicylic acid	⁶
6	m-Hydroxyphenol	⁶
7	p-Hydroxyquinone	⁶
8	C-Veratroylglycol	⁶
9	Evofolin B	⁶
10	4-Hydroxy-3-methoxy cinnamic acid methyl ester	⁶
11	2-Phenylethanol	⁷
12	5-O-(β-Apiofuranosyl-(1→2)-O-β-xylopyranosyl)gentisic acid	⁸
13	1-O-(β-Apiofuranosyl-(1→6)-O-β-glucopyranosyl)-3-O-methylphloroglucinol	⁸
Isoflavonoid
14	Daidzin	⁶
15	Sphaerobioside	⁶
16	Genistin	^6,9
17	Ononin	^6,10
18	2′-Hydroxygenistein	⁶
19	Daidzein	^6,11
20	Genistein	^6,9,11,12
21	Formononetin sodium	^6,9
22	Formononetin	^6,9–11
23	Biochanin A	^6,10,13
24	4′,8-Dimethoxy-7-O-β-d-glucopyranosyl isoflavone	^6,10
25	8-O-Methylretusin	⁶
26	Butesuperin A	⁶
27	3′-Methoxydaidzein	⁶
28	Calycosin	⁶
29	Odoratin	⁶
30	7,4′-Dihydroxy-8-methoxy-isoflavone	⁷
31	Glycyroside	⁷
32	Pseudobaptigenin	¹²
33	Afromosin	¹⁰
34	Spasuberoside B	^6,9
35	Spasuberol B	^6,9,10
36	Spasuberol A	^6,9
37	47,2′-Trihydroxy-4′-methoxy isoflavanol	^6,7,9,10
38	Sativan	^6,13
39	Dihydrocajanin	⁶
40	Spasuberoside A	⁹
41	(3S)-7-Hydroxy-8,2′,4′-trimethoxyisoflavane	¹³
42	(3S)-7-Hydroxy-8,2′-dimethoxy-4′,5′-methylenedioxyisoflavane	¹³
43	(3S)-7,2′-Dihydroxy-8,4′-dimethoxyisoflavane	¹³
44	7,4′-Dihydroxy-8,2′,3′-trimethoxyisoflavane	¹⁴
45	7,2′,4′-Trihydroxy-8,3′-dimethoxyisoflavane	¹⁴
46	(6aR, 11aR)-Methylnissolin 3-O-glucoside	⁶
47	(6aR, 11aR)-Maackiain	^6,12
48	Medicarpin	^11,12
Euflavonoid
49	Liquiritigenin	^6,9,11,15
50	Naringenin	⁶
51	7-Hydroxyflavanone	^6,10,11
52	(2S)-7,3′,5′-trihydroxyflavanone	¹⁵
53	Butin	¹⁵
54	(2R, 3R)-Buteaspermanol	⁷
55	(2R, 3R)-3,7-Dihydroxyflavanone	⁷
56	(2S)-67,3′,5′-Tetrahydroxyflavanone	¹⁵
57	3,5,7,3′,5′-Pentahydroxyflavanone	⁷
58	(2S)-7-Hydroxy-6-methoxyflavanone	¹²
59	Isoliquiritigenin	^9,11
60	Butein	¹⁶
61	7,3′,4′-Trihydroxyflavone	¹⁶
62	Methylenebisisoliquiritigenin	¹⁶
63	(+)-Gallocatechin	^6,17
64	(+)-Catechin	^6,17
65	(+)-Epicatechin	^6,17
66	(−)-Epiafzelechin	⁶
67	Spasuberol C	⁹
68	7,2′-Dihydroxy-8-methoxy-4′,5′-methylenedioxyisoflavan-3-en-2-one	¹³
69	5,7-Dihydroxycoumarin	⁶
70	Aloe-emodin	⁸
71	15-O-(α-ʟ-Rhamnopyranosyl)aloe-emodin	⁸
72	2,6-Dimethoxy-1,4-benzoquinone	⁹
73	7-Hydroxychromone	⁶
74	Homovanillyl-4-oxo-nonanoate	⁹
75	Prestegane B	⁷
76	(+)-Medioresinol	^6,7
77	(+)-Syringaresinol	⁶
78	(+)-Pinoresinol	⁶
79	(+)-Epipinoresinol	⁶
80	Isolariciresinol	⁶
81	Procyanidin B2	¹⁰
82	Procyanidin B4	¹⁸
83	(+)-Catechin-(4→8)-(+)-catechin-(4→8)-(−)-epicatechin	¹⁸
84	8,9-Dihydroxy megastigma-4,6-dien-3-one	⁶
85	Abscisic acid	⁶
86	6,9-Dihydroxy megastigma-4,7-dien-3-one	⁶
87	Nicotinic acid	⁶
88	Allantoin	⁶

Simple Phenolics

Simple phenolics (1–13) were identified from S suberectus stems. These compounds possess a simple benzene ring with different functionalities, such as hydroxyl (–OH), methoxyl (–OCH₃), carboxyl (–COOH), occasionally either propyl (4, 8, and 10) or sugar chains (12 and 13) (Figure 1).

Figure 1.

Structures of simple phenolics from Spatholobus suberectus stem.

Isoflavonoids

Isoflavonoid is a subclass of flavonoid, which possesses a 3-phenylchromane backbone, instead of the 2-phenylchromane backbone of euflavonoid. Phytochemical investigation revealed that isoflavonoids can be abundantly found in S suberectus stems with nearly 40 reported compounds (14–49). They can be classified into 3 types, that is, isoflavone (14–34), isoflavane (35–46), and pterocarpan (47–49) (Figure 2).

Figure 2.

Structures of isoflavonoids from Spatholobus suberectus stem.

Besides the common isoflavone scaffold with hydroxyls at C-5 and C-7, S suberectus compounds usually lack hydroxyl at C-5. Occasionally, methoxylation at C-8 can occur (24, 25, and 30). In the case of glycosides, the sugar chain is more likely to bind with C-7 of the aglycon moiety via an O-glycosidic bond (14–17 and 31). The monosaccharide components are of common type, that is, glucopyranose, rhamnopyranose, and apiofuranose.

Isoflavane derivatives were also obtained from S suberectus stems (34–45). The compounds usually lack a hydroxyl at C-5 and possess a methoxyl at C-8 (36 and 41–45). Some substances are hydroxylated at C-4 (35–37 and 40). The C-4′ position is usually methoxylated or a methylenedioxy group can be formed at C-4′ and C-5′ (35, 36, and 42). As for absolute configuration, circular dichroism (CD) spectra and specific optical rotations should be measured to determine the stereochemistry of the C-3 position. According to empirical rules, a positive Cotton effect at ∼240 nm and a negative Cotton effect at ∼280 nm are the signs of 3S-isoflavane,¹³ while the 3R-isomer should show a negative Cotton effect in the 215 to 240 nm region.¹⁴ For complex derivatives, electronic CD simulation is required to assign the absolute configuration of compounds.⁹ Pterocarpan-type isoflavonoids were also found in S suberectus stems (46–48). They are isoflavane derivatives with a ring disclosure formed by a C-4 carbonyl and the C-2′ position of the B-ring with the absolute configuration usually assigned 6aR, 11aR.

Euflavonoid

Euflavonoids are also present in S suberectus stems with a flavanone scaffold. The characteristics of flavanones from this plant are (1) lack of hydroxyl at C-5 (49, 51–56, and 58) and (2) the stereochemistry of C-2 is usually S. Occasionally, compounds with an unsubstituted B-ring or β-hydroxylated at C-3 can be found (51, 54, 55, and 58). Common catechin derivatives were also isolated and structurally identified (63–66). Chalcone derivatives (59, 60, and 62) were also found in the plant with an exceptional structure of a methylene-bridged bichalcone (62), which is a dimeric chalcone with a methylene bridge between 2 identical isoliquiritigenin molecules (Figure 3).

Figure 3.

Structures of euflavonoids from Spatholobus suberectus stem.

Other Compounds

A number of phenolic compounds belonging to other types have also been reported, including coumarins (67–76), quinones (70–73), lignans (75–80), and procyanidins (81–83). Besides that, some megastigmane-type sesquiterpenes were also isolated (84–86) (Figures 4 and 5).

Figure 4.

Structures of other compounds from Spatholobus suberectus stem.

Figure 5.

Structures of other compounds from Spatholobus suberectus stem.

From published phytochemical results, it is observed that the main chemical constituents of S suberectus stems are isoflavonoids with different substitution types. In addition, simple phenolics and other phenolic classes (ie, coumarin, lignan, quinone, and procyanidin) were also reported. With nearly 90 compounds reported with full chemical structure and stereochemistry, the chemical profile of S suberectus stem is clarified, which has paved the way for further analytical and pharmacological studies.

Phytochemical Analysis

To quantify the content of bioactive compounds from S suberectus stems, a number of analyses were performed on various instruments. Five batches of stem samples were analyzed by high-performance liquid chromatography–MS (HPLC–MS), which resulted in the identification of mainly procyanidins and catechins with stark examples of procyanidin B2, epicatechin, and propelargonidin dimer. The total phenolic content was estimated from 1.81% to 2.60% dry weight (d.w) by a colorimetric method.¹⁷ The combination of matrix-assisted laser desorption ionization (MALDI)–time-of-flight (TOF)–MS and electrospray ionization-tandom mass spectrometry (MS/MS) analyses on fractions from S suberectus extracts resulted in the identification of procyanidin oligomers (3–11 monomers) with catechin and epicatechin as the main components, B-type interflavane linkage, and these compounds belonged to a structural heterogeneity type. The oligomeric procyanidin (mean degree polymerization 1–6) can be considered as the bioactive component of S suberectus with a mass distribution of 70.4%.¹⁹ Another study on procyanidin composition indicated that the major type in S suberectus stems is of a homogenous B-type with flavan-3-ol units linking with each other via C–C bonds.¹⁷

In an analytical work with ultra-performance liquid chromatography and MS/MS, Liu's group successfully detected and quantified 57 compounds from 12 batches of S suberectus stem collected in different cultivation places in China, Myanmar, Vietnam, and Indonesia. As a result, isoflavones and flavonols were found in high abundance in the plant (40%–50% of all analytes) and their contents showed high dependence on geographic location, latitude, and local climate. According to S- and VIP plots, compounds 23 and 60 have greater VIP values, proving that they can be used as characteristic compounds for quality control of S suberectus samples from different areas.⁶ In herbal medicine practice, Spatholobi caulis and Sargentodoxae caulis are usually mistaken due to their similar physical appearance. Because of that, an HPLC–MS study with principal component analysis (PCA) support was conducted to develop an effective method to distinguish the 2 herbal medicines, which was based on 16 small-molecule analytes and 37 different sample batches. The analytical results showed that the PCA scores of the 2 species clustered in different regions in the scatter plot, which implied that HPLC–MS/MS with PCA add-on could be a useful tool for distinguishing the 2 physically similar plants.²⁰ Chemical compositions of Spatholobi Caulis can be analyzed quickly by employing ultrafast liquid chromatography (UFLC)-triple TOF-MS/MS and UFLC–quadrupole ion trap (QTRAP)–MS/MS, which can simultaneously identify 50 constituents within 25 min based on MS fragmentation. The study results also showed the correlation between the phloem ring number of the stem and flavonoid content, which provided scientific evidence for appearance-based quality evaluation of Spatholobi Caulis in traditional Chinese medicine.²¹

As for flavonoid components, liquiritigenin (49), genistein (20), isoliquiritigenin (59), and 7-hydroxyflavanone (51) were identified as major constituents of S suberectus stems from Daegu market (Korea). Their contents were determined as 0.654, 0.166, 0.665, and 0.193% (d.w.), respectively, by a validated HPLC quantification method.²² Besides the HPLC method, capillary electrophoresis was also chosen to analyze secondary metabolites from S suberectus stems due to its advantages, such as greener technology (using only buffer solutions instead of organic solvents), fast analysis, and high reproducibility. Zeng et al. developed a capillary electrophoresis method with electrochemical detection to quantify the contents of epicatechin, emodin, syringic acid, vanillic acid, rhein, and protocatechuic acid from S suberectus stem and its herbal preparations. However, the limitation of the method is that the secondary metabolites from the plant should be ionizable to move with the electric field, and neutral compounds will not be affected by the applied voltage.²³

S suberectus stem is listed as a monograph in the Chinese and Vietnamese Pharmacopoeias. The crude drug is prepared by collecting the lianoid stem of S suberectus, removing branches and leaves, cutting it into slices, and drying it under the sun. The shape of the crude drug is elliptical (0.3–1 cm of thickness) with a greyish-brown cork, reddish-brown texture, and a number of branch vessels. The identification tests include physical appearance and thin-layer chromatography (TLC; with standard plant materials).^1,2 In the Hong Kong Chinese Materia Medica Standards, it is required to perform HPLC fingerprinting of the plant methanolic extract. There are 6 chemical markers required in the fingerprinting chromatogram and the standard is formononetin (22). As for quantification assay, a reverse-phase HPLC method is used with (−)-epicatechin as standard and plant samples should not contain <0.021% of (−)-epicatechin.²⁴ Liu et al.²⁵ also developed a UFLC–MS/MS method to detect and quantify the major compounds of S suberectus extracts in rat serum. Twenty-five major compounds peaked at about 10 to 15 min after oral administration and gradually decreased to low levels after 6 to 12 h, which proved the rapid absorption of the active compounds via the gastrointestinal route.

The current analytical research on S suberectus stems indicated the high content of phenolic compounds and oligomeric procyanidins. Different approaches were utilized to determine the concentration of major constituents, such as HPLC, LC–MS, and capillary electrophoresis. Since the chemical composition of S suberectus stems in different regions is very diverse, it is recommended to identify marker compounds from the investigated samples for quality control procedures.

Pharmacology

Effects on the Blood Circulation System

S suberectus is well-known for its blood-activating and blood-stasis-dispelling properties in traditional Chinese medicine, which is usually associated with the red of the decoction. There have been a handful of pharmacological studies to demonstrate the medicinal effects. Hematopoietic studies often run into some difficulties, including (1) ethical issues because of using human embryonic cells; (2) mouse models cannot mimic the effects on the human body and they might be very expensive and time-consuming; (3) mouse hematopoiesis is different from that of humans; and (4) hematogenesis is considered a very complicated procedure for designing bioassays. Chang et al. developed an AGM-S3 coculture system (cells derived from the aorta-gonad-mesonephros region in mouse embryos) to test the hematopoietic effects of either crude extracts or isolated compounds in vitro. Catechin (65) appeared as the most active compound by increasing the population of blood cell surface markers such as CD34⁺, CD45⁺, and GPA⁺CD71⁺ by 2.5-fold and 1.5-fold at day 14 at a concentration of 10 µM, respectively. In a hematopoietic colony assay, the addition of catechin caused a remarkable increase in the formation of colony-forming unit-erythroid (CFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-Mix colonies, indicating that catechin strongly enhanced the hematopoietic process. Dimethyl sulfoxide (DMSO) was used as a negative control to remove its influence on hematopoiesis.²⁶ Another study by Wang's group clarified the effects of catechin on hematopoietic growth factors in mouse spleen and bone marrow cells. Specifically, catechin treatment was shown to promote the proliferation of colony-forming unit (CFU)-GM, BFU-E, and CFU-Meg, therefore producing granulocyte-macrophage-colony-stimulating activity, burst-promoting activity (BPA), and megakaryocyte-colony-stimulating activity in both in vitro and in vivo experiments, when compared to those of control groups.²⁷ Caulis Spatholobi aqueous extract was also found to be a strong HMAPM (hepcidin-encoding gene) expression inhibitor, which affected hepcidin production, therefore improving the iron content for iron deficiency anemia.²⁸

Furthermore, the role of S suberectus stem extract in preventing platelet aggregation was also investigated. The ethanolic extract (200–400 μg/mL) showed inhibitory effects on collagen- and adenosine diphosphate (ADP)-exposed platelet aggregation in human PRP (platelet-rich plasma), which was comparable to that of aspirin at 1 mM concentration, without affecting the clotting time. Mechanistically speaking, it was found that the extract blocked the binding of fibrinogen to the glycoprotein (GP) IIb/IIIa receptor and partly suppressed the formation of thromboxane A2, leading to the antiplatelet aggregating effects. However, the chemical characterization of the plant extract was not mentioned in the study.²⁹ Pharmacological studies were also expanded to the protective effects on the circulatory system from radiation. When compared to those of the untreated control group, oral administration of S suberectus stem ethanolic extract was capable of improving the blood cell profile and reducing the intracellular reactive oxygen species (ROS) serum level in irradiated mice by the end of the 28-day experiment. The mechanism might be due to the upregulation of phospho-Janus kinase 2 (pJAK2)/JAK2, phospho-signal transducer and activator of transcription 5a (pSTAT5a)/STAT5a, and B-cell lymphoma 2 (Bcl-2) in bone marrow tissue, thereby activating the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which holds the key role in functional hematopoiesis. The study did not mention the chemical characterization of the plant extract.³⁰

The role of S suberectus in the treatment of deep vein thrombosis (DVT) was also investigated in the inferior vena cava stenosis-induced DVT rat model. The authors decided to use 2 doses (0.125 and 0.25 g Spatholobi Caulis dispensing granule/kg/day), which was equal to 1.56 g crude drug/kg/day for rats, or 15 g crude drug/day for humans.² The rats were orally given the extract once a day for 7 consecutive days. As a result, the extract (0.125 and 0.25 g/kg/day) was shown to reduce thrombus weight by 23.1% and 44.1%, respectively, which was comparable to the effects of the positive controls, heparin (200 U/kg/day, i.v) and clopidogrel (25 mg/kg/day, p.o). The extract exhibited no effects on prothrombin time, ADP-induced maximum platelet aggregation, and blood profile. However, the prolonged activated partial thromboplastin time (APTT) in the extract-treated group was observed to increase significantly (20.2 ± 0.9 s and 20.6 ± 1.1 s), when compared to the vehicle group (18.6 ± 0.3 s). The S suberectus extract also ameliorated the total protein expression of tissue factor in the vein wall and thrombus. In-depth pharmacological assays have shown that Spatholobi Caulis dispensing granule might show anti-DVT activity through antiinflammation via Sirtuin 1 (SIRT1) and nuclear factor erythroid 2-related factor 2 (Nrf2).³¹

Past study results provided evidence for supporting the traditional uses of S suberectus stems, thereby partly explaining the ethnopharmacology of this plant's “blood-activating” and “blood-stasis-dispelling” properties. The extracts showed clear benefits for promoting the hematopoietic processes, preventing blood clots by inhibiting platelet aggregation, reducing thrombus, and increasing the prolonged APTT in DVT. Moreover, the plant was also able to protect the hematopoietic system in bone marrow from harmful factors like γ-radiation. These promising pharmacological data prompted researchers to conduct further research to clarify the benefits of using S suberectus in other hematological diseases.

Effects on Skin Conditions

The extracts of S suberectus stems were proven beneficial for skin care and healing. The aqueous and ethanolic extracts were capable of protecting human keratinocytes from ultraviolet B (UVB) irradiation at 30 µg/mL with low cytotoxicity (80% cell viability) in human keratinocyte cells (HaCaT). Further evaluations also indicated that both extracts suppressed the breakdown of elastin by inhibiting elastase activity, and upregulated the protein levels of elastin, type I collagen (COL1A1), and hyaluronan synthase 2 (HAS2), as well as downregulated the expression of matrix metalloproteinases 1 and 2 (MMP-1 and -2), which resulted in increasing the collagen and elastin content, thereby improving skin wrinkles and elasticity. It was also found that phenolics, major bioactive compounds in S suberectus extract, could produce synergistic effects in certain ratios, for example, syringic acid: epicatechin: vanillic acid = 2:1:1 (w/w) with a combination index of 0.28, which partly explained the significantly higher effects of the extract than those of the single isolated compounds.³² The plant extract was also active in the tyrosinase inhibitory bioassay, which is commonly used for the evaluation of skin-whitening properties. Bioassay-guided isolation led to the identification of 12 compounds in the most active fraction in mushroom tyrosinase inhibitory testing. Among the isolated flavonoids, compound 53 (butin) displayed the strongest antityrosinase activity (IC₅₀ = 35.9 µM), which was shown to be even better than that of the positive control, arbutin (IC₅₀ = 2.9 mM). The 5-deoxy flavanone derivative also reduced the melanin content (29.3% at 100 μM), which was explained by the inhibition of TRP1 and TRP2 encoding genes in human epidermal melanocytes.³³ In 2017, Dai and colleagues evaluated the wound-healing properties of a gel containing 10% to 20% S suberectus extract in second-degree burns in a rat. A histological examination revealed that topical treatment of the plant extract significantly reduced congestion or edema in the epidermal layer and it also increased fibroblast proliferation, angiogenesis, and epithelialization after 14 days when compared to the untreated group. Lipid peroxidation (LPO) and plasma cytokine levels were also measured to evaluate the inflammatory condition of the burn wounds. S suberectus extract was found to decrease the malondialdehyde activity and reduce the secretion of inflammatory mediators, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which can explain the antiinflammatory effects in burn wound treatment.³⁴

Previous study results confirmed the potential application of S. suberectus in skin care and wound healing. The extracts were found to promote the content of collagen, elastin, and hyaluronic acid, therefore improving skin elasticity and reducing wrinkles. Furthermore, the extract also inhibited the enzymatic activity of tyrosinase and reduced melanin content, which was essential for skin whitening. The extract was also effective in healing second-degree burn wounds. Commercial products containing plant extracts for skin conditions are popular, for example, Centella asiatica, Aloe vera, and Curcuma longa. This can be considered a new direction for pharmacological research of the plant.

Antiinflammatory Activities

The decoction of S suberectus stems has been used for the treatment of bone-and-joint-related diseases, such as osteoarthritis and rheumatism in traditional medicines. To provide scientific evidence for medicinal purposes, a number of pharmacological studies were conducted. Lipopolysaccharide-induced RAW264.7 macrophage cells are frequently used to evaluate the antiinflammatory effects of either crude plant extracts or purified natural compounds. Mohibbullah and colleagues (2017) reported that the hot-water extract of S suberectus stems significantly reduced NO production and downregulated the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in comparison with the untreated lipopolysaccharide (LPS) control. However, the inhibitory effects on COX-2 were not in a dose-dependent manner in the reported data. The study also revealed gallic acid as a major compound (22.77 mg/g dried plant materials), which was attributed to the antiinflammatory activities of the plant.³⁵ Liu et al.⁹ performed a bioactivity-guided isolation to discover antiinflammatory agents from S suberectus stems. As a result, compounds 73 and 59 showed the strongest activity with IC₅₀ values of 5.69 ± 0.28 and 6.78 ± 0.59 μM, which were even lower than those of the positive controls, ʟ-N₆-(1-iminoethyl) lysine hydrochloride (ʟ-NIL) and indomethacin. In-depth analysis indicated that isoflavone derivatives were mainly responsible for the antiinflammatory activities by downregulating the mRNA expression of IL-6, IL-1β, TNF-α, iNOS, and COX-2.⁹ Liu's research group evaluated the inhibitory effects on NO production of 25 major compounds from S suberectus stem, which resulted in the identification of isoflavones 21–23 as the most potential compounds (IC₅₀ = 22.75, 21.11, and 48.29 μM, respectively), in comparison with positive controls (ʟ-NIL with IC₅₀ = 19.08 μM and indomethacin with IC₅₀ = 55.44 μM).²⁵ 3′,4′,7-Trihydroxyflavone (61) was shown to inhibit either LPS- or interferon γ (IFN-γ)-induced NO production by suppressing iNOS via the STAT1 pathway in MG6 microglial cells, thus showing promising neuroprotective effects from oxidative stress and β-amyloid aggregation in neurodegenerative diseases, such as Alzheimer's disease.³⁶

Human neutrophil elastase (HNE) is a serine proteinase, which belongs to the same family as chymotrypsin. This enzyme plays a crucial role in causing inflammatory conditions, such as arthritis and rheumatism.³⁷ The total phenolic fraction of S. suberectus also inhibited HNE activity in a dose-dependent manner (IC₅₀ = 1.33 μg/mL). The HNE inhibitory bioassay used sivelestat as the positive control. However, its IC₅₀ value was not reported in the study. As for fraction characterization, 18 phenolic compounds were identified in the phenolic-enriched fraction by HPLC-diode array detection(DAD)-MSⁿ with the majority being B-type procyanidins, which were mainly responsible for the bioactivity.³⁸ S suberectus aqueous extract was also studied for its potential activities in atopic dermatitis. Oral administration of the water extract (30–300 mg/kg in 3 weeks) was shown to significantly reduce ear and epidermal thickness, dermatitis score, mast, and T-cells infiltration, as well as inflammatory signpost production (immunoglobulin E [IgE], histamine, and proinflammatory cytokines) in the male NC/Nga mouse model, which were comparable to those of the positive control, dexamethasone (3 mg/kg). The in-depth cellular bioassay also indicated that the extract exhibited antiinflammatory activity by regulating proinflammatory chemokines production via the mitogen-activated protein kinase (MAPK)/STAT1/nuclear factor-κB (NF-κB) pathway in IFN-γ and TNF-α-induced HaCaT cells. The water extract was also characterized by HPLC analysis with the identification of gallic acid, (+)-catechin, procyanidin B2, and epicatechin as major compounds.³⁹

Another study focused on the effects of liquiritigenin (49), which is one of the major isoflavones from S suberectus, in atopic dermatitis. Liquiritigenin (49) was shown to block T-cell activation by reducing IL-2 production and CD69 expression via the NF-κB and MAPK pathways. In vivo experiments on dinitrochlorobenzene-exposed atopic dermatitis in mice also indicated the benefits of oral treatment with liquiritigenin (1 mg/mL, 5 times × 4 weeks), such as reduction of ear dermis and epidermis thickness, downregulation of IgE level, and recovery of tissue structure, when compared to the untreated group.⁴⁰ Another molecular target, 5-Lipoxygenase (5-LOX), was also studied to develop antiinflammatory drugs. Jiang's group designed a virtual screening to predict potential 5-LOX inhibitors from S suberectus stems by Autodock and comparative molecular field analysis. The study suggested that liquiritigenin (49), catechin (65), butin (53), and gallocatechin (64) were the most potential candidates for inhibiting 5-LOX activity.⁴¹

Recent studies provided important evidence for the antiinflammatory effects of S suberectus stems, which is ethnopharmacologically relevant to traditional medicine. Molecular bioassays confirmed the mechanism of action via downregulation of inflammatory mediators and animal studies showed positive results on atopic dermatitis in the mouse ear model. Researchers also found a relationship between isoflavone derivatives (such as liquiritigenin and 3′,4′,7-trihydroxyflavone) and antiinflammatory properties, implying that these compounds can be considered bioactive markers in quality control procedures. Further animal studies should be performed before initiating any clinical investigations.

Antioxidant Activities

The stem extract of S suberectus was studied for radical scavenging effects in different systems, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, FeSO₄-induced LPO inhibition, and hepatoprotectivity in CCl₄-induced liver damage. Accordingly, the plant extract showed 86.7% and 91.2% DPPH radical scavenging activities at 100 and 1000 μg/mL, respectively. The bioactivity was also investigated in FeSO₄-induced LPO in rat liver homogenates with 83% inhibition at the concentration of 1000 μg/mL. However, the tests used neither negative nor positive controls, making it difficult to evaluate the antioxidant potential of the plant extract. In the CCl₄-induced hepatotoxicity test, the extract (30–300 mg/kg) was shown to reduce significantly the serum levels of liver enzymes (alanine transaminase [ALT], aspartate transaminase [AST], and alkaline phosphatase [ALP]), when compared to those of the vehicle-treated group.⁴² Another oxidative stress model is using RAW264.7 cells infected with porcine circovirus type 2 (PCV2), which can cause elevated levels of intracellular oxidants (NO, ROS, and glutathione) and intracellular oxidases (xanthine oxidase [XOD], myeloperoxidase [MPO]) while the content of antioxidants (glutathione [GSH]) and antioxidases will be in decline. The total flavonoid fraction of S suberectus (25–100 µg/mL) was capable of scavenging free radicals and balancing the ratio of oxidant/antioxidant, as well as that of oxidase/antioxidase, which was comparable to the effects of vitamin C (100 µg/mL) as a positive control.⁴³ In a mouse model, the flavonoid-enriched fraction (25–100 µg/mL) exhibited similar antioxidant effects in PCV2-infected mice, thereby remarkably improving thymus and spleen indices, when compared to the untreated control group. The fraction contained formononetin (0.44%, w/w) and isoliquiritigenin (0.20%, w/w).⁴⁴

Antioxidant bioassays are commonly used in phytopharmacological research. Hence, it is necessary to understand the purpose and extrapolability of each experimental setup to perform further studies, for example, animal studies and clinical settings. DPPH radical scavenging can measure the electron donation of tested extracts or compounds; however, since the assay is performed in a nonphysiological pH environment, further cellular or animal studies should be conducted to confirm the antioxidant effects. S suberectus extracts or flavonoid fractions were effective in protecting cells from oxidative stress caused by toxic substances or viral infection.

Anticancer Activities

Breast cancer is the most common type of cancer and is the leading cause of high morbidity and mortality for women on a global scale. With a high content of isoflavonoid, which is a so-called phytoestrogen due to its similar structure to the woman's hormone, S suberectus stem was expected to be beneficial for breast cancer. The prepared fraction of stem extract from a polyamide column was shown to be cytotoxic against an estrogen receptor-positive breast cancer cell line MCF-7 by activating the apoptotic mechanism, arresting the G₀/G₁ phase of the cell cycle, and inhibiting cell migration. HPLC analysis indicated that the main components of the fraction were formononetin (39.4%), daidzein (9.5%), and genistein (7.2%), implying that these compounds might be responsible for the cytotoxicity.⁴⁵ Another pharmacological study focused on the efficacy of S suberectus extract on triple-negative breast cancer (negative test results for estrogen receptors, progesterone receptors, and excess HER2 protein), which is considered an aggressive and poor prognostic type of breast cancer. In a xenograft-bearing mouse model, oral treatment with the crude extract (equivalent to 0.4–0.8 g plant materials/kg body weight) was observed to significantly reduce the size of the tumor after 22 days (55.7% reduction), compared to the vehicle group. The underlying mechanism was via inhibiting the activity of lactate dehydrogenase A,^17,19 suppression of the MAPK PI3K/protein kinase B (Akt) pathway,⁴⁵ and via ROS-activated noncanonical inflammasome pyroptosis,⁴⁶ which eventually led to cell cycle arrest at different phases and cell migration reduction in breast cancer. Extracts of Caulis Spatholobi were known for their inhibitory effects on platelet aggregation in an ADP-stimulation model. The plant extracts also showed effects in a tumor cell-induced platelet aggregation (TCIPA) experiment, which was found to be effective in preventing metastasis of the cancer cells. Chen et al.⁴⁷ reported the preventive effects of Caulis Spatholobi alcoholic extracts on lung metastasis of breast cancer in a mouse tail vein injection model. The extract-treated group showed fewer metastases and a higher survival rate when compared with the control group in a small animal in vivo imaging system and hematoxylin and eosin staining assay (aspirin used as positive control).⁴⁷ Meanwhile, Sun et al.⁴⁸ reported the antimetastatic activities of the ethyl acetate fraction of Caulis Spatholobi on colorectal cancer, which was even better than the positive control, aspirin. These studies showed promising results in tumor metastasis regulation via an anti-TCIPA mechanism.⁴⁸

A network pharmacology-based strategy was also used to discover the underlying mechanism of the antibreast cancer effects of S suberectus vine stem. A total of 16 potential compounds and 252 targets were analyzed to suggest that the possible mechanisms might include peroxisome proliferator-activated receptor γ (PPARγ) activation, PI3K/AKT inhibition, apoptosis induction, and G₂ phase arrest in breast cancer cells. The authors also confirmed that the plant extract showed cytotoxicity by increasing PPARγ activation and decreasing the expression of PI3K and AKT phosphorylation in both BT-549 and MCF-7 cell lines, which matched the prediction from network pharmacology screening.⁴⁹ By employing the same technique, Xie et al.⁵⁰ predicted that S suberectus extract could prevent lung cancer metastasis by targeting several pathways, including oxidative stress, AGE-RAGE signaling, and microRNAs in cancer. However, the authors did not report any experimental results within the work.⁵⁰

Since the plant extract showed clear and potent anticancer effects, it is important to identify the responsible compounds, which play a key role in bioactivity. Bioguided isolation using a lactate dehydrogenase A (LDH-A) inhibition assay has led to the identification of 4 major catechin derivatives (63–66). LDH-A has a role in maintaining energy production in cancer cells, hence LDH-A suppression will directly target tumor energy supply, leading to the inhibition of cell growth, angiogenesis, and metastasis. Among those isolates, epigallocatechin (64) might be the key compound in the LDH-A inhibitory effect due to its strongest inhibition and synergistic effects with other catechins in the mixture. Further studies indicated that epigallocatechin (64) promoted the degradation of hypoxia-inducible factor-1α (HIF-1α; an LDH-A regulator) and inhibited the cancer cell proliferation and LDH-A expression in a xenograft model.¹⁷ Another study focused on the inhibitory effects of sativan (38) on triple-negative breast tumor growth. Sativan was found to positively regulate miR-200c, and inhibit the epithelial-to-mesenchymal transition (EMT), thereby inhibiting cancer cell proliferation, migration, and invasion. In a xenograft model, the isoflavane derivative at doses of 25 to 50 mg/kg/day was also capable of reducing the size of the tumor (in terms of weight and volume) without any significant influence on internal organs such as heart, liver, and lungs, when compared to the control group.⁵¹ A study on structure–activity relationship of isoliquiritigenin analogs was carried out on the 2 breast cancer cell lines (MCF-7 and MDA-MB-231). The following modification can result in a reduction or even loss of activity, including (1) reduction of α, β-double bond; (2) cyclization of 2′-OH of chalcone scaffold; (3) introduction of more than 1 hydroxyl to either A- or B-ring; (4) replacement of 4-OH by 5-OH; (5) introduction of sugar moieties to the aglycone; (6) methylation of 4′-OH in B-ring. The authors also reported structural modification that increased the inhibitory effects on breast cancer cell growth, which are (1) methylation of 2,4-di-OH in A-ring and most noticeably (2) methoxylation at C-3,4′,5 positions in A- and B-rings with the lowest IC₅₀ values (0.71 ± 0.17 μM for MCG-7 and 6.5 ± 0.83 μM for MDA-MB-231 cells), which showed promise when compared to paclitaxel (IC₅₀ = 7.4 ± 1.92 and 3.9 ± 0.69 nM, respectively).¹⁶ Meanwhile, natural isoflavonoids from S suberectus only exhibited moderate cytotoxicity against the same cancer cell lines (IC₅₀ ∼ 25–95 μM).¹³

The high-pressure hot-water extract of S suberectus vine stem was studied for its potential antiglioma activity. In rat C6 glioma cells, the extract showed strong cytotoxicity with an IC₅₀ value of only 1.65 ± 0.52 ng/mL without affecting noncancerous cells, including chicken DF-1 and NIH-3T3 fibroblast cells. In addition to cytotoxicity, the extract also inhibited proliferation of the glioma cell line by 49.19 ± 6.40% at a concentration of 7 ng/mL after 3 days of growth. Mechanistically, the extract acted via dose-dependent reduction of the intracellular ROS serum level, induction of mitochondrial depolarization, and suppression of Bcl-2 expression. The total phenolic and catechin contents were measured as 91.92 ± 14.54 and 4.31 ± 0.14 mg/g, respectively, in the water extract.⁵² As for hematological cancers, S suberectus ethanolic extract (10–20 μg/mL) was shown to induce apoptosis in U266 (multiple myeloma) and U937 (myeloid leukemia) cell lines, which was evidenced by upregulating the protein expression of Bax and some endoplasmic reticulum stress-related proteins in Western blot analysis. Furthermore, the extract also increased ROS generation and reduced the expression of miR-657, which is a micro-RNA holding a key role in several types of cancer.⁵³

There was also a study on the DNA damage protective effects of some major flavonoids from the S suberectus vine stem. Accordingly, genistein (20), isoliquiritigenin (59), medicarpin (48), and naringenin (50) showed antimutagenic activity against N-methyl-N-nitrosourea in Salmonella typhimurium strain and hydroxyl radical scavenging activity, which suggested their roles in chemoprevention. Future studies could focus on the chemopreventive effects of S suberectus extracts or isolated phenolics on cancer development.⁵⁴

There have been a considerable number of studies of S suberectus and its bioactive compounds on anticancer properties (Table 3). The reported data strongly indicate the potent effects of the plant extract against cancer cells, specifically breast cancer via different mechanisms. Furthermore, it is also noticeable that all chemical components, especially catechins and isoflavonoids, contributed to synergistic effects, rather than a few single responsible compounds. The cytotoxicity was also confirmed in animal experiments with positive results. Besides that, the plant extract also showed promising results in several types of cancer, such as glioma and leukemia, which await researchers to shed light on. Further clinical studies are required to support the use of S suberectus stem in cancer treatment.

Table 3.

Pharmacological Activities of Spatholobus suberectus Extracts, Fractions and Their Isolated Compounds.

Pharmacological activity group	Extract/compound	Chemical profile of ext. (Yes/No)	In vitro/in vivo	Activity	Dose range/IC₅₀/EC₅₀	Control	Ref
Effects on a circular system
Enhance hemopoietic process	Catechin (65)	—	In vitro	↑CD34⁺CD45⁺ and GPA⁺CD71⁺ ↑Formation of CFU-E, CFU-GM, BFU-E, and CFU-Mix	2–50 µM	Vehicle	²⁶
Enhance hemopoietic process	Catechin (65)	–	In vitro In vivo	↑CFU-GM, BFU-E and CFU-Meg	0.08–2 mg/mL (i.p)	Saline	²⁷
Antihepcidin	SC aqueous ext.		In vitro	↓HMAP → ↓hepcidin	20–200 μg/mL	Vehicle	²⁸
Antiplatelet aggregation	SC ethanol ext.	Yes	In vitro	↓collagen- and ADP-exposed platelet aggregation (↓GP IIb/IIIa)	200–400 μg/mL (p.o)	Aspirin	²⁹
Protective effects on radiation-induced hematopoietic alteration and oxidative stress	SC ethanol ext.	No	In vivo	↑blood cell profile and ↓ROS (↑pJAK2/JAK2, pSTAT5a/STAT5a, and Bcl-2 → ↑JAK/STAT)	40 g crude drug/kg (p.o)	Amifostine	³⁰
Antideep vein thrombosis	SC dispensing granule	Yes	In vivo	↓Thrombus weight, ↑prolonged APTT, ↑TF protein expression ↑SIRT1 protein expression, ↓Ace-p65, ↑Nrf2 and HO-1, ↓p-p65	0.125 and 0.25 g/kg/day in 7 days (p.o)	Heparin and clopidogrel	³¹
Effects on skin conditions
Skin protection from UVB irradiation	SC aqueous and ethanol ext.	Yes	In vitro	↑survival of human keratinocyte (HaCaT) ↓elastase; ↑COL1A1, ↑HAS2, ↓MMP-1/2, ↓NF-κB, AP-1, MAPK	10–300 µg/mL	EGCG, gallic acid	³²
Skin whitening	Butin (53)	–	In vitro	↓Tyrosinase, ↓melanin, ↓TRP1 and TRP2	IC₅₀ = 35.9 µM	Arbutin	³³
Wound healing from secondary burns	SC aqueous ext.	No	In vivo	↓Congestion, ↑fibroblast, angiogenesis, and epithelialization; ↓MDH, ↓IL-6 and TNF-α	Gel with 10% and 20% SC extract (topical)	Silver sulfadiazine	³⁴
Antiinflammatory activities
Inhibitory effects on inflammatory mediators	SC aqueous ext.	Yes	In vitro	↓iNOS and COX-2	10–500 µg/mL	Vehicle	³⁵
	7-hydroxy chromone (73) Isoliquiritigenin (59)	–	In vitro	↓IL-6, IL-1β, TNF-α, iNOS, and COX-2	IC₅₀ = 5.69 µM (73) IC₅₀ = 6.78 µM (59)	ʟ-NIL and indomethacin	⁹
	Formononetin sodium (21) Formononetin (22) Biochanin A (23)	–	In vitro	↓NO production	IC₅₀ = 22.75 µM (21) IC₅₀ = 21.11 µM (22) IC₅₀ = 48.29 µM (23)	ʟ-NIL and indomethacin	²⁵
	3′,4′,7-trihydroxyflavone (61)	–	In vitro	↓iNOS via JNK–STAT1	0.1–10 μM	Vehicle	³⁶
	SC phenolic fr.	Yes	In vitro	↓HNE	IC₅₀ = 1.33 μg/mL	Sivelestat	³⁸
Antiatopic dermatitis	SC aqueous ext.	Yes	In vivo	↓Ear thickness, dermatitis score ↓IgE, histamine, ↓IL-8, RANTES, MCP-1, MDC, TARC via MAPK/STAT1/NF-κB	30–300 mg/kg in 3 weeks	Dexamethasone	³⁹
	Liquiritigenin (49)	–	In vitro In vivo	↓IL-2 and CD69, ↓CD40L and CD25 via NF-κB and MAPK; ↓ear thickness, IgE	5–40 μM (in vitro) 1 mg/mL, 5 times × 4 weeks (in vivo)	Vehicle	⁴⁰
Antioxidant activities
Radical scavenging and protective effects on CCl₄-induced hepatotoxicity	SC methanol ext.	No	In vitro	DPPH radical scavenging	86.7% (100 µg/mL) 91.2% (1000 µg/mL)	–	⁴²
			In vitro	FeSO₄-induced LPO inhibition	83% (1000 µg/mL)	–
			In vivo	↓ALT, AST, ALP; ↓LPO, MDA	30–300 mg/kg (3 times/day × 3 days)	Vehicle
Antioxidative stress	SC flavonoid fr.	Yes	In vitro	↓NO, ROS, GSSG; ↑GSH, SOD, MPO	25–500 µg/mL	Vitamin C	⁴³
Antioxidative stress	SC flavonoid fr.	Yes	In vivo	↑Thymus and spleen indices ↑GSH/GSSG ratio, ↑XOD, MPO	25–100 mg/kg	Vitamin C	⁴⁴
Anticancer activities
Antibreast cancer	SC aqueous ext.	Yes	In vitro	↓MCF-7 proliferation; ↑apoptosis, G2/M arrest, ↑ROS, ↓LDH-A	20–80 μg/mL	Vehicle	¹⁷
	SC aqueous ext.	Yes	In vivo	↓Tumor growth; ↓HIF-1a/LDH-A; ↑apoptosis	40 mg/kg (p.o)	Vehicle	¹⁷
	SC aqueous ext.	Yes	In vitro	↓MCF-7 proliferation; G₀/G₁ arrest, ↓migration; ↑cytochrome C, Bax; ↓Bcl-2; ↓ERE	80–320 μg/mL	Vehicle	⁴⁵
	SC ethanol ext.	Yes	In vitro	↓MDA-MB-231 and BT-549 proliferation	IC₅₀ = 52.58 (MDA-MB-231) and 10.89 (BT-549) μg/mL	Docetaxel	⁴⁶
	SC ethanol ext.	Yes	In vivo	↓Tumor growth, size; ↑ROS; ↑caspase-4, cleaved caspase-9, GSDME	0.4–0.8 g/kg (p.o)	Docetaxel	⁴⁶
	Sativan (38)	–	In vitro	↑miR-200c, ↓EMT	1–100 μM	Vehicle	⁵¹
	Sativan (38)		In vivo	↓tumor weight and volume	25–50 mg/kg/day (p.o)	Vehicle	⁵¹
	Sativan (38) Compound 41 Compound 42	–	In vitro	MCF-7 cytotoxicity	IC₅₀ values 41: 59.0 μM 42: 93.6 μM 38: 60.1 μM	Paclitaxel	¹³
	38, 47	–	In vitro	MDA-MB-231 cytotoxicity	IC₅₀ values 38: 34.1 μM 47: 25.1 μM	Paclitaxel	¹³
Antiglioma	SC water extract	Yes	In vitro	↓C6 glioma cells proliferation, ↓ROS, ↓Bcl-2	IC₅₀ = 1.65 ng/mL		⁵²
Antihematological cancer	SC ethanol extract	Yes	In vitro	↑U266 and U937 apoptosis, ↑Bax, ↑ROS, ↓miR-657	10–20 μg/mL	Vehicle	⁵³
Topoisomerase-II-inhibition	Compound 83 Compound 84	–	In vitro	↓DNA topoisomerase	IC₅₀ values 83: 22.5 nM 84: 21.9 nM	Etoposide	¹⁸
Proteasome inhibition	Genistein (20) Naringenin (51) Isoliquiritigenin (59)	–	In vitro	↓20S-proteasome	IC₅₀ values 59: 4.88 μM 20: 9.26 μM 51: 5.21 μM	Epoximicin	¹¹
Antibacterial activities
Anti-Staphylococcus aureus and Streptococcus mutans	7-hydroxy-6-methoxyflavanone (58) Formononetin (22)	–	In vitro	↓Sortase activity, ↓clumping	IC₅₀ values 58: 46.1 μM 22: 41.8 μM	Berberine chloride and curcumin	⁵⁵
Antibiotics synergism	SC acetone ext.	No	In vitro In vivo	Streptomycin synergism	–	streptomycin	⁵⁷
Antiviral activities
Anticoxsackievirus B3	SC water ext.	No	In vitro	↓Virus titers	5–10 μg/mL	Ribavirin	⁵⁸
Anticoxsackievirus B3	SC water ext.	No	In vivo	↓Heart virus titers, ↓mortality rate	50–100 mg/kg	Ribavirin	⁵⁸
Anti-HCV	SC fr.	Yes	In vitro	↓Viral replication, ↓NS3, NS5A, and NS5B, ↓RNA translation	EC₅₀ = 0.45 μg/mL	Telaprevir, sofosbuvir, daclatasvir, simeprevir	⁵⁹
Antiviral	SC ethanol ext.	Yes	In vitro	Inhibition of pseudo-typed SARS-CoV-1 and 2, HIV-1_{ADA and HXB2}, and H5N1 Block SARS-CoV-2 S-protein and ACE2	EC₅₀∼3.6–5.1 μg/mL	Vehicle	⁶⁰
Protective effects against diabetic complications
Antidiabetic	SC water and ethanol ext.	Yes	In vitro	↓α-Glucosidase activity	IC₅₀ = 6.42 ± 1.45 and 2.81 ± 0.48 μg/mL	Acarbose	⁶¹
			In vitro	↑Glucose uptake via Akt and AMPK; ↑GLUT4	10–300 μg/mL	Vehicle
			In vivo	↓α-Glucosidase activity, ↓postprandial blood glucose; insulin-mimetic effects and ↓Gluconeogenesis (↓PEPCK and G-6-Pase)	100 mg/kg	Acarbose
	SC ethanol ext.	No	In vitro	↓Apoptosis in HUVEC, ↓lncRNA MALAT1 via VEGF pathway	1–2 mg/mL	Vehicle	⁶²
Protective effects on diabetic nephropathy	SC ext.	Yes	In vivo	↓Serum Tg, free FA, and LDL; ↑Glo1, NQO1,Nrf2; ↓AGE accumulation	50 mg/kg	Aminoguanidine and alagebrium	⁶³
Antiglycative effects	SC ethanol ext.	Yes	In vitro	↓AGE formation	0–1000 μg/mL	Aminoguanidine, gallic acid, rutin	⁶⁴
Other bioactivities
Antiosteoarthritis	SC fr.	No	In vitro	↓MMP-1, -3, -9 and TIMP-1 ↓RANKL-induced osteoclast differentiation (↓TRAP)	12.5–200 μg/mL	Vehicle	⁶⁶
Antiosteoarthritis	SC water ext.	No	In vitro	↓Osteoclast differentiation (↓c-Fos and NFATc1); ↓bone resorption (disrupting actin ring structure)	10–50 μg/mL	Vehicle	⁶⁵
Antiobesity	SC water ext.	Yes	In vivo	↓Bodyweight gain; ↓fat accumulation; ↑Glucose homeostasis (AMPK and MAPK pathway) Modulate intestinal microbiota	0.25–1% (w/w) solution for drinking	Vehicle	⁶⁷
Antiparasite	Ononin (17)	–	In vitro In vivo	Toxicity against Dactylogyrus intermedius	EC₅₀ = 0.655 mg/L 100% anthelmintic efficacy at 3 mg/mL	DMSO	⁶⁸
Protective effects on brain tissue in cerebral ischemic events	SC water ext.	No	In vivo	↓MDH and NO; ↑SOD, GPx ↑IL-10, brain ATP; ↓TNF-α, caspase-3, NF-κB	100–200 mg/kg	Vehicle	⁶⁹
Effects on benign prostatic hyperplasia	Formononetin (22)	–	In vitro	Blocking α1A adrenergic receptors	–	Tamsulosin	⁷⁰

Abbreviations: ACE2, angiotensin-converting enzyme 2; ADP, adenosine diphosphate; AGE, advanced glycation end-product; Akt, protein kinase B; ALP, alkaline phosphatase; ALT, alanine transaminase; AMPK, AMP-activated protein kinase; AP-1, activator protein-1; APTT, activated partial thromboplastin time; AST, aspartate transaminase; ATP, adenosine triphosphate; Bcl-2, B-cell lymphoma 2; BFU-E, burst-forming unit-erythroid; CFU-E, colony-forming unit-erythroid; CFU-GM, colony-forming unit granulocyte-macrophage; CFU-Meg, colony-forming unit-megakaryocyte; COX-2, cyclooxygenase-2; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EMT, epithelial-to-mesenchymal transition; ERE, estrogen response element; ext., extract; FA, fatty acid; fr., fraction; GLUT4, glucose transporter 4; GP, glycoprotein; GPx, glutathione peroxidase; GSDME, gasdermin E; GSH, glutathione; GSSG, glutathione disulfide; HAS2, hyaluronan synthase 2; HCV, hepatitis C virus; HIF-1a, hypoxia-inducible factor-1a; HNE, human neutrophil elastase; HO-1, heme oxygenase-1; HUVEC, human umbilical vein endothelial cells; IgE, immunoglobulin E; IL, interleukin; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; LDH-A, lactate dehydrogenase A; LDL, low-density lipoprotein; lncRNA, long noncoding RNA; LPO, lipid peroxidation; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; MDC, macrophage-derived chemokine; MMP-1, matrix metalloproteinase 1; MPO, myeloperoxidase; NFATc1, nuclear factor of activated T-cells 1; NF-κB, nuclear factor-κB; NO, nitric oxide; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf2, nuclear factor erythroid 2-related factor 2; PEPCK, phosphoenolpyruvate carboxykinase; pJAK2, phospho-Janus kinase 2; RANKL, receptor activator of nuclear factor kappa-Β ligand; RANTES, regulated upon activation, normal T-cell expressed and presumably secreted; ROS, reactive oxygen species; SARS-CoV-1, severe acute respiratory syndrome-coronavirus-1; SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; SC, Spatholobi caulis; SIRT1, Sirtuin 1; SOD, superoxide dismutase; STAT, signal transducer and activator of transcription; TARC, thymus- and activation-regulation chemokine; TF, transcription factor; Tg, thyroglobulin; TIMP-1, TIMP metallopeptidase inhibitor 1; TNF-α, tumor necrosis factor-α; TRAP, thyroid hormone receptor-associated protein; VEGF, vascular endothelial growth factor; XOD, xanthine oxidase.

Antibacterial Activities

Bioguided fractionation of S suberectus stem extract resulted in the identification of highly bioactive flavonoids in the bacterial sortase A activity assay. Sortase A is a key transpeptidase, which is responsible for anchoring bacteria to the cell wall. Among those isolated compounds, 7-hydroxy-6-methoxyflavanone (58) and formononetin (22) exhibited the most significant inhibition on sortase A isolated from Staphylococcus aureus and Streptococcus mutans without affecting cell viability (IC₅₀ = 46.1 and 41.8 µM, respectively), which was comparable to those of berberine chloride and curcumin (positive controls; IC₅₀ = 56.9 and 45.8 µM, respectively). Furthermore, the 2 flavonoids also reduced the clumping capacity of the S aureus wild strain with fibrinogen and saliva-induced aggregation of S mutans. Interestingly, the tests on bacterial growth indicated that these compounds did not exhibit significant bactericidal activities on the mentioned strains, which implied that these flavonoids could be developed as potential sortase A inhibitory leads.^55,56 S suberectus acetone extract also showed in vitro antibacterial synergism against Streptococcus agalactiae with streptomycin sulfate in the optimal ratio of 42.5:1 with a significant reduction of 80% in MIC values for both components. Furthermore, the combination also broadened the antibacterial spectrum of antibiotics, including both Gram-positive and Gram-negative strains. The study also reported no observed oral acute toxicity and toxicological symptoms at 5000 mg/kg in mice. However, the chemical details of the plant extract were not mentioned in the study.⁵⁷

Although there have only been initial studies on the antibacterial activities of S suberectus extracts on sortase A inhibition and synergism when in combination with antibiotics, the study results showed a promising direction for future research on antibacterial activities of the plant.

Antiviral Activities

The aqueous extract of S suberectus stems was investigated for its bioactivity against coxsackievirus B3, which is a small RNA virus belonging to the Picornaviridae family, causing pericarditis and myocarditis in humans. In myocardial cells, the water extract showed strong inhibitory effects against the virus at concentrations of 5 and 10 μg/mL, which was equal to that of the positive control, ribavirin, at 1 mg/mL. In vivo experiments also revealed a significant reduction in mouse mortality (40%–50% mortality) at oral doses of 50 to 100 mg extract/kg when compared with the results of the untreated group (70%–80% mortality). The mortality rate and virus titer of the extract-treated group were shown to be better than those of the positive control, ribavirin, at a concentration of 10 mg/kg. Further studies also revealed the low virus titers in mouse heart and a significant reduction in necrosis and mononuclear cell infiltration. However, the chemical characterization of the plant extract was not described in the study.⁵⁸ Another study also reported the potential inhibitory effects of crude S suberectus extract against the hepatitis C virus (HCV). Fractions with different polarities produced by macroporous resin column chromatography were investigated for their anti-HCV activity. The 25% ethanol fraction was shown to be the strongest with an EC₅₀ value of 0.45 ± 0.08 µg/mL, with no observed cytotoxicity at 100 μg/mL. The active fractions were also shown to prevent viral replication, protein expression, and RNA translation. HPLC analysis indicated that the main chemical compositions of the investigated fractions were gallic acid, catechins, and tannins. The extracts were hypothesized to exhibit their bioactivity as a whole with synergistic and additive effects of all present components, rather than of a single compound.⁵⁹ The percolated extract of S suberectus stem using 60% ethanol showed strong inhibitory activities against pseudo-typed severe acute respiratory syndrome-coronavirus-1 and -2 (SARS-CoV-1 and -2), HIV-1_{ADA and HXB2}, and H5N1 with an EC₅₀ range of 3.6 to 5.1 μg/mL. The total extract acted via blocking SARS-CoV-2 spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. As for toxicity, the extract showed low cytotoxicity in various cell lines (CC₅₀ = 181.2–339.8 μg/mL) and the extract also appeared safe in a rat model, with no observed mortality, organ damage, and biochemical parameter abnormality within the tested doses (2–6 g/kg body weight). Positive and negative controls were used for antiviral and mechanistic tests. The extract was also characterized by TLC and HPLC analyses with catechin derivatives as major components.⁶⁰

Preliminary results promised S suberectus stems as a potential herb for the prevention and treatment of viral infection. Further studies may focus on bioactivities against other types of viruses, or enhanced effects when using the plant extract and other antiviral drugs in combination.

Protective Effects Against Diabetic Complications

The extracts of S suberectus stems were investigated in various antidiabetic bioassays. The aqueous and ethanolic extracts were shown to significantly inhibit α-glucosidase activity with IC₅₀ values of 6.42 ± 1.45 and 2.81 ± 0.48 μg/mL, respectively, which was even stronger than the positive control, acarbose (IC₅₀ = 217.87 ± 0.21 μg/mL). In the cellular assay, the extracts also promoted glucose uptake in C2C12 myotube cells without causing any cytotoxicity at concentrations of 10 to 300 μg/mL. The underlying mechanism was determined by activating the Akt and AMP-activated protein kinase (AMPK) signaling pathways. In streptozocin-induced diabetic mice, the S suberectus stem ethanolic extract also exhibited hypoglycemic activities at 200 mg/kg by showing insulin-mimetic effects and abolishing the gluconeogenesis process in the mice.⁶¹

S suberectus 50% ethanolic extract was studied for its protective effects on palmitic acid-induced apoptosis of human umbilical vein endothelial cells (HUVEC). Diabetic patients often develop abnormal angiogenesis, which might lead to cardiovascular complications. In addition, it is also found that the level of palmitic acid in diabetic patients is significantly higher than that of normal people, which eventually could damage vascular endothelial cells and have a negative influence on the insulin signaling pathway, leading to an insulin-resistant condition in diabetic patients. The research of Gu's group (2020) showed that S suberectus extract inhibited the apoptotic process in HUVEC under diabetic simulation and also promoted the proliferation and epithelial-mesenchymal transition of the same cells. In the in-depth mechanistic study, the extract was found to inhibit metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) via the vascular endothelial growth factor (VEGF) signaling pathway.⁶²

In the db/db mouse model, S suberectus extract (50 mg/kg) was shown to remarkably reduce the serum levels of triglyceride, free fatty acids, and low-density lipoprotein (LDL)-cholesterol, slowing down the progression of diabetic nephropathy. Moreover, the extract treatment also reduced the accumulation of advanced glycation end products (AGEs) and the interaction with its receptor via the Glo1 and Nrf2 pathways, which are well-known causes leading to renal cell death.⁶³ In a screening study of 6 selected plants for free radical scavenging activities, S suberectus was shown to be the best, with IC₅₀ values comparable to those of positive controls, including gallic acid, rutin, and ursolic acid in the ferric-reducing ability of plasma (FRAP), DPPH, and Trolox equivalent antioxidant capacity (TEAC) assays. The ethanolic extract also showed better antiglycative activities than the positive controls (aminoguanidine, gallic acid, and rutin) in bovine serum albumin (BSA)-methylglyoxal (MGO) and BSA-glucose assays. Therefore, S suberectus extracts were shown to have the potential for preventing AGEs formation and oxidative stress for type 2 diabetic patients.⁶⁴

Recent studies demonstrated that S suberectus extracts were beneficial not only in mitigating blood sugar levels, but also for protecting the cells from hyperglycemia-induced renal or cardiovascular complications. However, the responsible active compounds have not been discovered yet, which leads to the need of identifying the most bioactive compounds, in order to control the quality and maximize the medicinal properties of the herb. Since long-term hyperglycemia in diabetic patients can lead to other secondary complications, such as stroke, retinopathy, and neuropathy, it is expected that further studies should be performed to discover whether the plant can provide such protective effects for diabetic patients or not.

Other Bioactivities

To explain the treatment of osteoarthritis, S suberectus stem extracts were evaluated for their effects on osteoclast and osteoblast balance in bone remodeling. The water extract was found to suppress the differentiation of osteoclast cells via the receptor activator of nuclear factor kappa-Β ligand (RANKL)-mediated pathway and reduce bone resorption activity by breaking down the actin ring structure.⁶⁵ The extract also protected articular cartilage by inhibiting the expression levels of metalloproteinase in TNF-α-treated SW1353 cells and reduced the destruction of proteoglycan, collagen, and chondrocytes, which directly showed therapeutic benefits for osteoarthritis.⁶⁶

The water extract of Caulis Spatholobi was also tested for antiobesity effects in dietary obese mice. The study results indicated that the extract significantly reduced body weight gain, fat tissue accumulation, and promoter glucose homeostasis by upregulation of the MAPK and AMPK pathways in white and brown adipose tissues. Furthermore, the extract was also shown to modulate the composition of intestinal microbiota in the direction of ameliorating obesity.⁶⁷

Ononin (17), a 5-deoxyflavone glycoside, was reported to possess promising antiparasitic activities against Dactylogyrus intermedius (causing hemorrhaging and metaplasia in fish gills) from a bioactivity-guided isolation study. The isoflavone was shown to have strong parasiticidal activity with an EC₅₀ value of 0.655 mg/L and 100% anthelmintic efficacy at 3 mg/mL, at which concentration, the extract showed no harm to goldfish.⁶⁸

A study by Zhang's group was conducted to examine the protective effects of S suberectus aqueous extract on cerebral ischemia in a rat model. Cerebral ischemia is caused by an arterial blockage, leading to a blood shortage in the brain, which eventually causes the death of neuronal cells. Scavenging free radicals and reducing the production of cytokines might be beneficial for patients with cerebral ischemia. The oral administration of plant extract (100–200 mg/kg) was found to significantly decrease the malondialdehyde and nitric oxide serum levels while those of superoxide dismutase and glutathione peroxidase were observed to increase. In addition, the levels of inflammatory cytokines and NF-κB were decreased in the extract-treated group. Collectively, it was evidenced that S suberectus aqueous extract was effective in protecting brain tissue in cerebral ischemic rats.⁶⁹

Benign prostatic hyperplasia is also commonly called prostate gland enlargement, which is a common health condition for older men, causing uncomfortable kidney and urinary problems. Blocking α1A adrenergic receptors is one of the common ways of treatment with a typical example of such a drug being tamsulosin. Han's group developed a cell membrane chromatography with α1A adrenergic receptor highly expressed human embryonic kidney 293 (HEK 293) cells, to screen active compounds from S suberectus vine stem extract. As a result, formononetin was identified as a potential α1A adrenergic blocker, which shared the same binding position as tamsulosin.⁷⁰

Conclusion

S suberectus is an important medicinal plant in Asian countries, specifically China, Vietnam, and South Korea. It can be used either alone or in combination with other herbs to treat blood disorders, such as anemia, irregular menstrual period, and dysmenorrhea, as well as rheumatic and bone pain. There have been extensive efforts to shed light on the chemical composition and pharmacological activities of plant extracts and their respective isolated compounds. The main chemical constituents of the S suberectus vine stem were phenolic compounds, including flavonoids and catechins (monomeric and oligomeric types). However, it is difficult to identify exactly the most abundant compounds in the crude drug since there were significant differences in different Spatholobi Caulis samples collected from different locations. Therefore, quality control and extract characterization are very important in pharmacological research and clinical application.

As for pharmacology, S suberectus stem extracts were investigated in a variety of hematological assays to explain their ethnopharmacological relevance in traditional uses for activating and dispelling blood properties according to the 5-element theory of traditional Chinese medicine. The published results highly indicated the extract's potential in stimulating erythroid development by increasing blood cell surface markers and various erythroid forming units in both in vitro and in vivo experiments. In addition, the extract was also shown to inhibit platelet aggregation in cellular assays. Collectively, recent studies support the traditional uses of the plant, but further animal and clinical studies should be on the way to confirm the benefits in certain blood disorders in humans. Besides that, S suberectus stem extract also exhibited antiinflammatory effects by downregulating the expression of inflammatory mediators in RAW264.7 macrophages, which provides evidence for the traditional uses in osteoarthritic and rheumatic treatment. In China, traditional medicine physicians tend to utilize plants to support breast cancer treatment, including S suberectus stems. The aqueous and ethanolic extracts showed cytotoxic and antiproliferative activities toward some breast cancer cell lines via multiple mechanisms of action. However, some conducted studies did not characterize the chemical constituents of the plant extracts, which made challenges to future investigations on responsible compounds for bioactivities and experimental replication. Further works on isolated compounds have also been conducted to discover new leads in anticancer therapy with initially promising results, such as sativan as a potential inhibitor for triple-negative breast tumors, epigallocatechin as a promising LDH-A inhibitor, and isoliquiritigenin as a potential lead for antibreast cancer drug development.

S suberectus stem has a common name of “Ji-xue-teng” in China, “Gye-hyeol-deung” in South Korea, and “Kê huyết đằng” in Vietnam, which can be literally translated as “chicken's blood vine.” The plant was named after the characteristic red color of its decoction. However, there are many plants sharing the same name due to the similarities in physical appearance and they are being interchangeably used in traditional medicines. This phenomenon raises an urgent issue for developing analytical methods for quality control and distinguishing counterfeit plants, such as an HPLC–MS/MS method combined with PCA tools.

In conclusion, the findings of S suberectus research suggest the high medicinal application of the plant. Published pharmacological results strongly support its traditional usage for blood disorders and osteoarthritic diseases. However, further long-term toxicity and clinical studies are required to promote its safe and effective use in resolving and controlling diseases.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Hieu Nguyen-Ngoc

Tung Nguyen-Huu

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