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Phytochemistry, Ethnopharmacological Uses, Biological Activities, and Therapeutic Applications of Cassia obtusifolia L.: A Comprehensive Review

Cassia obtusifolia L., of the Leguminosae family, is used as a diuretic, laxative, tonic, purgative, and natural remedy for treating headache, dizziness, constipation, tophobia, and lacrimation and for improving eyesight. It is commonly used in tea in Korea. Various anthraquinone derivatives make up its main chemical constituents: emodin, chrysophanol, physcion, obtusifolin, obtusin, au rantio-obtusin, chryso-obtusin, alaternin, questin, aloe-emodin, gluco-aurantio-obtusin, gluco-obtusifolin, naphthopyrone glycosides, toralactone-9-β-gentiobioside, toralactone gentiobioside, and cassiaside. C. obtusifolia L. possesses a wide range of pharmacological properties (e.g., antidiabetic, antimicrobial, anti-inflammatory, hepatoprotective, and neuroprotective properties) and may be used to treat Alzheimer’s disease, Parkinson’s disease, and cancer. In addition, C. obtusifolia L. contributes to histamine release and antiplatelet aggregation. This review summarizes the botanical, phytochemical, and pharmacological features of C. obtusifolia and its therapeutic uses.


Introduction
Cassia (family Caesalpiniaceae) is a large tropical genus with~600 species of herbs, shrubs, and trees. Cassia obtusifolia (sicklepod) Linn., a member of the genus Cassia (Leguminosae), is a well-known traditional Chinese medicinal plant. It belongs to the medically and economically important family Leguminosae (syn. Fabaceae; subfamily Caesalpinioideae). C. obtusifolia L. is found mainly in China, Korea, India, and the western tropical regions. It is an annual semi-shrubby herb that ranges in height from~0.5 to 2 m. It has two or three pairs of round-tipped leaflets with one to three flowers on a short axillary peduncle with pedicels up to 2 cm; the yellow petals (0.8-1.5 cm) wilt by midday. The pods are linear (up to 20 cm in length), curve gently downward, and contain numerous shiny, dark brown seeds (~0.5 cm in length). The seeds of C. obtusifolia L. are rhomboidal or slightly flat, with linear concave ramps on each side. Cassia tora L. is considered synonymous with C. obtusifolia L., but differs in its botanical and morphological characteristics [1,2]. The main distinguishing morphological feature between the two is the seed coat, which is marked with an obliquely symmetrical dented line on each side of the rib (C. obtusifolia L.) or has broad bands on both sides of the rib (C. tora L.).
Cassia species are of medicinal interest because of their therapeutic value in traditional medicine. The dry seeds are processed as a crude drug for clinical use or as a dietary supplement. The cultured plants are important sources of Semen Cassiae-derived commercial products in the market. C. obtusifolia L. seeds are a well-known medicinal plant in East Asia In traditional Oriental medicine, the whole plant of C. obtusifolia has been used for treatment of Laxative, eye infections, diarrhea, urinary tract infections, gingivitis, fever, and cough remedy [13].

Roots
Root is considered bitter, tonic, stomachic and is antidote against snake bite. Other uses are in treatment of fungal diseases, worm infection, abdominal tumors, bronchitis, and asthma. The roots of C. obtusifolia are also usually crushed, mixed with lime juice, and applied to ringworms [14].

Seeds
The seeds of C. obtusifolia are used to treat dizziness and to benefit the eyes by anchoring and nourishing the liver. The dried and roasted seeds are also used as brew a tea. Seeds of C. obtusifolia were also used for the treatment of headache, ophthalmic diseases, constipation, hypertension, and hyperlipidemia. In Korea, the hot extract of seeds is taken orally for protection of liver [10,15].

Leaves
C. obtusifolia leaves and pods have been widely used as purgatives and laxatives. In Indian traditional ayurveda system, the leaves and Pods are used as digestible, laxative, diuretic, stomachic, antipyretic, improves the appetite, biliousness, blood diseases, burning sensation, leprosy, bronchitis, piles, and leucorrhoea [16,17].

Stem bark
In Indian traditional ayurveda system, Stem bark extract is used for various skin ailments, rheumatic diseases, and as laxative [18]. 6 Pods and fruits Pods are used in dysentery, in eye diseases and pains in the joints. The unripe fruits are also cooked and eaten [14].

Bioactivity
Numerous researchers have investigated the pharmacological activities of various C. obtusifolia L. extracts. Table 2 summarizes the pharmacological features that have been observed. They include: antidiabetic, anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, neuroprotective, immune-modulatory, anti-Parkinson's disease, anti-Alzheimer's disease, and larvicidal properties. The anthraquinones and naphthopyrones isolated from C. obtusifolia L. are structurally diverse and exhibit multiple pharmacological properties, which suggests that these compounds contribute to its therapeutic effects ( Table 3). C. obtusifolia L. and its major constituents display a vast number of biological activities ( Figure 2). Natural products are highly promising sources for antioxidant and anti-inflammatory agents. A wide range of bioactive constituents of plants have antioxidant and anti-inflammatory activities. Based on various assay methods and activity indices, antioxidant or anti-inflammatory activities and nutraceutical and therapeutic effects of traditional Chinese medicines as well as the mechanisms underlying such activities and effects have been investigated. The generation of free radicals can result in damage to the cellular machinery. The seeds of C. obtusifolia L. are widely used in Chinese folk medicine and have been demonstrated to exhibit significant antioxidant and anti-inflammatory. Over the past century, natural products, especially anthraquinone compounds, have become valuable products for achieving chemical diversity in the molecules used for inflammation relief. In addition, COE has traditionally been used in Korea to treat eye inflammation, photophobia, and lacrimation.   (c) Significantly increased the levels of LDL-R at 2 g/kg [38] Anti-diabetic Activity Antidiabetic activity In vitro (a) PTP 1B inhibitory activity (b) α-glucosidase inhibitory activity (c) Stimulation of glucose uptake in HepG2 cells -(a) 0-100 µg/mL (b) 0-400 µg/mL (c) 3.12-12.5 µM (a) IC 50 = 3.51 µg/mL (b) IC 50 = 1.02 µg/mL (c) glucose uptake [9] Platelet anti-aggregatory activity In vitro (a) Adenosine 5 -diphosphate inhibitory activity (b) Arachidonic-acid inhibitory activity (c) Collagen inhibitory activity -0-1 mg/mL 1 mg/mL [47]   Obtusifolin

Neuroprotective activity In vivo
Significantly reversed scopolamine-induced cognitive impairments in the passive avoidance test, improved escape latencies, swimming times in the target quadrant, and crossing numbers in the zone in Morris water maze test Orally 0.25-2 mg/kg 0.5 mg/kg [49] Hyperlipidemia and antioxidant activity In vivo Reduced body weight, TC, TG, LDL-C and increased HDL-C levels, as well as increased SOD and NO, and reduced MDA levels in hyperlipidemic rats.
Orally 5 and 20 mg/kg 20 mg/kg [50] Neuropathic and anti-inflammatory activity In vivo Inhibition of TNF-α, IL-1β, IL-6 and NF-kB up-regulation in the spinal cord in mice and rat models Intraperitoneal injection 0.25-2 mg/kg 1 and 2 mg/kg [51]     Anti-Alzheimer's activity In vitro

Neuroprotective Activity
Various studies have demonstrated the direct neuroprotective activities of the C. obtusifolia L. seed extract (COE) and its major constituents (anthraquinones). More detailed studies are required to clarify the compositional features and neuroprotective activities of the anthraquinones. The ethanolic COE (25, 50, or 100 mg/kg) ameliorates scopolamine or bilateral common carotid artery occlusion (2VO)-induced memory impairment by inhibiting acetylcholinesterase [8]. COE (10 or 50 mg/kg/day) reduced memory impairment and neuronal damage caused by 2VO in a mouse model of transient global ischemia; it was suggested that the neuroprotective effects of COE are attributable to its anti-inflammatory properties resulting in decreased expression of inducible nitric oxide synthase (iNOX) and cyclooxygenase-2 (COX-2) and increased expression of the neurotrophic factors pCREB and BDNF [33]. Alaternin, the active compound in C. obtusifolia L., exhibits neuroprotective activity after transient cerebral hypoperfusion induced by bilateral common carotid artery occlusion. Administration of alaternin (10 mg/kg) prevented or reduced nitrotyrosine and lipid peroxidation, bilateral common carotid artery occlusion (BCCAO)-induced iNOS expression, and microglial activation [48]. Drever et al. [11] reported that ethanolic COE is neuroprotective against NMDA-induced calcium dysregulation and 3-nitropropionic acid-induced cell death in mouse hippocampal cultures. Recently, Paudel et al. [56] also reported that four major compounds (cassiaside, rubrofusarin gentiobioside, aurantioobtusin, and 2-hydroxyemodin 1-methylether) exhibited neuroprotective effects; among them, aurantio-obtusin showed promising neuroprotective effects via targeting various G-protein-coupled receptors and transient brain ischemia/reperfusion injury C57BL/6 mice model.

Prevention and Treatment of Parkinson's Disease
A neuroprotective effect of COE was observed in both in vitro and in vivo models of Parkinson's disease [34]. In PC12 cells, COE reduced cell damage induced by 100 µM 6hydroxydopamine and inhibited the overproduction of reactive oxygen species, glutathione depletion, mitochondrial membrane depolarization, and caspase-3 activation at 0.1 to 10 µg/mL. In addition, COE displayed radical scavenging effects in DPPH and ABTS assays, which suggests that COE may be useful for treating Parkinson's disease [34].

Hepatoprotective Activity
Few studies have demonstrated the hepatoprotective activities of COE [15]. Further studies are required to establish the hepatoprotective mechanisms of major COE anthraquinones. The protective effects of ethanolic COE against the cytotoxicity induced by CCl 4 liver in mice were evaluated by assessing aminotransferase activities, histopathological changes, hepatic and mitochondrial antioxidant indices, and cytochrome P450 2E1(CYP2E1) activity. Administration of COE (0.5, 1, 2 g/kg) markedly reduced ALT and AST release, Ca 2+ -induced mitochondria membrane permeability transition, and CYP2E1 activity. In addition, COE significantly reduced hepatic and mitochondrial malondialdehyde levels, increased hepatic and mitochondrial glutathione levels, and restored superoxide dismutase, glutathione reductase, and glutathione S-transferase activities [15]. Meng et al. [38] reported the hepatoprotective effects of ethanolic COE on non-alcoholic fatty liver disease (NAFLD). Administration of COE (0.5, 1, 2 g/kg) markedly reduced the levels of AST, ALT, TG, TC, TNF-a, IL-6, IL-8, and MDA. COE treatments also increased the levels of SOD, GSH, and the expression of LDL-R mRNA [38]. Seo et al. [12] observed hepatoprotective effects of ethanolic COE and its components (e.g., toralactone glycoside) in t-BHP-induced cell death in HepG2 cells. Cassia anthraquinones, aurantio-obtusin, and obtusifolin also protected against tacrine-induced cytotoxicity in HepG2 cells [36]. Recently, Ali et al. [37] investigated the hepatoprotective effects of different soluble fractions of methanolic derived COE and its active components in t-BHP-induced oxidative stress in HepG2 cells. The possible mechanism was that alaternin, aloe emodin, and cassiaside potently scavenge ROS in t-BHP-induced HepG2 cells and the decrease in ROS generation parallels the up-regulation of glutathione (GSH). Very recently, Paudel et al. [57] investigated the hepatoprotective activity of an anthraquinone (1-desmethylaurantio-obtusin 2-O-β-D-glucopyranoside) and two naphthopyrone glycosides (rubrofusarin 6-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside and rubrofusarin 6-O-β-gentiobioside) isolated from the butanol fraction of COE in the t-BHP-induced oxidative stress in HepG2 cells through up-regulated HO-1 via the nuclear factor erythroid-2-related factor 2 (Nrf2) activation and modulation of the JNK/ERK/MAPK signaling pathway.

Anti-Inflammatory and Antioxidant Activity
COE has traditionally been used in Korea to treat eye inflammation, photophobia, and lacrimation. Pretreatment with the aqueous extract of C. obtusifolia L. inhibited interleukin (IL)-6 and cyclooxygenase-2 (COX-2) and reduced the activation of transcription nuclear factor-kB p65 in colon tissues treated with dextran sulfate sodium [40]. Two major anthraquinones from C. obtusifolia, obtusifolin and gluco-obtusifolin, reduced neuropathic and inflammatory pain [40]. Pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and activation of NF-kB have been strongly implicated in the initiation and development of inflammatory and neuropathic pain, and the administration of obtusifolin and glucoobtusifolin (1 and 2 mg/kg) significantly inhibited this upregulation. This finding suggests that obtusifolin and gluco-obtusifolin inhibited the overexpression of spinal TNF-α, IL-1β, IL-6, and NF-κB p65 associated with inflammatory and neuropathic pain, which involves the regulation of neuroinflammatory processes and the neuroimmune system [51]. In another study, water-extracted polysaccharides (CP) from the whole seeds of C. obtusifolia L. and its two subfractions CP-30 and CP-40 were obtained. CP, CP-30, and CP-40 possessed immunomodulation activity by promoting phagocytosis and stimulating the production of nitric oxide (NO) and cytokines TNF-α and IL-6 [41]. Methanolic COE was investigated for antioxidant and health-relevant functionality. The extract exhibited 1292 mM Fe[II] per 1 mg/mL extract of antioxidant power, 49.92% inhibition of β-carotene degradation, 65.79% of scavenging activity against DPPH, and 50.78% of superoxide radicals (at a concentration 1 mg/mL). These antioxidant properties may be attributed to the total free phenolic content of the raw seeds, which was 13.33 ± 1.73 g catechin equivalent/100 g extract [14]. Recently, Kwon et al. [58] investigated the anti-inflammatory activity of major anthraquinone derivatives; among them, aurantio-obtusin inhibited iNOS expression without affecting iNOS enzyme activity and down-regulation mechanisms included interruption of the JNK/IKK/NF-κB activation and proinflammatory cytokine production from the lungrelated cells. Additionally, aurantio-obtusin also dose-dependently (10 and 100 mg/kg) inhibited the inflammatory responses in a mouse model of airway inflammation, LPSinduced acute lung injury. Very recently, Hou et al. [54] reported anti-inflammatory activity by decreasing the production of NO, PGE2, and inhibiting iNOS, COX-2, TNF-α, and IL-6. Additionally, there was a reduction in the LPS-induced activation of nuclear factor-κB in RAW264.7 cells [54].

Antidiabetic Activity
Two key enzymes, protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, are effective in treating diabetes mellitus. The effects of methanolic COE revealed inhibitory activities against PTP1B and α-glucosidase. Out of 15 anthraquinones from the extract, compounds with alaternin, physcion, chrysophanol, emodin, obtusin, questin, chryso-obtusin, aurantio-obtusin, 2-hydroxyemodin-1 methylether, gluco-obtusifolin, gluco-aurantio obtusin, and naphthalene glycoside aloe-emodin exhibited the highest inhibitory activities against PTP1B and α-glucosidase in vitro [9]. The effects of alaternin and emodin on the stimulation of glucose uptake by insulin-resistant human HepG2 cells were examined at concentrations ranging from 12.5 to 50 µM and 3.12 to 12.5 µM, respectively. In another study, five new anthraquinones were isolated from ethanol seed extracts of C. obtusifolia L. and evaluated for their antidiabetic activities through the inhibition of α-glucosidase in vitro [39]. Obtusifolin isolated from C. obtusifolia L. may have an antihyperlipidemic effect; an intraperitoneal obtusifolin injection reduced blood lipid levels in streptozotocin-induced diabetic rats [59]. Results from another study indicated that oral administration of obtusifolin significantly reversed the changes induced by hyperlipidemia in body weight, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol; increased serum superoxide dismutase, and nitric oxide, and reduced malondialdehyde [50].

Larvicidal Activity
The larvicidal activity of methanol COE against early fourth-stage larvae of Aedes aegypti and Culex pipiens pallens was investigated [43]. At 200 ppm, extracts of C. obtusifolia L. caused more than 90% mortality in larvae of Ae. aegypti and Cx. pipiens pallens. At 40 ppm, extracts of C. obtusifolia L. caused 51.4% and 68.5% mortality in fourth-stage larvae of Ae. aegypti and Cx. pipiens pallens, respectively. Larvicidal activity of C. obtusifolia extract at 20 ppm was significantly reduced [43]. In another study, COE obtained in different fractions showed mosquito larvicidal activity against fourth instar larvae of A. aegypti, Aedes togoi, and Cx. pipiens pallens [44]. However, the chloroform fraction of C. obtusifolia extracts exhibited a strong larvicidal activity of 100% mortality (at a concentration 25 mg/L), and the isolated active compound emodin showed strong larvicidal activity, with LC 50 values of 1.4, 1.9, and 2.2 mg/L against C. pipiens pallens, A. aegypti, and A. togoi, respectively [44]. The ethanolic leaf extract of C. obtusifolia L. was also investigated for larvicidal and oviposition deterrence effects against late third instar larvae of Anopheles stephensi [45]. Extracts from the leaf displayed significant larvicidal activity, with LC 50 and LC 90 values of 52.2 and 108.7 mg/L, respectively (at concentrations of 25 mg/L). In addition, the oviposition study indicated that different concentrations of leaf extract greatly reduced the number of eggs deposited by gravid A. stephensi. At concentrations of 100, 200, 300, and 400 mg/L, the maximum percentages of effective repellency against oviposition were 75.5%, 83.0%, 87.2%, and 92.5%, respectively [45].

Other Activities
The methanol extract of C. obtusifolia L. and its isolated naphthopyrones cassiaside B2 and cassiaside C2 inhibited histamine release from rat peritoneal exudate mast cells induced by antigen-antibody reaction [6]. The anti-angiogenic activity of two polysaccharides, COB1B1S2 and COB1B1S2-Sul, from C. obtusifolia L. seeds was evaluated by tube formation of HMEC-1 cells on Matrigel. COB1B1S2 at 50 or 100 µg/mL did not impair tube formation, but COB1B1S2-Sul at 50 or 100 µg/mL significantly disrupted tube formation; even at 50 µg/mL, COB1B1S2-Sul could potentially completely inhibit tube formation in HMEC-1 cells [61]. Water-soluble polysaccharides (WSPs) from C. obtusifolia L. (pectic polysaccharides and hemicellulose) were identified. These WSPs reduced pancreatic αamylase activity by 20.5% and 28.9% (at concentrations of 20 and 80 mg/mL, respectively), reduced pancreatic lipase activity by about 18.9% (at a concentration of 80 mg/mL), and increased protease activity 5-to 7-fold (at concentrations of 20 and 80 mg/mL, respectively). These WSPs were also able to bind bile acids and reduce the amount of cholesterol available for absorption [63]. The simultaneous determination and pharmacokinetic study of seven anthraquinones (chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin, and aurantio-obtusin) in rat plasma after oral administration of C. obtusifolia L. extract was investigated and may help to explain the bioactivity and clinical applications of C. obtusifolia L. [64]. The effects of COE and its anthraquinones on muscle mitochondrial function were evaluated in vivo in rats and in vitro using mitochondrial energy metabolism models. The organic extract of C. obtusifolia L. and emodin significantly inhibited NADH: cytochrome c oxidoreductase activity of bovine heart mitochondrial particles and NADH: coenzyme Q oxidoreductase activity of porcine heart mitochondrial NADH dehydrogenase and exhibited protective effects of coenzyme Q against enzyme inhibition by anthraquinones [65]. Inhibition of trypsin activity by C. obtusifolia L. seeds was investigated [66]. A Kunitz-type trypsin inhibitor showed strong resistance against the midgut trypsin-like protease of Pieris rapae. In addition, a trypsin inhibitor gene (CoTI1) was isolated from C. obtusifolia L. and exhibited dominant inhibitory activities against trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura [67]. Moreover, Dong et al. [68], has been also reported that Cassia semen (C. obtusifolia and C. tora) and its major constituents possesses a wide spectrum of pharmacological properties.

Conclusions and Perspectives
As presented in this review, pharmacological studies on C. obtusifolia L. and its putative active compounds, especially anthraquinones and naphthopyrone, support that several biological activities of C. obtusifolia can potentially impact human health. Anthraquinones and naphthopyrone can be effectively isolated and purified from C. obtusifolia seeds, leaves, root and its whole plant with various extraction analytical methods, mainly separation-based methods using TLC, HPLC, high-speed counter-current chromatography (HSCCC), and column chromatography (silica gel, reverse-phase, and Sephadex). The semi-shrubby herb C. obtusifolia L., which belongs to the family Leguminosae, has gained popularity because of its medicinal and historical importance. It has been widely used in traditional medicine to treat headaches, dizziness, dysentery, and eye disease. In addition, C. obtusifolia L. is important to the food industry and possesses a wide spectrum of pharmacological properties (e.g., anti-allergic, antidiabetic, anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, neuroprotective, anti-Alzheimer's disease, antiplatelet aggregation, and larvicidal activities) that are associated with its diverse chemical constituents (e.g., anthraquinones, naphthopyrone, terpenoid, flavonoid, polysaccharides, and lipids). The number of modern studies on bioactive compounds is increasing in biomedicine, suggesting that these compounds might have great medical significance in the future. Although the bioactivities of seed extracts or compounds isolated from C. obtusifolia L. have been substantiated using in vitro and in vivo studies, the mechanisms of action remain unknown. Thus, there are still opportunities and challenges for research of seed extracts or compounds. Therefore, additional studies are required before C. obtusifolia L. and its components can be considered for further clinical use. In conclusion, C. obtusifolia L. is an edible medicinal plant that is important to the food industry and has a wide range of potential pharmacological uses. This review presents a summary of studies published to date on this promising plant.

Conflicts of Interest:
All authors agree to the authorship and submission of the manuscript for peer review.