Scrophulariae Radix: An Overview of Its Biological Activities and Nutraceutical and Pharmaceutical Applications

Scrophulariae Radix (SR) has an important role as a medicinal plant, the roots of which are recorded used to cure fever, swelling, constipation, pharyngitis, laryngitis, neuritis, sore throat, rheumatism, and arthritis in Asia for more than two thousand years. In this paper, the studies published on Scrophularia buergeriana (SB) and Scrophularia ningpoensis (SN) in the latest 20 years were reviewed, and the biological activities of SB and SN were evaluated based on in vitro and in vivo studies. SB presented anti-inflammatory activities, immune-enhancing effects, bone disorder prevention activity, neuroprotective effect, anti-amnesic effect, and anti-allergic effect; SN showed a neuroprotective effect, anti-apoptotic effect, anti-amnesic effect, and anti-depressant effect; and SR exhibited an immune-enhancing effect and cardioprotective effects through in vitro and in vivo experiments. SB and SN are both known to exert neuroprotective and anti-amensice effects. This review investigated their applicability in the nutraceutical, functional foods, and pharmaceutical industries. Further studies, such as toxicological studies and clinical trials, on the efficacy and safety of SR, including SB and SN, need to be conducted.


Introduction
The genus Scrophularia consists of more than 300 different herbs, and Scrophularia buergeriana (SB) and Scrophularia ningpoensis (SN) are representative plants plants of this genus. SB is called "Hyun-sam" in Korea; it is a perennial plant with a strong fragrance that grows up to 1.8 m [1,2]. It is native to Korea, North China, and Japan, and it has an important role as a traditional medicinal herb. The SB root has been used to treat fever, swelling, constipation, pharyngitis, laryngitis, neuritis, sore throat, rheumatism, and arthritis, and it is also used for fire pursing, blood cooling, and toxin removal as oriental medicine [3][4][5]. SN is called "Xuan shen" in China, and it has been mainly used as a tea in traditional medicine. The SN root has been used to treat laryngitis, swelling, fever, constipation, and neuritis and it also used for immune enhancement [1,6]. In this paper, we reviewed the physiological activities of Scrophulariae Radix (SR), specifically, SB and SN. The physiological properties discussed in this review have been verified using in vitro and in vivo studies, and these results are the scientific basis for the development of health foods or therapeutics.
Gong et al. [58] demonstrated that 85% ethanol extract of SR (ERS, 0.001-10 mg/mL) demonstrated the immune-enhancing effects in lymphocytes isolated from ICR mice spleen. The cAMP/cGMP ratio is known as an indicator of deficiency in immunity. In addition, MDA is an indicator that reflects oxidative stress, and SOD is an enzyme that converts superoxide radicals into molecular oxygen and hydrogen peroxide. ERS (1, 10 mg/mL: p < 0.01) treatment increased the cell viability in lymphocytes. In addition, ERS markedly decreased cAMP/cGMP (1 mg/mL: p < 0.05), IFN-γ/IL-10 (1 mg/mL: p < 0.01), and MDA content (1 mg/mL: p < 0.01) and increased the SOD content (1 mg/mL: p < 0.01) in lymphocytes isolated from ICR mice spleen.

Prevention of Bone Disorders
Nam et al. [5] reported that SBE (50-200 µg/mL) prevented the bone disorder in bone marrow macrophage. TRAP is an enzyme expressed in osteoclasts, and an increase in TRAP indicates mature and differentiated osteoclasts. SBE statistically (p < 0.001) suppressed TRAP-positive cell formation at 200 µg/mL concentration and had a resorption inhibition effect on mature osteoclasts. The resorption area (p < 0.001) was also observed to decrease after SB treatment.

Neuroprotective Effects
Lee et al. [7] demonstrated that SB 70% ethanol extract (SBE, 125-500 µg/mL) treatment showed the neuroprotective activity in SH-SY5Y cells. Acetylcholinesterase is an enzyme that the neurotransmitter acetylcholine, and increased activity of this enzyme affects the concentration of acetylcholine. SBE increased the cell viability (SBE: p < 0.01) in SH-SY5Y cells with glutamate-induced cell toxicity. Acetylcholinesterase activity (SBE 250 µg/mL: p < 0.05, SBE 500 µg/mL: p < 0.01) was decreased, and total glutathione content (SBE: p < 0.01) was increased in a dose-dependent manner. Glutamate-induced cell morphology changes and DNA fragmentation were measured using DAPI staining and TUNEL assays, and SBE was observed to reduce glutamate-induced fragmentation (SBE: p < 0.01).

Anti-Apoptotic Effects
Shen et al. [1] demonstrated that Scrophularia ningpoensis water extract (RSN) presented the anti-apoptotic activity in HaCaT cells. RSN showed the IC50 value at 0.032 mg/mL concentration. TNF-a stimulation activates the NF-κB pathway and induces inflammation. In addition, ERK upstream of NF-κB affects cell proliferation and induces apoptosis. Pretreatment with 0.032 mg/mL RSN inhibited NF-κB translocation induced by TNF-α, which was observed using immunofluorescence staining of HaCaT cells. RSN decreased ERK phosphorylation, and ERK increased gradually with dose and time. RSN did not affect the cell cycle phase in G1/G0, S, and G2/M (at 0.016-0.064 mg/mL for 6-96 h) in HaCaT cells, and a tumor-preferred effect was not detected in Colo 38, SK-Mel-28, and MRI-221 cells. These results suggest that RSN regulates ERK and NF-κB signaling.
These results suggest that SBE exhibits anti-inflammatory activity by inhibition of NF-κB phosphorylation, and it could be applied as an effective therapeutic agent against allergic asthma.

Anti-Amnesic Effects
Jeong et al. [8] reported that 70% ethanol extract of SB root (KD-501) possesses cognition-enhancing activity. Male ICR mice administered scopolamine, a substance known to cause short-term memory loss, exhibited induced amnesia. In these mice, KD-501 (3, 10 These results showed that KD-501 may be used for the prevention and therapeutics of Alzheimer's disease. Lee et al. [9,69] demonstrated that SBE has a neuroprotective effect on mice with memory impairment induced by scopolamine and β-amyloid. The mice were orally administered 30 and 100 mg/kg SBE for 28 days. The step-through latency (SBE: p < 0.05) in the passive avoidance test that decreased after scopolamine injection was significantly increased by treatment with 30 and 100 mg/kg SBE, and escape latency and swim distance were decreased in the Morris water maze test. Moreover, in the probe trial conducted on day 28, administration of SBE (30 and 100 mg/kg) significantly increased the crossing number (SBE: p < 0.01). The decreased acetylcholine (SBE 30 mg/kg: p < 0.05, SBE 100 mg/kg: p < 0.01) level was increased, while increased acetylcholinesterase (SBE 100 mg/kg: p < 0.01) activity was decreased in the hippocampus after SBE (30 and 100 mg/kg) administration.
SBE indicated its potential for development as a health functional food for memory improvement and as a treatment for Alzheimer's disease.
Meng et al. [6] reported that RSAE has a neuroprotective effect on the middle cerebral artery occlusion and reperfusion (MCAO/R) mouse model. Oral administration of RSAE (2.4 g/kg) for 7 days significantly decreased the brain water content (RSAE 2.4 g/kg: p < 0.05) and MDA (RSAE 2.4 g/kg: p < 0.01) and NO (RSAE 2.4 g/kg: p < 0.01) levels in the ischemic hemisphere. 2,3,5-Triphenyltetrazolium chloride (TTC) staining presented that the corrected infarct volume increased by MCAO/R operation was significantly decreased by RSAE (2.4 g/kg: p < 0.0001) administration. In the cortex and CA1 region, significantly increased neuronal damage due to MCAO/R was observed compared to the sham group. RSAE treatment significantly decreased neuronal damage by increasing neuron density in the ischemic cortex (RSAE 2.4 g/kg: p < 0.01) and hippocampus CA1 region (RSAE 2.4 g/kg: p < 0.01).
These results suggest that RSAE could be a new therapeutic target for ischemic stroke patients.

Anti-Depressant Effects
Xu et al. [70] demonstrated that SN EtOAc extract (II) has an anti-depressive effect. Male ICR mice were orally treated with II (5, 10, 15, and 20 mg/kg) for 5 consecutive days, and the avoidance-escape test was performed. The extract of II (15 mg/kg: p < 0.01, 20 mg/kg: p < 0.005) remarkably decreased the number of escape failures and had an anti-depressive effect on the animal model.
These results suggest that II could be used for the treatment of depression.

Anti-Allergic Effects
Kim et al. [3] reported that SBE possesses anti-allergic activity. Dinitrofluorobenzene was used to induce the hypersensitivity reaction in BALB/c mice. SBE (10, 100, and 1000 µg/ear) was topically administered to the ear for 3 days.
According to the previous in vitro and in vivo studies, SBE showed anti-allergic activity and may be effective in treating rhinitis and asthma.
These results suggest that ERRS exhibits preventive effects against cardiac fibrosis and attenuates ventricular remodeling. It could be used to treat early ventricular remodeling and heart failure.
Zhang et al. [72] showed that the active extract of Scrophularia Radix (ACRS) possesses ventricular remodeling inhibition effects. Spontaneously hypertensive (SHR) male rats were used for the experiment and orally treated with 70 These results show that ACRS inhibited ventricular remodeling through MAPK pathway regulation, and it may be used to prevent and treat heart failure.
Gu et al. [73] showed that ERS possesses cardioprotective effects. Experimental ventricular remodeling was induced in the rats that were orally administered 8 (L) and 16 (H) g/kg ERS for 4 weeks. Cardiac weight indexes such as LVWI (ERS-L: NS, ERS-H: p < 0.05), HWI (ERS-L: NS, ERS-H: p < 0.05), myocardium Ang II (ERS-L: p < 0.05, ERS-H: p < 0.01), and Hyp (ERS-L and H: p < 0.01) decreased with the ERS treatment. To analyze the cardiocyte cross-section area (ERS-L and H: p < 0.01) by H&E staining, ERS administration was reduced similarly to that in the captopril group used as the positive control. In addition, PVCA (ERS-L and H: p < 0.01) and CVF (ERS-L and H: p < 0.01), which are increased by ventricular remodeling, were significantly decreased by the ERS treatment.
These results suggest that ERS may be used for the treatment of myocardial infarction by decreasing the progression of left ventricular remodeling.

Immune-Enhancing Effects
Gong et al. [58] reported that ERS has immune-enhancing activity. The mice were administered 2 g/kg ERS intragastrically for 14 days, and the exterior signs of the mice with ERS were measured. Body weight (ERS: p < 0.01) was observed to significantly increase, and body temperature (ERS: p < 0.05), heart rate (ERS: p < 0.05), average speed (ERS: p < 0.01), and upright time (ERS: p < 0.01) were found to markedly decrease. Measurement of serum MDA and SOD levels and cAMP/cGMP suggested that ERS treatment decreased the MDA (ERS: p < 0.05) level and cAMP/cGMP (ERS: p < 0.05) and increased SOD (ERS: p < 0.05) level. The ERS-treated mice showed a statistical decrease in IL-6 (ERS: p < 0.01) expression level in the serum and Na + -K + ATP enzyme content (ERS: p < 0.01) in the liver homogenates.
According to the in vitro and in vivo studies, ERS showed its immune regulation and antioxidant activities, and it can be used as dietary supplement for better health.

Conclusions
In this review, we summarized the biological effects of SB, SN, and SR based on the previous in vitro and in vivo studies. SB was administered orally at a minimum dose of 20 mg/kg to a maximum of 200 mg/kg (mice), SN was administered orally at a minimum dose of 5 mg/kg to a maximum of 2.4 g/kg (mice), and SR was orally administered at 2 g/kg (rat). SB showed anti-inflammation activity, immune-enhancing effects, bone disorder prevention activity, neuroprotective effect, anti-amnesic effect, and anti-allergic effect. SN exhibited neuroprotective effect, anti-apoptotic effect, anti-amnesic effect, cardioprotective effect, and anti-depressant effect; and SR showed immune-enhancing activity and cardioprotective effects. However, clinical trials need to be conducted to investigate the efficacy and safety of SR, including SB and SN.
As per studies published on SB, SN, and SR in the last 20 years, these plants have been processed using water or ethanol as a solvent. This is thought to reduce side effects when administered orally, and will help increase their applicability to various formulations when developing health functional foods or therapeutics in the future.
Taken together, we suggested the possibility of utilizing SB, SN, and SR for developing health functional foods or therapeutics for various applications on the basis of previously reported literature.