Approach of Supercritical Carbon Dioxide for the Extraction of Kleeb Bua Daeng Formula.

Supercritical fluid extraction (SFE) is an innovative green technology for the extraction of phytochemicals from plants. Therefore, this study aimed to evaluate the application of SFE and to optimize the extraction conditions of the Thai herbal formula, Kleeb Bua Daeng (KBD). A Box-Behnken design (BBD) with response surface methodology (RMS) was used to determine the effect of the extraction time (30-90 min), temperature (30-60 °C), and pressure (200-300 bar) on response variables including the extraction yield, total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TCC), and total anthocyanin content (TAC) of the KBD formula. The highest percentage extraction yield (3.81%) was achieved at 60 °C, 300 bar, and 60 min of the extraction time. The highest TPC (464.56 mg gallic acid equivalents/g extract), TFC (217.19 mg quercetin equivalents/g extract), and TCC (22.26 mg ß-carotene equivalents/g extract) were all achieved at 60 °C, 250 bar, and 90 min of the extraction time. On the contrary, it was not possible to quantify the total anthocyanin content as anthocyanins were not extracted by this method. The results indicated that SFE-CO2 is a suitable method of extraction for a green recovery of phytochemicals with low and moderate polarity from the KBD formula.

Effect of Yakae-Prajamduen-Jamod Traditional Thai Remedy on Cognitive Impairment in an Ovariectomized Mouse Model and Its Mechanism of Action.

Cognitive impairment is a neurological symptom caused by reduced estrogen levels in menopausal women. The Thai traditional medicine, Yakae-Prajamduen-Jamod (YPJ), is a formula consisting of 23 medicinal herbs and has long been used to treat menopausal symptoms in Thailand. In the present study, we investigated the effects of YPJ on cognitive deficits and its underlying mechanisms of action in ovariectomized (OVX) mice, an animal model of menopause. OVX mice showed cognitive deficits in the Y-maze, the novel object recognition test, and the Morris water maze. The serum corticosterone (CORT) level was significantly increased in OVX mice. Superoxide dismutase and catalase activities were reduced, while the mRNA expression of IL-1ß, IL-6, and TNF-α inflammatory cytokines were up-regulated in the frontal cortex and hippocampus of OVX mice. These alterations were attenuated by daily treatment with either YPJ or 17ß-estradiol. HPLC analysis revealed that YPJ contained antioxidant and phytoestrogen constituents including gallic acid, myricetin, quercetin, luteolin, genistein, and coumestrol. These results suggest that YPJ exerts its ameliorative effects on OVX-induced cognitive deficits in part by mitigating HPA axis overactivation, neuroinflammation, and oxidative brain damage. Therefore, YPJ may be a novel alternative therapeutic medicine suitable for the treatment of cognitive deficits during the menopausal transition.

Anti-SARS-CoV-2 Activity of Andrographis paniculata Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 µg/mL and 0.034 µM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 µg/mL for A. paniculata extract and 13.2-81.5 µM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

Kleeb Bua Daeng, a Thai Traditional Herbal Formula, Ameliorated Unpredictable Chronic Mild Stress-Induced Cognitive Impairment in ICR Mice.

Thai traditional herbal formula ''Kleeb Bua Daeng (KBD)''consists of a 1:1:1 ratio (dry weight) of three medicinal plants: Piper nigrum fruit, the aerial part of Centella asiatica and the petals of Nelumbo nucifera. Oral administration of KBD to unpredictable chronic mild stress (UCMS) mice significantly improved their cognitive function caused by chronic mild stress. Daily administration of KBD significantly decreased the serum corticosterone (CORT) and malondialdehyde (MDA) levels but increased the catalase and superoxide dismutase activities in both frontal cortex and hippocampus. The effects of KBD were similar to those caused by oral administration of vitamin E. HPLC analysis of the KBD extract revealed the presence of piperine, madecassoside, asiaticoside, luteolin-7-O-glucoside, rutin, kaempferol-3-glucoside, quercetin, kaempferol and ferulic acid as major constituents.

Prophylactic management of radiation-induced mucositis using herbal mouthwash in patients with head and neck cancer: an assessor-blinded randomized controlled trial.

OBJECTIVES: Radiation-induced mucositis (RIOM) is one of the most common side effects from head and neck radiotherapy. Several reagents have been introduced to manage the symptom; however, there is still a limited number of effective reagents. Herbal mouthwashes with payayor (Clinacanthus nutans Lindau) and fingerroot (Boesenbergia rotunda) were tested their efficacies in preventing and reducing severity of RIOM in comparison with normal saline with sodium bicarbonate. METHODS: One hundred twenty patients with head and neck cancer undergoing radiotherapy participated in the study and were randomly assigned into three treatment groups using block randomization method. The participants were assigned one of the three mouthwashes for use throughout their radiotherapy course and were assessed for their mucositis scores from week one to six into their radiotherapy course as well as at one-month follow-up. Body mass index was also measured for comparison of nutritional status. RESULTS: The two mouthwashes were similarly effective in prophylaxis of RIOM in term of severity. The averaged mucositis scores were less than two for all groups. For the onset of RIOM, both herbal mouthwashes could slightly delay the symptom but not statistically significant. Patients' body mass index across the three treatment groups was also comparable. The patients were largely satisfied with all the mouthwashes with no clear preference on any of them. CONCLUSIONS: Prophylactic treatment of RIOM using herbal mouthwashes could substitute the current standard of normal saline with bicarbonate. A different formulation of the two herbs could potentially improve the prophylactic outcome. TRIAL REGISTRATION NO: NCT03359187.

Efficacy and Safety of Kleeb Bua Daeng Formula in Mild Cognitive Impairment Patients: A Phase I Randomized, Double-Blind, Placebo-Controlled Trial.

Individuals with mild cognitive impairment (MCI) were at increased risk of conversion to dementia. The Kleeb Bua Daeng (KBD) formula could be the alternative treatment option for MCI through multitarget activities. Lacking of clinical trial information brought about the study in our research. Forty patients with MCI were randomly assigned to receive the KBD capsule or placebo at a dose of 1,000 mg twice a day for three months. Their cognitive functions were monitored by the Montreal Cognitive Assessment (MoCA) and blood chemistry assessment every one month. We found that the KBD-treated group had no significant differences in the MoCA test compared to placebo. Moreover, there was no alteration in biochemical parameters of the liver and renal function was observed which could confirm the safety of this KBD formula.

Standardization of the ethanolic extract of Crinum latifolium leaves by two bioactive markers with antiproliferative activity against TGF-ß-promoted prostate stromal cells (WPMY-1).

BACKGROUND: Crinum latifolium L. (Amaryllidaceae) has been used in Southeast Asian traditional medicine to alleviate the symptoms of benign prostatic hyperplasia (BPH). The pathological mechanism of BPH is associated with the induction of prostate stromal cell proliferation through transforming growth factor-beta (TGF-ß). Standardization as well as investigation of the potential anti-BPH activity of C. latifolium extract could benefit the further development of BPH-related analyses and provide evidence to support the application of this extract for BPH treatment. This study aimed to standardize and investigate the antiproliferative activity of the ethanolic extract of C. latifolium leaves. The major alkaloids isolated from C. latifolium were also explored for their potential use as bioactive markers. METHODS: Two major alkaloids were isolated from the ethanolic extract of C. latifolium leaves by chromatographic techniques, identified by NMR and MS, and quantified by a validated UHPLC method. Their antiproliferative activity was studied in human prostate stromal cells (WPMY-1) induced by TGF-ß. The synergistic effect of combining the two major isolated alkaloids was analyzed by the zero interaction potency (ZIP) model. RESULTS: Two alkaloids, lycorine (1) and 6α-hydroxybuphanidrine (2), were isolated from the ethanolic leaf extract of C. latifolium. A UHPLC method for the quantification of (1) and (2) was developed and validated in terms of linearity, precision, and accuracy. The C. latifolium leaf extract contained 0.279 ± 0.003% (1) and 0.232 ± 0.004% (2). The crude extract was more potent than either (1) and (2) alone against TGF-ß-treated WPMY-1 cell proliferation. The drug combination study revealed that the greatest synergistic effect of (1) and (2) was achieved at a 1:1 ratio. CONCLUSIONS: The results of this study support the anti-BPH activity of C. latifolium in traditional medicine and suggest that these the two isolated alkaloids may promote the efficacy of the C. latifolium extract. Additionally, major alkaloids (1) and (2) can be used as bioactive markers for the standardization of C. latifolium extracts.

Toxicity Profiles of Kleeb Bua Daeng Formula, a Traditional Thai Medicine, and Its Protective Effects on Memory Impairment in Animals.

Kleeb Bua Daeng (KBD) formula has long been used in Thailand as a traditional herbal medicine for promoting brain health. Our recent reports illustrated that KBD demonstrates multiple modes of action against several targets in the pathological cascade of Alzheimer's disease (AD). The main purpose of the present study was to determine the protective effect and mechanism of KBD in amyloid beta (Aß)-induced AD rats and its toxicity profiles. Pretreatment with the KBD formula for 14 days significantly improved the short- and long-term memory performance of Aß-induced AD rats as assessed by the Morris Water Maze (MWM) and object-recognition tests. KBD treatment increased the activities of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase; reduced the malondialdehyde content, and; decreased the acetylcholinesterase activity in the rat brain. An acute toxicity test revealed that the maximum dose of 2000 mg/kg did not cause any mortality or symptoms of toxicity. An oral, subchronic toxicity assessment of KBD at doses of 125, 250, and 500 mg/kg body weight/day for 90 days showed no adverse effects on behavior, mortality, hematology, or serum biochemistry. Our investigations indicate that KBD is a nontoxic traditional medicine with good potential for the prevention and treatment of AD.

Antioxidant Activity and Anti-Photoaging Effects on UVA-Irradiated Human Fibroblasts of Rosmarinic Acid Enriched Extract Prepared from Thunbergia laurifolia Leaves.

The current study investigated the inhibiting effect on reactive oxygen species (ROS), reactive nitrogen species (RNS), and matrix metalloproteinase-1 (MMP-1) production in a cell-based study of standardized rosmarinic acid enriched extract (SRAEE) prepared from Thunbergia laurifolia leaves. HPLC chromatogram revealed that rosmarinic acid is a major component in prepared SRAEE, followed by caffeic acid. SRAEE exhibited antioxidant activity both in vitro and cell-based studies. SRAEE showed scavenging effects on nitric oxide and superoxide anion and inhibition effects on lipid peroxidation in vitro. SRAEE also inhibited ROS and MMP-1 production in normal human dermal fibroblast cells induced by H2O2 and UVA, respectively, without exerted cytotoxicity. Additionally, collagen degradation was protected by SRAEE induced by UVA. Nitric oxide and inducible nitric oxide synthase (iNOS) productions were also inhibited by SRAEE in RAW264.7 mouse macrophage cells induced by combined lipopolysaccharide (LPS)-interferon-γ (IFN-γ). The results indicated that SRAEE is a potential candidate as a natural pharmaceutical active ingredient for cosmeceutical product application.

Merging the Multi-Target Effects of Kleeb Bua Daeng, a Thai Traditional Herbal Formula in Unpredictable Chronic Mild Stress-Induced Depression.

Major depressive disorder (MDD) is a common and debilitating psychiatric disease characterized by persistent low mood, lack of energy, hypoactivity, anhedonia, decreased libido, and impaired cognitive and social functions. However, the multifactorial etiology of MDD remains largely unknown due the complex interaction between genetics and environment involved. Kleeb Bua Daeng (KBD) is a Thai traditional herbal formula that has been used to promote brain health. It consists of a 1:1:1 ratio of the aerial part of Centella asiatica, Piper nigrum fruit, and the petals of Nelumbo nucifera. According to the pharmacological activities of the individual medicinal plants, KBD has good potential as a treatment for MDD. The present study investigated the antidepressant activity of KBD in an unpredictable chronic mild stress (UCMS) mouse model. Daily administration of KBD to UCMS mice ameliorated both anhedonia, by increasing 2% sucrose intake, and hopeless behavior, by reducing immobility times in the forced swimming test (FST) and tail suspension test (TST) without any effect on locomotor activity. The mechanism of KBD activity was multi-modal. KBD promoted neurogenesis by upregulation of brain-derived neurotrophic factor (BDNF) and cyclic AMP-responsive element binding (CREB) mRNA expression in the frontal cortex and hippocampus. Daily treatment with KBD significantly reversed UCMS-induced HPA axis dysregulation by upregulating the glucocorticoid receptor (GR) while downregulating serum- and glucocorticoid-inducible kinase 1 (SGK1) and FK506 binding protein 5 (FKBP5) mRNA expression. KBD treatment also normalized proinflammatory cytokine expression including tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-1ß and IL-6. KBD and its component extracts also exhibited an inhibitory effect in vitro on monoamine oxidase (MAO) A and B. The multiple antidepressant actions of KBD emphasize its potential as an effective, novel treatment for MDD.

Prophylaxis of Radiation-Induced Dermatitis in Patients With Breast Cancer Using Herbal Creams: A Prospective Randomized Controlled Trial.

Radiation-induced toxicity is a major limiting factor for prescribing radiation dose in cancer radiotherapy. Skin reaction to radiation is one of the primary concerns, which could affect quality of life of the patients both physically and mentally. Reviews of the literature show limited number of effective reagents for its prophylaxis. In this study, we attempted to determine whether prophylactic treatment of the 3 different herbal creams containing Centella asiatica, Cucumis sativus, and Thunbergia laurifolia extracts as well as a commercial moisturizing cream could reduce acute skin reaction in breast cancer patients undergoing radiotherapy. A total of 153 breast cancer patients undergoing radiotherapy were randomly assigned into 5 different groups with one group receiving no treatment. The patients were instructed to apply their designated creams once daily from their first radiotherapy session until 1-month post-irradiation. Their skins were graded by a radiation oncologist on a weekly basis until 1-month post-irradiation to identify any skin reactions. The results showed that the administration of the herbal creams or the moisturizing cream could neither reduce the severity nor delay the onset of dermatitis compared with the no treatment group. However, despite the limited benefits from the prophylaxis, the Cucumis sativus cream was shown to help with the skin recovery post-irradiation. These results suggested that breast cancer patients undergoing radiotherapy should be advised to apply moisturizing cream to the area of irradiated skin.

Multitarget Activities of Kleeb Bua Daeng, a Thai Traditional Herbal Formula, Against Alzheimer's Disease.

The Kleeb Bua Daeng formula (KBD) is a Thai traditional medicine for brain health promotion. On the basis of the activities of its individual components, the KBD could have good potential for the treatment of Alzheimer's disease (AD). Herein, we investigated the KBD as an AD treatment. The ethanol extracts of KBD and its components, i.e., Nelumbo nucifera (NN), Piper nigrum fruits (BP), and the aerial part of Centella asiatica (CA) exhibited antioxidant activity, as determined by both ABTS and DPPH assays. The Ellman's assay revealed that the KBD, NN, and BP showed an ability to inhibit acetylcholinesterase. The thioflavin T assay indicated that the KBD, NN, BP, and CA inhibited beta-amyloid aggregation. The neuroprotection and Western blot analysis revealed that the KBD reduced H2O2-induced neuronal cell death by inhibiting the expression of pro-apoptotic factors, i.e., cleaved caspase-9 and -3, p-P65, p-JNK, and p-GSK-3ß, as well as by inducing expression of anti-apoptotic factors, i.e., MCl1, BClxl, and survivin. Furthermore, the KBD could improve scopolamine induced memory deficit in mice. Our results illustrate that the KBD with multimode action has the potential to be employed in AD treatment. Thus, the KBD could be used as an alternative novel choice for the prevention and treatment of patients with AD.

High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 µg/mL (CC50 = 28.06 µg/mL) and 0.81 µΜ (CC50 = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50 = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.) Urban.

Centella asiatica-a medicinal plant that produces high-value active triterpenoids-is in increasing demand by the pharmaceutical and cosmetic industries. The aim of this study was to field-test one induced tetraploid and three diploid C. asiatica lines for the selection of high-quality plants with high phytomass and triterpenoid content and to determine their optimal harvesting time. All tested C. asiatica were micropropagated using an established protocol. One-month-old plantlets were acclimatized for the field experiment. The plants were grown in a randomized complete block design (RCBD) with three replications, ten plantlets per replication, and the experimental bed site was 0.6 × 1.0 m. Growth parameters, phytomass and the amounts of four active triterpenoids were evaluated. All lines exhibited the highest growth, yields and triterpenoids at 4 months after cultivation. The tetraploid line showed significantly better characteristics, i.e., larger leaf area, leaf width, petiole length, and greater yields, than diploid lines. Dry weight per cultivated area (77.53 ± 3.07 g/m(2)) and total triterpenoids (15.38 ± 0.76 % dry weight) were increased significantly in tetraploid plants of C. asiatica. Furthermore, the harvesting time had an effect on the yield and triterpenoid content (P < 0.001). In all tetraploid and diploid lines, the yields and triterpenoid content per cultivated area reached their maximum at 4 months after planting. Our results demonstrated that polyploidy induction is a beneficial tool that can be used to improve the medicinal value of C. asiatica.

Relief of radiation-induced oral mucositis in head and neck cancer.

This study was a prospective, randomized clinical trial carried out to explore the efficacy of payayor in the prevention and relief of radiation-induced oral mucositis compared with benzydamine. Sixty patients with head and neck cancer, who have started to receive radiotherapy and met predetermined criteria, were randomly assigned into each group to use assigned products 3 times a day from the first to the last day of radiation. The first group used glycerin payayor, a Thai prepared herbal product, by dripping it into the mouth. Another group rinsed their mouths with benzydamine hydrochloride. The World Health Organization Mucositis Grading System was used to assess oral status every week and 2 weeks after radiation. Comparison of time to the onset, pain, severity, xerostomia, postponement of treatment, satisfaction of the solution, and body weight between the 2 groups was performed by t test. The average time to the onset of oral mucositis in the payayor group was significantly later, and its severity and pain score were less than those of the benzydamine group throughout the study period. Significantly higher satisfaction with the solution and higher body weight at the end of the study were shown in the payayor group. Payayor seemed to be superior to benzydamine for preventing and relieving radiation-induced oral mucositis.