Utilization of UPLC-PDA and GC-MS/MS coupled with metabolomics analysis to identify bioactive metabolites in medicinal turmeric at different ages for the quality assurance.

BACKGROUND: Available monographs often suggest measurement of curcumin or curcuminoids for quality control of turmeric-based medicines/products. However, one compound is not enough to indicate the quality of traditional herbal medicines due to the holistic approach. Furthermore, to ensure high quality of such products, good harvesting practice plays an important role in the quality control of turmeric raw materials. PURPOSE: This study aimed to indicate quality markers of turmeric and to suggest optimum harvesting times for turmeric used for medicinal purposes by integration of Thai traditional medicine knowledge. METHODS: Turmeric rhizomes at 4, 6, 8, 9, 10, and 11 months old were analyzed. UPLC-PDA was used for quantitation of curcumin. GC-MS/MS was used to obtain chemical profiles of turmeric volatile oil. PCA, Volcano plot, and HCA were performed to identify similarities or differences of the data. RESULTS: Turmeric aged between 6 and 10 months old contained > 5%w/w of curcumin, which complied with Thai Herbal Pharmacopoeia 2021. GC-MS/MS analysis suggested suitable chemical markers, namely Ar-turmerone, turmerone, curlone, and zingiberene, because they exhibited pharmacological activities related to the traditional uses of turmeric. Eucalyptol, santalene, ß-caryophyllene, cis-ß-farnesene, α-caryophyllene, curcumene, ß-bisabolene, ß-sesquiphellandrene, and cis-sesquisabinene hydrate were also tentatively identified. Later, the multivariate analysis revealed that turmeric aged between 6 and 10 months old showed similar metabolite profiles. CONCLUSION: Based on curcumin content and chemical profiles, turmeric aged between 6 and 10 months old are appropriate for uses in Thai traditional medicine. Our study suggests additional information in the quality control of turmeric to assure its efficacy, especially for uses in traditional medicines.

The Protective Effect of Polyherbal Formulation, Harak Formula, on UVA-Induced Photoaging of Human Dermal Fibroblasts and Mouse Skin via Promoting Nrf2-Regulated Antioxidant Defense.

Polyherbal formulation combining multiple herbs is suggested to achieve enhanced therapeutic effects and reduce toxicity. Harak herbal formula (HRF) extracts were proposed to regulate skin responses to UVR through their ability to suppress UVA-induced matrix metalloproteinase-1 (MMP-1) and pigmentation via promoting antioxidant defenses in in vitro models. Therefore, natural products targeting Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated antioxidant response might represent promising anti-photoaging candidates. Hesperetin (HSP) was suggested as a putative bioactive compound of the HRF, as previously shown by its chemical profiling using the liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In this study, we explored the anti-photoaging effects of HRF extracts and HSP on normal human dermal fibroblasts (NHDFs) and mouse skin exposed to UVA irradiation. Pretreatment of NHDFs with HRF extracts and HSP protected against UVA (8 J/cm2)-mediated cytotoxicity and reactive oxygen species (ROS) formation. The HRF and HSP pretreatment also attenuated the UVA-induced MMP-1 activity and collagen depletion concomitant with an upregulation of Nrf2 activity and its downstream genes (GST and NQO-1). Moreover, our findings provided the in vivo relevance to the in vitro anti-photoaging effects of HRF as topical application of the extracts (10, 30 and 100 mg/cm2) and HSP (0.3, 1, and 3 mg/cm2) 1 h before UVA exposure 3 times per week for 2 weeks (a total dose of 60 J/cm2) mitigated MMP-1 upregulation, collagen loss in correlation with enhanced Nrf2 nuclear accumulation and its target protein GST and NQO-1 as well as reduced 8-hydroxy-2'-deoxyguanosine (8-OHdG) in irradiated mouse skin. Thus, our findings revealed that HRF extracts and HSP attenuated UVA-induced photoaging via upregulating Nrf2, together with their abilities to reduce ROS formation and oxidative damage. Our study concluded that the HRF and its bioactive ingredient HSP may represent potential candidates for preventing UVA-induced photoaging via restoration of redox balance.

In vitro protective effects of plants frequently used traditionally in cancer prevention in Thai traditional medicine: An ethnopharmacological study.

ETHNOPHARMACOLOGICAL RELEVANCE: Thai traditional medicine (TTM) has been used widely in cancer management in Thailand. Although several Thai medicinal plants were screened for pharmacological activities related to cancer treatment, such evidence still suffers from the lack of linking with TTM knowledge. AIM OF THE STUDY: To document knowledge and species used in cancer prevention in TTM and to preliminary investigate pharmacological activities related to the documented knowledge of twenty-six herbal drugs used in cancer/mareng prevention. METHODS: Fieldwork gathering data on TTM concept and herbal medicines used in cancer prevention was performed with TTM practitioners across Thailand. Later, water and ethanol extracts from twenty-six herbal drugs mentioned as being used in cancer prevention were screened for their protective effect against tert-butyl hydroperoxide-induced cell death in HepG2 cells. Then active extracts were investigated for their effects on NQO1 activity, glutathione level, and safety in normal rat hepatocytes. RESULTS: The fieldwork helped in the development of TTM cancer prevention strategy and possible experimental models to test the pharmacological activities of selected medicinal plants. Fifteen plant extracts showed significant protective effect by restoring the cell viability to 40-59.3%, which were comparable or better than the positive control EGCG. Among them, ethanol extracts from S. rugata and T. laurifolia showed the most promising chemopreventive properties by significantly increased NQO1 activity, restored GSH level from oxidative damage, as well as showed non-toxic effect in normal rat hepatocytes. CONCLUSION: TTM knowledge in cancer prevention was documented and used in the planning of pharmacological experiment to study herbal medicines, especially in cancer, inflammation, and other chronic diseases. The proposed strategy should be applied to in vivo and clinical studies in order to further confirm the validity of such a strategy. Other traditional medical systems that use integrated approaches could also apply our strategy to develop evidence that supports a more rational uses in traditional medicine.

Caucasian endemic medicinal and nutraceutical plants: in-vitro antioxidant and cytotoxic activities and bioactive compounds.

OBJECTIVES: In order to assess traditional claims about the therapeutic potential of Caucasian endemic medicinal plants and to select plants for phytochemical research, nine plant species were selected and assessed for their in-vitro antioxidant and cytotoxic activities. The metabolite profiles of some priority plants were analysed. METHODS: Antioxidant effects were assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) system, lipid peroxidation and Folin-Ciocalteu methods. Cytotoxic activities were examined against human liver cancer cells (HepG2) using the Alamar Blue assay. Terpenoids of selected species were analysed by GC and GC-MS. Polyphenols were separated by HPLC. KEY FINDINGS: Thymus transcaucasicus, Heracleum transcaucasicum, Ribes armenum, Crataegus armena showed most promising antioxidant activity in ABTS model system. Moreover, Rubus takhtadjanii, C. armena, T. transcaucasicus showed a high level of antioxidant activity by inhibiting lipid peroxidation. C. armena and T. transcaucasicus expressed high-to-moderate cytotoxicity against HepG2 cells. The main terpenoids and polyphenols of Centaurea hajastana, C. armena and T. transcaucasicus were quantified. CONCLUSIONS: This study provides in-vitro data relevant for assessing the use of Caucasian endemic medicinal plants, most importantly C. armena and T. transcaucasicus as traditional antioxidant supplements and potential anticancer remedies. Metabolite profiles of some lead plants showed the nature of bioactive compounds of medical interest.

Understanding cancer and its treatment in Thai traditional medicine: An ethnopharmacological-anthropological investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Thai traditional medicine (TTM) is widely practiced in Thailand and continues to gain importance in cancer management, but little is known about the TTM practitioners' emic concepts and practice. AIM OF THE STUDY: With this study we firstly aim to document the practice of cancer treatment and prevention by TTM practitioners and, secondly, to evaluate how such traditional concepts and practices are correlated with biomedical ones. This in turn can form the basis for developing novel strategies for designing pharmacological experiments and longer term strategies to develop TTM practice. METHODS: Semi-structured interviews with 33 TTM practitioners were performed in five provinces in different regions of Thailand. The following information were recorded; basic information of informants, descriptions of cancer (mareng in Thai), causes, diagnosis, treatment, and prevention. Plants used in the treatment and prevention of mareng were also collected. RESULTS: Using an in depth ethnographic approach four representative case studies to assist in a better understanding of the characteristics of mareng, its diagnosis, treatment, and prevention are reported here. Five characteristics of mareng - waste accumulation (khong sia), chronic illnesses (krasai), inflammation (kan aksep), bad blood (luead) and lymph (namlueang), and the imbalance of four basic elements (dhatu si) - have been identified. Explanatory models of cancer in TTM were linked with biomedical concepts and relevant pharmacological actions. Traditional uses and available scientific evidence of medicinal plants mentioned in the case studies for the treatment or prevention of mareng are presented and discussed. CONCLUSION: Here for the first time five main characteristics of cancer based on Thai traditional medical concepts are analysed. Our findings are relevant not only for the planning of clinical studies or pharmacological experiment in the search for novel compounds for cancer treatment and prevention, but also for the integration of Thai traditional medicine in cancer care.

Effect of Moringa oleifera Leaf Capsules on Glycemic Control in Therapy-Naïve Type 2 Diabetes Patients: A Randomized Placebo Controlled Study.

BACKGROUND: Studies showed effects of Moringa oleifera (MO) on lowering blood sugar levels in animal and diabetes patients. The aims of this study were to determine the effect of MO leaf capsules on glucose control in therapy-naïve type 2 diabetes mellitus (T2DM) and to evaluate its safety. METHOD: This was a prospective randomized placebo controlled study. Therapy-naïve T2DM was randomly assigned to receive either 8 grams per day of MO leaf capsules (MO leaf group) or placebo for 4 weeks. Clinical and laboratory characteristics were recorded at screening and at the end of 4-week study. 9-point plasma glucose was obtained before and every week during the study. RESULTS: Thirty-two T2DM patients were enrolled. The mean age was 55 years and the mean HbA1C was 7.0%. There was no significant difference in FPG and HbA1C between groups. MO leaf group had SBP reduction by 5 mmHg as compared to baseline but this difference had no statistical significance. There were no adverse effects of MO leaf. CONCLUSIONS: Moringa oleifera leaf had no effect on glycemic control and no adverse effects in T2DM. Interestingly, this study demonstrated that MO leaf had a tendency on blood pressure reduction in T2DM, and this result needs further investigation.

Moringa Oleifera Leaf Increases Insulin Secretion after Single Dose Administration: A Preliminary Study in Healthy Subjects.

BACKGROUND: Herbal medicine has long been used as an alternative medicine for treatment of type 2 diabetes mellitus (T2DM). Recently, Moringa oleifera (MO or ma-rum in Thai) leaf has been widely used in T2DM patients. Several studies in diabetes rat model have shown that MO had effect on glucose metabolism. However study in humans is lacking. OBJECTIVE: Examine effects of MO on plasma glucose and insulin secretion. MATERIAL AND METHOD: Ten healthy volunteers were enrolled in this study (mean age 29 ± 5 years; BMI 20.6 ± 1.5 kg/m2; FPG 81 ± 5 mg/dl). After an overnight fast and every two weeks, subjects received an oral dose of MO at increasing dosages of 0, 1, 2, and 4 g. Plasma glucose (PG) and insulin were collected at baseline and at 0.5, 1, 1.5, 2, 4, and 6 hours after each MO dosage administration. Insulin secretion rate was measured using area under the curve (AUC) of insulin and AUC of insulin/glucose ratio. RESULTS: After doses of 0, 1, 2, and 4 g MO, mean plasma insulin increased (2.3 ± 0.9, 2.7 ± 1.0, 3.3 ± 1.4, and 4.1 ± 1.7 µU/ml, respectively) despite there being no differences in mean PG (77 ± 6, 78 ± 5, 79 ± 6, and 79 ± 5 mg/dl, respectively). AUC of insulin was greater after high-dose MO (4 g) than after baseline or low-dose MO capsule (1 g) (24.0 ± 3.5 vs. 14.5 ± 1.8 or 16.1 ± 2.0, respectively; p = 0.03), while there was no difference in AUC of glucose. Accordingly, insulin secretion rate represented by AUC of insulin/glucose ratio after high-dose MO was significantly increased by 74% (P = 0.041), as compared with that of baseline. CONCLUSION: We concluded that high-dose (4 g) MO leaf powder capsules significantly increased insulin secretion in healthy subjects. These results suggest that MO leaf may be a potential agent in the treatment of type 2 diabetes. Further studies of MO in patients with T2DM are needed.

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 µg/ml; 241.2 µM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

The safety of Homnawakod herbal formula containing Aristolochia tagala Cham. in Wistar rats.

BACKGROUND: A dried root of Aristolochia tagala Cham. (ATC) is often used in Thai traditional medicine as an antipyretic, anti-inflammatory agent, muscle relaxant, appetite-enhancing agent, and analeptic. Homnawakod, an important herbal recipe, originally contains ATC in its formula, however, some Aristolochia species have been reported to cause nephrotoxicity due to aristolochic acid (AA) and its derivatives, resulting in ATC removal from all formulae. Therefore, this study investigates the chemical profiles of ATC, the original (HNK+ATC) and the present Homnawakod Ayurved Siriraj Herbal Formulary™ (HNK), and investigates whether they could cause nephrotoxicity or aggravate LPS-induced organ injuries in vivo. METHODS: HPLC and LC/MS were used for chemical profile study. Male Wistar rats were randomly divided into groups in which the rats were intragastrically administered distilled water (2 groups), ATC (10 or 30 mg/kg), HNK+ATC (540 or 1,620 mg/kg), or HNK (1,590 mg/kg) for 21 days. A positive control group was administered with single dose 100 mg/kg standard AA-I intragastrically at day 1. Serum creatinine and urea were measured at baseline and at 7, 14 and 21 days of the treatment. On day 22, a model of lipopolysaccharide (LPS)-induced endotoxemia was used. One-way and two-way analyses of variance were performed and a P value of less than 0.05 was considered to be significant. RESULTS: The similarity of the HPLC chromatograms of HNK+ATC and HNK could suggest that the qualities of both formulae are nearly the same in terms of chemical profile. The amount of AA-I found in ATC is 0.24%w/w. All experimental groups exhibited similar levels of serum urea at baseline and 7 and 14 days of the treatment. At 21 days, rats received AA exhibited a significant increase in serum urea, whereas the others did not exhibit such toxicity. On day 22, there were no significant changes in LPS-induced renal and liver dysfunction, or LPS-induced mean arterial pressure (MAP) reduction upon administration of ATC, HNK+ATC, HNK or AA-I. CONCLUSIONS: These results suggest that ATC, HNK+ATC or HNK, at the animal dose equivalent to that used in human, do not cause the acute nephrotoxicity in rats and do not aggravate LPS-induced organ injuries even further.