Tropical Journal of Pharmaceutical Research

Self-nanoemulsifying drug delivery system (SNEDDS) prepared from Begonia medicinalis and Moringa oleifera ethanol extracts

Muhammad Zubair — 2025-02-11

Purpose: To investigate the activity of self-nanoemulsifying drug delivery system (SNEDDS) containing B. medicinalis and M. oleifera ethanolic extracts. Method: The SNEDDS was prepared using propylene glycol (40 %), Tween 80 (50 %), and isopropyl myristate (10 %). Transmittance percentage, particle size, polydispersity index, and zeta potential were evaluated. In vitro immunostimulant activity was measured by assessing the macrophages' phagocytic capacity and proliferation of lymphocyte cells using MTT assay. In vivo immunostimulant activity was determined by measuring the number (%) of leukocytes, neutrophils, and lymphocytes in Wistar male rats over 14 days following administration of the SNEDDS after induction with Staphylococcus aureus. Results: The SNEDDS formula of combined extract of B. medicinalis and M. oleifera demonstrated polydispersity index (0.46 ± 0.00), particle size (24.4 ± 0.1 nm), zeta potential (-37.73 ± 0.32 mV) and percentage transmittance (86.81 ± 0.16 %). In vitro and in vivo tests revealed that SNEDDS significantly increased macrophage phagocytic capacity, number of leukocytes, neutrophils and lymphocytes over 14 days of treatment (p < 0.05) following induction with S. aureus. Conclusion: Self-nanoemulsifying drug delivery system of combined ethanol extract of B. medicinalis and M. oleifera demonstrates stable physicochemical properties, and increases macrophage phagocytic activity, leukocytes, neutrophils and lymphocyte count.

Influence of DMSO on gene expression in bovine embryos: Exploring solvent-specific effect on substance dissolution in fertilization medium

Ramzi Amran — 2025-02-11

Purpose: To investigate the role of dimethyl sulfoxide (DMSO) as a cryoprotectant and solvent in bovine embryogenesis, with a particular focus on its impact on gene expression during early and late developmental stages. Method: Bovine embryos were used to evaluate effects of DMSO on expression of genes critical to apoptosis and developmental processes. Gene expression analysis was performed to assess changes in both pro-apoptotic and anti-apoptotic markers, as well as genes essential for growth and survival. Results: Dimethyl sulfoxide influenced gene expression in a stage-specific manner. During early development, DMSO induced overexpression of the pro-apoptotic gene, BAX, and downregulated the anti-apoptotic gene, BCL2, indicating increased apoptotic activity. Furthermore, the expression of GDF9 and IGF1, which are crucial for growth and survival, was altered, suggesting interference with key developmental pathways. In contrast, late-stage embryos exhibited elevated levels of BCL2 and HSPB1, markers of anti-apoptotic activity, indicating a more complex regulatory role of DMSO at advanced stages of embryogenesis. Conclusion: While DMSO is effective as a cryoprotectant, its impact on gene expression raises concerns about potential developmental consequences. These findings highlight the need for further investigation to better understand the specific effects of DMSO in the context of assisted reproductive technologies (ART).

Evaluation of the California mastitis test, pH and milk colour as indicators for subclinical mastitis detection in local dairy cows

Ramzi Amran — 2025-02-11

Purpose: To evaluate the utilization of diagnostic tools such as the California Mastitis Test (CMT) and milk pH and colour assessments for detecting subclinical mastitis (SCM). Method: A total of 60 milk samples were collected from 60 apparently healthy local dairy cows in Dhamar City, Yemen, to investigate the incidence of SCM. The samples were subjected to California Mastitis Test (CMT) as well as tests for pH and colour. Results: According to CMT results, 45 % of the cows had SCM. The rate of prevalence in (+), (++) and (+++) CMT results was 17 (28 %), 7 (12 %) and 3 (5 %), respectively. In terms of milk pH, 27 (45 %) of the samples had a pH between 6.6 and 6.8, indicating no signs of SCM. However, 20 (33 %) of the samples exhibited a pH < 6.6, reflecting increased acidity, while 13 (22 %) had a pH > 6.8, indicating alkaline milk, a marker of severe inflammation and tissue damage. The assessment of milk colour showed that 34 (57 %) of the samples had normal white milk, indicating no mastitis, while 17 (28 %) were yellow, suggesting mild to moderate inflammation or infection. Finally, 9 (15 %) of the samples were pink, indicating the presence of blood and severe inflammation with udder damage. Conclusion: These findings underscore the reliability of CMT, pH and milk colour assessments as effective diagnostic tools for detecting SCM in dairy cows, thus providing valuable insights for early intervention and management strategies.

Synergistic anticancer activity of Juniperus indica Bertol extract plus 5-fluorouracil against oral squamous cell carcinoma in vitro

Ju-Huei Chien — 2025-02-11

Purpose: To investigate the potential effect and underlying mechanism of Juniperus indica Bertol extract (JIB) in combination with 5-fluorouracil (5-FU) for the treatment of oral squamous cell carcinoma (OSCC). Methods: The OECM-1, and OSCC cells were exposed to JIB and/or 5-FU for 24–72 h, and subsequently, cell viability, cell cycle distribution, caspase activity, apoptotic cells, and protein expression were determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide; MTT) assay. Flow cytometry, fluorometric caspase activity assay kit, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and western blotting were carried out. Results: The combination of JIB and 5-FU significantly inhibited the growth of OECM-1 cells compared to JIB or 5-FU alone (p < 0.05). After treatment with JIB plus 5-FU, the cell cycle was significantly impeded by downregulating levels of CKD2/cyclin A and CDK4/cyclin D1 (p < 0.05). Furthermore, the combination treatment triggered apoptosis through activation of caspases and the regulation of Bax/caspase 9/caspase 3. In particular, JIB significantly suppressed the regrowth of 5-FU-treated cells and diminished the development of 5-FU resistance during OSCC treatment (p < 0.05) Conclusion: Juniperus indica Bertol extract (JIB) in combination with 5-FU synergistically inhibits cell proliferation, alters cell cycle distribution, and triggers apoptosis compared to single drug. There is need to further explore the potential use of JIB-based combination therapies to improve the clinical application of 5-FU in OSCC.

Anti-obesity potential of Porelis, a standardized extract of purple tea (Camellia sinensis), via modulation of obesity-related inflammation in RAW264.7 macrophages and anti-adipogenesis in 3T3-L1 cells

Kuluvar Gouthamchandra — 2025-02-11

Purpose: To assess the effect of Porelis, a standardized extract of purple tea (Camellia sinensis) on inflammation in RAW 264.7 cells, anti-adipogenesis and lipid accumulation in 3T3-L1 cells. Methods: Viability of RAW 264.7 and 3T3L1 cells was determined by MTT assay. Lipopolysaccharide (LPS) was used to induce inflammation in RAW264.7 cells. Nitric oxide (NO) generation and levels of anti-inflammatory cytokines (TNF-α and IL-6) were determined using Gries reagent and ELISA, respectively. In addition, Oil-O red staining was carried out to assess the differentiation of 3T3L cells. Furthermore, Western blotting was used to quantify the protein expressions of PPAR- γ, C/EBP and SREBP-1 while RT-PCR was used to determine leptin and FAS mRNA expression. Results: Porelis comprised 3 – 5 % of 1,2-di-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-β-D-glucose (GHG). Its use significantly inhibited the generation of NO, IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells (p < 0.0001). Interestingly, there was a significantly inhibited adipocyte differentiation, fat accumulation and protein expression of PPARγ and CEBPα in a dose-dependent manner (p < 0.0001). The mRNA levels of leptin and FAS were significantly (p < 0.0001) upregulated in differentiated adipocytes compared to preadipocytes. Furthermore, treatment with Porelis significantly inhibited mRNA expression levels of leptin and FAS. Conclusion: Porelis may exert anti-inflammatory activity by inhibiting inflammatory responses in RAW264.7 macrophages as well as anti‑obesity activity by blocking fat accumulation via inhibition of adipogenesis in 3T3L adipocytes. This study shows that Porelis could be a promising dietary supplement for prevention of inflammation and obesity.

Efficacy of atorvastatin in the management of atherosclerotic cardiovascular disease (ASCVD)

Cuijun Hao — 2025-02-11

Purpose: To investigate the efficacy of atorvastatin in the treatment of atherosclerotic cardiovascular disease (ASCVD) Methods: A total of 158 patients who underwent coronary angiography in the Department of Cardiology, First Affiliated Hospital of Hebei North University, China were divided into acute myocardial infarction (AMI, n = 42), unstable angina pectoris (UAP, n = 70), and control (n = 46) groups. Atorvastatin (40 mg) was administered to the patients in AMI and UAP groups for 40 days. Triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, phospholipase A2 (PLA2), YKL40, and clinical efficacy were evaluated after treatment. Results: Acute myocardial infarction group (AMI) had significantly higher TC, LDL-C, non-HDL-C, PLA2, and YKL40 levels compared to UAP and control (p < 0.05). Also, HDL-C, TG, and TG/HDL-C were significantly higher in AMI and UAP compared to control group (p < 0.05). In coronary heart disease, TG/HDL-C had the highest sensitivity, and LDL-C had the highest specificity. Furthermore, TC, LDL-C, and TG significantly decreased in both AMI and UAP groups (p < 0.05), while HDL-C remained unchanged (p > 0.05). Conclusion: Atorvastatin effectively improves lipid profiles in ASCVD patients, and markers such as PLA2 and YKL40 effectively predict ASCVD risk. Further larger-scale, long-term studies are needed to validate the effectiveness of combining lipid and inflammatory biomarkers for ASCVD risk prediction and management.

Synergistic effect of Allium sativum and gliclazide on pharmacokinetic parameters in rabbit models

Nimmathota Madhavi — 2025-02-11

Purpose: To assess gliclazide hypoglycemic effect in the presence of Allium sativum and determine the possible pharmacokinetics interactions of gliclazide and Allium sativa extract. Methods: A set of 6 Wistar rabbits weighing between 1.35 kg - 1.75 kg was used. Gliclazide (3.7 mg/kg) was administered orally. After a washout period of 1 week, the same group of animals received Allium sativum extract (56 mg/kg) with the required quantity of water. After a further washout period of 1 week, the same group received Allium sativum (56 mg/kg) 30 min prior to the administration of gliclazide (3.7 mg/kg). Blood samples were withdrawn at 0, 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24 h and were analyzed for blood glucose using the glucose oxidase (GO)/peroxidase (PO) method. The mean significance of the pharmacokinetic parameters such as area under the curve (AUC)0–24, biological half-life (t1/2), maximum peak plasma concentration (Cmax), maximum time to reach Cmax (Tmax), and elimination rate constant (KE) of gliclazide was estimated with and without Allium sativum in rabbits. Results: Allium sativum significantly increased AUC0-24, AUC0-a and AUMC0-24 and Cmax of gliclazide (p < 0.05). The results revealed that Allium sativum significantly enhanced the hypoglycemic effect of gliclazide (p < 0.05). Conclusion: Allium sativum significantly enhances the hypoglycemic effect of gliclazide by improving pharmacokinetic parameters.

Therapeutic potential of methanol extracts of Calocybe indica (mushroom) on cadmium chloride-induced hepato-renal toxicity in rats

Ebuka Onyeyilim — 2025-02-11

Purpose: To investigate the efficacy of Calocybe indica extract (CIE) in alleviating liver and kidney toxicity caused by cadmium chloride (CdCl₂) in rats. Methods: Six groups of five rats each were used in this study. Group A was the control while groups B to F received 3 mg/kg CdCl2 subcutaneously. Group B was induced with CdCl2 alone for 21 days. Orally, 100 mg/kg of vitamin C, and 200, 400, and 800 mg/kg of CIE were used to treat groups C, D, E, and F respectively. Data were analyzed using Statistical Packages for Social Sciences (SPSS), and results were presented as mean ± standard deviation (SD). Results: Group B had higher liver and kidney weights, and lower body weight compared to control group (p ≥ 0.05). Treatment with CIE increased body weight in CdCl2-induced rats lowers serum levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and total bilirubin, improves liver and kidney function, and significantly increased superoxide dismutase (SOD) activity (p < 0.05). Conclusion: Calocybe indica (CIE) extract increases body weight, lowers serum levels of liver enzymes, improves kidney function and significantly lowers SOD activity. Calocybe indica extract may serve as a potential pharmacological candidate or therapeutic alternative for managing hepato-renal injuries. Subsequent molecular studies to ascertain its bioactive compounds will pave the way for the discovery of drug candidates.

Alleviation of cognitive impairments in ischemic stroke rat treated with baicalein-enriched fraction via JAKMIP1/GABRA6 pathway

Farah Othman — 2025-02-11

Purpose: To investigate the therapeutic potential of baicalein-enriched fraction (BEF) extracted from Oroxylum indicum leaves on ischemic stroke in rat models induced with endothelin-1 (ET-1). Methods: A total of 10 male Sprague Dawley rats were randomly assigned to the study (n = 5) and control (n = 5) groups. Study group received 50 mg/kg BEF treatment via oral gavage administration for 4 days prior to the induction of ischemic stroke using ET-1, while control group (n = 5) received normal saline. Neurological deficits, total infarct volume, histological scoring and expression of targeted genes were evaluated. Results: The study group exhibited significantly improved motor function test, motor coordination and balance test, motor asymmetry test, and contralateral sensory test (p < 0.05) compared to control group. However, the reflex test was not significantly different between the study and control groups (p > 0.05). Furthermore, infarct volume, infarction area, and relative mRNA GABRA 6 expression were significantly lower in study group compared to control group while the relative mRNA expression of JAKMIP 1 was significantly higher (p < 0.05). Conclusion: Baicalein-enriched fraction (BEF) extracted from O. indicum leaves improves motor coordination, lowers infarction parameters and therefore, may be a potential therapeutic agent for future clinical approaches to treat ischemic stroke disease.

Anxiety-like behavior associated with alcohol withdrawal syndrome in mice and possible antagonistic effect of Polycephalomyces nipponicus aqueous extract

Sirinapa Rungruang — 2025-02-11

Purpose: To investigate the effect of Polycephalomyces nipponicus (P. nipponicus) extract on ethanol withdrawal syndrome in mice. Methods: Male Institute of Cancer Research (ICR) mice were divided into 3 groups of 10 animals per group. Mice were intraperitoneally injected with ethanol (2 g/kg/day) or normal saline solution 0.9 % (0.05 mL/kg) as control for 10 consecutive days. Anxiety-like behavior associated with alcohol withdrawal syndroms (AWS) was assessed at 12, 24, and 36 h after the last dose of ethanol or normal saline using the light-dark box, open field, and elevated plus maze tests. Thereafter, the time that showed the most prominent AWS was chosen to determine the effect of P. nipponicus extract. P. nipponicus extract (600 mg/kg, orally) or diazepam (4 mg/kg, i.p., as a positive control), was administered 1 h before the tests. Results: Polycephalomyces nipponicus extract significantly mitigated anxiety-like behavior in alcohol-withdrawn mice across all evaluated models (p < 0.05). The results suggest that P. nipponicus reduced alcohol withdrawal syndrome, especially anxiety-like behavior. Conclusion: This study provides evidence that P. nipponicus may be useful in the treatment of alcohol withdrawal syndrome, especially anxiety-like behavior. This study shows the potential of P. nipponicus as a new intervention for alleviating neurochemical imbalances linked to alcohol withdrawal.

In vivo investigation of Roselle flower (Hibiscus sabdariffa L.) and Bee pollen (Tetragonula laeviceps) on carbon tetrachloride-induced spermatozoa damage

Ni Watiniasih — 2025-02-11

Purpose: To determine the effect of roselle flower and bee pollen on carbon tetrachloride (CCl4)-induced spermatozoa damage. Methods: Roselle flowers (Hibiscus sabdariffa L.) and bee pollen (Tetragonula laeviceps) were obtained from Bali, Indonesia. A total of 24 BALB/c (Mus musculus) mice were used and randomly assigned into control, negative control (CCl4-induced), positive control (CCl4 + ascorbic acid) and treatment groups (25, 50, and 100 mg/20 g). Spermatozoa-induced damage was done using carbon tetrachloride (CCl4). Sperm levels, movement, viability, and morphology of the spermatozoa were evaluated. Results: Roselle and bee pollen granules at 50 mg/20 g (11.2 mg of roselle extract and 28 mg of pollen extract), demonstrated the most significant improvement in spermatozoa levels, movement, viability, and morphological characteristics compared to negative controls (p < 0.05). Conclusion: Roselle and bee pollen granular combination offers a promising natural intervention for mitigating chemically induced reproductive toxicity. It suggests potential therapeutic applications in preserving male reproductive health and counteracting oxidative stress-related spermatogenic damage.

Isolation and characterization of terrestrial Streptomyces strains with antimicrobial and anti-ulcer activities

Martin-Luther Okolo — 2025-02-11

Purpose: To determine the antimicrobial, anti-ulcer and cytotoxic activities of secondary metabolites isolated from Streptomyces spp in soil samples obtained from Anyigba, Kogi State, Nigeria. Methods: The cytotoxic activity of Streptomyces secondary metabolites (concentration range: 62.5 – 1000 mg/mL) was assessed using brine shrimp lethality assay on different concentrations (62.5, 125, 250, 500, 1000 mg/mL) of the secondary metabolites or the reference K2Cr2O7. The anti-ulcer activity was evaluated using aspirin and ethanol models of ulcers in albino rats. Five groups of animals viz three pretreated groups with the extracts at two different oral concentrations of 100 and 200 mg/kg, one pretreated group with omeprazole (30 mg/kg) as standard and another group with oral 2 mL/kg normal saline (control). Antibacterial and antifungal activities were investigated using the microdilution method. Results: Cytotoxicity test showed that extract concentrations of 12.5 to 62.5 mg/mL exerted mild toxicity compared to control. For antiulcer activity, animals in group 1 showed mucosal infiltration of leucocytes, massive exfoliation of epithelial cells and cellular hemorrhage while group 2 had mild tissue erosion and small ulcer. In groups 3 to 5, extract at 200 mg/kg showed excellent cytoprotection and proficient curative capacity in contrast to the 100 mg/kg, without significant side effects while the standard group showed some side effects and a significant reduction in the mucus cells (p < 0.05). The secondary metabolites inhibited ulcer-implicated organisms (Helicobacter pylori, Escherichia coli, Campylobacter spp and Streptococcus spp), exerted curative effect and protected the rats from ulcers. Conclusion: Secondary metabolites isolated from Streptomyces spp were not toxic to epithelial tissue and exhibit antimicrobial and anti-ulcer activities and thus has potential as an important source of drugs against peptic ulcers.

Recovery of phenolic-rich extracts from Handeuleum leaves using mixture design modeling

Feda Makiyyah — 2025-02-11

which are rich in phenolic compounds. Methods: In optimizing the extraction solvent, the simplex centroid mixture design was used together with the extractor components, namely water, acetone, methanol, and ethanol, which were designed to produce 15 formulas. Total phenolic analysis was carried out based on the Follin-Ciocalteu method, and the results were expressed in milligrams of gallic acid equivalent per gram of dry weight of the sample. Results: The quadratic model with an R2 value of 0.8407 is relied upon for total phenolic recovery. The best-mixed formula yielded a desirability value of 0.913 with a quarternary mixture of water (0.6910), acetone (0.3054), methanol (0.0002), and ethanol (0.0031) as solvents, obtaining a total phenolic content of 2.9613 mg GAE/g dry weight. The optimal condition validation results are also stated to be very accurate, with a residual standard error (RSE) of 0.905 %. Conclusion: The optimized solvent combination for extracting high amounts of phenolic compounds from Handeuleum leaves has been reported which may be upscaled for commercial purposes.

A questionnaire-based study to assess the knowledge, attitude, and practices of antimicrobial resistance among PharmD students

Bhupalam Kumar — 2025-02-11

Purpose: To evaluate the knowledge, attitude, and practices (KAP) regarding antibiotic use and antimicrobial resistance (AMR) among Doctor of Pharmacy (PharmD) students in selected institutions in India. Methods: A cross-sectional study using a structured questionnaire was conducted among 292 fourth-, fifth-, and sixth-year PharmD students across various institutions. Results: The findings revealed adequate knowledge (mean score 5.87 ± 1.34) and a positive attitude (mean score 7.05 ± 1.20) among participants, but practices were notably poor (mean score 4.08 ± 1.24). Conclusion: These results highlight the need for enhanced educational interventions and practical training for PharmD students to improve antibiotic stewardship and patient care outcomes.

A case-control study on factors associated with hypoglycemia unawareness among the ambulatory type 2 diabetes mellitus patients in Bali, Indonesia

Made Jaya — 2025-02-11

Purpose: To determine the factors related to hypoglycemia unawareness (HU) in patients with type 2 diabetes mellitus (T2DM) in Bali-Indonesia. Methods: A case-control study was conducted across three hospitals to investigate the incidence of HU as a primary outcome. Medical record data were collected to obtain information for further analysis. The primary data were analyzed using chi-square analysis, and variables with p-value ≤ 0.10 were further evaluated using multivariate logistic regression, with odds ratio (OR) parameters at 95 % confidence interval (CI). A two-tailed statistical analysis was then conducted, and p < 0.05 was considered statistically significant. Results: Results of the multivariate analysis showed that five variables were significantly associated with HU incidents. These included insulin users (OR: 6.15 (CI 95: 1.65 - 22.86)), chronic kidney diseases (CKD) (OR: 6.56 (CI 95: 1.41 - 30.39)), diabetic neuropathy (OR: 24.61 (CI 95: 5.17 - 117.11)), hypertension (OR: 3.76 (CI 95: 1.01 - 13.96)), and dyslipidemia (OR: 6.44 (CI 95: 1.62 - 25.71)). Conclusion: Variables in this study are in line with the characteristics of the ambulatory T2DM population in Bali-Indonesia. These factors are used as evidence by health workers in managing and mitigating the risk of HU, thereby reducing the associated health burden.

Assessment of community pharmacists’ knowledge, attitudes and practices regarding garlic and ginger as herbal medicines

Elijah Mohammed — 2025-02-11

Purpose: To assess the knowledge, attitudes and practices of community pharmacists regarding the use of garlic and ginger as herbal medicines. Methods: This was a cross-sectional study among community pharmacists practicing in various urban, semi-urban and rural communities in Federal Capital Territory, Abuja. Semi-structured questionnaires containing both open-ended and closed-ended questions in electronic Google Forms® were used to assess the respondents’ knowledge, attitudes and practices regarding the use of garlic and ginger as herbal medicines. Data were analyzed using Microsoft Excel® 2016. Results: Community pharmacists stocked and dispensed garlic and ginger as herbal medicines in their pharmacy and have positive attitude/disposition towards the use and practice of complementary and alternative medicines (CAM). They exhibited average knowledge of garlic and ginger as herbal medicines, though knowledge of their interactions with conventional medicines was poor. Few belonged to a networking and multi-level marketing company that specializes in herbal medicines and supplements. Conclusion: Community pharmacists demonstrate fair knowledge about garlic and ginger as herbal remedies. Though knowledge of herb-drug interactions was poor, community pharmacists need to be sufficiently knowledgeable about herbal products to better advise patients who are seeking or using alternative remedies. Purpose: To assess the knowledge, attitudes and practices of community pharmacists regarding the
use of garlic and ginger as herbal medicines.
Methods: This was a cross-sectional study among community pharmacists practicing in various urban,
semi-urban and rural communities in Federal Capital Territory, Abuja. Semi-structured questionnaires
containing both open-ended and closed-ended questions in electronic Google Forms® were used to
assess the respondents’ knowledge, attitudes and practices regarding the use of garlic and ginger as
herbal medicines. Data were analyzed using Microsoft Excel® 2016.
Results: Community pharmacists stocked and dispensed garlic and ginger as herbal medicines in their
pharmacy and have positive attitude/disposition towards the use and practice of complementary and
alternative medicines. They exhibited average knowledge of garlic and ginger as herbal medicines,
though knowledge of their interactions with conventional medicines was poor. Few belonged to a
networking and multi-level marketing company that specializes in herbal medicines and supplements.
Conclusion: Community pharmacists demonstrate fair knowledge about garlic and ginger as herbal
remedies. Though knowledge of herb-drug interactions was poor, community pharmacists need to be
sufficiently knowledgeable about herbal products to better advise patients who are seeking or using
alternative remedies.