Bovine whey protein and other biological fluids as alternative to fetal bovine serum in supplementing cell culture media

Fetal bovine serum (FBS) is still the mainstay as supplement to cell culture media for cell-culture practices. The collection method of FBS is a violent, ethically objectionable and repugnant. Moreover, FBS is cost-prohibitive for biomedical research. Here, we explored easily procurable and less expensive bovine whey protein (BWP) and other putative biological fluids for their growth promoting activity. BWP with minimal amount of FBS effectively supported cell growth in short term (72 h) and long term (21 days) cultures in Chinese hamster ovary and Jurkat E6.1 cells. The combination also protected the cells during cryopreservation and facilitated revival of the cell culture. However, BWP without FBS didn’t support the growth for longer and the cells changed their morphology. Further, BWP was enriched with human platelet lysate and it maintained the cell growth and morphology at a very low concentration.

Comparative study of amrutbhallataka and glucosamine sulphate in osteoarthritis: Six months open label randomized controlled clinical trial.

Background: AmrutBhallatak (ABFN02), a ‘rasayana’ drug from Ayurveda is indicated in degenerative diseases and arthritis. Objective: To evaluate safety and effi cacy of ABFN02 in osteoarthritis (OA) and compare it with Glucosamine sulphate (GS) Materials and Methods: This was a randomized open comparative study. Ambulant OPD patients of OA knees (n = 112) were enrolled for 24 weeks. Tablets (750mg each) of GS and ABFN02 were matched. Three groups of patients: (A) GS, one tablet × twice/day × 24 weeks. (B) ABFN02, incremental pulse dosage (one tablet x twice/day × two weeks, two tablets × twice/day × two weeks, three tablets × twice/day × two weeks), two such cycles of drug and non-drug phases alternately for six weeks each (C) ABFN02 continuous dosage akin to GS. Pain visual analogue score (Pain-VAS) and Western Ontario and Mc-Master University Osteoarthritis Index (WOMAC) were the primary outcome measures. Secondary outcome measures were Health assessment questionnaire (HAQ), paracetamol consumption, 50 feet walking, physician and patient global assessment, knee stiffness, knee status, urinary CTX II, serum TNFa-SRI, SRII and MRI knee in randomly selected patients. Results: ABFNO2 and GS demonstrated, adherence to treatment 87.75% and 74.3%, reduction in Pain-VAS at rest 61.05% and 57.1%, reduction in pain-VAS on activity 57.4% and 59.8%, WOMAC score drop 62.8% and 59.1% respectively. Secondary outcome measures were comparable in all groups. Safety measures were also comparable. No serious adverse events reported. However, asymptomatic reversible rise in liver enzymes was noted in the ABFNO2 group. Conclusions: ABFN02 has signifi cant activity in OA; the formulation needs further investigation.

Shirodhara: A psycho-physiological profi le in healthy volunteers.

Background: Shirodhara is a classical and a well-established ayurvedic procedure of slowly and steadily dripping medicated oil or other liquids on the forehead. This procedure induces a relaxed state of awareness that results in a dynamic psycho-somatic balance. Objectives: The objective of the study is to evaluate the psychological and physiological effects of Shirodhara in healthy volunteers by monitoring the rating of mood and levels of stress, electrocardiogram (ECG), electroencephalogram (EEG), and selected biochemical markers of stress. Materials and Methods: The study was conducted in the human pharmacology laboratory. The study design was open labeled, comparing the baseline variables with values after Shirodhara. The subjects (n = 16) chosen were healthy human volunteers who gave an informed consent. Shirodhara was preceded by Abhyanga – whole body massage. The Shirodhara method was standardized for rate of dripping with peristaltic pump and temperature was controlled with a thermostat. Mood and stress levels were assessed by validated rating scales. The pre- and post-Shirodhara ECG and EEG records were evaluated. Results: Student’s paired “t” test was applied to the means + SE of the variables to calculate statistical signifi cance at P <0.05. There was a signifi cant improvement in mood scores and the level of stress (P <0.001). These changes were accompanied by signifi cant decrease in rate of breathing and reduction in diastolic blood pressure along with reduction in heart rate. The relaxed alert state, after Shirodhara, was co-related with an increase in alfa rhythm in EEG. Conclusion: A standardized Shirodhara leads to a state of alert calmness similar to the relaxation response observed in meditation. The clinical benefi ts observed with Shirodhara in anxiety neurosis, hypertension, and stress aggravation due to chronic degenerative diseases could be mediated through these adaptive physiological effects.

Exploratory study to evaluate tolerability, safety, and activity of Ashwagandha (Withania somnifera) in healthy volunteers.

Ashwagandha (Withania somnifera) (WS), a “rasayana” drug, is recommended for balavardhan and mamsavardhan. The study was intended to evaluate dose-related tolerability, safety, and activity of WS formulation in normal individuals. The design was prospective, open-labeled, variable doses in volunteers. Eighteen apparently healthy volunteers (12M:6F, age:18-30 years, and BMI: 19-30) were enrolled. After baseline investigations, they received WS capsules (Rx) (aqueous extract, 8:1) daily in two divided doses with increase in daily dosage every 10 days for 30 days (750 mg/day x10 days, 1 000 mg/day x 10 days, 1 250 mg/day x 10 days). Volunteers were assessed for symptoms/signs, vital functions, hematological and biochemical organ function tests. Muscle activity was measured by hand grip strength, quadriceps strength, and back extensor force. Exercise tolerance was determined using cycle ergometry. Lean body weight and fat% were computed from skin fold thickness measurement. Adverse events were recorded, as volunteered by the subjects. Repeated measures ANOVA, McNemar’s test, and paired t test were employed. All but one volunteer tolerated WS without any adverse event. One volunteer showed increased appetite, libido, and hallucinogenic effects with vertigo at the lowest dose and was withdrawn from study. In six subjects, improvement in quality of sleep was found. Organ function tests were in normal range before and after the intervention. Reduction in total- and LDL- cholesterol and increase of strength in muscle activity was signifi cant. Total body fat percentage showed a reduction trend. WS, in escalated dose, was tolerated well. The formulation appeared safe and strengthened muscle activity. In view of its traditional Rasayana use, further studies are planned to evaluate potential of this drug in patients of sarcopenia.

A study of standardized extracts of Picrorhiza kurroa Royle ex Benth in experimental nonalcoholic fatty liver disease.

As a major organ of intermediary metabolism, the liver is exposed to a variety of metabolic insults due to diseases and xenobiotics viz., insulin resistance (IR) drugs, toxins, microbial products, etc. One of the consequences of these metabolic insults including obesity and type 2 diabetes mellitus is the development of non-alcoholic fatty liver disease (NAFLD). The recent alarming increase in the prevalence of NAFLD compels the need to develop an appropriate animal model of the disease so as to evolve effective interventions. In this study, we have developed, in the rat, a new model of NAFLD showing several key features akin to the disease in humans. Male Wistar rats were challenged with 30% high fat diet (HFD) – butter, for 2 weeks to induce NAFLD. A hydroalcoholic extract of Picrorhiza kurroa was administered to study the possible reversal of fatty changes in the liver. The extract was given in two doses viz., 200mg/kg and 400 mg/kg b.i.d., p.o. for a period of 4 weeks. There were three control groups (n = 6/group) – vehicle with a regular diet, vehicle with HFD, and HFD with silymarin – a known hepatoprotective. Histopathology showed that the P. kurroa extract brought about a reversal of the fatty infiltration of the liver (mg/g) and a lowering of the quantity of hepatic lipids (mg/g) compared to that in the HFD control group (38.33 ± 5.35 for 200mg/ kg; 29.44 ± 8.49 for 400mg/kg of P. kurroa vs.130.07 ± 6.36mg/g of liver tissue in the HFD control group; P<0.001). Compared to the standard dose of the known hepatoprotective silymarin, P. kurroa reduced the lipid content (mg/g) of the liver more significantly at the dose of 400mg/kg (57.71 ± 12.45mg/kg vs. 29.44 ± 8.49 for the silymarin group vs. 400mg/kg of P. kurroa, P<0.001). In view of the increasing prevalence of metabolic syndrome and NAFLD, P. kurroa should be investigated by the reverse pharmacology path as a potential drug for the treatment of NAFLD, and essential safety studies and preformulation research for concentration of the putative actives should be carried out.

Natural products drug discovery: Accelerating the clinical candidate development using reverse pharmacology approaches.

The pharmaceutical industry is facing serious challenges as the drug discovery process is becoming extremely expensive, riskier and critically inefficient. A significant shift from single to multi targeted drugs especially for polygenic syndromes is being witnessed. Strategic options based on natural product drug discovery, ethnopharmacology and traditional medicines are re-emerging to offer good base as an attractive discovery engine. Approaches based on reverse pharmacology may offer efficient development platforms for herbal formulations. Relevant case studies from India and other countries where such approaches have expedited the drug discovery and development process by reducing time and economizing investments with better safety are discussed.

Early human safety study of turmeric oil (Curcuma longa oil) administered orally in healthy volunteers.

OBJECTIVE: Turmeric extract and turmeric oil have shown chemoprotective effect against chemically-induced malignancies in experimental animals. They can reverse precancerous changes in oral submucous fibrosis in humans. The use of turmeric or Curcuma longa Linn as a spice and household remedy has been known to be safe for centuries. In view of the long term administration required for cancer prevention a Phase I clinical trial of turmeric oil (TO) was designed to study the safety and tolerance of TO in volunteers for a period of 3 months. MATERIAL AND METHODS: Nine healthy volunteers between 20 and 33 years of age were tested for haemoglobin, blood counts, liver and kidney functions, bleeding and clotting time and serum electrolytes initially and at 1 and 3 months of treatment. They were administered 0.6 ml of TO three times a day for 1 month and 1 ml in 3 divided doses for 2 months. The acute tolerability study on Day 1 was conducted in a Clinical Pharmacology daycare Unit. Blood pressure and pulse were recorded frequently on Day 1 and at 24, 48, 72 and 96 hours and fortnightly till 12 weeks. Volunteers were daily supervised for TO intake as well as for any side effects throughout the study period. RESULTS: Nine volunteers were enrolled for the study. One discontinued on 3rd day for allergic skin rashes which, on discontinuation of TO, gradually disappeared by two weeks. Another discontinued on 7th day for intercurrent fever requiring antibiotic treatment. Seven volunteers completed the study. There was no effect of TO, in two doses, on pulse and blood pressure and no side effects in acute tolerability study on Day 1. There was no effect of TO intake on weight, blood pressure, symptoms and signs upto 12 weeks. There was no clinical, haematological, renal or hepatic-toxicity of TO at 1 month and 3 months. Serum lipids did not show significant change except in one volunteer (reversible). CONCLUSIONS: In view of the potential for reversing oral submucous fibrosis, a precancerous condition for oral cancer, TO, can be recommended directly for a Phase II trial in patients.