Modulation of programmed cell death by medicinal plants.

Programmed cell death (apoptosis), a form of cell death, described by Kerr and Wyllie some 20 years ago, has generated considerable interest in recent years. The mechanisms by which this mode of cell death (seen both in animal and plant cells), takes place have been examined in detail. Extracellular signals and intracellular events have been elaborated. Of interest to the clinician, is the concentrated effort to study pharmacological modulation of programmed cell death. The attempt to influence the natural phenomenon of programmed cell death stems from the fact that it is reduced (like in cancer) or increased (like in neurodegenerative diseases) in several clinical situations. Thus, chemicals that can modify programmed cell death are likely to be potentially useful drugs. From foxglove, which gave digitalis to the Pacific Yew from which came taxol, plants have been a source of research material for useful drugs. Recently, a variety of plant extracts have been investigated for their ability to influence the apoptotic process. This article discusses some of the interesting data. The ability of plants to influence programmed cell death in cancerous cells in an attempt to arrest their proliferation has been the topic of much research. Various cell-lines like HL60, human hepatocellular carcinoma cell line (KIM-1), a cholangiocarcinoma cell-line (KMC-1), B-cell hybridomas, U937 a monocytic cell-line, HeLa cells, human lymphoid leukemia (MOLT-4B) cells and K562 cells have been studied. The agents found to induce programmed cell death (measured either morphologically or flow cytometrically) included extracts of plants like mistletoe and Semicarpus anacardium. Isolated compounds like bryonolic acid (from Trichosanthes kirilowii var. Japonica, crocin (from saffron) and allicin (from Allium sativum) have also been found to induce programmed cell death and therefore arrest proliferation. Even Chinese herbal medicine "Sho-saiko-to" induces programmed cell death in selected cancerous cell lines. Of considerable interest is the finding that Panax ginseng prevents irradiation-induced programmed cell death in hair follicles, suggesting important therapeutic implications. Nutraceuticals (dietary plants) like soya bean, garlic, ginger, green tea, etc. which have been suggested, in epidemiological studies, to reduce the incidence of cancer may do so by inducing programmed cell death. Soy bean extracts have been shown to prevent development of diseases like polycystic kidneys, while Artemisia asiatica attenuates cerulein-induced pancreatitis in rats. Interestingly enough, a number of food items as well as herbal medicines have been reported to produce toxic effects by inducing programmed cell death. For example, programmed cell death in isolated rat hepatocytes has been implicated in the hepatitis induced by a herbal medicine containing diterpinoids from germander. Other studies suggest that rapid progression of the betel- and tobacco-related oral squamous cell carcinomas may be associated with a simultaneous involvement of p53 and c-myc leading to inhibition of programmed cell death. Several mechanisms have been identified to underlie the modulation of programmed cell death by plants including endonuclease activation, induction of p53, activation of caspase 3 protease via a Bcl-2-insensitive pathway, potentiate free-radical formation and accumulation of sphinganine. Programmed cell death is a highly conserved mechanism of self-defense, also found to occur in plants. Hence, it is natural to assume that chemicals must exist in them to regulate programmed cell death in them. Thus, plants are likely to prove to be important sources of agents that will modulate programmed cell death.

Adaptogenic properties of six rasayana herbs used in Ayurvedic medicine.

Plants from all over the world such as Eleutherococcus senticosus, Panax ginseng, Raponticum carthamoides, Rhodiola rosea, Withania somnifera and Ocimum sanctum have been extensively evaluated for their adaptogenic potential. However, none of them has been successfully introduced as an adaptogen in the clinic. This paper discusses some of the problems in evaluation of adaptogens which have precluded their inclusion as clinically useful drugs. We further discuss our results with six rasayana plants from Ayurveda, which were studied for their adaptogenic potential. The whole, aqueous, standardized extracts of selected plants (Tinospora cordifolia, Asparagus racemosus, Emblica officinalis, Withania somnifera, Piper longum and Terminalia chebula) were administered orally to experimental animals, in a dose extrapolated from the human dose, following which they were exposed to a variety of biological, physical and chemical stressors. These plants were found to offer protection against these stressors, as judged by using markers of stress responses and objective parameters for stress manifestations. Using a model of cisplatin induced alterations in gastrointestinal motility, the ability of these plants to exert a normalizing effect, irrespective of direction of pathological change was tested. All the plants reversed the effects of cisplatin on gastric emptying, while Tinospora cordifolia and Asparagus racemosus also normalized cisplatin induced intestinal hypermotility. Tinospora cordifolia was also tested for its ability to modulate the changes occurring in the phagocytic activity of peritoneal macrophages after exposure of rats to either carbon tetrachloride or horse serum. It was found to normalize the phagocytic function irrespective to the direction of change, complying to the definition of an adaptogen. All the plant drugs were found to be safe in both acute and subacute toxicity studies. Studies on the mechanisms of action of the plants revealed that they all produced immunostimulation. The protection offered by Tinospora cordifolia against stress induced gastric mucosal damage was lost if macrophage activity was blocked. Emblica officinalis strengthened the defence mechanisms against free radical damage induced during stress. The effect of Emblica officinalis appeared to depend on the ability of target tissues to synthesize prostaglandins. Recent data obtained with Tinospora cordifolia suggest that it may induce genotypic adaptation, further opening the arena for more research and experimentation.

The Mexican poppy poisons the Indian mustard facts and figures.

Argemone seeds are mixed with mustard seeds either accidentally or purposefully, and, ingestion of this contaminated oil can lead to often fatal "epidemic dropsy". The liver, heart, kidney and lungs are the major target organs of the toxins (the alkaloids, sanguinarine and dihydrosanguinarine) and damage is mostly caused by free radical (singlet oxygen and hydroxyl radical) to the cell membranes. Treatment at present is mainly symptomatic but therapy with anti-secretory agents for glaucoma and anti-oxidants/free radical scavengers for systemic manifestations appear to be logical.

Leech therapy for complicated varicose veins.

Several methods of limb bandaging have been described to reduce the oedema and enhance ulcer healing in complicated varicose veins, with varying success rates. Leech therapy has never before been tried for the same. We evaluated the effectiveness of medicinal leech therapy in producing venous decongestion, reversal of oedema, hyperpigmentation and healing of varicose ulcer(s). Whether the leech selectively sucks venous blood was also investigated. Hirudo medicinalis (medicinal leech) was applied to the area surrounding the varicose ulcer(s) in 20 patients with varicose veins with complications and the patients monitored for ulcer healing, and decrease in hyperpigmentation, oedema and limb girth. The partial pressure of O2 (pO2) of 7 patients' arterial and venous blood was compared to that sucked by the leech. After leech therapy all the ulcers showed healing, while 95 per cent of patients showed a decrease in oedema and limb girth. Seventy five per cent patients demonstrated a decrease in hyperpigmentation. The mean pO2 of blood sucked by the leech was 40.05 +/- 7.24 mmHg, which was similar to the mean pO2 of the patients' venous blood (34.33 +/- 8.4 mmHg). Thus it appears from this study that the medicinal leech sucks venous blood and aids ulcer healing, and can probably therefore be used as an effective adjunct in the management of complicated varicose veins. This however requires further evaluation by controlled trials.

Effect of prazosin GITS, atenolol, nifedipine SR, and enalapril on ADP-induced platelet aggregation.

A randomized, observer-blind, parallel-group study was carried out to compare the effect of prazosin GITS, atenolol, nifedipine SR, and enalapril on platelet aggregation, measured at a time expected to coincide with trough plasma levels of these drugs. 24 patients (age-30 to 60 yrs) with uncomplicated mild to moderate hypertension who completed a placebo run-in phase successfully were recruited in this study. They were randomly allocated to one of the 4 treatments: prazosin GITS 2.5 mg OD (Group 1), atenolol 50 mg OD (Group II), nifedipine SR 20 mg BD (Group III), and enalapril 5 mg OD (Group IV). All the drugs were given for 7 days, and blood samples were collected at 0 hr on day 1 (pre-treatment) and day 8 (post-treatment). Based on the dose (incremental concentrations of ADP)--response (% maximum aggregation) curve obtained, 2.5 microM/L of ADP was used to compare % inhibition of platelet aggregation among the 4 groups. We found that prazosin GITS inhibited % maximum aggregation significantly (p = 0.02) at 2.5 microM/L of ADP. Such inhibitory effect was not seen in any of the other groups. The inhibition produced by prazosin GITS differed significantly from the action of the other 3 drugs (p < 0.05). This antiplatelet effect of prazosin GITS bears more clinical relevance in view of the fact that it was seen at a time which is expected to coincide with the trough plasma levels of prazosin.

Apoptosis: clinical relevance and pharmacological manipulation.

Apoptosis, often synonymously used with the term 'programmed cell death', is an active, genetically controlled process that removes unwanted or damaged cells. Suppression, overexpression or mutation of a number of genes which orchestrate the apoptotic process are associated with disease. The diseases in which apoptosis has been implicated can be grouped into 2 broad groups: those in which there is increased cell survival (i.e. associated with inhibition of apoptosis) and those in which there is excess cell death (where apoptosis is overactive). Diseases in which there is an excessive accumulation of cells include cancer, autoimmune disorders and viral infections. Deprivation of trophic factors is known to induce apoptosis in cells dependent on them for survival. This fact has been exploited in the use of antiandrogens or antiestrogens in the management of prostate or breast cancer. Haemopoietic growth factors like granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 prevent apoptosis in target cells and modulation of levels of these factors has been tried in the prevention of chemotherapy-induced myelosuppression. Until recently, it was thought that cytotoxic drugs killed target cells directly by interfering with some life-maintaining function. However, of late, it has been shown that exposure to several cytotoxic drugs with disparate mechanisms of action induces apoptosis in both malignant and normal cells. Physiological regulation of cell death is essential for the removal of potentially autoreactive lymphocytes during development and the removal of excess cells after the completion of an immune response. Recent work has clearly demonstrated that dysregulation of apoptosis may underlie the pathogenesis of autoimmune diseases by allowing abnormal autoreactive lymphocytes to survive. AIDS and neurodegenerative disorders like Alzheimer's or Parkinson's disease represent the most widely studied group of disorders where an excess of apoptosis has been implicated. Amyotrophic lateral sclerosis, retinitis pigmentosa, epilepsy and alcoholic brain damage are other neurological disorders in which apoptosis has been implicated. Apoptosis has been reported to occur in conditions characterised by ischaemia, e.g. myocardial infarction and stroke. The liver is a site where apoptosis occurs normally. This process has also been implicated in a number of liver disorders including obstructive jaundice. Hepatic damage due to toxins and drugs is also associated with apoptosis in hepatocytes. Apoptosis has also been identified as a key phenomenon in some diseases of the kidney, i.e. polycystic kidney, as well as in disorders of the pancreas like alcohol-induced pancreatitis and diabetes.

Effect of Y virilin on male infertility.

A prospective placebo controlled double blind study was conducted in patients attending male infertility clinics of our hospital to evaluate effects of a herbal formulation for male infertility--'Y-virilin'. In phase 1 forty patients with oligospermia with or without asthenospermia were randomly allocated to 2 treatment groups--Treatment Group A i.e. formulation under test and treatment Group B (Placebo). Therapy with these agents was given twice a day for 6 months. In phase 2, 12 patients with azospermia were administered either 'Y virilin' or the placebo (n = 6/Gp). In all patients along with semen analysis (sperm count, percentage of motile sperms and grade of motility) was done monthly for 6 months. Serum FSH levels were estimated before and at the end of therapy. A significant increase in sperm count was observed from 2-3 months in oligospermics receiving Y virilin as compared to basal values (p < 0.05). In Group B the follow-up sperm counts were either comparable to basal values or were lesser. However, the percentage and grade of motility did not differ in two groups at the end of respective treatment. No change was found in mean FSH value. During the therapy period incidence of conception was 20% in treatment Group A and 5% in Group B. Of the azospermic receiving 'Y-virilin' 50% showed a count of 10-20 millions/cmm while none from the placebo group. This study highlights the therapeutic potential of the tested formulation in the patients with infertility.

Effect of oral administration of Terminalia chebula on gastric emptying: an experimental study.

Terminalia chebula is a commonly advocated agent in Ayurveda for improving gastrointestinal motility. Charles Foster rats (150-200 gms of either sex) were divided into four groups as follows--Group 1 (n = 15) normal animals; Group II (n = 6) rats administered metoclopramide (1.35 mg/kg); Group III (n = 8) rats given atropine (0.45 mg/kg). These agents were injected intramuscularly, 30 mins before the experiment. Rats from Group IV (n = 8) were administered Terminalia chebula (100 mg/kg/day for 15 days orally). Metoclopramide and atropine have established prokinetic and antikinetic activities respectively and are therefore included for comparison. All rats were then given a test meal of methyl cellulose (1.5%) mixed with phenol red (50 mg/100 ml) orally and gastric emptying was measured 20 mins later. Gastric emptying of normal rats (Group I) was found to be 51.6 +/- 7.79%. Metoclopramide significantly increased the gastric emptying (76.33 +/- 12.37%; p < 0.01) and atropine inhibited the motility (% gastric emptying being 7.26 +/- 19.76%; p < 0.01). Terminalia chebula was found to increase the percent gastric emptying (86.57 +/- 6.65%; p < 0.01). Thus from this study it appears that Terminalia chebula can serve as an useful alternative to prokinetic drugs available today.

Cardiac surgery and plasma digoxin levels.

Cardiopulmonary bypass (CPB) can induce several haemodynamic alterations and therefore influence pharmacokinetics of various drugs. In order to assess the effect of CPB on plasma digoxin levels, these were monitored in patients undergoing open heart surgery involving CPB (n = 11), over a 24 hour period, starting just prior to commencement of surgery. For comparison, plasma digoxin was also monitored in a group of patients (n = 10) who underwent cardiac surgery not involving CPB. In 7 of the 11 patients in the CPB group, plasma digoxin levels (ng/ml) were significantly (p < 0.01) lower at the end of 24 hours (0.654 +/- 0.094) than basal levels (1.3114 +/- 0.2498). In contrast, in the non CPB group, 7 of 10 patients showed significantly higher (p < 0.001) plasma levels (ng/ml) at the end of 24 hours (0.477 +/- 0.125) as compared to basal levels (0.26 +/- 0.098). Thus, rather than the type of surgery, it appears that the pre-operative levels of plasma digoxin influence its pharmacokinetics.

The influence of surgical stress on the psychoneuro-endocrine-immune axis.

Stress is known to depress the immune system severely. This study was done to evaluate whether surgical stress influenced polymorphonuclear (PMN) and monocyte functions in association with serum cortisol and the anxiety score as measured on the HARS Rating Scale. We found that surgery (irrespective of whether it was major or minor) significantly depressed PMN and monocyte functions and increased serum cortisol levels. PMN phagocytosis correlated significantly (p < 0.05) with the rise in serum cortisol. In spite of these changes, postoperative clinical recovery was uneventful. No major alterations in the HARS scores were noted pre and post operatively. This study demonstrates that surgical stress depresses the immune system with a concomitant rise in cortisol.

Can we do away with PTBD?

Percutaneous Transhepatic Biliary Drainage (PTBD) is performed in surgical jaundice to decompress the biliary tree and improve hepatic functions. However, the risk of sepsis is high in these patients due to immunosuppression and surgical outcome remains poor. This raises a question--can we do away with PTBD? To answer this query a study was carried out in 4 groups of patients bearing in mind the high incidence of sepsis and our earlier studies, which have demonstrated immunotherapeutic potential of Tinospora cordifolia (TC): (A) those undergoing surgery without PTBD (n = 14), (B) those undergoing surgery after PTBD (n = 13). The mortality was 57.14% in Group A as compared to 61.54% in Group B. Serial estimations of bilirubin levels carried out during the course of drainage (3 Wks) revealed a gradual and significant decrease from 12.52 +/- 8.3 mg% to 5.85 +/- 3.0 mg%. Antipyrine half-life did not change significantly (18.35 +/- 4.2 hrs compared to basal values 21.96 +/- 3.78 hrs). The phagocytic and intracellular killing (ICK) capacities of PMN remained suppressed (Basal: 22.13 +/- 3.68% phago. and 19.1 +/- 4.49% ICK; Post drainage: 20 +/- 8.48% Phago and 11.15 +/- 3.05% ICK). Thus PTBD did not improve the metabolic capacity of the liver and mortality was higher due to sepsis. Group (C) patients received TC during PTBD (n = 16) and Group (D) patients received TC without PTBD (n = 14). A significant improvement in PMN functions occurred by 3 weeks in both groups (30.29 +/- 4.68% phago, 30 +/- 4.84% ICK in Group C and 30.4 +/- 2.99% phago, 27.15 +/- 6.19% ICK in Group D). The mortality in Groups C and D was 25% and 14.2% respectively during the preoperative period. There was no mortality after surgery. It appears from this study that host defenses as reflected by PMN functions play an important role in influencing prognosis. Further decompression of the biliary tree by PTBD seems unwarranted.

Emblica officinalis: a novel therapy for acute pancreatitis--an experimental study.

Acute necrotising pancreatitis is associated with an unacceptably high mortality for which no satisfactory remedy exists. Emblica officinalis (E.o.) is a plant prescribed in Ayurveda, the Indian traditional system of medicine, for pancreas-related disorders. This study was carried out to evaluate the protective effect of E.o. against acute necrotising pancreatitis in dogs. Pancreatitis was induced by injecting a mixture of trypsin, bile and blood into the duodenal opening of the pancreatic duct. Twenty eight dogs were divided into 4 groups (n = 6-8 each): GpI--control, GpII--acute pancreatitis, GpIII--sham-operated, GpIV--pretreatment with 28 mg E.o./kg/day for 15 days before inducing pancreatitis. Serum amylase increased from 541.99 +/- 129.13 IU/ml to 1592.63 +/- 327.83 IU (p <0.02) 2 hrs after the induction of pancreatitis in GpII. The rise in serum amylase in both GpIII and GpIV was not significant. On light microscopic examination, acinar cell damage was less and the total inflammatory score was significantly lower in the E.o. treated group as compared to GpII. Electron microscopy confirmed this and showed an increased amount of smooth endoplasmic reticulum and small, condensed granules embedded in a vacuole. More studies are needed to explore the clinical potential of E.o. and its mechanism of action.

Tinospora cordifolia induces colony stimulating activity in serum.

Tinospora cordifolia (Tc) is an Indian medicinal plant with proven immunomodulatory activity. This study was performed to elucidate its possible mechanism of action. We measured CFU-GM Cotony forming units of the granulocyte-macrophage series in serum of mice treated with Tc. We found that 10 days treatment with Tc (100 mg/ kg/d) induced a significant (p < 0.01) increase in the number of CFU-GM (255 +/- 49.32 vs 38.51 +/- 9.98) This suggests that activation of macrophages by Tc leads to increase in GM-CSF which leads to leucocytosis and improved neutrophil function.

Inhibition of phagocytes by cyclosporin in vitro.

Cyclosporin is an immunosuppressant that acts by selectively inhibiting the activation of T lymphocytes. Its effects on monocytes and neutrophils are not well explored. We investigated the in vitro effects of cyclosporin on these cells, harvested from venous blood from nine healthy, non-smoking volunteers. In vitro incubation of monocytes with increasing concentrations of cyclosporin (5, 25 and 625 micrograms) depressed their phagocytosis by 22%, 32% and 49%, respectively, compared to the control values. The intracellular killing capacity of monocytes decreased by 26%, 31% and 43% with these doses, and neutrophil phagocytosis was depressed in a similar manner (16%, 30% and 40%). Patients receiving cyclosporin are susceptible to infections, and inhibition of these phagocytic cells by cyclosporin may be partly responsible for this. Neutrophil chemotaxis is reduced in patients with impaired renal function. Treating these patients with cyclosporin may in addition suppress the phagocytic function of these cells.

Modulation of Kupffer cell activity by Tinospora cordifolia in liver damage.

Kupffer cells are major determinants of outcome of liver injury. Their activity was therefore studied in a model of chronic liver disease. The effect of Tinospora cordifolia, an indigenous agent with proven hepatoprotective activity, was evaluated on Kupffer cell function, using carbon clearance test as a parameter. Rats were divided into two major groups. In Gp I which served as normal control t1/2 of carbon was 9.48 +/- 4.14 min. GpII received horse-serum in a dose of 0.5 ml/100 gm b.w. i.p. for a period of 12 weeks and was divided into three sub-groups. In Gp IIA at the end of 12 weeks half-life of carbon was found to be significantly increased to 19.86 +/- 7.95 min (p < 0.01). Indicating suppressed Kupffer cell function in chronic liver damage. In Gp IIB treated with vehicle for 4 more weeks there was significant prolongation of half-life to 38.32 +/- 10.61 min (p < 0.01), indicating perpetuation of damage in absence of damaging agent. Whereas in Gp IIc, treated with Tinospora cordifolia t 1/2 was decreased to 14.24 7.74 min (p < .01), as compared to vehicle control indicating a significant improvement in Kupffer cell function and a trend towards normalization.