Scientific basis for the use of Indian ayurvedic medicinal plants in the treatment of neurodegenerative disorders: ashwagandha.

Ayurveda is a Sanskrit word, which means "the scripture for longevity". It represents an ancient system of traditional medicine prevalent in India and in several other south Asian countries. It is based on a holistic view of treatment which is believed to cure human diseases through establishment of equilibrium in the different elements of human life, the body, the mind, the intellect and the soul [1]. Ayurveda dates back to the period of the Indus Valley civilization (about 3000 B.C) and has been passed on through generations of oral tradition, like the other four sacred texts (Rigveda, Yajurveda, Samaveda and Atharvanaveda) which were composed between 12(th) and 7(th) century B.C [2, 3]. References to the herbal medicines of Ayurveda are found in all of the other four Vedas, suggesting that Ayurveda predates the other Vedas by at least several centuries. It was already in full practice at the time of Buddha (6(th) century B.C) and had produced two of the greatest physicians of ancient India, Charaka and Shushrutha who composed the basic texts of their trade, the Samhitas. By this time, ayurveda had already developed eight different subspecialties of medical treatment, named Ashtanga, which included surgery, internal medicine, ENT, pediatrics, toxicology, health and longevity, and spiritual healing [4]. Ayurvedic medicine was mainly composed of herbal preparations which were occasionally combined with different levels of other compounds, as supplements [5]. In the Ayurvedic system, the herbs used for medicinal purposes are classed as brain tonics or rejuvenators. Among the plants most often used in Ayurveda are, in the descending order of importance: (a) Ashwagandha, (b) Brahmi, (c) Jatamansi, (d) Jyotishmati, (e) Mandukparni, (f) Shankhapushpi, and (g) Vacha. The general appearance of these seven plants is shown in Fig.1. Their corresponding Latin names, as employed in current scientific literature, the botanical families that each of them belongs to, their normal habitats in different areas of the world, as well as the common synonyms by which they are known, are shown in the Table 1. The scientific investigations concerning the best known and most scientifically investigated of these herbs, Ashwagandha will be discussed in detail in this review. Ashwagandha (Withania somnifera, WS), also commonly known, in different parts of the world, as Indian ginseng, Winter cherry, Ajagandha, Kanaje Hindi and Samm Al Ferakh, is a plant belonging to the Solanaceae family. It is also known in different linguistic areas in India by its local vernacular names [6]. It grows prolifically in dry regions of South Asia, Central Asia and Africa, particularly in India, Pakistan, Bangladesh, Sri Lanka, Afghanistan, South Africa, Egypt, Morocco, Congo and Jordon [7]. In India, it is cultivated, on a commercial scale, in the states of Madhya Pradesh, Uttar Pradesh, Punjab, Gujarat and Rajasthan [6]. In Sanskrit, ashwagandha, the Indian name for WS, means "odor of the horse", probably originating from the odor of its root which resembles that of a sweaty horse. The name"somnifera" in Latin means "sleep-inducer" which probably refers to its extensive use as a remedy against stress from a variety of daily chores. Some herbalists refer to ashwagandha as Indian ginseng, since it is used in India, in a way similar to how ginseng is used in traditional Chinese medicine to treat a large variety of human diseases [8]. Ashwagandha is a shrub whose various parts (berries, leaves and roots) have been used by Ayurvedic practitioners as folk remedies, or as aphrodisiacs and diuretics. The fresh roots are sometimes boiled in milk, in order to leach out undesirable constituents. The berries are sometimes used as a substitute to coagulate milk in cheese making. In Ayurveda, the herbal preparation is referred to as a "rasayana", an elixir that works, in a nonspecific, global fashion, to increase human health and longevity. It is also considered an adaptogen, a nontoxic medication that normalizes physiological functions, disturbed by chronic stress, through correction of imbalances in the neuroendocrine and immune systems [9, 10]. The scientific research that has been carried out on Ashwagandha and other ayurvedic herbal medicines may be classified into three major categories, taking into consideration the endogenous or exogenous phenomena that are known to cause physiological disequilibrium leading to the pathological state; (A) pharmacological and therapeutic effects of extracts, purified compounds or multi-herbal mixtures on specific non-neurological diseases; (B) pharmacological and therapeutic effects of extracts, purified compounds or multi-herbal mixtures on neurodegenerative disorders; and (C) biochemical, physiological and genetic studies on the herbal plants themselves, in order to distinguish between those originating from different habitats, or to improve the known medicinal quality of the indigenous plant. Some of the major points on its use in the treatment of neurodegenerative disorders are described below.

Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants.

Reactive oxygen species (ROS) are produced in the course of normal metabolism and they serve important physiological functions. However, because of their high reactivity, accumulation of ROS beyond the immediate needs of the cell may affect cellular structure and functional integrity, by bringing about oxidative degradation of critical molecules, such as the DNA, proteins, and lipids. Although cells possess an intricate network of defense mechanisms to neutralize excess ROS and reduce oxidative stress, some tissues, especially the brain, are much more vulnerable to oxidative stress because of their elevated consumption of oxygen and the consequent generation of large amounts of ROS. For the same reason, the mitochondrial DNA (mtDNA) of brain cells is highly susceptible to structural alterations resulting in mitochondrial dysfunction. Several lines of evidence strongly suggest that these effects of ROS may be etiologically related to a number of neurodegenerative disorders. Nutraceutical antioxidants are dietary supplements that can exert positive pharmacological effects on specific human diseases by neutralizing the negative effects of ROS. The present communication concentrates on a review of recent concepts and methodological developments, some of them based on the results of work from our own laboratory, on the following aspects: (1) the complex interactions and complementary interrelationships between oxidative stress, mitochondrial dysfunction, and various forms of neural degeneration; (2) fractionation and isolation of substances with antioxidant properties from plant materials, which are extensively used in the human diet and, therefore, can be expected to be less toxic in any pharmacological intervention; (3) recent developments in methodologies that can be used for the assay of oxidative stress and determination of biological activities of exogenous and endogenous antioxidants; and (4) presentation of simple procedures based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the resulting amplicon for investigations of structural alterations in mtDNA.

Mitochondrial thioredoxin system: effects of TrxR2 overexpression on redox balance, cell growth, and apoptosis.

Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (DeltaPsi(m)), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of DeltaPsi(m) dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against beta-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells.

Requirement for enzymatically active lipoprotein lipase in neuronal differentiation: a site-directed mutagenesis study.

Lipoprotein lipase (LPL) is well known for its role in the catabolism of plasma triglyceride (Tg)-rich lipoproteins, such as very low density lipoproteins (VLDL) and chylomicrons. The action of LPL on Tg-rich lipoproteins provides free fatty acids to skeletal muscle and adipose tissues, the main sites of LPL synthesis. Several studies have demonstrated that LPL is widely expressed in the parenchyma of brain tissues. We have recently shown that LPL expression is essential for promoting VLDL-stimulated differentiation of Neuro-2A cells. In the present study, we have generated stably transfected Neuro-2A cell lines expressing either wild-type LPL or various LPL mutants, including three enzymatically inactive variants (Asp156Asn, Gly188Glu and Pro207Leu), an enzymatically defective variant (Asn291Ser) and a variant known to express increased LPL activity (Ser447Ter). In Neuro-2A cells expressing enzymatically inactive LPL variants, VLDL-stimulated differentiation and neurite extension were not observed. However, in Neuro-2A cells expressing partially active or overactive LPL variants, VLDL added to the cultured medium was able to induce the phenotypic differentiation similar to that observed in Neuro-2A cells expressing wild-type LPL. In summary, these data show that the availability of fatty acids, resulting from the catabolism of VLDL by LPL, is required to promote the phenotypical differentiation of neuroblastoma cells. These findings may have significant relevance to lipoprotein metabolism in the brain as well as to the maturation and regeneration of nervous tissues in carriers of mutant LPL.

Lipoprotein lipase affects the survival and differentiation of neural cells exposed to very low density lipoprotein.

Lipoprotein lipase (LPL) is a key enzyme involved in the metabolism of lipoproteins, providing tissues like adipose tissue or skeletal muscle with fatty acids. LPL is also expressed in the brain, fulfilling yet unknown functions. Using a neuroblastoma cell line transfected with a NEO- or a LPL-expression vector, we have developed a model to study the function of LPL in neurons exposed to native or copper-oxidized lipoproteins. The addition to the culture media of VLDL with 10 microm copper sulfate led to a significant reduction in the viability of NEO transfectants whereas LPL-transfectants were protected from this injury. In the presence of VLDL and CuSO(4), LPL transfectants were even able to display significant neurite extension. This neuritogenic effect was also observed in LPL transfectants exposed to native lipoproteins. However, addition of VLDL particles oxidized with CuSO(4) prior to their addition to the culture media resulted in neurotoxic effects on LPL transfectants. These findings suggest that the presence of LPL in cultured neuronal cells modulates the physiological response of neurons following exposure to native or oxidized lipoproteins. LPL could thus play a key role in the differentiation of Neuro-2A cells and in the pathophysiological effects of oxidative stress in several neurodegenerative disorders.

Hyperinsulinemia and abdominal obesity affect the expression of hypertriglyceridemia in heterozygous familial lipoprotein lipase deficiency.

We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterol-rich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.

A monoclonal antibody against human lipoprotein lipase inhibiting heparin binding without affecting catalytic activity.

A fragment of the human lipoprotein lipase (LPL) cDNA (405 bp, 5' terminal end) was cloned in an expression vector to produce a approximately 17 kDa fusion peptide and was used as antigen to produce a high titre anti-LPL monoclonal antibody (10C3 MAb). This antibody reacts with both native and denatured forms of LPL from different tissue and animal sources. Competition studies with heparin indicate that 10C3 MAb is specific for an epitope at a heparin binding site. The antibody does not inhibit LPL enzyme activity, indicating that the antigenic epitope is not situated within or in the proximity of the LPL catalytic region. With these characteristics, 10C3 MAb should prove to be a useful immunochemical tool in clinical as well as in fundamental investigations on the metabolism of triglyceride-rich lipoproteins and in studies on the functional anatomy of LPL.

Human lipoprotein lipase deficiency: does chronic dyslipidemia lead to increased oxidative stress and mitochondrial DNA damage in blood cells?

Lipoprotein lipase (LPL) is a key enzyme in the metabolism of lipoproteins and their balanced distribution in the plasma. A deficiency of this enzyme due to gene mutations leads to severe dyslipidemia. In this report, we describe the major LPL gene mutations that are prevalent in the French-Canadian population of Québec and the nature of dyslipidemia caused by the resulting enzyme deficiency. We discuss the possibility that dyslipidemia caused by LPL deficiency may enhance oxidative stress in the blood cells, bring about increased fluidity of the membrane components of these cells and increase the susceptibility of their mitochondrial DNA to structural alterations. Some preliminary experimental results in verification of this hypothesis are presented.

Hemolysis in primary lipoprotein lipase deficiency.

A slight to moderate hemolysis is often present in plasma from patients with primary lipoprotein lipase (LPL) deficiency. To determine the nature of this hemolysis, we measured erythrocyte hypo-osmotic fragility, plasma free hemoglobin, and phospholipid composition in 26 patients with primary LPL deficiency and 21 unrelated controls. In some patients, these investigations were completed by erythrocyte cytoskeletal protein determinations and abdominal echography. Osmotic fragility was similar between control subjects and patients. However, there was a significantly increased concentration of plasma free hemoglobin in primary LPL deficiency (0.282 +/- 0.331 v 0.048 +/- 0.038 g/L in controls, P < .005). In LPL-deficient patients, an increase of plasma lysophosphatidylcholine concentration (12.6% +/- 5.8% v 6.4% +/- 1.9% in controls, P < .0001) was also found. The protein composition of the erythrocyte membrane skeleton was abnormal in some LPL-deficient patients and splenomegaly was present in 12, but these abnormalities did not correlate with plasma free hemoglobin levels. Bilirubin and haptoglobin levels were also within physiologic ranges in these patients, suggesting that the observed hemolysis did not result from hypersplenism. It appears likely that the accumulation of lysophosphatidylcholine was due to an impairment in the reverse metabolic pathway converting lysophosphatidylcholine back to phosphatidylcholine. Collectively, these data, along with a positive correlation between plasma free hemoglobin and lysophosphatidylcholine levels (r = .58, P = .0001), suggest that the hemolysis observed in primary LPL deficiency is mediated to some extent by the abnormally elevated concentration of lysophosphatidylcholine.

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers.

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.

Adipose cell size and distribution in familial lipoprotein lipase deficiency.

To determine the effect of lipoprotein lipase deficiency on the size distribution of fat cell populations in human adipose tissues, abdominal and femoral subcutaneous fat tissue biopsies were obtained from seven patients affected by familial hyperchylomicronaemia. These patients were characterized by massive accumulation of chylomicrons in the fasting state due to defective catabolism of plasma triglyceride-rich lipoproteins. They had no post-heparin plasma lipoprotein lipase activity and their fat tissues were deficient in lipoprotein lipase activity. The size distribution of adipocytes examined by scanning electron microscopy were similar to distributions observed in control subjects. Patient fat cell diameters were not statistically different from control fat cells obtained from subjects of similar body mass index. Mature fat cells contributed to 99% of the total fat tissue mass in lipoprotein lipase deficiency. Normal adiposity in lipoprotein lipase deficiency can thus be attributed to mature adipocytes and not to hyperplastic growth of immature fat cells. It is concluded that normal adipose tissue homeostasis is maintained in these patients in spite of the deficiency in lipoprotein lipase activity.

Geographic distribution and genealogy of mutation 207 of the lipoprotein lipase gene in the French Canadian population of Québec.

Mutations in the lipoprotein lipase (LPL) gene, leading to partial or total inactivation of the enzyme, result in a hereditary clinical syndrome called familial LPL deficiency. The French Canadian population, which is primarily and historically located in the province of Québec, has the highest worldwide frequency of LPL-deficient patients. We have analyzed the prevalence, spatial distribution, and genealogy in the Québec population of a LPL gene mutation, M-207 (P207L in conventional notation), which changes the amino acid proline to leucine in position 207 of the LPL protein and inactivates the enzyme. Our results show that M-207 is the most prevalent LPL gene mutation among French Canadians and accounts for the largest proportion of LPL-deficient patients in this population. Genealogical reconstruction of French Canadian LPL-deficient patients point to 16 founders of M-207, all of whom migrated to Québec in the early seventeenth century from the north-western part of France, especially from the region of Perche. Most of the carriers of M-207 are, at present, found in Charlevoix, Saguenay-Lac-St-Jean regions of eastern Québec. On the basis of the number of homozygote M-207 LPL-deficient patients so far identified, we estimate that there are at least 31,000 carriers of this mutation in the province of Québec. This constitutes a large pool of individuals at risk for atherosclerosis and other lipid-related diseases, since LPL deficiency is considered to be a significant contributing factor in the etiology and development of these diseases.

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.

Prevalence, geographical distribution and genealogical investigations of mutation 188 of lipoprotein lipase gene in the French Canadian population of Québec.

Familial lipoprotein lipase deficiency (FLD) is of particular interest to the French Canadian population of Québec since the largest concentration of homozygotes and carriers of this genetic disease in the world resides in this area. We have previously described a missense mutation (M-188) in the lipoprotein lipase (LPL) gene which was present in FLD patients belonging to different ancestries, including a number of French Canadians (Monsalve MV et al. J Clin Invest 1990: 86: 728-734). In the present report, we show that this mutation, although found in largest absolute numbers among French Canadians as compared to other groups in the world, accounts for only a small proportion (24%) of all the LPL mutant alleles in this population. The M-188 occurs either in the homozygote state or as a compound heterozygote with another LPL mutation. Analysis of geographic distribution indicates that the M-188 is more prevalent in western Québec, with the highest carrier rate in the Mauricie region. Genealogical reconstruction leads to the recognition of four founders for M-188, all emigrants from France to Québec in the 17th century.

Founder effect in familial hyperchylomicronemia among French Canadians of Quebec.

Familial hyperchylomicronemia has reached a high prevalence in the French Canadian population of eastern Quebec. The birth places of 58 carriers identified through the birth of one affected child clustered in three regions. The genealogies of these 58 individuals showed that no founder was common to all of them. Three sets of founders were found, one for each region, with little overlapping between two regions. These results strongly suggest that more than one mutation, introduced by the French migrants in the 17th century, are segregating in the French Canadian population. Perche, a region situated between Paris and Normandy, appeared to be the most likely putative center of diffusion of at least one mutation in the lipoprotein lipase gene segregating in the modern-day French Canadian population of Quebec.

Preparation and purification of gamma gamma enolase (neuron-specific enolase) using high performance anion exchange chromatography.

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of gamma gamma enolase isoenzymes from human and beef brain extracts. In the first step, a crude gamma gamma enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. gamma gamma enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of gamma gamma enolase by this method was 7-8 mg of pure enzyme per 100 g of brain.

Efficacy of RNase inhibitors during brain polysome isolation.

We have investigated the efficiency of heparin, polyvinyl sulfate and yeast RNA (as competitive RNase inhibitors), liver extract (as crude preparation of liver RNase inhibitors) and DEPC (as irreversible non-competitive inhibitor) for the preparation of rat brain polysomes. Sucrose gradient sedimentation profiles, obtained from PMS, were used to determine the optimal concentration of each inhibitor. Diethylpyrocarbonate, whatever the composition of isolation buffer, was found detrimental for brain polysomes. Most of the other inhibitors where found useless or even harmful. A slight positive effect was observed with heparin 0.75 mg/mL both for total yield and sedimentation pattern. It is concluded that the utilisation of most of the widely used RNase inhibitors is of questionnable effectiveness for brain polysome preparation.