Amla Therapy as a Potential Modulator of Alzheimer’s Disease Risk Factors and Physiological Change

INTRODUCTION

Alzheimer’s disease (AD), the most common form of dementia, is a progressive degenerative disease of the brain which mainly affects the elderly over 60 years of age. The prevalence of AD and dementia is rapidly growing due to increased lifespan associated with improved healthcare and nutrition. It is estimated that approximately 35 million people worldwide have dementia [1], with this number expected to rise to more than 120 million by 2050 [2].

Progression from initial mild symptoms to severe dementia and eventually death, occurs over approximately eight years. However, it is known that the disease has a very long preclinical phase, of approximately two decades. A clinical diagnosis is routinely made once symptoms have manifest, by which stage considerable, and currently irreversible, synaptic and neuronal loss has already occurred [3]. A greater understanding of disease development, together with sophisticated neuroimaging techniques, is making diagnosis at preclinical stages increasingly feasible. However, the development of a widely accessible diagnostic test is essential to identify at risk individuals and facilitate the development and early implementation of effective disease-delaying therapies.

Indeed, no cure for AD currently exists, despite many years of research. However, with such a long preclinical phase, research focusing on slowing or preventing pathogenesis is gaining momentum. Even a modest delay in onset of symptoms would be of tremendous significance to affected individuals and their families; further, the resultant savings to healthcare providers would also be substantial.

An increased risk of AD and dementia has been attributed to a number of genetic and lifestyle risk factors. Furthermore, type 2 diabetes (T2D), hyperlipidemia, obesity, hypertension, and other cardiovascular disease risk factors have also been shown to increase AD and dementia risk [4].

Oxidative stress is an early contributing factor to AD pathogenesis, and is also a common event observed in several cardiovascular diseases. Oxidative stress is partly a result of compromised mitochondrial function, leading to excess reactive oxygen species, oxidized lipids, and reduced antioxidant defense mechanisms, as well as metabolic disturbances [5]. Reducing oxidative stress is a major target of potential anti-AD therapies, and foods rich in antioxidants represent one potential therapeutic contributor. In this regard, the Amla plant (Emblica officinalis Gaertn), specifically the Amla fruit (also known as Indian gooseberry), has received attention. Extracts from the Amla plant are known to be particularly high in antioxidants, and to contain both anti-microbial and anti-inflammatory substances. Consequently, such extracts are being investigated as potential treatments for reducing the risk of cardiovascular risk factors as well as AD. Here we review the evidence suggesting that Amla extracts could be of value as part of an effective disease-delaying treatment for AD.

ALZHEIMER’S DISEASE PATHOGENESIS

The pathological hallmarks of AD seen in the brain at autopsy include cortical shrinkage, extensive synaptic and neuronal loss, extracellular amyloid plaques, and intracellular neurofibrillary tangles (NFT). It now appears that these pathological changes start to develop many years before symptoms manifest. The accumulation of the key proteins, amyloid-β (Aβ) and tau, leads to the formation of amyloid plaques and NFT, respectively. Together these components have central roles in driving the neurodegenerative process in the AD brain, through promoting inflammatory and oxidative stress processes.

OXIDATIVE STRESS AND INFLAMMATION IN ALZHEIMER’S DISEASE

Evidence from animal and human studies suggests that oxidative stress and inflammation are associated with AD pathology [16], although it remains to be determined whether these processes are causative or if they occur downstream of AD-related pathological events. Nevertheless, it should be noted that signs of oxidative stress and inflammation are detectable at the earliest stages of AD pathogenesis, and there is also some evidence to suggest that oxidative stress may in fact be a disease initiator. For example, mitochondrial dysfunction, which increases with age, can be a trigger for increased Aβ production, which then itself increases mitochondrial damage and causes further oxidative stress [17], as well as increased vascular nitric oxide (NO) activity, thought to lead to brain chronic hypoperfusion, which has been hypothesized to occur early in disease pathogenesis [18]. As mentioned earlier, there is also a growing body of literature which suggests cardiovascular disease, obesity, dyslipidemia, insulin resistance, and T2D increase AD risk [19], and all of these conditions have been associated with oxidative stress and chronic inflammation. Thus, a strategy that targets oxidative stress and chronic inflammation may be beneficial in slowing AD progression.

Many mechanisms for the deleterious oxidative stress and chronic inflammatory changes and how they link to AD pathogenesis have been suggested. For example, the over-nutrition that often leads to obesity, insulin resistance, and T2D is accompanied by chronic high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which promote oxidative stress by free radical damage. High ROS levels in particular arise from (and cause further) mitochondrial dysfunction, inflammation, increased cytosolic Ca^2 +, and increased levels of advanced glycation end-products; all causes of oxidative stress in T2D as well as AD [20]. Furthermore, levels of the receptor for advanced glycation end-products (RAGE) are increased in AD and T2D; this receptor is involved in the transport of Aβ from the periphery into the brain, and binding of RAGE to Aβ can induce cerebrovascular dysfunction, as well as promote the release of cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) from microglia, potentially through inducing neuronal expression of macrophage-colony stimulating factor (M-CSF) which is followed by neuronal damage [21, 22].

Inflammation is associated with aging, and both of these are risk factors for AD; however, as with the other early changes, it is not known whether inflammatory changes such as increased levels of the inflammatory cytokines interleukins (IL) 1β and 6, TNF-α, and C-reactive protein play a causal role in the other biochemical changes associated with AD, or whether they are simply a consequence of the normal aging process [23]. Furthermore, increases in inflammatory cytokines may also be the result of obesity, hyperinsulinemia, and insulin resistance, which as mentioned earlier are themselves risk factors for AD, and which can all lead to chronic inflammation. The release of IL-6, IL-1β, and TNF-α and subsequent receptor binding, can, for example, activate the nuclear factor-kappa β pathway, which stimulates inflammation [22]. Moreover, Aβ peptide oligomers can exacerbate this inflammation, with in vitro experiments demonstrating rapid Aβ-induced increases in IL-1β and TNF-α; whereas IL-6 and IL-1β can directly regulate AβPP processing, thereby increasing production of Aβ₄₂ [24].

In peripheral insulin resistance, the TNF-α/JNK pathway is activated, leading to subsequent activation of major stress and inflammation signaling pathways, causing endoplasmic reticulum (ER) stress and higher levels of the stress kinases IkBα kinase, and double stranded RNA-dependent protein kinase (PKR). In T2D, high TNF-α causes serine phosphorylation of insulin receptor substrate-1 (IRS-1), thereby blocking insulin signaling. Similar changes occur in the brain as pro-inflammatory signaling appears to mediate impaired neuronal insulin signaling, synapse deterioration, and memory loss. A key biochemical event believed to lead to this deterioration involves microglia releasing high levels of TNF-α early in AD, and it has been shown that IkBα kinase and PKR, as well as ER stress, which are reported to be elevated in AD brains [25], mediate Aβ-oligomer-induced IRS-1 inhibition in hippocampal neurons [23, 26].

It has also been shown that hypothalamic dysfunction including hypothalamic insulin resistance occurs in obesity, and that inflammation underlies this change, particularly ER stress in the hypothalamus [27]. A recent review has suggested that cellular pathways that link AD neuroinflammation to metabolic changes in T2D revolve around the activation of neuronal stress-related protein kinases and excess phosphorylation of the eukaryotic translation initiation factor 2α (elF2α; a key enzyme in protein translation), which leads to synapse dysfunction and neurodegeneration [28].

Similarly, in both AD and T2D, changes to metabolic pathways lead to lower production of glutathione (GSH), an important free radical scavenger that is generated from the reduction of glutathione disulphide (GSSG) by the enzyme glutathione reductase. Lower levels of GSH have been linked to AD and T2D; in fact, low GSH in plasma and a lower GSH:GSSG ratio have been suggested as peripheral markers of neurodegeneration [29]. Another reason GSH has been linked to AD relates to production from its precursor amino acids: glutamate, cysteine, and glycine. Cysteine transport into cells requires the amino acid antiporter system Xc, which also simultaneously transports glutamate out of cells. This is relevant as glutamate excitotoxicity is a component of AD pathogenesis, and disturbances in this transport system have been shown to lead to decreased intracellular glutathione, yet also to excess extracellular glutamate. Upregulation of this amino acid exchange system by retinoic acid has been shown to confer neuroprotection [30].

An additional link between metabolic pathway changes and AD or T2D involves the conversion of glucose-6-phosphate to ribose-5-phosphate by the enzyme glucose-6-phosphate dehydrogenase (G6PD) during glycolysis. The reaction produces nicotinamide adenine dinucleotide phosphate (NADPH); however, in hyperglycemia, G6PD activity decreases dramatically, thus reducing NADPH levels. Another source of NADPH in the brain is the malic enzyme, which catalyzes the reversible formation of pyruvate CO₂ and NADPH from malate and NADP [31]. Malic enzyme levels have been reported to be diminished in T2D [32]. Other changes can add to the reduction in NADPH production, which in turn favors oxidative stress, and also causes a diminished capacity to maintain GSH levels in the body [20]. Interestingly, the enzyme responsible for lower G6PD activity is protein kinase A (PKA), an enzyme which has been linked to abnormal tau phosphorylation in AD [33].

LIPID AND PROTEIN DAMAGE IN ALZHEIMER’S DISEASE

Another sign of oxidative damage and stress in AD is lipid peroxidation, as demonstrated by increased levels of relevant markers which have been found in plasma, urine, and brain tissue of AD transgenic mouse models. Mitochondrial dysfunction and ER stress also appear; and high levels of carbonyls, which are markers of protein oxidation, are detected in AD brains [34]. High ceramide levels are detected in AD, indicative of perturbed sphingomyelin metabolism, which is believed to lead to degeneration of neurons [35]: ceramides promote the production of Aβ and lead to further oxidative stress. Thus, the oxidation of lipids to produce ceramides is particularly important in AD [36], and decreases in certain plasma sphingomyelin species, as well as increases in plasma ceramide (N16 : 0 and N21 : 0) levels, are considered good potential biomarkers of AD [37]. Ceramides are important second messengers associated with apoptosis, and since these lipid molecules can regulate neurotransmitter release and synaptic vesicle fusion, higher levels may lead to synaptic dysfunction [38]. Other links between sphingolipids and AD include the autophagocytic clearance of Aβ which involves sphingomyelinase activities [39], and disturbances in sphingolipid metabolism caused by food restriction have been linked to learning and memory [40].

DIABETES, DYSLIPIDEMIA, CARDIOVASCULAR DISEASE, AND ALZHEIMER’S DISEASE

As reviewed previously, diabetes increases AD risk, potentially through multiple mechanisms that may also involve modulating Aβ production and clearance, and/or by promoting oxidative stress and inflammation. Importantly, levels of the low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) are reduced in T2D, and LRP-1 is involved in the clearance of Aβ, as it clears Aβ from plasma, and mediates transport out of the brain [41]. In fact, the similarities between AD and diabetes, including changes to glucose and lipid metabolism, higher oxidative stress, and chronic inflammation, have led to AD being labelled, by some, as type 3 diabetes [20].

Cholesterol and lipid metabolism were first linked to AD more than 20 years ago when it was found that possession of one or more copies of the apolipoprotein E ɛ4 (APOE ɛ4) allele was linked to a higher risk of developing the disease [42, 43]. Dyslipidemia is a major cause of cardiovascular disease, leading to an increased risk of myocardial infarction [44, 45]; in particular, low plasma levels of high density lipoproteins (HDL) and high levels of LDL have been linked to cardiovascular disease. HDL is a crucial player in the reverse cholesterol transport pathway; HDL also plays a significant role in reducing oxidation and inflammation, as well as promoting pro-endothelial function, anti-thrombotic functions, and modulating immune function [46]. High levels of HDL (often labelled as ‘good cholesterol’) have been associated with lower risk of cognitive decline and AD [47, 48]. By contrast, LDL, often referred to as ‘bad cholesterol’, transports lipids and cholesterol to cells, is able to invade the endothelium, and once present, if these LDL become oxidized or modified by glycosylation, carbamylation, and glycoxidation, can lead to atherosclerosis within artery walls [49]. LDL levels have been shown to positively correlate with cognitive decline [50].

THE POTENTIAL OF ANTIOXIDANT THERAPIES

With the knowledge that the preclinical phase of AD progresses for up to two decades before clinical symptoms manifest, focus has shifted to developing preventative strategies and medications. One such strategy has been to significantly increase the levels of antioxidants in the diet, to combat age- and disease-associated oxidative stress. This would have the effect of reducing the risk of cardiovascular disease and T2D, or, reducing the damage caused by such diseases, both of which, as detailed above, are known to increase AD risk. Other strategies include improving aerobic fitness, as this has also been linked to reduced incidence of many of the conditions associated with increased AD risk (T2D, cardiovascular disease, insulin resistance, obesity), as well as a reduced risk of AD itself. Combinations of complementary antioxidants such as vitamin E, vitamin C, and curcumin, together with other nutrients such as choline, folate and omega-3-fatty acids, have produced mixed results, with some studies showing benefits by conferring a degree of protection against cognitive decline. However, there have been few large longitudinal studies of combination antioxidant therapy, and further research is needed [51, 52]. Pharmaceutical and nutraceutical companies have designed various supplements aimed at reducing cognitive decline, based on the concept of reducing oxidative stress and inflammation. One such supplement is Souvenaid, a nutrient mixture which has shown improved memory performance in patients with mild AD in clinical trials, as well as improvement in plasma micronutrient levels and fatty acid profiles [53]. Souvenaid studies have so far focused on individuals who already have cognitive decline, and although some aspects of cognition improved, a systematic review and meta-analysis of randomized clinical trials did not find any significant improvement in global clinical function [54]. Ideally, a supplement would be able to prevent AD symptom onset, and in doing so, be suitable for long-term use. Many other potential dietary supplements or dietary components known to be naturally rich in antioxidant and anti-inflammatory properties warrant investigation. One such dietary supplement is Amla.

AMLA AND ITS POTENTIAL BENEFITS

Amla, an Indian gooseberry (Emblica officinalis Gaertn; synonym: Phyllanthus emblica Eunphorbia), is a member of the small genus of Emblica (Euphorbiaceae). Amla grows in tropical and subtropical parts of China, Indonesia, the Malay Peninsula, and India [55, 56]. Amla preparations have been used for centuries in traditional Indian medicine systems such as Ayurveda [55–57]. Various parts of the plant are used to treat a variety of diseases; however, the most important part is the fruit, which is used either alone or mixed with other plants.

Accumulating evidence suggests that Amla exhibits several pharmacological properties, including anti-cancer activities, anti-microbial, anti-inflammatory, and antioxidant properties. It has also been used in the treatment of hypercholesterolemia, atherosclerosis, and diabetes [58, 59]. Furthermore, the fruit has been used alone, or in combination with other plants, to treat ailments including common colds and fever, inflammation, peptic ulcers and dyspepsia, constipation, liver dysfunction, as well as various cancers [60].

The pharmacological properties may be attributable to individual constituents of Amla, or, more likely, to the synergistic activity of multiple components. Amla preparations have been so widely used that extracts from this plant are now the focus of research in many laboratories. In the following sections of this review, Amla preparations are described, followed by an extensive review of the evidence that Amla can influence AD and AD-related risk factors.

AMLA PREPARATIONS

A variety of beneficial compounds have been reported to be present in the Amla fruit, including several tannins (such as pyrogallol, ellagic acid, and gallic acid), and the flavonoid quercetin [61–64]. The constituents of Amla extract depend on the nature of the preparation, with various forms containing different concentrations of the active compounds in existence. One example is the enzymic Amla fruit juice SunAmla, which is a polyphenol-rich fraction of Amla fruits. SunAmla is derived from ethyl acetate (EtOAc) extraction of air-dried Amla fruit pieces in water–EtOAc (1 : 4) at room temperature for 24 hours. The extract is evaporated under reduced pressure followed by lyophilization [56]. Amla is also often used in the form of triphala; an herbal formulation containing fruits of Amla, terminalia belerica, and erminalia chebula in equal proportions [65, 66]. An alternative preparation of Amla, named Amlamax™ also exists, comprising a reconstituted, purified, standardized dried extract of Amla [58].

These Amla extracts contain many compounds, and the active compounds have been reported to have antioxidant, anti-inflammatory, immuno-modulatory, pro-apoptotic, and/or pro-autophagy properties [61]. Some of the active constituents of Amla include hydrolysable ellagitannins (type of polyphenol tannins) such as emblicanin A, emblicanin B, punigluconin, and pedunculagin. Amla is also reported to contain the polyphenolic compounds punicafolin, phyllanemblinin A, phyllanemblin, flavonoids (such as quercetin), kmpferol, ellagic acid, and gallic acid [62, 68]. As alluded to above, the percentage of each constituent varies depending on the preparation; for example, Amlamax™ is reported to contain 35% ellagitannins [69]. It is therefore unsurprising that a study of four commercial Amla extracts reported that each preparation demonstrated varying degrees of antioxidant efficacy; although all were found to contain flavonoids, tannins, ellagic and gallic acids, and corilagin [62]. By contrast, a high-performance liquid chromatography study of a methanolic extract of Amla found it to contain gallic acid (2.10%), mucic acid (4.90%), ellagic acid (2.10%), quercetin (28.00%), rutin (3.89%), and β-glucogallin (1.46%) [70]. There is active debate as to whether Amla contains high amounts of ascorbic acid (vitamin C) [71], although recent reports suggest that the antioxidant value of Amla may lie in other components. For example, one study found that the free radical scavenging strength of some of the antioxidant components ranked in decreasing order was emblicanin B > emblicanin A > gallic acid > ellagic acid > ascorbic acid [72]. Moreover, a recent study isolated 58 phenolic compounds from Phyllanthus emblica and found that at least 20 were potent antioxidants due to their strong scavenging activity in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Danio rerio ROS assays [73]. The existence of this variety of Amla preparations makes it difficult to compare extracts, their constituents, and the effects of different extracts, with more systematic research and standardized preparations required to fully evaluate the pharmacological effects of the extracts and their components [59].

EFFICACY OF AMLA IN THE MANAGEMENT OF DYSLIPIDEMIA

Both animal and clinical studies have demonstrated the effectiveness of Amla in treating dyslipidemia and atherosclerosis [58, 74], and accumulating evidence suggests Amla is a safer alternative for the management of dyslipidemia when compared to pharmaceutical drugs, as there appears to be a lower risk of serious side effects. In early studies, Thakur et al. reported improved lipid profiles in a rabbit model of cholesterol-induced dyslipidemia and atherosclerosis, after being fed Amla (1 g/kg body weight) for 16 weeks. Serum cholesterol levels and the cholesterol contents of the liver and aorta were significantly reduced in the Amla treatment group compared to control. Aortic atherosclerosis was also significantly reduced in the Amla treatment group, with an average reduction of 26% compared to control. No reduction in the level of serum triglycerides was found, and Amla was not found to increase excretion of cholesterol, leading the authors to propose enhanced enzymatic clearance as the likely mechanism [75]. In other similar dyslipidemia rabbit models, Amla administered as a juice (at a dose of 5 ml/kg body weight for 60 days) was also shown to have potent anti-atherosclerotic and lipid lowering effects. In contrast to the Thakur study, serum cholesterol, triglycerides, phospholipid, and LDL levels were lowered by 82%, 66%, 77%, and 90%, respectively, in the Amla treatment group. Levels of lipids in the liver, heart, and aorta were also significantly lowered by Amla treatment, while atherosclerotic plaques were markedly reduced in size compared to control animals. In addition, unlike the results reported by Thakur et al., increased cholesterol excretion was observed among the rabbits fed Amla juice, suggesting decreased cholesterol absorption as an alternative mechanism of action of this Amla formulation [76].

Supplementation of butter and beef fat diets with Amla powder (5% of total diet) for 3 months significantly reduced the atherogenic and dyslipidemic properties of these diets in Sprague Dawley rats: a positive effect on serum and tissue (heart, liver, and kidney) lipid profiles was observed, with Amla treatment decreasing triacylglycerol, total cholesterol, very low density lipoprotein (VLDL), and LDL [77]. The Amla treatment also significantly increased serum and tissue levels of HDL cholesterol, again resulting in a reduced atherogenic index (ratio of total cholesterol to HDL cholesterol) in these animals compared to control. The authors observed reductions in the liver levels and activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase following Amla treatment, leading them to propose decreased synthesis of cholesterol as a significant mechanism underlying the anti- dyslipidemic effects of Amla [77]. In another study, involving dyslipidemic male rats fed Amla extract at a dose of 10 mg/kg for 90 days, the decreased synthesis of cholesterol via reduced hepatic HMG-CoA reductase activity was again implicated as a potential mechanism underlying the reduced serum and tissue lipid levels observed. In addition, plasma lecithin cholesterol acyl transferase (LCAT) levels were significantly elevated in the Amla treatment group. LCAT is implicated in the reverse cholesterol transport pathway which involves HDL-mediated removal of excess cholesterol from peripheral tissue. Thus, the authors concluded that the anti-dyslipidemic activity of Amla was due to both its ability to decrease synthesis and upregulate degradation of cholesterol [78].

Later studies of male dyslipidemic NZ white rabbits treated with Amla at doses of 10 mg or 20 mg/kg per day for 4 months reported significantly lowered serum total cholesterol, HDL, LDL, and triglyceride levels. Furthermore, postmortem tissue analysis demonstrated significantly reduced cholesterol content of liver, heart, and kidney among the Amla-treated animals compared to controls. The authors also proposed decreased synthesis of cholesterol as the anti-dyslipidemic mechanism, as they too found significantly reduced levels of HMG-CoA reductase activity in the liver of the Amla-treated animals [79]. Similarly, studies of male Wistar rats found that age-related dyslipidemia could be reduced following administration of either SunAmla (40 mg/kg body weight per day) or a polyphenol-rich ethyl acetate extract of Amla (10 mg/kg body weight per day) for 100 days. Levels of total cholesterol, non-esterified cholesterol, and esterified cholesterol in both the serum and liver were significantly reduced following this Amla treatment. Interestingly, the authors also reported a reversal of age-related inhibition of peroxisome proliferator-activated receptor-α (PPARα) protein levels in the hepatic nuclei of Amla-treated animals compared to controls. Given that PPARα is intimately involved in the regulation of the transcription of genes implicated in lipid and cholesterol metabolism, these results allude to a potential alternative mechanism underlying the anti-dyslipidemic characteristics of Amla [56].

In more recent studies, the oral administration of an ethyl acetate extract of Amla (10 or 20 mg/kg body weight per day for two weeks) to male Wistar rats, decreased fructose diet-induced hyper-triacylglycerolemia and dyslipidemia, in a dose-dependent fashion. Similar to the Mathur study mentioned earlier, the authors reported reductions in both serum and hepatic levels of triglycerides and total cholesterol following Amla treatment [80]. Furthermore, Amla extract administered over a period of 18 weeks at a dose of 100 mg/kg of body weight was shown to both decrease LDL cholesterol and significantly increase HDL cholesterol in ovariectomized rats that had been fed chow and fructose; resulting in an improved atherogenic and dyslipidemic profile [81]. The same group, Koshy et al., recently reported that Amla treatment of ovariectomized rats increases protein expression of liver farnesoid X receptor (a nuclear receptor which suppresses cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis from cholesterol), and liver X receptor (another nuclear receptor, which regulates lipid and glucose homeostasis) [82]. Amla was also found to increase the protein expression of ATP binding cassette transporter A1 (ABCA1), involved in HDL synthesis, as well as the LDL receptor (LDLR), responsible for uptake of LDL cholesterol. Such effects on protein expression suggest further candidate mechanisms of action, and support the potential therapeutic role of Amla as a preventative agent for dyslipidemia.

While there are several studies reporting anti-dyslipidemic effects of Amla in animals, there is a paucity of data regarding these effects in humans. Antony et al. administered a purified, standardized, dried extract of Amla (Amlamax™) orally to men and women at a dose of either 500 (n = 22) or 1000 mg (n = 17) per day for 6 months. Both doses induced significant reductions in the serum levels of total cholesterol, LDL, VLDL, and triglycerides, and a significant increase in serum HDL level was also observed, although there were no significant differences in efficacy between the two doses administered [58]. The authors extended their studies, prescribing a dose of 500 mg Amlamax™ to dyslipidemic patients (n = 15) aged 35–45 years for 4 months. A significant reduction in total serum cholesterol (17%), LDL cholesterol (21%), and triglycerides (24%), as well as a 14% increase in HDL cholesterol levels was seen following administration of Amla [74]. The authors subsequently proposed Amlamax™ as a potential therapy for the management of dyslipidemia, and therefore that it may be beneficial in reducing cardiovascular disease. In a more recent clinical study in obese adults, 12 weeks of supplementation with Amla extract was found to lower the levels of LDL and C-reactive protein, as well as downregulate adenosine diphosphate (ADP)- and collagen-induced platelet aggregation; overall suggesting a lowering of cardiovascular risk factors [83]. Collectively, these data indicate an anti-dyslipidemic effect of Amla, and since dyslipidemia and cardiovascular disease are risk factors for AD, Amla therapy may also indirectly help to decrease the risk of AD.

THE EFFECT OF AMLA ON INDICES OF TYPE 2 DIABETES

The hydrolysis of starch by α-amylase in the pancreas, coupled with glucose uptake mediated by intestinal α-glucosidase, induces hyperglycemia in T2D. Nampoothiri and colleagues reported that significant inhibition of both of these enzymes in vitro could be induced by a reconstituted methanol extract of Amla [84]. In this study, protein glycation, another pathological change seen in diabetes, was also reported to be reduced following Amla treatment. Amla and some of its constituents have also demonstrated anti-diabetic effects, purportedly via antioxidant- and free radical-scavenging properties. Studies using a variety of animal models have shown Amla to prevent or reduce hyperglycemia, the development of cataracts, diabetic nephropathy, neuropathy, and cardiac complications [85]. Some of these studies are described below.

One animal model of diabetes is the alloxan-treated rat, with alloxan being a glucose analogue that is toxic to insulin-producing beta-cells of the pancreas. Using rats with alloxan-induced diabetes, it was shown that oral administration of a methanolic extract of Amla, at a dose of 100 mg/kg body weight per day for 11 days, significantly lowered fasting blood glucose levels. The glucose-lowering effects were observed from day 3 of the feeding study [86]. Later studies using the streptozotocin (STZ)-induced diabetic rat model demonstrated that the oral administration of a hydro-methanolic extract of Amla leaves, at a concentration of 100–400 mg/kg body weight, induced a dose-dependent decrease in fasting blood glucose and a dose-dependent increase in plasma insulin levels. The hypoglycemic effects were observed at day 22—interestingly though, the effect size was greater at day 45 [87]. Another study of this diabetic rat model showed that Amla treatment improved oral glucose tolerance, reduced fasting serum glucose levels, and raised glutathione levels [88]. Moreover, Amla administered to STZ-induced diabetic rats, orally for 20 days, as either the commercial enzymatic extract SunAmla (20 or 40 mg/kg of body weight/day), or a polyphenol-rich fraction of ethyl acetate extract (10 or 20 mg/kg of body weight/day), improved indices of oxidative stress, such as lipid peroxidation marker levels. The authors also reported reduced protein glycation. Furthermore, increased serum adiponectin levels were observed in the Amla treatment group [89]. Adiponectin is an adipokine, secreted by white and brown adipose tissue, which influences glucose and lipid metabolism: decreased levels of this hormone are observed in obese, insulin-resistant and T2D patients, and raising adiponectin levels induces improved insulin sensitivity and glucose tolerance. Most studies indicate insulin resistance and obesity are linked to reduced adiponectin. However, insulin favors adiponectin production possibly via inhibition of Forkhead box protein O1 (FOXO1), which then favors production of PPAR-γ (a ligand-activated transcription factor), which in turn favors adiponectin production. Furthermore, patients with type 1 diabetes have elevated adiponectin levels, despite having almost no insulin. This appears to contradict many other findings concerning the effect of insulin on adiponectin, and possibly suggests the influence of other metabolic factors on adiponectin levels [90].

Diabetes-associated chronic hyperglycemia can result in complications affecting the eye which include retinopathy and the development of cataracts. In other studies of STZ-induced diabetic rats, treatment with Amla extract (administered as Amla-derived tannoids or Amla-derived tannoids + Amla fruit), for 8 weeks, did not reverse the decreased insulin and elevated blood glucose profile; however, both forms of supplementation delayed cataract progression. The authors observed the lowering of sorbitol levels and aldose reductase activity, as well as reduced protein aggregation and insolubilization in the lens of the Amla-treated animals: factors which would normally contribute to cataract development [91].

Neuropathy is a common complication of T2D, as it occurs in more than 50% of cases. The complex etiology of neuropathy, and ineffective drug therapies, results in frequently unsuccessful management of neuropathic pain. The effect of Amla treatment (10–1000 mg/kg/day for 8 weeks) on neuropathy has also been investigated in STZ-treated rats. Dose-dependent improvements in neuropathic pain and attenuated diabetes-induced axonal degeneration were observed following Amla administration. The beneficial effects observed were attributed to Amla’s antioxidant capacity [92, 93]. Clinical trials are needed, however, to determine Amla’s potential in treating this aspect of diabetes in humans.

In one of the few diabetes-related clinical trials of Amla, it was found that Amla powder administered at doses of 1, 2, or 3 g per day, for 21 days, significantly decreased fasting and post-prandial blood glucose levels in both diabetic and non-diabetic individuals. Dose-dependent improvements in blood lipid profiles were also observed, at day 21, in the two patient groups, with reductions in total cholesterol, triglycerides, and LDL-cholesterol, and increased HDL levels recorded [94]. Another clinical trial, a randomized double-blind study, compared the effects of an aqueous extract of Amla (250 or 500 mg twice daily), with atorvastatin (cholesterol lowering medication) treatment, and placebo, in patients with T2D over 12 weeks (n = 20 per treatment group). Treatment with Amla, at either dose, significantly improved endothelial function at 12 weeks, to levels comparable with those induced by atorvastatin therapy. Furthermore, significant improvements in lipid and systemic inflammation biomarker profiles were observed in both the Amla and atorvastatin treatment groups, compared to placebo [95], and significant improvements in oxidative stress biomarker profiles were also observed in the Amla and atorvastatin treatment groups, as determined by reduced malondialdehyde (MDA) levels and increased glutathione levels [95].

Like diabetes, end-stage renal failure (uremia), and its treatment with hemodialysis, are associated with increased ROS production and subsequent oxidative stress. Chen et al. (2009) reported a reduction in oxidative stress among uremic patients (n = 17), following the ingestion of Amla at a dose of 450 mg/day for four months. Plasma levels of the oxidative marker 8-isoprostaglandin were significantly reduced after the four months of treatment, while plasma total antioxidant status was observed to be increased as early as one month, and to remain increased for the four months [96]. In later studies, Chen and colleagues administered Amla extract to 13 uremic diabetic patients for three months, in a 1 : 1 ratio with the antioxidant polyphenolic compound epigallocatechin gallate (EGCG; a type of catechin, and the most abundant catechin in green tea), at a dose of 100 mg of each constituent combined in tablet form and ingested three times per day. Antioxidant power and markers of oxidative stress were measured in the blood of patients pre- and post-treatment. The authors reported significantly increased antioxidant capacity following Amla/EGCG therapy, determined by the ferric reducing/antioxidant power assay. However, no suppression of plasma nitrogen oxide levels (an additional marker of oxidative stress) was observed [97]. The authors concluded that Amla/EGCG therapy is capable of enhancing antioxidant defense in diabetic uremic patients, and they suggested that the polyphenolic content of the extracts is likely to induce these beneficial effects. However, it is not possible to determine from this study whether the Amla or the EGCG extract, or indeed a synergy of their combination, augments antioxidant status.

Considering that T2D induces insulin dysregulation and chronic hyperglycemia, and given the link with impaired Aβ clearance, tau hyperphosphorylation, oxidative stress, inflammation, and neuronal dysfunction [98, 99], it seems plausible that the positive effects of Amla therapy on many of the metabolic and pathological changes that occur in diabetes may also have benefits in AD: this warrants further investigation.

INFLUENCE OF AMLA ON INFLAMMATORY INDICES

The current clinical treatment of inflammation involves non-steroidal anti-inflammatory drugs (NSAIDS), mostly for chronic conditions such as rheumatoid arthritis and osteoarthritis. This class of compounds acts by inhibiting the activity of cyclooxygenase (COX) enzymes, which are responsible for synthesizing mediators of inflammation, such as prostaglandins. However, there are common side effects associated with NSAID use, such as indigestion and stomach pain, and long-term use is often linked with adverse side effects, such as gastric mucosal damage, occult blood loss, elevation of serum hepatic transaminases, salt and water retention [100]. To avoid these problems, selective inhibitors of the COX-2 isoform have been developed; unfortunately, however, the use of these compounds has resulted in the occurrence of serious adverse reactions, to the extent that interest in these drugs has diminished [101]. Partly as a consequence, investigation into the potential of medicinal plants and plant extracts for the treatment of inflammation has increased in recent years. Amla is one such plant to be investigated specifically for its potential anti-inflammatory properties [98].

Early studies in a rat model of (adjuvant-induced) arthritis found that the intraperitoneal administration of Amla extract, for 25 days at a dose of 25 mg/kg, resulted in marked reductions in inflammation and edema, with reduced lymphocyte proliferation activity and synovial hyperplasia evident in the Amla-treated animals [102]. Animal studies have also shown that intraperitoneal administration of a hydroalcoholic extract of Amla significantly reduced inflammation and edema (induced acutely by carrageenan, histamine, serotonin, or prostaglandin E2, and chronically by cotton pellet granuloma), increased glutathione, superoxide dismutase (SOD), and catalase activity, and subsequently lowered lipid peroxidation; all further evidence of Amla’s anti-inflammatory as well as antioxidant activity [103].

The anti-inflammatory effects of Amla extracts containing free and bound phenolic compounds (20 and 40 mg/kg) have been assessed in other rat studies, using carrageenan-induced acute inflammation and cotton pellet-induced chronic inflammation. Pre-treatment of the rats with Amla extracts, for 1 hour, induced a dose-dependent reduction in acute inflammatory response, as determined by decreased paw volume. Importantly, the higher dose of Amla extracts induced anti-inflammatory responses equivalent to that produced by the NSAID Diclofenac (administered at a dose of 25 mg/kg). Pre-treatment of the rats with Amla extracts for 8 days was also shown to attenuate the chronic inflammatory response, with significant inhibition of granulomatous tissue mass formation observed on day 16. Positive effects on the levels of tissue biomarkers of inflammation (e.g., myeloperoxidase) were also observed. The anti-inflammatory response was again dose-dependent, with only the higher Amla extract dose producing equivalent effect sizes to those observed in the Diclofenac treatment group [104].

Acute and chronic rodent models of inflammation have also been used to assess the effects of a standardized water extract of Amla fruit. For example, one study of acute inflammation reported positive effects, as Amla administered orally to mice at a dose of 780 mg/kg significantly reduced acetic acid-induced peritonitis [105]. In another study, the extract significantly improved ethyl phenylpropiolate-induced ear edema, when applied externally at a dose of 1 mg/ear, and produced a dose-dependent inhibition of carrageenan-induced paw edema when administered orally (150–600 mg/kg), suggesting beneficial effects of the extract with respect to an acute inflammatory response. However, unlike in the experiments conducted by Muthuraman et al. [104], no beneficial effect was observed among the Amla treatment group in relation to a cotton pellet-induced granuloma model of chronic inflammation [106]: although it should be noted this was primarily a study of anti-cancer activity, and the aqueous Amla extract did inhibit invasiveness, reduce tumor numbers and/or promote apoptosis of certain cancer cell lines (with DNA fragmentation, increased caspase activity, and up-regulation of Fas protein detected).

Using a diabetic rat model to study inflammation, it was found that the oral administration of Amla extract at doses of 250–1000 mg/kg/day could modulate the inflammatory cascade through induction of dose-dependent decreases in both serum and neural levels of the pro-inflammatory cytokines TNFα, IL-1β, and transforming growth factor beta 1 (TGF-β1) [93]. A reduction in serum levels of the pro-inflammatory cytokines TNFα and IL-6, measured over a 24-hour period, has also been demonstrated following Amla pre-treatment (50 mg/kg body weight) in a rat model of endotoxemia—a condition usually caused by gram-negative bacterial infections that results in endotoxins being present in the blood. The same authors also reported that Amla (1–100 mg/ml) attenuated inflammatory responses in an in vitro leucocyte adhesion model of inflammation [107].

Amla administered to male rats, at a dose of 100 mg/kg by oral gavage for up to 8 weeks, has also been shown to confer a degree of protection against hepatotoxicity induced by the potent carcinogenic nitrosamine N-Nitrosodiethylamine. This study suggested that Amla produced its beneficial effects via multiple mechanisms including downregulation of inflammation through attenuation of neutrophil and monocyte infiltration, determined by postmortem histological examination [108]. Moreover, in recent studies of mice treated with benzo(a)pyrene to induce pre-cancerous lung lesions and inflammation, it was found that feeding the mice Amla extract significantly reduced the number of nodes on the lung surface, and attenuated the benzo(a)pyrene-induced levels of the proinflammatory cytokines MIP-2, TNF-α, IL-6, and IL-1β in lung tissue; suggesting Amla could protect the lung tissue from the cancerous changes as well as from inflammation [109].

In clinical studies, researchers investigated the effect of oral administration of the Amla extract Amlamax™, at doses of 500 or 1000 mg/day for 6 months, in individuals with mild hypercholesterolemia. The authors reported significantly reduced blood levels of the inflammatory marker C-reactive protein (CRP). Interestingly, the lower dose demonstrated greater anti-inflammatory capacity, reducing CRP levels by approximately 40% at 6 months compared to the 27% reduction observed in the higher dose treatment group [58]. Similarly, another clinical study observed significant reductions in serum CRP levels in patients with T2D, after aqueous Amla extract was administered for 12 weeks. However, in this study, patients receiving a daily dose of 500 mg demonstrated a 45% mean reduction in high sensitivity CRP levels compared with placebo, while a 63% reduction was observed among those ingesting 1000 mg per day [95]. A more recent clinical study of metabolic syndrome subjects found that both 250 mg and 500 mg Amla aqueous extract given twice daily for 12 weeks significantly improved endothelial function and lipid profiles, as well as reduced signs of oxidative stress and systemic inflammation [110]. It was found that the higher dose was more effective, and some of the changes included higher NO (+41.89%, +50.7%), higher GSH (+24.31%, +200953.22%), lower MDA (oxidative stress marker) (–21.02%, –31.44%), and lower levels of the systemic inflammation biomarker, high sensitivity CRP (–39.68%, –53.77%) (p < 0.001) with the 250 mg and 500 mg twice daily dosages, respectively. Untreated metabolic syndrome often leads to T2D and/or cardiovascular disease, known risk factors for AD, and inflammation and oxidative stress are involved in all these conditions.

A recent cell culture study used quantitative rtPCR and western blotting to reveal that Amla fruit extract could reduce lipopolysaccharide-induced inflammation in RAW 264.7 cells, as treatment with the extract dose-dependently reduced nuclear factor-κB (NF-κB), inducible nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2) [111] in the cells. The study showed that the extract could not only reduce oxidative stress damage, it could also inhibit the inflammatory reaction.

EFFECTS OF AMLA ON OXIDATIVE STRESS

Many of the studies reporting the variety of therapeutic effects above, attribute the mechanism of action to Amla’s antioxidant capacity. Indeed, oxidative stress manifests in parallel with diabetes, and contributes to the long-term complications associated with the disease. Lipid peroxidation, stimulation of protein glycation, and inactivation of antioxidant enzymes constitute some of the contributory mechanisms associated with oxidative stress in diabetes. Several studies conducted in rodent models of diabetes have suggested that, in addition to improving diabetic indices such as fasting glucose and plasma insulin levels, and ameliorating diabetes-associated complications such as neuropathic pain, Amla treatment improved measures of oxidative stress which most likely contribute to the overall beneficial effects observed. For example, in a rat model of diabetic neuropathy, in addition to improving neuropathic pain and attenuating axonal degeneration, Amla treatment (10–1000 mg/kg/day for 8 weeks) induced a significant dose-dependent increase in levels of the antioxidant enzymes GSH and SOD, and a decrease in levels of lipid peroxidation, an important marker of oxidative stress [93]. Moreover, the administration of Amla to STZ-induced diabetic rats significantly reduced the level of serum thiobarbituric acid reactive substance (TBARS) [89]. TBARS, which is formed as a by-product of lipid peroxidation (i.e., as a degradation product of fats), and detected by assay using thiobarbituric acid as a reagent, provides an indirect measure of the damage produced by oxidative stress: thus, the beneficial effects observed in this study were attributed to Amla’s anti-oxidant capacity [92]. In later studies, the administration of a hydro-methanolic extract of Amla (100–400 mg/kg/day for 45 days) to STZ-induced diabetic rats significantly ameliorated reductions in liver and kidney levels of GSH, glutathione peroxidase, SOD, and catalase, and also attenuated elevated levels of TBARS in a dose-dependent manner [87]. Furthermore, in a recent study of rats fed a high-fat diet for 21 days,+/–an ethanolic extract of Amla, it was found that the extract supplementation reduced the high-fat diet-induced increases in MDA and NO, and improved the diet-induced changes to the myocardium and coronary arterial architecture, indicating cardio-protective effects [112].

The growing interest in Amla’s antioxidant properties is leading to a greater interest in carrying out clinical studies. In one clinical study of impaired platelet function associated with T2D, it was found that Amla extract could significantly prolong bleeding and clotting time in diabetic patients, and the authors suggested that the extract’s antioxidant properties were responsible for this effect [113]. The recent clinical study of metabolic syndrome mentioned in the previous section also measured indices of oxidative stress such as GSH, NO, and MDA and as mentioned above, twice daily treatments of 250 mg or 500 mg Amla aqueous extract could significantly reduce levels of these biomarkers [110]. Recent clinical studies of metabolic syndrome (discussed above) have shown that 3 months of Amla extract treatment can improve lipid profiles [110], and in a separate recent study of patients with dyslipidemia, 500 mg of Amla extract given twice daily for 3 months was shown to significantly reduce total cholesterol (p = 0.0003), triglyceride (p = 0.0003), LDL cholesterol (p = 0.0064), and VLDL cholesterol (p = 0.0001) [114], with a non-significant reduction in the ratio of ApoB to ApoA1. Considering there were also signs the extract might reduce fasting blood glucose, the authors concluded that such treatment might improve both dyslipidemia and T2D symptoms, though further studies of the glucose benefit are required. One small clinical study investigated a multicomponent nutraceutical which included Amla as one of the components (other components were hydroxytyrosol, maqui, monacolin K, berberine, astaxanthin, coenzyme Q10, and folic acid). Twenty-one people took the supplements twice daily for 60 days, and changes from baseline in various parameters were investigated. A significant improvement in most atherogenesis and oxidative stress biomarkers was recorded, including total cholesterol levels, serum antioxidant capacity, LDL levels, and blood glucose levels [115]. The promising effects of this polyphenol-based treatment on oxidative stress and atherogenesis parameters, with no adverse side-effects reported, suggests that more clinical studies of individual as well as combined nutraceuticals should be carried out.

When considering studies of dyslipidemia, Augusti et al. [77] demonstrated that Amla reduced the atherogenic and dyslipidemic profiles induced by butter and beef fat diets in rats, with reduced cholesterol synthesis implicated as the primary mechanism. Additionally, the authors reported partially ameliorated elevations in levels of TBARS in the liver, heart, and kidney within the Amla treatment group [77]. Kim et al. (2010) reported many beneficial effects of Amla extract therapy (10 or 20 mg/kg body weight per day for two weeks), administered to fructose diet-induced dyslipidemic male Wistar rats. In addition to improving serum and hepatic lipid profiles, the higher Amla dose also significantly reduced serum and hepatic mitochondrial TBARS levels by 21.1 and 43.1%, respectively [80]. Significantly reduced levels of TBARS have also been reported in serum, renal homogenate, and mitochondria in aged rats, leading the authors to conclude that Amla is a useful antioxidant for the prevention of age-related renal disease [55]. Furthermore, in a rat model of hypertension induced by deoxycorticosterone acetate/1% NaCl high salt, a hydroalcoholic lyophilized Amla extract was found to prevent the development and progression of hypertension, as well as renal and cardiac hypertrophy, via the modulation of endogenous antioxidants, activated eNOS, raised serum nitric oxide and K+ levels, and lowered Na⁺ levels [116]. Further recent studies of rats, using a hypothyroid model fed a high-fat diet for 6 weeks, showed that when supplemented with Amla fruit extract 100 mg/kg bw/day), the redox imbalance and inflammatory signaling in the hypothyroid rats, which was exacerbated by the diet, could be mostly restored to normal levels, as judged by measurement of biomarkers such as GSH, MDA, IL6, COX2, and NOX-4 [117], suggesting this extract might be a good supplement for the treatment of both dyslipidemia and hypothyroidism. Another study of rats fed a high fat diet+/–an ethanolic extract of Amla for 21 days showed that the high dietary fat-induced abnormalities in lipid profile and cardiac autonomic functions could be reduced by the Emblica officinalis supplementation [118]. In recent studies of a cell culture model of age-related macular degeneration, Amla extract was found to improve mitochondrial membrane potential, reduce apoptosis and oxidative stress, downregulate VEGF and upregulate peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α; plays a central role in the regulation of cellular energy metabolism) [119]. This adds to the evidence of a cytoprotective and antioxidant effect of Amla.

Other recent rat studies attempting to identify the active ingredient(s) in Amla used several hyperlipidemic rat models to compare the effects of Amla, as well as one its constituents, gallic acid [120]. Both treatments were found to increase PPARα expression and increase activity of lipid oxidation through carnitine palmitoyl transferase along with decreased activity of hepatic lipogenic enzymes, i.e., G6PD, fatty acid synthase, and malic enzyme. The treatment also improved liver cholesterol uptake and helped to restore glucose homeostasis through increased Glut4 and PPARγ protein expression in adipose tissue. The study supports the use of Amla extract as an anti-dyslipidemia agent, and suggests gallic acid could be the major active ingredient.

INFLUENCE OF AMLA ON APOPTOSIS AND AUTOPHAGY

Apoptosis is programmed cell death that activates a series of cellular processes in order to eliminate damaged, unwanted, or infected cells in multi-cellular organisms [121, 122]. By contrast, autophagy degrades and recycles damaged, dysfunctional, or unwanted cellular components within cells: this involves the formation of vesicles known as autophagosomes around the targeted cytoplasmic constituents, followed by fusion of the autophagosomes with lysosomes, and then degradation of the components [123]. Researchers have confirmed oxidative stress plays a fundamental role in the induction of apoptosis, under both physiological and pathological conditions [124]. As detailed above, many studies have shown that Amla decreases oxidative stress, thus, it is not surprising that Amla has also been shown to have significant effects on apoptosis.

Interestingly, Amla induces apoptosis at a higher dose and attenuates apoptosis at a lower dose [108]. For example, the apoptotic effect of Amla on cancer cells was investigated through qualitative and quantitative analysis of DNA fragmentation, and measurement of the mono- and oligo-nucleosomes released into the cytoplasm after induced cell death [106]. At high concentrations, Amla is cytotoxic and inhibits the in vitro proliferation of various tumor cell lines by inducing G2/M phase cell cycle arrest and apoptosis [106, 126]. Moreover, higher doses of Amla have been shown to induce membrane blebbing, chromatin condensation, and internucleosomal breaks, as evident from the morphology and DNA ladder pattern obtained in gel electrophoresis [127]. As there is evidence that the anti-tumor activity of Amla extract occurs principally because of its interaction with cell cycle regulation [106, 128], Amla has been suggested to have potential in cancer prevention and cellular protection. However, some studies have indicated that antioxidants slow down or block the apoptotic process via stabilizing mitochondrial functions [129, 130]; further studies are therefore needed to clarify Amla’s effects on apoptosis.

To date, Amla has demonstrated differential effects, depending on cell type and dosage. For example, in a study of several human cancer cell lines, as well as mouse skin tumorigenesis, and in vitro invasiveness, aqueous Amla extract at 50–100μg/ml was found to inhibit growth of 5 of the 6 cancer cell lines tested. Amla treatment showed a 50% reduction in tumor growth in a skin tumor model, and reduced tumor growth in animals treated with 7,12-Dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA, which results in 2-stage accelerated carcinogenesis) [106]. Some of the cells tested were HeLa cells, where apoptosis was observed following treatment with Amla, and these cells also demonstrated increased caspase-3/7 activity and caspase-8 activity, and upregulation of the Fas protein, indicating a death receptor-mediated apoptosis mechanism [55, 131].

In cancer cells, activation of caspase activity is desirable, whereas in AD, higher caspase activity (particularly caspase-3) has been linked to increased tau truncation, aggregation, and tangle formation [132–134]. Recent rat studies have shown that oral gavage with an aqueous Amla solution (100 mg/kg body weight), as a treatment for myocardial ischemia-reperfusion injury, causes an upregulation of the PI3K/Akt/GSK3β/β-catenin cardio-protective pathway, an upregulation of the anti-apoptotic protein Bcl-2, and a reduction in 3-nitrotyrosine and caspase-3 expression, thereby preserving cardiac tissue [135]. Similarly, in other studies of rats treated with N-nitrosodiethylamine to induce liver injury, it was found that Amla (100 mg/kg body weight) was more potent than vitamin C in scavenging O₂^–, hydrogen peroxide and nitric oxide. Amla also significantly preserved Mn-SOD and catalase expression, and decreased iNOS and CYP2E1 protein expression [108, 124].

The protein Bax is a mammalian cell-death protein that targets mitochondrial membranes, to induce mitochondrial damage and cell death, whereas Bcl-2 protein is an anti-apoptotic protein. Bax can form a heterodimer with Bcl-2, thus functioning as an apoptotic activator. It has been shown that Bcl-2 overexpression reduces lipid peroxidation in cells exposed to oxidative stress [56]. The oral administration of Amla extract has been shown to modulate levels of these proteins, reducing cell death [56], and more recent studies have shown that Amla can upregulate Bcl-2 levels, and decrease the Bax/Bcl-2 ratio, poly (ADP-ribose) polymerase (PARP) fragment-related apoptosis, as well as Beclin-1-induced autophagy in rat livers [108]. A study of fructose-induced metabolic syndrome in rats also found that an Amla extract, fed at 10 or 20 mg/kg/day for two weeks, inhibited the increase of COX-2, accompanied by regulation of NF-κB and Bcl-2 proteins in the liver, while the elevated expression level of Bax was significantly decreased [80]. These effects were attributed to the polyphenolic content of the Amla extract. In other studies using a murine skeletal muscle cell model, Amla treatment resulted in cytoprotective effects and lowered ROS levels in cells subjected to t-butylhydroperoxide-induced oxidative stress; mitochondrial biogenesis and antioxidant systems were also stimulated, and polyphenols in the extract were found to be the functional components [136]. A recent study of the neurotoxicity induced by a high salt and high cholesterol diet in rats showed that a tannin-enriched extract of Amla fruit fed to the rats for 7 weeks could reduce the cognitive impairment induced by the diet (passive avoidance tests), and reduce the overexpression of NF-κB in the brain [137].

Lymphocyte proliferation is a very sensitive test that is often used as a biomarker for toxic exposure. The treatment of lymphocytes with chromium, a toxic metal, causes immunosuppression, resulting in increased DNA fragmentation and a characteristic DNA ladder which is a hallmark of apoptosis [138, 139]. Chromium also induces free radical production, lipid peroxidation, and lower GSH levels. In a study of chromium-treated lymphocytes, Amla treatment was shown to reduce signs of toxicity: these signs included improved cytoprotection, reduced DNA fragmentation, enhanced lymphocyte proliferation, and increased interleukin and γ-interferon production. In addition, Amla significantly inhibited the chromium-induced free radical production and restored the antioxidant status back to control levels [139].

A study of ovarian cancer cells found that Amla extract had anti-proliferative effects, both in vitro and in vivo. The Amla extract did not induce apoptotic cell death, but did significantly increase the expression of the autophagic proteins Beclin-1 and LC3B-II, under in vitro conditions. In a recent study which used the neuronal cell lines PC12 and SH-SY5Y cells, markers of inflammation and ER-stress-mediated apoptosis induced by aluminum maltolate were found to be reduced by tannoid constituents of Amla extracts (EoT) [140]. In particular, the EoT reduced ROS levels, mitochondrial membrane dysfunction, and apoptosis (protein expressions of Bax; Bcl-2; cleaved caspases 3, 6, 9, 12; and cytochrome c) by regulating endoplasmic reticulum stress. Markers of inflammation (NF-κB, IL-1β, IL-6, and TNF-α) and Aβ toxicity (Aβ_1 - 42) triggered by the aluminum maltolate were also significantly normalized by the EoT treatment. Further studies are needed to determine if these results are relevant in in vivo models.

Quercetin, one of the components of Amla extract, has also been shown to increase the expression of the autophagic proteins Beclin-1 and LC3B-II, under in vitro conditions. In mouse xenograft tumors, Amla extract had anti-proliferative effects, and induced the expression of Beclin-1 and LC3B-II, it also reduced endothelial cell antigen - CD31 positive blood vessels and hypoxia-inducible factor-1α (HIF-1α, part of a transcription factor implicated in cancer biology) expression, suggesting that Amla extract may inhibit ovarian cancer growth via inhibition of angiogenesis, and activation of autophagy [63].

However, as stated earlier, the effect of Amla on these cellular processes appears to be dose-dependent (e.g., [108]). Whilst induction of programmed cell death is of benefit in cases of cancer for example, in other scenarios, it would not necessarily be desirable, and may in fact be to the detriment of the organism. Indeed, in a toxicological evaluation of Amla extract, performed in vivo, the LD₅₀ (dose required to kill half the animals) was found to be approximately 1125 mg/kg body weight, yet doses of 200 and 400 mg/kg, yielded potent anti-inflammatory effects [70]. Thus, close attention must be paid to the dose administered in order to achieve the greatest benefit.

EFFECT OF AMLA ON COGNITION

The results of the above studies all point to a multimodal beneficial effect of Amla, with direct effects on modulators of lipid synthesis and homeostasis, T2D indices, inflammation, oxidative stress, and apoptosis all being reported. This thereby suggests Amla has the potential to reduce a plethora of AD pathogenic mechanisms. Indeed, in recent animal studies relevant to AD, rats treated intraperitoneally with aluminum chloride, to induce toxicity in the brain, showed that co-administration of Amla tannoids prevented tau hyperphosphorylation, by targeting the GSK-3β/Akt signaling pathway, and reduced signs of oxidative stress [141]. Hallmarks of AD pathology are known to accumulate in the human brain decades before cognitive deficits manifest. Thus, given the evidence above, which suggests that Amla can modify AD risk factors, and more directly, AD hallmarks such as tau hyperphosphorylation, the question remains: do such beneficial effects culminate in the prevention or slowing of cognitive decline? Indeed, accumulating evidence suggests that Amla does show promise for the slowing and prevention of cognitive decline. For example, a range of doses of Amla (50, 100, and 200 mg/kg), administered orally for 15 days, have been shown to reverse scopolamine and diazepam-induced amnesia in rats. Moreover, Amla treatment of aged rats resulted in a dose-dependent improvement in memory scores, determined using elevated plus- and Hebb-Williams maze behavioral tests [57]. The beneficial effects of Amla in this study were attributed to reduced brain cholinesterase activity and total cholesterol levels [57]. Triphala, which as mentioned previously is made with three fruits including Amla, is reported to possess anti-aging properties, it has also been associated with improved mental faculties [57, 142]. More recently, the pre-treatment of rats with Amla extract for 7 days, administered via intra-peritoneal injection at a dose of 500 or 700 mg/kg, significantly improved kainate-induced cognitive deficit, as evidenced by increased latency in the passive avoidance behavioral test. Further, the effect of Amla on cognition was dose-dependent, with 700 mg/kg producing the greatest effect. The authors proposed that the observed beneficial effects may be due to its antioxidant and anti-inflammatory properties [143]. Similar results have been obtained by the same authors studying pentylenetetrazole (PTZ)-induced seizures in rats: Amla extract (500 and 700 mg/kg intra-peritoneally) was found to reduce PTZ-induced oxidative stress, tonic seizures, and to improve retention latency in passive avoidance tasks [144]. The intra-peritoneal administration of Amla extract for 7 consecutive days at a dose of 150–600 mg/kg has also been shown to reverse scopolamine-induced amnesia in Swiss albino mice. Memory performance, as assessed using elevated plus-maze and passive avoidance tests, was most improved in the 600 mg/kg treatment group. Importantly, Amla pre-treatment was shown to reverse scopolamine-induced increases in brain levels of acetylcholinesterase (AChE; the enzyme responsible for rapidly degrading the neurotransmitter acetylcholine; ACh). Given that cognitive dysfunction and AD are associated with decreases in brain levels of ACh, the influence of Amla on AChE activity reported here suggests that this compound may hold promise in the treatment of cognitive impairment due to AD [145]. Furthermore, Amla treatment reversed kainate- and scopolamine-induced reductions in brain GSH levels [143, 145] and increased brain MDA levels [145], indicating a concomitant reduction in oxidative stress; thus providing further evidence that reducing oxidative stress is important in maintaining brain health and brain function.

Another study compared ethanolic extracts of ripe and unripe Amla fruit, by feeding either 100 or 200 mg/kg to Swiss albino male rats for 12 days. Passive avoidance tests and reward alternation tests were carried out to assess changes to cognitive function, and it was found that Amla extracts significantly increased the correct responses, particularly the extracts from unripe fruit [146]. The higher dose of either fruit extract also increased levels of superoxide dismutase, GSH, catalase and glutathione peroxidase, and decreased the TBARS level compared to the control group; and both the ripe and unripe fruit extract also decreased levels of AChE activity, compared to the control group. The authors concluded that these fruit extracts not only reduce oxidative stress parameters, but also act as cognitive enhancers and may help in the treatment of AD and other neurodegenerative diseases. However, there is a dearth of human studies, and randomized controlled trials are required to definitively determine the effect of Amla treatment on cognition, cognitive decline, and subsequently AD.

CONCLUSION

In AD, it is difficult to decipher which metabolic abnormality, or change (if there is just one), is the trigger for other damage, which initiates a destructive cycle of oxidative stress, glucose and lipid dyshomeostasis, inflammation, and the development of AD-specific pathology. T2D, chronic obesity, and AD all have insulin resistance in common, which is associated with inflammation and lipid dyshomeostasis. Chronic inflammation is mediated by activation of pro-inflammatory cytokines, such as TNF-α and IL-6. Lipid dyshomeostasis results in increased lipid peroxidation, ceramide generation in adipose tissue and liver, and abnormal lipid profiles, including HDL and LDL levels. Insulin resistance, inflammation, and ceramide accumulation promote oxidative and ER stress, which impair mitochondrial function, energy balance, and membrane integrity, and worsen insulin resistance, inflammation, and ceramide generation. Unchecked, the rates of oxidative and inflammatory injury eventually exceed those of repair. Slowing or halting the development of oxidative stress, inflammation, and other changes, with medicinal plants or plant extracts such as Amla which contain concentrated antioxidants, may be a highly effective treatment in the slowing of development of several conditions, particularly AD, to lessen the growing epidemic of disease of the elderly.

In this review, we have discussed many animal-based studies, and some clinical trials, which have shown that Amla, and its extracts, exert many positive effects on dyslipidemia, hyperglycemia, inflammation, oxidative stress, apoptosis, and autophagy, that contribute to AD risk. This growing body of knowledge on the potential benefits of Amla therapy in reducing AD risk, and slowing disease development, supports theory that reducing oxidative stress and inflammation are crucial in decelerating disease development. However, no double-blind placebo-controlled longitudinal clinical trials of Amla have been conducted to date, to definitively assess its beneficial effects in the context of AD. Further work is therefore needed, with the aim of reducing the incidence of this debilitating disease.