Introduction

Despite all the advances in medical sciences, cancer, a disease as old as humankind, is globally a major health problem (^{Arora, 2010}). Recent reports from the International Agency for Cancer Research indicate that in 2008, approximately 12.7 million new cancer cases and 7.6 million cancer deaths occurred and of these, 56% of all new cancer cases and 63% of cancer deaths were in the less developed regions of the world (^{Ferlay et al., 2010}). Projections are that by 2020, the incidence of cancer will increase three-fold, and that there will be a disproportionate rise in cancer cases and deaths from the developing countries that have limited resources to tackle the problem (^{Are et al., 2010}).

Conventionally, when localized, cancer may be treated with either surgery (if operable), or with ionizing radiation (when inoperable), or by combining both these modalities. However, in the advanced stage, and more importantly, when metastasis is observed, the use of cytotoxic chemotherapeutic agents is obligatory (^{DeVita et al., 2004}). Unfortunately, the use of chemotherapy and ionizing radiation is associated with deleterious side effects as their cytotoxic effects are unbiased, and in association with neoplastic cells it can also affect normal tissues (^{Hall, 2000; DeVita et al., 2004}). In addition, the treatment of cancer and its complications is very expensive, and to patients in developing countries, where general health care in itself is beyond the reach of most people, the cost is exorbitant and unaffordable (^{Arora, 2010}).

In the light of these observations, a large number of patients, especially in the developing countries, prefer complementary and alternative medicines for treating and managing the symptoms of cancer and pain (^{Arora, 2010}). Ayurveda, the traditional Indian system of medicine, is one of the oldest systems of medicine and is practised in the Indian subcontinent (^{Arora, 2010}). Emphasis in Ayurveda is on disease prevention and promotion of good health by adopting a proper lifestyle and following therapeutic measures, which will rejuvenate the body (^{Kulkarni, 1997}). The Ayurvedic remedies, which are both preventive and therapeutic, are mostly made of plants and when compared with their synthetic counterparts are either nontoxic or less toxic (^{Arora, 2010}).

Some of the Ayurvedic formulations and plants used in these preparations are globally receiving increasing attention. In the recent past, these plants have been investigated for their pharmacological effects in accordance with modern medicine (^{Arora, 2010}). One such plant that has been extensively studied is the medium-sized deciduous tree Emblica officinalis Gaertn. or Phyllanthus emblica Linn belonging to the family Euphorbiaceae. The plant species, which was originally native to India, is today found growing in Pakistan, Uzbekistan, Sri Lanka, South-East Asia, China, and Malaysia (^{Warrier et al., 1996; Zhang et al., 2003; Khan, 2009}). Colloquially, they are known as Indian gooseberry tree, emblic myrobalans, and Malacca tree in English and amla in Hindi. The other vernacular names have been listed in Table 1.

All parts of the plant are of use in treating various ailments, but the fruit, which is yellowish-green in color, globular in shape, fleshy and smooth, striated with an obovate, obtusely triangular six-celled nut, is of immense use in various folk and traditional systems of medicine (^{Warrier et al., 1996; Zhang et al., 2003; Khan, 2009}) (Fig. 1). The fruit is also of culinary use in making pickles, chutneys, and vegetable dishes. Amla is also used to prepare a sweet delicacy called murabbah, in which the ripe fruit is soaked in concentrated sugar syrup for extended period till the aroma of the fruits exudes into the sugar syrup. The ripe fruit is also used to prepare fresh juice and has been recently marketed as a concentrate to prepare readily usable diluted juice (^{Warrier et al., 1996}).

Phytochemistry

Amla is one of the most extensively studied plants and reports suggest that it contains tannins, alkaloids, and phenolic compounds. Amla is a rich source of vitamin C (478.56 mg/100 ml) and the levels are more than those in oranges, tangerines, or lemons (^{Khan, 2009}). The fruit also contains gallic acid, ellagic acid, chebulinic acid, chebulagic acid, emblicanin A, emblicanin B, punigluconin, pedunculagin, citric acid, ellagotannin, trigallayl glucose, pectin, 1-O-galloyl-β-D-glucose, 3,6-di-O-galloyl-D-glucose, chebulagic acid, corilagin, 1,6-di-O-galloyl-β-D-glucose, 3 ethylgallic acid (3 ethoxy 4,5 dihydroxy benzoic acid), and isostrictiniin (^{Zhang et al., 2003}). It also contains flavonoids such as quercetin, kaempferol 3 O-α-L (6″ methyl) rhamnopyranoside and kaempferol 3 O-α-L (6″ ethyl) rhamnopyranoside (^{Habib-ur-Rehman et al., 2007; Khan, 2009; Krishnaveni and Mirunalini, 2010}). Some of the phytochemicals are shown in Fig. 2.

Traditional uses

A number of medicinal properties are ascribed to amla and it is a necessary constituent of many Ayurvedic medicines (^{Warrier et al., 1996; Poltanov et al., 2009}). Various polyherbal formulations, such as Amlakadi gritha, Amlakadi Tailya, Alakyadi churna, Aamalaki Rasayanam, Asokarista, Avipatikara Churnam, Chyavananaprasa Leham, Dasamularishta, Dhatri lauha, Dhatryarista, Kumaryasava, Panchatika guggulu Ghritam, Thriphala Lepam, Thriphala Guggulu, Thriphala Ghritam, and Thriphala Churnam, are commonly used to treat various ailments (^{Warrier et al., 1996; Kulkarni, 1997}).

It is also of use in Siddha, Unani Tibetan, Sri Lankan, and Chinese systems of medicine (^{Warrier et al., 1996; Poltanov et al., 2009}). In Ayurveda, amla is considered to be a potent rasayana (rejuvenator) and to be useful in stalling the degenerative and senescence process, to promote longevity, enhance digestion, to treat constipation, reduce fever, purify the blood, reduce cough, alleviate asthma, strengthen the heart, benefit the eyes, stimulate hair growth, enliven the body, and enhance the intellect (^{Pandey, 2002}).

In various folk medicines the fruits, which are astringent, are useful in treating ophthalmic problems, dyspepsia, gastritis, hyperacidity, constipation, colitis, hemorrhoids, hematuria, menorrhagia, treat anemia, diabetes, cough, asthma, osteoporosis, premature graying of hair, weakness and fatigue. Amla is also reported to possess hepatoprotective, cardioprotective, diuretic, laxative, refrigerant, stomachic, restorative, alterative, antipyretic, anti-inflammatory properties, is a hair tonic, prevents peptic ulcer dyspepsia, and is a digestive medicine (^{Pandey, 2002}). It is used for a variety of ailments such as anemia, hyperacidity, diarrhea, eye inflammation, leucorrhea, jaundice, nerve debility, liver complaints, cough, and anomalies of urine (^{Pandey, 2002}).

Scientifically validated studies

Preclinical studies carried out in the past three decades have validated many of the traditional uses of amla. Experiments have shown that amla possesses antibacterial, antifungal, antiviral, antidiabetic, hypolipidemic, antiulcerogenic, free radical scavenging, antioxidant, antimutagenic, anti-inflammatory and immunomodulatory, antipyretic, analgesic, antitussive, antiatherogenic, adaptogenic, snake venom neutralizing, gastroprotective, antianemia, antihypercholesterolemia, wound healing, antidiarrheal, antiatherosclerotic, hepatoprotective, nephroprotective, and neuroprotective properties (^{Khan, 2009; Krishnaveni and Mirunalini, 2010}). Compelling preclinical studies with both in-vitro and in-vivo systems have shown that amla possesses anticancer, chemopreventive, cytoprotective, and radioprotective effects. Here, an attempt is made to analyze the role of amla in the treatment and prevention of cancer.

Amla as an antineoplastic agent

Preclinical studies have shown that the aqueous extract of amla causes a concentration-dependent cytotoxic effect on L 929 cells in vitro and that the IC₅₀ was observed to be 16.5 μg/ml (^{Jose et al., 2001}). The extract also caused apoptosis in Dalton's lymphoma ascites and CeHa cell lines (^{Rajeshkumar et al., 2003}). ^{Khan et al. (2002)} studied the antiproliferative activity of the extract in the human tumor cell lines of different histological orgins (human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL) and observed it to be effective.

Recently, ^{Ngamkitidechakul et al. (2010)} have observed that the aqueous extract of amla, which contains tannins (43%), uronic acid (11%), and gallic acid (21%), inhibited the growth of A549 (lung), HepG2 (liver), HeLa (cervical), MDA-MB-231 (breast), SK-OV3 (ovarian), and SW620 (colorectal) cells in vitro. However, at the same concentration the extract did not cause similar level of cytotoxicity in the MRC5, normal lung fibroblast, suggesting it to be safe for normal cells (^{Ngamkitidechakul et al., 2010}). The extract also induced apoptosis in HeLa, A549, MDA-MB-231, and SK-OV3 cells (^{Ngamkitidechakul et al., 2010}).

An amla extract possesses antiproliferative activity in MCF7 and MDA-MB-231 breast cancer cell lines and also induces an increase in ERαmRNA in these cells (^{Lambertini et al., 2004}). The extract was devoid of cytotoxic effects on the normal Chinese hamster ovary cell line, suggesting it to be selectively cytotoxic to only neoplastic cells (^{Sumantran et al., 2007}). Administering the extract to Dalton's lymphoma-bearing mice caused a reduction in ascitic volume (when the tumor cells were inoculated in the peritoneum) and solid tumor growth (when inoculated subcutaneously). The amla extract significantly reduced the solid tumors and prolonged survival time. At a molecular level, the extract was observed to inhibit the cell cycle-regulating enzyme, Cdc25 phosphatase, in a dose-dependent manner and the IC₅₀ was observed to be 5 μg/ml (^{Jose et al., 2001}).

Studies have also shown that some of the compounds present in amla are effective in inhibiting the proliferation of neoplastic cells in vitro and also in tumor-bearing animals. The hydrolyzable tannins of amla are also reported to possess selective cytotoxicity to the human oral squamous cell carcinoma and salivary gland tumor cell lines, while they were nontoxic to the normal human gingival fibroblasts. The dimeric compounds, oenothein B, woodfordin C, and woodfordin D, were more effective than the monomeric compounds, while the macrocyclic ellagitannin oligomers were more effective than gallic acid and epigallocatechin gallate. These compounds also induced apoptosis in the neoplastic cells and mechanistic studies showed that the effect was mediated by the prooxidant actions, but not through the generation of hydrogen peroxide (^{Sakagami et al., 2000}).

^{Zhang et al. (2004)} evaluated the antiproliferative effects of 18 phytochemicals of amla (norsesquiterpenoids, phenolic compounds, and proanthocyanidin polymers) in B16F10, HeLa, and MK-1 cells in vitro. Among the norsesquiterpenoids, it was observed that the glycoside phyllaemblicins B and C were highly potent in all the three cells [B16F10 (GI50 at 2.0, 3.5 μg/ml, respectively), HeLa (GI50 at 3.0, 12.0 μg/ml, respectively), and MK-1 (GI50 at 7.0 μg/ml for both compounds)]. However, with respect to the phenolic compounds, all showed inhibitory activity against the three tumor cell lines (at a concentration of <68 μg/ml), and were more effective against B16F10 than against HeLa and MK-1 cells. The highest activity was observed with corilagin, geraniin, elaeocarpusin, and prodelphinidins B1 and B2 against B16F10 (^{Zhang et al., 2004}).

Pyrogallol, a catechin compound of amla, is also reported to possess a potent antiproliferative effect on human lung cancer cell lines and, to a lesser degree, on the human bronchial epithelium cell line. Detailed studies with the human lung cancer cell lines H441 (lung adenocarcinoma) and H520 (lung squamous cell carcinoma) have shown that pyrogallol inhibited the growth of these cells, triggered apoptosis by increasing Bax and concomitantly decreasing Bcl-2, arrested the cells in the G2/M phase by affecting the cyclin B1, Cdc25C and increasing the phosphorylation of Cdc2 (Thr14). The in-vitro observations also extended into in-vivo studies with xenograft nude mice (^{Yang et al., 2009}).

Gallic acid, another chief constituent of amla, is also shown to cause a concentration- and time-dependent inhibition of proliferation, and to induce apoptosis in BEL-7404 cells (^{Zhong et al., 2009}). Gallic acid is also shown to cause apoptosis in human non-small-cell lung cancer NCI-H460 cells (^{Ji et al., 2009}), A375.S2 human melanoma cells (^{Ji et al., 2009}), human bladder transitional carcinoma cell line (TSGH-8301 cell) (^{Lo et al., 2010}) and HeLa cervical cancer cells (^{You et al., 2010}). Consuming gallic acid (0.3–1% in drinking water) inhibited the growth of prostate cancer and retarded the progression to advanced-stage adenocarcinoma in mice with transgenic adenocarcinoma of the prostate by suppressing cell cycle progression and cell proliferation and, concomitantly, increasing apoptosis (^{Raina et al., 2008}). Gallic acid also suppressed lung xenograft tumor growth (^{Ji et al., 2009}). Some of the other phytochemicals such as quercetin and kampferol also possess antineoplastic effects in the various cultured cell lines (Table 2) and their presence may have also resulted in the observed antineoplastic effect.

Chemomodulatory effects

Chemotherapy is known to possess deleterious effects on normal cells. At times, the effects can be extremely severe and can compel the physician to discontinue or reduce the dose of treatment. This will affect cancer control and ultimately the survival of the patient. In addition, the development of drug resistance is another major problem in the treatment of cancer as chemoresistance can lead to unabated proliferation of the defiant tumor cells and the administered antineoplastic agent can cause nonspecific toxicity to the normal cells. Accordingly, an agent that can selectively protect the normal cells against the deleterious effects of chemotherapy (chemoprotective agents), or can sensitize the tumor cells to anticancer drugs (chemosensetizers), is an attractive proposition in cancer treatment and the goal of researchers (^{Coleman et al., 1988}).

The aqueous extract of amla has been observed to be effective at reducing cyclophosphamide-induced suppression of humoral immunity and to restore the levels of glutathione and the antioxidant enzymes in the kidneys and liver of mice (^{Haque et al., 2001}). Amla is reported to decrease cyclophosphamide-induced DNA damage as measured by a reduction in both micronuclei and chromosomal aberration in the bone marrow cells of mice (^{Sharma et al., 2000a}). Amla reduced the levels of cytochrome (Cyt) P450, increased the levels of the antioxidant glutathione, antioxidant enzymes [glutathione peroxidase (GPx), glutathione reductase], and increased the detoxification enzyme glutathione-S-transferase (GST), which thereby contributed to these observations (^{Sharma et al., 2000a}).

In-vitro studies have shown that amla effectively suppressed the proliferation of the human hepatocellular carcinoma (HepG2) and lung carcinoma (A549) cells and synergized the cytotoxic effects of doxorubicin and cisplatin, two important clinically used antineoplastic drugs (^{Pinmai et al., 2008}). The ethanolic extract of amla also protected the cardiac myoblasts H9c2 cells against doxorubicin-induced toxicity (^{Wattanapitayakul et al., 2005}). Together these observations suggest that it is quite possible that amla prevents doxorubicin-induced cardiotoxicity to the normal cardiac myoblasts and, concomitantly, sensitizes the antineoplastic effects on cancer cells. However, detailed studies are required for this hypothesis to be validated, especially in the relevant animal models of study.

Amla as a radioprotective agent

Since the discovery of the deleterious effects of ionizing radiation, studies have been focused on developing chemical radioprotectors that have the ability to decrease the ill effects of radiation on normal tissues (^{Arora et al., 2005}). The thiol compound amifostine is credited with being the only radioprotector to have been approved by the Food and Drug Administration to reduce the incidence and severity of xerostomia in head and neck cancer patients undergoing radiotherapy (^{Arora et al., 2005}). Unfortunately, the application of this drug has so far been less than hoped for, owing to its untoward toxicity often being evidenced at the optimal radioprotective doses (^{Arora et al., 2005}).

With regard to the radioprotective effects of amla, studies have shown that administering (50, 100, 200, 400, and 800 mg/kg b.wt./day) amla once daily for 7 consecutive days before exposure to sublethal dose of γ-radiation (9 Gy) protected mice against the radiation-induced sickness and mortality (^{Singh et al., 2005}). Among all the doses studied, the optimal effect was observed at 100 mg/kg b.w. as it delayed the radiation-induced lethality and caused a survival of 87.5% when compared with placebo-treated irradiated cohorts in which no survivors were observed (^{Singh et al., 2005}).

Administration of amla (100 mg/kg b.wt.) ameliorated the radiation (5 Gy)-induced gastrointestinal damage as evaluated by the histopathological studies, by quantifying the crypt cell population, mitotic figures, and villus length at all the assay points (12 h–30 days). Reports also suggest that amla ameliorated the radiation-induced hemopoietic damage (^{Hari Kumar et al., 2004}). Feeding mice with 2.5 g/kg b.wt. of amla for 10 consecutive days before exposure to a single dose of 7 Gy of radiation increased the total leukocyte count, bone marrow viability, and levels of hemoglobin. However, treatment with amla after exposure to irradiation (continuously for another 15 days) was not as effective when compared with administeration before radiation, suggesting it to be of use only when exposure to radiation is planned (^{Hari Kumar et al., 2004}).

Mechanistic studies have shown that feeding amla enhanced the activity of the various antioxidant enzymes (catalase, superoxide dismutase, and GPx), the phase II detoxifying enzyme, GST, and the antioxidant thiol, glutathione, in the blood, with a concomitant decrease in the levels of lipid peroxides (^{Hari Kumar et al., 2004}). Similar results were also observed by ^{Jindal et al. (2009)} in mice intestine and together both these studies confirm that amla significantly reduces the deleterious effects of radiation at least in part through its antioxidant and inhibition of lipid peroxidation activities. The phytochemicals ellagic acid, gallic acid, and quercetin (Fig. 2) present in amla also possess radioprotective effects and are shown in Table 3.

Amla as a chemopreventive agent

Cancer chemoprevention has traditionally been defined as a dietary or therapeutic approach for the prevention, delay, or reversal of carcinogenesis with nontoxic agents (^{Bonte, 1993; Pastorino, 1994; Sporn and Suh, 2002}). Epidemiological studies have provided convincing evidence that natural dietary compounds can modify the process of carcinogenesis, which includes the three decisive steps: namely initiation, promotion, and progression, in several types of human cancer (^{Sporn and Suh, 2002}). Experimental studies have also validated the efficacy of a number of bioactive dietary components, supporting the acceptance of natural dietary compounds as chemopreventive agents in the near future. Amla is reported to be effective in stopping initiation, promotion, and progression of cancer and the ability of amla to render chemopreventive effects is discussed in the following sections.

^{Sancheti et al. (2005)} investigated the chemopreventive effects of amla in two-stage carcinogenesis {[7,12-dimethylbenz(a)anthracene] (DMBA)-induced and croton oil promoted} in mice by considering the delay in tumorigenesis, cumulative number of papillomas, tumor incidence, tumor yield, and tumor burden as the end points. The researchers observed that feeding amla for 7 consecutive days before and after DMBA application was less effective than when administered during the promotion (starting from the time of croton oil treatment and continued till the end of experiment for 16 weeks). However, the best effect was observed when amla was fed throughout the experimental period, that is, before and after DMBA application and during the promotional stage.

These observations may be because of the various protective mechanisms that were operating. When amla is administered before DMBA treatment, there will be an increase in the levels of antioxidant and phase II enzymes, with a concomitant decrease in the phase I detoxifying enzymes, which cumulatively may prevent/reduce the process of carcinogenesis. However, when administered during the promotion, amla may trigger the selective apoptosis of the mutated and preneoplastic cells and decrease the carcinogenesis (explained later). The phytochemicals, such as ellagic acid, gallic acid, and quercetin, present in amla also possess chemopreventive effects and may have been responsible for the beneficial effects (Table 4).

Recently, ^{Ngamkitidechakul et al. (2010)} have also observed that the aqueous extract of amla containing tannins (43%), uronic acid (11%), and gallic acid (21%) was effective in delaying and reducing DMBA-induced and (12-otetradecanoylphorbol-13-acetate)-promoted skin carcinogenesis in mice. The topical application of the extract (1, 2, or 4 mg in 0.1 ml acetone) 1 h before each (12-otetradecanoylphorbol-13-acetate) application until the termination of the experiment caused a concentration-dependent decrease in the appearance and incidence of skin papillomas (^{Ngamkitidechakul et al., 2010}). These results clearly suggest the effectiveness of amla when applied topically and also its possible use as a skin care product.

In Ayurveda amla is considered to be a hepatoprotective agent and scientific studies have validated this traditional belief. Studies have shown that amla protects against chemical-induced carcinogenesis and oxidative stress. With regard to chemoprevention, studies by ^{Rajeshkumar et al. (2003)} have shown that feeding amla decreased the N-nitrosodiethylamine-induced liver tumors in rats. Amla decreased the levels of serum γ-glutamyl transpeptidase, alkaline phosphatase, glutamate pyruvate transaminase, and bilirubin (^{Rajeshkumar et al., 2003}). Similar observations were also made when the chemopreventive effects of amla were studied against diethylnitrosoamine-induced and 2-acetylaminoflourine-promoted hepato-carcinogenesis in rats (^{Sultana et al., 2008}).

Prophylactic treatment with amla for 7 consecutive days before the single administration of thioacetamide reverses the thioacetamide-induced oxidative stress and early promotional events of primary hepato-carcinogenesis in rats. Amla inhibited the serum levels of SGOT, SGPT, and GGT; decreased levels of lipid peroxide, inhibited aberrant synthesis of DNA; decreased the activities of GST, GR, G6PD, and ornithine decarboxylase; and concomitantly increased the glutathione content and GPx activity in the liver (^{Sultana et al., 2004}).

Studies have also shown that administering amla reduces the cytotoxic effects of the proven carcinogens such as 3,4-benzo(a)pyrene (^{Nandi et al., 1997}), benzo[a]pyrene (^{Sharma et al., 2000a}), DMBA (^{Banu et al., 2004}) by reducing the mutagenesis, oxidative stress, lipid peroxides, phase I enzymes [cytochrome (Cyt) P450 and Cyt b5], and concomitantly increasing the antioxidants (glutathione) and enzymes (GPx, glutathione reductase, and phase II detoxifying enzyme GST (^{Nandi et al., 1997; Sharma et al., 2000a; Banu et al., 2004}).

In addition to these observations, amla has been scientifically studied for its protective role against country liquor (^{Gulati et al., 1995}), ethanol (^{Pramyothin et al., 2006; Reddy et al., 2009}), carbon tetrachloride (^{Sultana et al., 2005; Lee et al., 2006; Mir et al., 2007}), ochratoxin (^{Verma and Chakraborty, 2008}), hexachlorocyclohexane (^{Anilakumar et al., 2007}), paracetamol (^{Gulati et al., 1995}), and the antituberculosis drugs (rifampicin, isoniazid, and pyrazinamide) (^{Tasduq et al., 2005; Panchabhai et al., 2008})-induced oxidative stress and damage to the liver. Most of these agents are known to be hepatotoxins and to initiate and promote carcinogenesis. By preventing oxidative stress and the resulting damage, amla protects against both hepatotoxicity and possible carcinogenesis.

Mechanisms of action (Fig. 3)

Amla is a free radical scavenger

Excess generation of free radicals, the reactive oxygen species [ROS superoxide anion radical (O₂^•−), hydroxyl radical (OH^•) and hydrogen peroxide (H₂O₂)], and the reactive nitrogen species [RNS nitric oxide (NO), peroxynitrite (ONOO⁻)], respectively, causes oxidative stress and nitrosative stress. The free radicals that are generated are highly reactive and cause damage to the membrane lipids, proteins, and DNA (^{Devasagayam et al., 2004}). Accordingly, their prevention is important in preventing cell damage, mutagenesis, and carcinogenesis.

In-vitro studies have shown that amla scavenges 2,2-diphenyl-1-picrylhydrazyl radicals (^{Naik et al., 2005; Hazra et al., 2010}), superoxide anions (^{Naik et al., 2005; Hazra et al., 2010}), hydroxyl radical (^{Hazra et al., 2010}), nitric oxide (^{Hazra et al., 2010}), hydrogen peroxide (^{Hazra et al., 2010}), peroxynitrite (^{Hazra et al., 2010}), singlet oxygen (^{Hazra et al., 2010}), and hypochlorous acid (^{Hazra et al., 2010}). The phytochemicals, such as gallic acid, ellagic acids, emblicanin A, and emblicanin B, are also reported to possess free-radical-scavenging effects in the 2,2-diphenyl-1-picrylhydrazyl assay and efficacy was as follows: A emblicanin greater than B emblicanin greater than gallic acid greater than ellagic acid greater than ascorbic acid (^{Pozharitskaya et al., 2007}).

Studies have also shown that the methanol extract of amla and its various fractions (hexane, ethyl acetate, and water fractions) possess NO scavenging effects. The isolated compounds, such as gallic acid, methyl gallate, corilagin, furosin, and geraniin, which were isolated from the ethyl acetate fraction that possessed the best NO-scavenging effect, were also effective. Gallic acid was found to be a major compound in the ethyl acetate extract and geraniin showed highest NO-scavenging activity among the isolated compounds (^{Kumaran and Karunakaran, 2006}).

Amla decreases phase I enzymes

Phase I drug-metabolizing enzymes, especially the CYP P450 mixed-function oxidases, which are involved in the biotransformation of xenobiotics, can transform a nontoxic chemical (procarcinogen) into a harmful toxic substance (ultimate carcinogen), which can induce damage to the nucleic acids and other macromolecules (^{Percival, 1997}). Studies have also shown that administering the ethanolic extract of amla reduced the hepatic levels of the activating enzymes, Cyt P450 and Cyt b5, which are important in converting the procarcinogen DMBA into ultimate carcinogen (^{Banu et al., 2004}). In addition, the inhibition of microsomal-activating enzymes, including Cyt P450, was also responsible for the antimutagenic effects of amla against 2-aminofluorene (^{Arora et al., 2003}), aflatoxin B1, and benzo[a]pyrene-induced mutagenesis in the Ames test (^{Sharma et al., 2000b}).

Amla increases glutathione S-transferase, a phase II enzyme

The reactive species formed by the phase I enzymes are often detoxified by phase II drug-metabolizing enzymes. In the reaction, the hydrophobic intermediates generated by the phase I enzymes are converted to a water-soluble group, thus decreasing their reactive nature, and allowing subsequent excretion (^{Jana and Mandlekar, 2009}). A properly functioning and balanced phase II system would detoxify the metabolically activated carcinogen, thereby preventing mutagenesis and carcinogenesis. Agents preferentially activating phase II over phase I enzymes can be more beneficial as chemopreventive agents (^{Percival, 1997; Jana and Mandlekar, 2009}).

Studies have shown that amla increases the level of GST and thereby reduces the toxic effects of N-nitrosodiethylamine (^{Jeena et al., 1999; Rajeshkumar et al., 2003}), benzo[a]pyrene (^{Sharma et al., 2000a}), cyclophosphamide (^{Sharma et al., 2000a}), thioacetamide (^{Sultana et al., 2004}), CCl4 (^{Sultana et al., 2005}), ionizing radiation (^{Hari Kumar et al., 2004}), hexachlorocyclohexane (^{Anilakumar et al., 2007}), arsenic (^{Panchabhai et al., 2008}), ethanol (^{Reddy et al., 2009}), and ochratoxin (^{Sultana et al., 2004}). Molecular studies have also shown that amla increased GSTP1 expression (^{Niture et al., 2006}), thereby validating the biochemical observation.

Amla decreases ornithine decarboxylase

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in polyamine synthesis. High levels of ODC are an adverse prognostic factor as it is observed to be important in tumor proliferation, progression, and metastasis and for the survival of cancer patients (^{Manni et al., 2002}).

Studies have shown that administering amla inhibited thioacetamide-induced hyper-proliferation in rat liver by decreasing the levels of ODC activity and thymidine incorporation in DNA (^{Sultana et al., 2004}). These observations clearly indicate the inhibitory effects of amla on ODC and DNA replication, steps that are important in tumor cell proliferation.

Amla increases the antioxidant enzymes

The antioxidant enzymes, superoxide dismutase, GPx, and catalase, cooperate or, in a synergistic method, work to protect cells against oxidative stress. The superoxide dismutase catalyses the dismutation of superoxide radicals, a major form of ROS, into hydrogen peroxide, which is acted on by the GPx and catalase to give water. When an appropriate balance exists between these three enzymes, oxidative stress is reduced and the cells are protected from the cytotoxic and mutagenic effects of the ROS (^{Devasagayam et al., 2004}).

Preclinical studies have conclusively shown that amla ameliorates the oxidative and xenobiotic-induced stress, mutagenesis, and carcinogenesis by increasing the antioxidant enzymes. Reports suggest that amla increases the antioxidant enzymes and prevents benzo[a]pyrene (^{Sharma et al., 2000a}), cyclophosphamide (^{Sharma et al., 2000a}), DMBA (^{Banu et al., 2004}), γ-radiation (^{Hari Kumar et al., 2004; Jindal et al., 2009}), hexachlorocyclohexane (^{Anilakumar et al., 2007}), and ethanol (^{Pramyothin et al., 2006})-induced toxic effects.

Amla decreases lipid peroxidation

Lipid peroxidation is one of the most evaluated consequences of free radicals on membrane structure. The polyunsaturated fatty acids are vulnerable to peroxidative attack and this can cause loss of fluidity, decreased membrane potential, increased permeability for protons and calcium ions and eventually loss of cell membranes, and result in pathological and toxicological processes (^{Devasagayam et al., 2004}). The major aldehydic end product of lipid peroxidation is malondialdehyde and is mutagenic in the bacterial and mammalian systems of studies.

Multiple studies have shown that amla possesses inhibitory effects on lipid peroxidation induced by various inducers. In-vitro studies have shown that amla prevents radiation-induced lipid peroxidation (^{Naik et al., 2005}) and this effect also extends to animal studies (^{Hari Kumar et al., 2004; Jindal et al., 2009}). Amla inhibits cadmium (^{Khandelwal et al., 2002}), carbon tetra chloride (^{Sultana et al., 2005}), arsenic (^{Panchabhai et al., 2008}), ethanol (^{Reddy et al., 2009}), ochratoxin (^{Chakraborty and Verma, 2010}), N-nitrosodiethylamine (^{Rajeshkumar et al., 2003}), and thioacetamide (^{Anilakumar et al., 2007})-induced lipid peroxidation. By inhibiting lipid peroxidation amla may contribute toward the observed beneficial effects, at least in part.

Amla possess anti-inflammatory effects

Chronic inflammation has been proved to cause free radicals and the resulting oxidative and nitrosative stress is known to directly or indirectly contribute toward malignant cell transformation by inducing genomic instability, alterations in epigenetic events, inappropriate gene expression, enhanced proliferation of mutated cells, resistance to apoptosis, tumor neovascularization, and metastasis (^{Kundu and Surh, 2005}).

Experiments have shown that the aqueous fraction of methanol extract of the leaves possesses anti-inflammatory effects in carrageenan-induced and dextran-induced rat hind paw edema. Mechanistically, it was observed that the extract inhibited migration of human polymorphonuclear cells and exerted its anti-inflammatory effects (^{Asmawi et al., 1993}). Studies have also shown that amla extract and the phytochemical pyrogallol also possess anti-inflammatory effects and inhibited the Pseudomonas aeruginosa laboratory strain PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-α, GRO-γ, of the adhesion molecule, ICAM-1, and of the pro-inflammatory cytokine, IL-6 (^{Nicolis et al., 2008}). Recently, ^{Muthuraman et al. (2010)} have also observed that the phenolic compounds from amla possess anti-inflammatory effects in the carrageenan and cotton pellet-induced acute and chronic inflammatory response in animal models of study. The effect was significant at high doses and was comparable to the positive control, diclofenac (^{Muthuraman et al., 2010}).

Antimutagenic effects

The initial step in the process of carcinogenesis is induction of mutation in the oncogenes or tumor-suppressor genes of the genome of a somatic cell. Therefore, its prevention is of great importance (^{Weisburger, 2001}). Multiple studies carried out in the last two decades have conclusively shown that amla prevents DNA damage against different carcinogens and mutagens. Using the standard Ames test, ^{Sharma et al. (2000b)} observed for the first time that the aqueous extract of amla inhibited aflatoxin B1 and benzo[a]pyrene-induced mutagenesis in the Salmonella typhimurium strains TA 98 and TA 100.

Amla is also reported to increase the levels and activities of O6-methylguanine-DNA methyltransferase, an enzyme important for removing the highly mutagenic adducts formed by alkylating agents in human lymphocytes (^{Niture et al., 2006}). Amla was also effective in preventing the radiation-induced damage in the plasmid DNA assay (^{Naik et al., 2005}), suggesting its effectiveness against different classes of mutagens.

In addition, studies with experimental animals have shown that amla prevents cadmium (^{Khandelwal et al., 2002}), lead (^{Dhir et al., 1990}), aluminium (^{Dhir et al., 1990}), nickel (^{Dhir et al., 1991}), cesium chloride (^{Ghosh et al., 1992}), arsenic (^{Biswas et al., 1999}), chromium (^{Sai Ram et al., 2003}), 3,4-benzo(a)pyrene (^{Nandi et al., 1997}), benzo[a]pyrene (^{Sharma et al., 2000a}), DMBA (^{Nandi et al., 1997}), and cyclophosphamide (^{Sharma et al., 2000a})-induced DNA damage. Together these observations clearly suggest the effectiveness of amla in preventing mutagenesis and DNA damage, which would inhibit/reduce the incidence and process of carcinogenesis, at least in part.

Amla possesses immunomodulatory effects

Immune activation is an effective protective approach against emerging infectious diseases and certain cancers. Immunostimulants enhance the overall immunity of the host, present a nonspecific immune response against microbial pathogens and increase humoral and cellular immune responses, by either enhancing cytokine secretion, or by directly stimulating B-lymphocytes or T-lymphocytes (^{Spelman et al., 2006}). In Ayurveda, amla is considered to be an immunostimulatory agent and scientific studies have validated this (^{Warrier et al., 1996; Kulkarni, 1997; Khan, 2009; Krishnaveni and Mirunalini, 2010}).

Studies have shown that amla enhances natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity in BALB/c mice bearing Dalton's lymphoma ascites tumor. Amla increases the life span of tumor-bearing animals and this was because of the increase in the activation of splenic NK cell activity and antibody dependent cellular cytotoxicity. However, the increase in survival was completely abrogated when the NK cell and killer cell activities were depleted, either by cyclophosphamide or anti-asialo-GM1 antibody treatment, validating that the observed effects were because of its immunomodulatory effects (^{Suresh and Vasudevan, 1994}).

Amla and its phytochemicals modulate the levels of proteins important in cell cycle progression

Cancer is frequently considered to be a disease of the cell cycle and a convincing body of data has proved that the disruption of the normal regulation of cell-cycle progression and division are important events in cancer development (^{Hanahan and Weinberg, 2000; Kastan and Bartek, 2004}). The progression of the cell cycle is a tightly regulated and highly ordered process involving multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity (^{Kastan and Bartek, 2004}). The cyclin-dependent kinases (CDKs) and their respective partners (cyclin) are responsible for the progression of the cell cycle, whereas the CDK inhibitors act as brakes to stop cell cycle progression (^{Hartwell and Weinert, 1989}). The genesis of cancer is principally because of the derailed expression or activation of positive regulators and functional suppression of negative regulators (^{Hartwell and Weinert, 1989; Kastan and Bartek, 2004}).

Studies by ^{Jose et al. (2001)} have shown for the first time that amla extract caused a dose-dependent inhibition of the cell cycle-regulating enzyme Cdc25 phosphatase in vitro, with an IC₅₀ of 5 μg/ml (^{Jose et al., 2001}). The phytochemical pentagalloylglucose is shown to cause G1 arrest in human Jurkat T cells by elevating p27Kip1 and p21Cip1/WAF1 proteins (^{Chen and Lin, 2004}). Gallic acid induces cell cycle arrest by decreasing CDKs and cyclins. It phosporylates Cip1/p21 and cell division cycle 2 (Cdc2), Cdc25A, and Cdc25C in DU145 cells (^{Sun et al., 2004}). It also induces G2/M phase cell cycle arrest by regulating 14-3-3β release from Cdc25C; activation of chk2; decreasing CDK1, cyclin B1, and Cdc25C; increasing phosphorylation of p-Cdc2 (Tyr-15), Cip1/p21 and Cdc25C in human bladder transitional carcinoma cells (TSGH-8301cells) (^{Ou et al., 2010}). Gallic acid feeding also reduces Cdc2, CDK2, CDK4, CDK6, cyclin B1, and E in the prostatic tissue of mice with transgenic adenocarcinoma of the mouse prostate (^{Raina et al., 2008}).

Amla and some of its constituents cause apoptosis and cytotoxicity of neoplastic cells

Apoptosis, a process by which the cell is committed to death by not initiating an inflammatory response, is vital in regulating tissue homeostasis (^{Sun et al., 2004; Ghobrial et al., 2005}). A large body of evidence has proved that the processes of neoplastic transformation, progression, and metastasis involve alterations of the normal apoptotic pathway and that the number of cell deaths is very low in these cells (^{Sun et al., 2004; Ghobrial et al., 2005}). Therefore, the induction of apoptosis is arguably the most potent defence against cancer as it effectively eliminates the mutated and severely damaged cells. Accordingly, agents that can eliminate mutated, preneoplastic, and neoplastic cells by sparing the normal cells are supposed to be an effective chemopreventive agent and to offer therapeutic advantage in the elimination of cancer cells (^{Sun et al., 2004; Ghobrial et al., 2005}).

The ability of the extract of amla and some of its phytochemicals to induce apoptosis in cancer cells contributes to the understanding of its anticancer and chemopreventive potential. Studies have shown that the aqueous extract of amla induces apoptosis and inhibits the growth of HeLa, MDA-MB-231, and SK-OV3 without affecting the normal lung fibroblast, MRC5 (^{Ngamkitidechakul et al., 2010}). The hydrolyzable tannins possess selective cytotoxicity to the human oral squamous cell carcinoma and salivary gland tumor cell lines, whereas they were nontoxic to the normal human gingival fibroblasts (^{Sakagami et al., 2000}). Studies have also shown that quercetin (^{Son et al., 2004}), gallic acid (^{Isuzugawa et al., 2001}), ellagic acid (^{Losso et al., 2004}), and pyrogallol (^{Yang et al., 2009}) also possess cytotoxic and apoptogenic effects on the neoplastic and transformed cells, but not in normal cells. Together, these observations clearly suggest that the presence of these compounds in amla resulted in the elimination of the mutated and neoplastic cells and resulted in the desired effects in both antineoplastic effects and chemoprevention.

Amla and some of its constituents prevent metastasis

Cancer cells differ from normal cells; the most important being the loss of differentiation, self-sufficiency in growth signals, limitless replicative potential, decreased drug sensitivity, increased invasiveness, and metastasis (^{Hanahan and Weinberg, 2000}). Metastasis, the process by which some of the neoplastic cells spread from the primary site to distant tissue, is the life-threatening aspect of cancer. It is the hallmark of cancer and is responsible for the failure of treatment and death. The process of tumor metastasis is extremely complex and involves myriad biochemical interactions operating concurrently or sequentially. The important steps in the process of metastasis are (i) invasion and migration, (ii) intravasation, (iii) circulation, (iv) extravasation, and (v) colonization, proliferation, and angiogenesis (^{Chiang and Massagué, 2008; Leber and Efferth, 2009}). Cell invasion is one of the fundamental processes required during tumor progression and metastasis and matrix metalloproteinases (MMPs), a group of enzymes that regulate cell-matrix composition, are important in this process (^{Chiang and Massagué, 2008; Leber and Efferth, 2009}).

Recent studies have suggested that the aqueous extract of amla was effective in preventing the invasion of MDA-MB-231 cells in the in-vitro matrigel invasion assay (^{Ngamkitidechakul et al., 2010}). The amla phytochemical, kaempferol, inhibited the expression of stromelysin 1 (MMP-3) in the MDA-MB-231 breast cancer cell line (^{Phromnoi et al., 2009}). The polyphenol gallic acid is also reported to possess inhibitory effects on gastric adenocarcinoma cell migration, decreased expression of MMP-2/9 in vitro (^{Ho et al., 2010}), and metastasis of P815 mastocytoma cells to the liver of DBA/2 mice (^{Ohno et al., 2001}). The flavanol, quercetin, decreased the expression of gelatinases A and B (MMP-2 and MMP-9) in the human metastatic prostate PC-3 cells (^{Vijayababu et al., 2006}) and stromelysin 1 (MMP-3) in the MDA-MB-231 breast cancer cell line (^{Phromnoi et al., 2009}) and inhibited the lung metastasis of murine colon 26-L5 carcinoma cells (^{Ogasawara et al., 2007}) and B16-BL6 murine melanoma metastasis in mice (^{Piantelli et al., 2006}).

Conclusion

Preclinical studies carried out in the past two decades have clearly shown that amla possesses antineoplastic, chemomodulatory, chemopreventive, and radioprotective effects. Several mechanisms are likely to be responsible for the observed effects, the most important being the induction of apoptosis of neoplastic and preneoplastic cells, free radical scavenging, antioxidant, antimutagenic, anti-inflammatory activities; increase in the antioxidant enzymes, modulation of phase I and II enzymes and immunomodulatory effects (Fig. 3). It is unlikely that all targets and cell biological effects found in vitro individually may be operating in the animal system, but studies should be attempted to understand whether the effects observed in vitro translate to the animal system.

Although studies on the effects of amla on some cancer cell lines and animals substantiate its effectiveness, countless possibilities for investigation still remain. Relevant animal and cell culture studies are required to understand the underlying mechanism of action, especially with the phytochemicals. In addition, rationally designed clinical trials are also needed to understand the maximum permissible dose and also to assess for its adverse effects, if any, following consumption over longer periods.

From a phytochemical perspective, there is considerable variation in the composition among various samples of amla. A quality control should be established for the authenticity of the plant and the presence of active phytochemicals, especially gallic acid, ellagic acid, chebulinic acid, quercetin, chebulagic acid, corilagin, kaempferol, apigenin, luteolin, emblicanin A, and emblicanin B in the required levels. Experiments should also be performed to understand which of the phytochemicals are effective and their mechanisms of action.

Studies indicate that amla and some of its phytochemicals (gallic acid, pentagalloylglucose, ellagic acid, quercetin, and kaempferol) are cytotoxic to neoplastic cells, whereas the normal cells are unaffected. It is quite possible that these compounds exert their effects on neoplastic cells that have aberrant cell cycle progression. It is observed that these molecules induce apoptosis and cytotoxicity by modulating the proteins involved in cell progression, and the observations of ^{Jose et al. (2001)} support the hypothesis. However, detailed studies are needed on this aspect with a range of cells encompassing normal, mutated, preneoplastic, and highly metastatic cell lines of different histological origins and cell doubling time.

Owing to its abundance, low cost, and safety in consumption, amla remains a species with tremendous potential and countless possibilities for further investigation. Amla has the potential to develop as a nontoxic anticancer, chemopreventive agent, and as an adjuvant to radiotherapy and chemotherapy when lacunas existing in knowledge are understood. The outcomes of such studies may be useful for the clinical applications of amla in humans against different cancers and may open up a new therapeutic avenue.

Acknowledgements

The authors are grateful to Rev. Fr. Patrick Rodrigus (Director), Rev. Fr. Denis D'Sa (Administrator), Dr Sanjeev Rai (Chief of Medical Services), and Dr Jaya Prakash Alva, (Dean) of Father Muller Medical College for their unstinted support. They also thank to Harshith P. Bhat for drawing the chemical structures. The authors dedicate this review to Professor Ramdasan Kuttan of Amala Cancer Centre, Thrissur, India. Professor Kuttan is a pioneer cancer researcher and his work on the radioprotective and chemopreventive effects with amla has been a source of inspiration to the authors. This study was not supported by any private or public funding body.

The authors declare that they do not have any conflict of interest.