INTRODUCTION:

Cancer occurs to be the second-largest cause of death worldwide with a mortality rate of 9.8 billion till 2018. Besides United States encompasses to be the most affected population globally¹. Cancer is a broad word that refers to a variety of malignant diseases that can affect various parts of the body². Cancer is group of disease characterised by abnormal cell proliferation that can spreadto other areas of the body³. According to the world health organisation, plants have the capacity to synthesise a wide range of chemical compounds that are used to treat a variety of diseases⁴.

In contrast to the chemically synthesized anticancer compounds which are extracted from plant sources, such as Catharanthus roseus, Taxus brevifolia, Cephalotaxus species, Curcuma longa, and many others, Amygdalin shows great therapeutic effect on both antitumor and anticancer activity. Amygdalin is one of the nitriloside, which contains abundant cyanide in the seeds of the prunasinin family which includes, black berries,plums, apples, blueberry, peaches, apricots etc^5,6, Amygdalin shows pharmacological action containing a cyanogenic component possessing a chemical formula C20H27O11 and a molecular mass of 457.42g/mol. Additionally Amygdalin is also called as laetrile (vitamin B17) which consists of hydrocyanic acid, benzaldehyde, and two glucose molecules (D-mandelonitrile-β-D-glucoside-6-β-glucoside).⁷

Amygdalin under amygdalase and prunasin is non-toxic in general but decomposed into benzaldehyde and hydrocyanic acid will hydrolyze and produces prunasin and mandelonitrile HCN) that is a poisonous product.^8,9 The various study states that amygdalin’s effect has a wide range of remedial actions as a supporting role in several diseases which includes bronchitis, leprosy, asthma, etc,^10,11. Alongside it also shows numerous pharmacological activities like analgesia, anti-inflammation, anticancer, antitussive, antiasthmatic, antiulcer, immunoregulation, antifibrosis, immunosuppression¹². Cancer is a severe illness that affects the majority of the world’s population¹³. Amygdalin is already using for anticancer activity in different countries like America, Japan, Germany, Italy which functions by decomposing carcinogenic substances, blocking the supplements for tumor cells thereby inhibiting cancer cell development^14,10. Additionally, this compound diminishes the disease manifestation, progressive stages of cancer, and also enhances the surveillance stretch. Thus amygdalin shows a potential effect on anti-cancer activity¹⁰. Furthermore, in recent years, studies reported beneficial action on several other cancers as well, which includes prostate cancer, breast cancer, lung cancer, cervical cancer, bladder cancer, and colon cancer. The main mechanism of action by which amygdalin shows its unique anticancer activity includes inducing apoptosis and inducing a reduction of the tumor by inhibiting the cell cycle and reducing tumor cell metastasis.

Pharmacological activity:

Amygdalin is one of the ingredients of the san she Dan Bei Ye formula used as Chinese medicine for the treatment of asthma, relieve cough, and relaxing the bowels, as well as for constipation¹⁵.

Antifibrosis effect:

Amygdalin has an anti-fibrosis action and has been shown its inhibitory effect on various fibrosis such as liver fibrosis, renal fibrosis, pancreatic fibrosis, pulmonary fibrosis, and renal interstitial fibrosis.

Renal fibrosis:

The pathogenicity of renal fibrosis can be described by excessive production and accumulation of extracellular matrix(ECM), thereby responsible for tissue scarring and fibrotic lesion¹⁶. Renal interstitial fibroblasts remain essential effector cells responsible for the overproduction of the extracellular matrix in the fibrotic kidney and their activation as a major event of chronic fibrosis pathogenesis¹⁷. Treatment with amygdalin of the cultured renal intestinal fibroblasts inhibited the production and proliferation of transforming growth factors (TGF-β). Delivering of amygdalin to rat model causes the elimination of extracellular matrix aggregation and reduces renal impairment on 3^rd week generally, additionally, it reduces kidney fibroblast stimulation and also rats renal interstitial fibrosis¹⁸.

Liver fibrosis:

Hepatic stellate cells are essentials for excretion and regulating the production of ECM who’s excessive generation and accumulation is the main reason for the pathophysiology of liver fibrosis.^12,19. TGF-β1 mediated pathway is responsible for the development of cells, HSC activation, ECM remodeling, collagen accumulation, also various hepatic fibrosis processes²⁰. Furthermore HSC activation can secrete TGF-β, give rise to the production of ECM, and release cytokines and chemokines associated with hepatic fibrogenesis²¹. TGF-β is a crucial intermediator for hepatic fibrosis and the connective tissue growth factor is the main tissue alteration and fibrosis mediator which functions for TGF-β downstream. As a consequence, both take part in the progression of liver fibrosis. Thus treatment with amygdalin reduces mRNA (200µg/ml), degree of CTGF and TGF-β, and protein expression as a result amygdalin could be an innovative treatment for liver fibrosis¹².

Anti-inflammatory:

Inflammation is a key physiologic defensive system that helps the body against infections, burns, harmful chemicals, allergens, and other noxious stimuli²². Macrophages induce the production of pro-inflammatory cytokines such as IL-1β, IL- 6, and IL- 12, tumor necrosis factor-alpha, IL-23, chemokine 2, and chemokine 5 which plays a vital role in innate immunity and also activates the downstream pathways of nuclear factor kappa B (NF-kB) and p38 throughout the inflammatory response. Overactivation of p38 MAPK/NF-B communication performs salient role in inflammatory disorders^23,24. Amygdalin reduces the synthesis of prostaglandins E2 , cyclooxygenase (COX1 and COX2), shows decreased expression of IL-23, IL-17A, chemokine 2, chemokine 5 mRNA, and p-p38 protein grade, also nitric oxide synthase mRNA in BV2 microglial cells of a mouse in in-vitro studies ¹⁶. In in-vivo, the amygdalin has a defensive effect against inflammation associated with disease and decreased TNF, IL-1, IL-6, soluble intracellular adhesion molecules-1, and NF-kB¹².

Toxicity of Amygdalin:

The oral route of administration has a lethal dose (LD50) of 880mg/kg body weight in mice²⁵. whereas the LD50 of intravenous injection was found to be 25gm/kg and for the intraperitoneal route, LD50 was found to be 8g/kg which shows the oral route of administration is more toxic when compared to intravenous and intraperitoneal²⁶. This effect of toxicity due to the formation of hydrocyanic acid which is produced by intestinal microbes that hydrolyze the amygdalin into hydrocyanic acid²⁷. This was proved when mice were treated by inhibition of intestinal microbes and administered 300 mg/kg had no death occurred when compared to untreated mice which have a mortality rate of 60% at the same dose. Humans when administered 4 gm of amygdalin per day continuously for half a month lead to systemic toxicity. In the case of intravenous, the toxicity occurred in a month. However, after the withdrawal of the drug this toxicity can be reversed. The ideal amount of oral dose of amygdalin daily without toxicity is 0.6 to 1gm⁸.

Antitumor activity of Amygdalin:

Amygdalin was initially extracted out of bitter almond during the 1830s by Robiquet and Boutron-charlard²⁸. This was first tried by a Russian doctor for the treatment of cancer in 1845 based on the rationale that amygdalin was hydrolyzed and treated for cancer cells because of their differential enzymatic profile and liberation of cyanide which causes the killing of cancer cells¹⁵^,29. Subsequently in the 1850s, intravenous amygdalin entitled as a patent. However USA national cancer institute analysis exhibits that, oral and IV type of amygdalin produced by Mexico didn’t comply with the American drug manufacture standard⁸. Consequently in 1892 the use of amygdalin for the treatment of cancer is removed in Germany, it was found to be too toxic and ineffective ¹⁵^,³⁰. Amygdalin at foremost utilized in America for cancer therapy during 1920s by Dr. Krebs and his son Ernst. They formulated a decreased toxic intravenous variety of amygdalin and laetrile, patented in 1950s. In 1961 an amygdalin derivative called hexuronic acid was granted permission and used as a preservative in food products. It has been recorded that 70,000 American cancer victims were reported to use amygdalin as a complementary and alternative medication in 1978 being the most common anticancer technique in the 1970s³¹. Later in 1979 amygdalin was found to be toxic by the American Food and Drug Administration (FDA). Subsequently in the year 1980 amygdalin was prescribed for the treatment of advanced stages of cancer⁸^,20. Mexico produces large amount of amygdalin towards the manifestation of cancer. The latest evidence has provided sufficient evidence for the activity of amygdalin in cancer treatment. cyanogenic sugar a glycoside derivative is also considered as an alternative for anticancer treatment.

Amygdalin produces antitumor activity by various mechanisms:

Apoptosis:

Apoptosis is a common defence mechanism that leads to programmed cell death. This mechanism takes place in different pathological situations which includes removal of dead /damaged cells, such as shrinkage of cells, condensation of chromatin, and inter-nucleosomal DNA fragments and causes the formation of apoptotic bodies¹⁰^,³². Members of the Bcl2 family and caspases (cysteine proteases) are 2 major classes of proteins that take part in apoptosis. However, bcl2 family is of two classified types which bcl2, antiapoptotic proteins, and Bax, a pro-apoptotic protein. One way to estimate the induction of apoptosis in several tissues is the ratio of bcl2 to Bax¹⁰^,21.In the aspect of caspases, caspases-3 activation is considered as the primary mechanism of apoptosis. Several studies proved that apoptosis of cancer cells results in the reduction of the tumor. Anti-tumor drugs are involved in apoptosis of cancer by damaging, inhibiting DNA synthesis, removal of intracellular nucleotide pool, and damaging mitotic apparatus. A study conducted by Chen et al and involved a decrease in the level of Bcl-2 and an increase in the level of Bax when treated with amygdalin in HeLa cells ¹⁰^,³³.

Role of Amygdalin in prostate cancer cells:

In developing countries, prostate cancer comes in second place in the structure of oncological diseases³⁴. A study was conducted by using DU1455 and LNCAP prostate cancer cells and treatment with amygdalin exhibited decreased level of Bcl-2 mRNA, protein expression and also increase of Bax mRNA and protein expression in a concentration-dependent manner additionally elevated the level of caspases 3 enzymes¹⁰.

Role of Amygdalin in breast cancer:

The uncontrolled growth of abnormal cells in the milk producing glands of the breast or in the passages that carry milk to the nipples characterises breast cancer³⁵. A study conducted by Lee and Moon on apoptotic and anti-tumor behavior of amygdalin on various cell lines such as ER-positive MCF-7, TNBC M5-MB-231, and HS578T³⁶ and found that mitochondrial activity triggers apoptosis by mechanisms such as molecular events such as ex-proteins binding with Bcl2 (Bax) and caspase 3 activations³⁷. PARP (poly (ADP-ribose)polymerase) acts as a substrate for caspase 3 in apoptosis and also in pp3821 involve in signaling mechanisms^38,39. Thus, by using the property of amygdalin to regulate apoptotic proteins and signaling molecules determination of PARP, procaspase 3 mitochondrial protein which involves apoptosis i.e, Bcl2 and Bax in TNBC-HS578T alls. Lee and Moon proved amygdalin increases the amount of pro-apoptotic Bax protein and caspase 3 and decreases the level of anti-apoptotic protein Bcl2. Also, the study proved the activation of the P38 MAPK signaling molecule, a pro-apoptotic protein, and also the activity of caspase 3 in HS578T cells of breast cancer⁵. Amygdalin suppresses the growth of cancer cells and activity is dependent on concentration and duration of treatment in a study by using T47D and MCI7 human breast cancer cell lines and found that MCF-7 was 6 times/6 fold higher in untreated cell lines by estimates the glutathione levels and similarly in case of TD7D it was 2.1 times higher in untreated cell lines⁴⁰. Amygdalin also inhabits several cells such as U87-MG brain glioblastoma, MDA-MB-231, MCF7 breast adenocarcinoma^{5, 41}.

Role of Amygdalin in cervical cancer:

Cervical cancer is the world’s fourth most common cancer among women⁴². According to estimates cervical cancer affects between 500,00 and 300,000 women per year⁴³. Studies also reported that Amygdalin mediated apoptosis by increasing the level of Bax and decreasing the level of Bcl2 and procaspase-3 in the concentration-dependent manner of HeLa cell lines in human cervical cancer⁴⁴.

Role of Amygdalin in the reduction of tumor by inhibition of cell cycle.

The pathway involved is protein kinase B(Akt) mammalian target of rapamycin, (MTOR), also plays a significant role in castration-resistant diseases and also in prostate cancer. During castration-resistant diseases the study conducted by PC3, DU145 cell line, and castration sensitive LNCaP cell lines, amygdalin reduced cell count in a concentration-dependent manner. This study showed a reduction in the growth of above cells and stop their colony formation without any signs of toxicity and found to inhibit G2/M phase and S phase of cell cycles and increased in several phases and G0/G1 which is estimated by flow cytometry, this led to the proof to downregulate the cell cycle proteins such as cyclin-dependent kinases1 (cdk1),(cdk2),cdk4 & cyclin A, B & D3 when administered for two weeks by Amygdalin⁴⁵. It reduced the mRNA levels of exonuclease-1, ATP binding cassette, subfamily F, member2 recombinant meiotic recombination 11 homology topoisomerase1 &FK506 binding protein 12-rapamycin associated protein 1 during a study of human colon cells (SNU-C4)and it led to the decrease of genes encoding the cell cycle in SNU-C4 cancer cells⁴⁶. Amygdalin reduced G2, M, S phase and led to the delay in cell progression and G0/G1 phase when studied in UMUC3, RT112&TCCSUP bladder cancer cell lines when given (1-10mg/ml), it may also suppress cancer growth by down-regulation of cdk2 & cycling⁴⁷^,⁴⁸. The cytotoxic and antiproliferative effect was seen in oral SCC cell lines when amygdalin was administered 10-200mg/ml and also decreased the viability of KB cells by concentration dependant manner¹². Amygdalin can also be used in bladder cancer treatment since it can act on bladder cancer cell lines such as UMUC-3, TCCSUP & RT112 during in-vitro studies, when administered after 24 h it arrests the G0/G1 phase in UMUC3 & TCCSUP cell lines in the RTT2cell lines it inhibits the S phase first after two weeks of treatment it alters G0/G1 as well and thus proves its effect on mitosis by altering the cell cycle⁵^,⁴⁹. Amygdalin was also used for renal cancer since it showed a promising effect on RCC cells by inhibiting its activity by altering the cell cycle and possibly by increasing the number of cells in the G1 phase and vise versa in the S phase ⁴⁷. In the RCC cell lines decrease in CDK1 & cyclin B which is the protein required for entering the mitotic phase is observed and it may also directly or indirectly influence change in CDK2 and cyclin A levels, which increase theG1 phase cells ⁴⁵. RCC cell activity inhibition is also based on a protein P19 which is responsible for the inhibition process of cells from the G1 to S phase which leads to the ceasing of the proliferation process⁵.

Amygdalin reducing metastasis of tumor cells:

The FAK is a protein that is linked with integrin was found necessary for the movement of urothelial cancer cells. The blocking of FAK could stop the movement of cells during migration in-vitro. Tumor metastasis is associated with integrin β1 and β4 and FAK can be triggered led to an increased β-catenin level further which stimulates, the release of ILK for the initiation of downstream signaling of AktmTOR, leading to a proliferation of cells, adhesion, and metastasis. Whereas β2 leads to activation of migration-associated protein by targeting the nucleus specifically and leads to the growth of cancer⁵⁰. Amygdalin downregulated the expression integrin β1, β4, βcatenin, FAK, p- FAK, ILK, and upregulated E-cadherin levels in lung cancer cells H1299/M and PA/M and lead to suppression of cell proliferation⁵⁰. Amygdalin reduces cell adhesion in cell lines of the bladder such as TCCSUP, UMUC3 and RT112 which leads to a decrease in the binding affinity to immobilized collagen⁴⁵. Amygdalin (1000 to 2000mg/kg i.p ) when administered in CD8F1 mice hybrid reduced tumor volume at an incidence of 33%, in an average period of 10 months the mammary tumor study also decreased the rate of lung matter metastasis from 90 to 22% ^12,15.

Recent Research Updates of Amygdalin on the antitumor effect:

Bladder cancer:

Makarewicz et al studied the effect of amygdalin in ILK, FAK and α-β subtypes of integrin detected binding of cancer cells to a vascular endothelial cell or immobilized collagen migration in cell lines such as RT112 and also with UMUC-3 for a period of 24hrs to 48hrs is treated by amygdalin. This study resulted in stopping the ability of integrin on cell migration and attachment with UMUC3 and RT112 cells after the treatment of amygdalin and also the amount of prevalence was found to be decreased the levels of ILK , integrin α and β levels, and decrease in FAK levels ^48,52. Makarevic J et al conducted an in vitro study by using cell lines such as TCCSUP, UMUC-3 and also RT112 when treated with various concentrations of amygdalin resulted in inhibition of cell proliferation in all of the above cells lines by blocking the G0&G1 phase in the cell cycle ⁴⁷. Amygdalin can be used for specific targeting on the tumor site by a therapy called antibody directed enzyme prodrug therapy (ADCPT). This treatment thus proves to show activity towards the treatment of bladder cancer with reduced side effects and also can be given as a combinational treatment with chemotherapeutics. To examine this Syringes et al combined β-glucosidase with mab (cancer-associated monoclonal and HMFGI) to determine the specificity and cytotoxicity using HT1376 cell lines. This study discovered that an increased quantity of amygdalin is required for cytotoxisc effect in HT1373 cell lines but when given with HMFG1-β glucosidase it showed 36-fold increased cytotoxic effect. The mab enzyme conjugate also found that, effect of amygdalin in the treatment of bladder cancer is by lowering the mobility of cancer cells and also arresting cell cycle by binding with HMFG-1 β-glucosidase ^53,54.

Renal cancer

Juengel et al conducted a study on renal carcinoma which is the second most renal cancer in china by using RCC A498, CAKT1, and KTC26 cell lines. When administered with amygdalin for 24 and 48 hrs, results were evident that, amygdalin targets cell cycle and blocks G2 and M phase in CKKAI1 A498 cell lines and s phase in KTC cell lines. Besides there was rise in the total amount of cells found in the s phase and reduction in the multiplication rate and growth of RCC cell. Sequentially it was found that amygdalin may also reduce cell cycle activators such as cyclinβ, CDK-1, E-cadherin, and N-cadherin and downgrade the chemo-tactic invasive activity of cancer cells, decreased the level of integrin α5 and α6, also showed a decreased level of binding of tumor cells to collagen. In this study, untreated cells used as control ^{52, 55}.

Lung cancer:

It has become commonly occurring recurrent class of disease globally. Lung cancer is now the leading cause of cancers-related death in the world⁵⁶. When concentrated mainly on amygdalin action on lung cancer and treated using H1299/M and PA/M in-vitro cell lines it inhibited cell multiplication, protrusion, and emigration. Further, it will suppress integrins like β1, β4, β- catenin, FAK, & ILK components that aid metastasis of cancer cells, elevate cadherin E factor which suppresses metastasis, and decrease AKT, RICTOR phosphorylation, thus by hindering AKT-mTOR activity. Thus this evidence supports that amygdalin shows antimetastatic action for NSCLC and reduces lung cancer by blocking AKT- mTOR pathway^{52, 57}.

Current clinical research on amygdalin:

Blood samples were collected from participants treated with Amygdalin during clinical trials and estimated high cyanide content that is 2.1 mg/ml and it showed few clinical side effects when given orally at the dose of (0.5gm). Few of the patients developed symptoms of cyanide poisoning when administered Amygdalin at an increased dose of 1gm orally. The concentration of cyanide in blood was increased to 3.5mg/ml. This study suggested that amygdalin would be administered only in the advanced stages of cancer ⁵²^,⁵⁸. The digestion parameter was studied for amygdalin from an in-vitro model for the evaluation of GIT for humans and lead to the in-depth knowledge that, it gets degraded by the presence of two main digestive enzyme products such as prunasin & glucose. Then, in the small intestine mandelonitrile was formed by conversion of prunasin which finally gets converted as hydroxyl mandelonitrile and this showed that amygdalin was nontoxic to human and cyanide formation was due to the type of microorganism in the intestine. However further research is needed in this field⁵²^,⁵⁹.

CONCLUSION:

Typically, Amygdalin has employed for the treatment of asthma, nausea, bronchitis, leprosy, etc. Along with in-vitro studies various other studies have suggested that amygdalin shows promising effect towards antitumor and anti-cancer activity, besides it also functions as antifibrotic, anti-inflammatory, immunosuppression, and analgesic medication by inducing apoptosis, inhibiting tumor cell growth, reducing tumor cell metastasis. Furthermore, number of observational studies reported that the compound shows a wide range of effects on breast cancer, lung cancer, renal cancer, bladder cancer, prostate cancer also. Hence Amygdalin could be a potential therapeutic agent for numerous cancer classes.

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Received on 02.03.2021 Modified on 12.08.2021

Research J. Pharm. and Tech 2022; 15(11):5373-5380.

DOI: 10.52711/0974-360X.2022.00906

Drug name	Type of cancer	Mode of action
	Prostate	Cdk1 ↑ , cdk2 ↓ ,cdk4 ↓ G0/G1phase ↑ ,G2/M ↓ Cyclin A ↓ , cyclin B ↓ s phase ↓ Cyclin D3 ↓ , p19 ↓, p27 ↓ p-AKT ↓, p-RICTOR ↓ , p-Raptor ↓ AKT/mTOR ↓ Bcl-2↓ , Bax ↑, Caspase-3↑
Amygdalin	Cervix uteri	Bcl2 ↓ , Bax ↑ , Caspase-3 ↑
	Colon	Cell cycle-related genes:EXO1 ↓ , ABCF2 ↓ , MRE11A ↓ TOPI ↓ , ATP- binding cassette ↓ , subfamily F ↓
	Hemopoietic system	Combined with β glucosidase Bcl-2 ↓ , Bax↑