CRINUM; AN ENDLESS SOURCE OF BIOACTIVE PRINCIPLES: A REVIEW. PART V. BIOLOGICAL PROFILE

CRINUM; AN ENDLESS SOURCE OF BIOACTIVE PRINCIPLES: A REVIEW. PART V. BIOLOGICAL PROFILE

John Refaat*¹, Mohamed S. Kamel ¹, Mahmoud A. Ramadan ² and Ahmed A. Ali ²

Pharmacognosy Department, Faculty of Pharmacy, Minia University ¹, 61519 Minia, Egypt

Pharmacognosy Department, Faculty of Pharmacy, Assiut University ², 71515 Assiut, Egypt

ABSTRACT: Crinum is a well-known traditional herb belongs to family Amaryllidaceae. Worldwide, different Crinum species are commonly used to treat various conditions due to their excellent medicinal values. Members of this genus are also best known biofactories for the unique Amaryllidaceae alkaloids. Due to the significant phytoconstituents produced by this plant as well as their therapeutic potentials, many Crinum species have been subjected to extensive chemical, cytological and pharmacological investigations. This part of our comprehensive review work on the chemical and biological profiles of Crinums describes the results of biological and toxicological studies conducted on different species. In addition, general analytical conclusions as well as some suggestions for future phytochemical and biological work on Crinums are discussed.

Keywords:

Amaryllidaceae, Biological Activities, Crinum, Toxicological Studies

INTRODUCTION: Long ago, natural products had attracted considerable phytochemical and pharma-cological attention. One of their chief sources is plants which are well-known untapped reservoirs of bioactive substances. In fact, medicinal plants can magically provide us with the key to our leading problem in life; diseases. They also found wide application in pharmaceutical, cosmetic and food industry.

Today, despite of the great advance in synthetic organic chemistry, there is a growing focus on the importance of natural products from plants in solving various health care problems by coupling traditional knowledge with scientific principles. Most used synthetic drugs have got a bad reputation due to their familiar side effects.

That's why, the development of naturally based potent, less toxic and cost effective drugs is urgent and medicinal plants appear to have these desired comparative advantages.

Amaryllidaceae is a great widely spread family all over the world containing about 90 genera and 1310 species ¹. The genus Crinum represents an important sector in family Amaryllidaceae with wide geographical distribution throughout the tropics, subtropics and warm temperate regions of the world ². The specific alkaloids produced by these plants have attracted considerable attention due to their interesting pharmacological activities.

In fact, up till now members of Amaryllidaceae continue to yield novel compounds having interesting biological activities and they can be considered a sleeping giant of drug development. Consequently, the previous parts of our review work provided a comprehensive overview of the phyto-chemical studies of the genus Crinum and finally, the current article highlights the possibility of development of this botanical drug into widely used remedies through a detailed account on various biological reports of different species studied so far as well as their toxicological aspects.

Folkloric significance of Crinums: Crinum species have a considerable medicinal reputation as potent folkloric remedies. Their use extended from the ancient times to nowadays especially in Africa, tropical Asia and South America. Several Crinums are traditionally used as emetics, laxatives, expectorants, tonics, antipyretics, diuretics, diaphoretics, anti-asthmatics, anti-malarial, anti-aging, anti-tumor and lactagogues ^3-10. In addition, they are commonly used in treatment of various painful and inflammatory disorders such as rheumatism, earache, lumbago, edema, headache, swelling, backache, wounds and haemorrhoids ^{3, 5, 11-13}.

Besides, they have important antimicrobial applications in parasitic skin diseases, suppurating sores, abscesses, otitis, tonsillitis, laryngitis, prostatitis, leprosy, anthrax; dysentery and sexually transmitted diseases ^{3, 7, 12, 14-16}. On the other hand, Crinums also found use in veterinary medicine for weight loss, low milk production, milk loss and for retained placenta among cattle ^{5, 17}. The detailed ethnomedical uses of Crinums were previously reviewed by Fennell and Van Staden ¹⁸.

Biological activities of Crinum: Different biological investigations carried out on various Crinum species showed that the total extracts together with many of the isolated compounds exhibited a wide range of interesting activities. The results of these enormous studies strongly account for the wide use of Crinums as folkloric medicines.

Analgesic and Anti-inflammatory activities: Pharmacological investigation of the effects of total extracts obtained from different parts of Crinums using many algesiometric and inflammatory models showed their potential for treatment of various pains and inflammatory processes. The aqueous extract of C. giganteum bulbs (100-200 mg/Kg) significantly inhibited formalin- and acetic acid-induced pain in rats and mice, respectively, in a dose-dependent manner. The effects were comparable to aspirin. In addition, oral administration of the extract (200 mg/Kg) resulted in a marked dose-dependent anti-inflammatory activity in cotton pellet-induced granuloma in rats ⁸.

Similarly, the aqueous extract of C. glaucum bulbs (100-400 mg/Kg, orally) showed significant activities in rat tail flick, mouse writhing and formalin pain models. Moreover, the extract (125-500 mg/Kg, orally) produced a dose-dependent inhibition of carrageenan-induced paw swelling in rats. The results obtained suggested both peripheral and central mechanisms for the analgesic and anti-inflammatory effects of the extract ¹⁹.

Ratnasooriya et al., studied the antinociceptive activity of the aqueous leaves' extract of C. bulbispermum orally administered to rats using the tail flick, hot plate and formalin tests. The results collectively suggested that the antinociception is mediated both spinally and supraspinally, as well as its effectiveness against phasic and continuous non-inflammatory/inflammatory pain ²⁰.

On the other hand, the chloroform and methanol extracts of C. asiaticum leaves (50 mg/Kg, orally) caused significant effect that was stronger than indomethacin on carrageenan-induced paw edema in mice. The anti-inflammatory activity could not be attributed to its anti-bradykinin activities, but may be partly due to its anti-histaminic properties ^{21, 22}. Consequently, new topical and cosmetic formulations comprising the extract of C. asiaticum L. as an active ingredient in an amount effective to treat and alleviate allergy and inflammatory diseases were patented ²³.

In another investigation of pain and inflammation relieving properties of C. augustum Rox., the alklaine chloroform- and ethyl acetate- soluble fractions (II and III) of the bulbs' total extracts at (400 mg/Kg, orally) showed the highest analgesic effects in mice using the hot plate test, whereas both fractions (II and III) together with the chloroformic fraction (IV) -obtained after saturation with Na₂CO₃ during total extract fractionation- exhibited the highest anti-inflammatory effects in the carrageenan-induced paw swelling in mice at (400 mg/Kg, orally) ²⁴.

Likewise, Lee et al. proved that the ethyl acetate fraction of C. folium possesses significant analgesic and anti-inflammatory actions by inhibition of prostanoids biosynthesis as one of its mechanism of action ²⁵. In other studies, the ethanolic bulbs' extract of C. defixum Ker Gawl showed significant inhibition of acetic acid-induced writhing and tail clip-induced algesia ²⁶, while the petroleum ether, dichloromethane, ethanol and 50% methanol extracts of C. moorei bulbs showed good inhibition against both COX-1 and COX-2 enzymes ²⁷.

Moreover, the methanolic extract of C. latifolium leaves exhibited strong to moderate inhibitory activity to nuclear factor-kappa B (NF-κB) which is an inducible and ubiquitous transcriptional factor required for gene expression of many inflammatory mediators ²⁸. A significant anti-inflammatory effects of C. latifolium leaves' extract were also shown by its potential to suppress indoleamine 2,3-dioxygenase mediated tryptophan degradation in unstimulated- and mitogen-stimulated peripheral blood mononuclear cells at IC₅₀ of 241 ± 57 μg/ml and 92 ± 20 μg/ml, respectively ²⁹.

It is worthy mentioned that the previous investigations attributed the observed activities of Crinums to their alkaloidal content. Furthermore, studies on the analgesic effects proposed the participation of opioid mechanisms ²⁰, and the resemblance of Amaryllidaceae alkaloids to morphine and codeine skeletons may account for their analgesic activity ³⁰ e.g. caranine, crinine, galanthamine and galanthine ^{31, 32} . Haemanthidine and lycorine are also analgesics and anti-inflammatory with activities greater than aspirin ³³ and indomethacin ³⁴, respectively, while narwedine and vittatine could potentiate the analgesic effects of caffeine and morphine ³³.

Effects on Central Nervous System: In 1960, Wildman had reported the action of Amaryllidaceae alkaloids on CNS ³¹. One of the most common and important alkaloids of this family is galanthamine which attracted many pharmacological, biochemical and clinical investigations due to its variable central effects ³⁵. Galanthamine is a tertiary amine and would be expected to be sufficiently lipid-soluble to cross the blood-brain barrier and act on the central nervous system. It exhibited reversible muscarinic and anticholinesterase activities and can be useful as a treatment for nervous diseases, neurological injuries, paralysis syndrome, schizophrenia, mania, and other forms of dementia as well as Alzheimer's disease ^35-37. This alkaloid acts by restocking acetylcholine levels in brain areas lacking cholinergic neurons by binding to the enzyme acetylcholinesterase.

In addition, it stimulates pre- and postsynaptic nicotinic receptors so increases the release of neurotransmitters like acetylcholine and glutamate, and stimulates neuronal function.

Moreover, Galanthamine was shown to have no hepatotoxicity; therefore, it has been approved as its HBr salt for the first time in Bulgaria under the name of Nivalin^® in the early 1960s, and later licensed as Razadyne^® (formerly Reminyl^®) in the United States and some European countries ³⁸.

Furthermore, galanthamine has a central stimulant action.  Early reports from Bulgaria and Russia claimed that recovery of consciousness after anesthesia was faster if galanthamine was used instead of neostigmine. This effect has been attributed to either a central stimulant action of galanthamine, or its ability to antagonize the actions of morphine-like analgesics ³⁵. In addition, Cozanitis and Toivakka concluded from EEG recordings that galanthamine was a mild analeptic, i.e. a central stimulant ³⁹.

In a study of sleep patterns in healthy volunteers, galanthamine HBr (10 or 15 mg, orally) shortened the latency to rapid eye movement sleep, but also increased the number of awakenings ⁴⁰. Additional information on central effects of galanthamine could be obtained from case reports in which galanthamine was used to reverse poisoning. It aided recovery after an overdose of the centrally acting muscarinic antagonist hyoscine (scopolamine) ⁴¹.  Moreover, it is also known to inhibit traumatic shock and has been patented for use in treatment of nicotine dependence ³⁷.

In the same way, ungeremine and lycorine also exhibited strong anticholinesterase activities. The former was found to be about 6-8 times more potent than galanthamine ^{38, 42}. Additionally, alkaloidal extracts of C. jagus and C. glaucum bulbs increased the depressed levels of acetylcholine in the brain associated with Alzheimer's disease. The most active alkaloids isolated were hamayne and lycorine ⁴³, whereas linoleic acid ethyl ester has been identified in the ethanolic extract of C. powellii bulbs as the compound responsible for acetylcholinesterase inhibition ⁴⁴. On the other hand, in spite of being slightly active when tested for anticholinesterase activity of their leaves' extracts, bulbs and roots of C. campanulatum, C. graminicola, C. macowanii, C. moorei and C. variabile contained several compounds with significant inhibitory activity ⁴⁵. In another study, lycorine and hamayne were found to reduce the production of amyloid β-peptide (Aβ) which can antagonize Alzheimer's progression ⁴⁶. On the other hand, galanthine -a lycorine-type alkaloid- found a great importance in treatment of myasthenia gravis, myopathy and CNS diseases due to its cholinergic effects, while, some derivatives of it are being evaluated as CNS depressants.

The aqueous extract of C. giganteum (6.25, 12.5 and 25 mg/kg, i.p) was found to have central inhibitory activity (sedative effect). It prolonged the duration of pentobarbital sleeping time in rats and reduced exploratory and spontaneous motor activities, in mice ⁴⁷. Similarly, the aqueous leaves' extract of C. bulbispermum (1, 1.5 and 3 g/Kg, orally) significantly and markedly impaired the four parameters of rat hold-board test indicating its sedative properties ²⁰. Narwedine has been found to potentiate the pharmacological effects of caffeine, carbazole, arecoline and nicotine, in laboratory animals ³³.

Mesembrine and its analog compounds are serotonin-uptake inhibitors and can be used for treatment of depressive states, psychiatric disorders with anxiety component, alcohol and drug dependence, and obsessive-compulsive disorders ⁴⁸. In addition, the crude extract of C. ornatum bulbs, lycorine and haemanthamine exhibited dose-dependent anticonvulsant effects using electrical stimulation test in rats ⁴⁹.

Effects on Cardiovascular system and blood: A number of Crinum alkaloids were reported to have hypotensive activity, e.g. ambelline, caranine, crinamine, crinine, lycorine, narwedine, tazettine, ismine and galanthine (50 mg/Kg, in rabbits) ^{31, 50}, while haemanthamine exhibited hypertensive properties ⁵¹. The alkaloid narwedine was found to decrease the frequency of cardiac contractions, while galanthamine is known to cause bradycardia or atrioventricular conduction disturbance ⁵². Galanthamine HBr was found to cause a fall in blood pressure in anaesthetized dogs, an effect apparently associated with a transient decrease in respiration ⁵³.

However, i.v. injection of galanthamine HBr (0.2-3 mg/kg) caused an increase in the mean arterial blood pressure which could last for 30 min in anaesthetized rats. The hypertensive action of galanthamine was not affected by ganglion blocking agents ⁵⁴. On the other hand, Wasicky had reported that C. pratense has Digitalis-like effects ⁵⁵.

The ethyl acetate fraction of C. folium was found to have a considerable platelet-aggregation inhibitory effect ²⁵, while the aqueous extract of C. giganteum bulbs caused a dose-dependent suppression of total leucocytes count, but an increase in neutrophils percentage was noticed, while monocytes, eosinophils and basophils were not significantly altered ⁸.

Respiratory system activities: Galanthamine at 3 mg/kg reversed the respiratory depression induced by dextromoramide in urethane-anesthetized rabbits ⁵⁶. Narwedine increases the amplitude and frequency of respiratory movements, while crinamine shows respiratory depressant activity ^{52, 57}.

Effects on skeletal and smooth muscles: Galanthamine is said to be widely used as a reversal agent in anesthetic practice in Eastern Europe. It was tested in 40 surgical patients for its ability to reverse neuromuscular blockades induced by alcuronium, pancuronium, gallamine, and tubocurarine. Galanthamine HBr at (5 mg doses to a total of 20 mg, i.v.) successfully reversed the muscular paralysis with all four relaxants, although the rate of recovery was slower than with neostigmine ⁵⁸.

On the other hand, C. glaucum aqueous extract was found to be a non-specific relaxant of the gastrointestinal smooth muscles. It produced a concentration-dependent, non-competitive inhibition of both contractions induced by acetylcholine and calcium chloride on the rat duodenum and contractions of the guinea-pig ileum induced by acetylcholine and histamine at (1-8 mg/mL) and (1-4 mg/mL), respectively. The extract (0.125-2 mg/mL) also, produced a concentration-dependent relaxation of the guinea-pig Taenia coli, precontracted with potassium chloride ⁵⁹. Similarly, Wiart attributed the vasorelaxing activity of lycorine to the resemblance of Amaryllidaceae alkaloids to isoquinoline alkaloids e.g. papaverine which are known smooth muscle relaxant ⁶⁰.

Effects on Sexual functions: Hippadine was reported to produce reversible inhibition of fertility by acting on germ cells at early stages of spermatogenesis in rats' tests ⁶¹.

Likewise, application of lycorine to the testes and ovaries of immature rats inhibited cell division in the spermatogonia or primary spermatocytes. No spermatid cells were also found in the tested animals, whereas follicles were found to be smaller and less in number in rats' ovaries ³¹. Moreover, Ghosal et al. reported that ungeremine significantly inhibited testicular metabolism in mice as well ⁶².

Endocrinological Effects: Galanthamine was found to increase plasma levels of hydrocortisone (cortisol) and ACTH.  A single dose of 20 mg (with 0.5 mg atropine) produced a sustained elevation of plasma hydrocortisone from 0.54 to 0.8 μmol/L ⁶³. It was assumed that such effect is due to the central action of galanthamine, although its mechanism is unknown. Another study reported that galanthamine HBr (0.3 mg/kg, i.v.) caused an increase in adrenocorticotrophic hormone (ACTH) plasma levels in a group of 8 patients undergoing surgery ⁶⁴.

Antiallergic and Antianaphylactic activities: The aqueous extract of C. glaucum was found to inhibit mast cell degranulation in normal and passively sensitized rats induced by dextran and antigen, as well as allergic bronchoconstriction, ileum contractions, and release of mediators from the lungs of sensitized guinea pigs. The effects of the extract observed were comparable to those of sodium cromoglycate. These results may account for its use as an anti-asthmatic in traditional medicine ^{65, 66}.

On the other hand, different combinations of glucan A and phosphatidyllycorine isolated from C. latifolium L. produced statistically significant in vitro protection against Tween 80-induced degranulation, as well as to sensitized mast cells challenged with an antigen (horse serum). The combination, in vivo, also provided protection against compound 48/80-induced degranulation of mast cells ⁶⁷. These findings may support the use of C. latifolium total extract in treatment of allergic disorders in Ayurvedic medicine.

In addition, lycoriside at (1-20 µg/ml) produced statistically significant protection against Tween 80-induced degranulation in vitro, as also to sensitized mast cells challenged with an antigen (horse serum). It also provided protection against compound 48/80-induced degranulation of mast cells when administered in vivo (1-5 mg/kg, p.o) ⁶⁸.

Immunological activities: Aqueous extract of C. latifolium showed immunomodulatory properties in human peripheral blood mononuclear cells. Extracts of C. latifolium slightly enhance neopterin production in unstimulated peripheral mononuclear cells, whereas an effective reduction of neopterin formation in cells stimulated with concanavalin A (Con A), phytohemagglutinin (PHA), or interferon-gamma (IFN-gamma) was observed ⁶⁹.

It also promoted human T-lymphocytes in vitro, particularly the cell-mediated immune response of CD4⁺T lymphocytes (T-helper cells) ⁷⁰. Moreover, aqueous extracts of C. latifolium L. and Camellia sinensis showed immunomodulatory properties in human peripheral blood mononuclear cells, whereas extracts of the former seemed to be more effective in reducing neopterin formation in stimulated cells than green and black tea extracts ⁶⁹.

1,2-β-epoxyambelline (5 μg/ml) either alone or in a mixture with ambelline (1:1) activated mouse spleen lymphocytes. The effect was comparable to that of the known mitogen, concanavalin A ⁷¹. Lycorine-1-O-glucoside is a potent immunostimulatory agent and has been found to activate spleen lymphocytes in mice ³⁰. In contrast, the aqueous extract of C. giganteum bulbs showed a pronounced dose-dependent anti-lymphocytic activity in rats ⁸. On the other hand, lycorine had been patented as an immunosuppressor and can be useful in suppression of the immune system of mammals for treatment of autoimmune diseases, immune complex diseases, allergic and rheumatic conditions, as well as for prophylaxis against transplant rejections ⁷².

Cytotoxic and Anticancer activities: Many preparations of Crinums have been used as anti-tumors ³¹. Recently, a large number of the isolated compounds as well as extracts of different Crinum species are known to have potent cytotoxic and anticancer activities. A study byNair et al. reported that lycorine, crinamine and 6-hydroxycrinamine isolated from C. delagoense bulbs are active against BL-6 mouse melanoma cells after substantiated reports confirmed the cure of a human cancer after oral intake of the hot aqueous extract of its bulbs ⁷³. Crinafolidine and crinafoline were found to produce remarkable reduction in the viability and in vivo growth of S-180 ascites tumor cells ⁷⁴.

In addition, criasbetaine, palmilycorine and lycorine showed significant anti-tumor activity in vitro and also caused cytolysis of sarcoma 180 ascites tumor cells, but the alkaloidal glycosides enhanced cells growth ^{75, 76}. Additionally, criasbetaine displayed significant activites against P-388 and KB tests in vitro, with ED₅₀ of 0.82 and 1.2 μg/ml, respectively ⁷⁶. Crinasiatine was also found to exhibit tumor-inhibiting properties ⁷⁵, whilst ungeremine significantly inhibited the activity of several test-tumor systems ^{62, 77}.

On the other hand, crinamine, lycorine and augustine from C. amabile bulbs demonstrated important cytotoxic activities in twelve cell lines ⁷⁸. Furthermore, lycorine was reported to inhibit the in vivo growth and synthesis of DNA and proteins in murine ascite tumor cells and reduce the viability of in vitro grown tumor cells ³⁰. It also reduces cellular activity in femoral bone marrow, which results in granulocytic leucopenia and erythrocytopenia ³¹. Abd El-Hafiz et al. reported that 4'-hydroxy-7-methoxyflavan have an important cytotoxic effect at 42 μg/ml while pratorinine and 6-α-hydroxy buphanisine showed a moderate activity when tested on human leukaemic Molt 4 cells ⁷⁹.

Additionally, precriwelline and pretazettine showed remarkable anti-leukaemic activity. The latter is also reported to be effective against Ehrlich ascites carcinoma, lymphocytic leukaemia and Lewis lung carcinoma ⁵², whereas haemanthamine, crinamine and 6-hydroxycrinamine were moderately active against Rauscher leukaemia ⁸⁰. The alkaloid lycobetaine was found to act as a selective topoisomerase IIβ poison. This mechanism causes or at least contributes to the antitumour activity ⁸¹.

Of all the Amaryllidaceae alkaloids, lycorenine was found to be the most cytotoxic against HepG2 hepatoma ⁸². Isoliquiritigenin was also found to inhibit cell proliferation and induce apoptotic cell death in human hepatoma cells (Hep G2) ⁸³. Furthermore, Min et al. reported that criasiaticidine A, pratorimine and lycorine have in vitro cytotoxicity against Meth-A (mouse sarcoma) and Lewis lung carcinoma (mouse lung carcinoma), whereas dihydrolycorine, haemanthamine, lycorine, narci-clasine, pretazettine and pseudolycorine halted protein synthesis in eukaryotic cells by inhibiting peptide bond formation step ^{30, 32}.

In addition, extracts of C. asiaticum leaves demonstrated cytotoxic activity against murine P388 D1 cells ¹¹. Some alkaloids isolated from bulbs of C. asiaticum L. var. sinicum Baker showed remarkable inhibition against tumor cell lines A549, LOVO, HL-60, and 6T-CEM ⁸⁵. Similarly, the ethyl acetate and alkaline ethyl acetate-soluble fractions of C. asiaticum L. var. japonicum bulbs showed significant cytotoxicity against Lewis lung carcinoma cells ⁸⁴, whereas hot water extracts of C. asiaticum showed strong inhibition of calprotectin-induced cytotoxicity in vitro using MM46 mouse mammary carcinoma cells as targets ⁸⁶. It was reported that many Amaryllidaceae alkaloids belonging to the crinane-type have selective apoptosis-inducing activity. Biological screening indicated that crinamine and haemanthamine are potent inducers of apoptosis in tumour cells at micromolar concentrations. An alpha C-2 bridge and a free hydroxyl at the C-11 position are important as pharmacophoric requirements for this activity ⁸⁷.

Crinine, 6-hydroxybuphanidrine and 6-ethoxy buphanidrine showed also antiproliferative effects against human tumor cell lines HL-60 and MDA-MB-231 with crinine being the most active. Hydrogenation of the double bond at C-1 – C-2 leads to activity loss, whereas substitutions at C-6, C-8 and C-11 affect their cytotoxicity ⁸⁸. The introduced per os (instead of water) cold-hot aqueous extract from the Vietnamese plant C. latifolium L., retarded in vivo the growth of chemically induced (by 20-methylcholanthrene) tumours (sarcomas) in rats.

Such inhibition of carcinogenesis has occurred probably due to the influence of immunomodulating and anti-tumour plant alkaloids and other biologically active components in the plant decoctions ⁸⁹. Recently, C. latifolium showed promise to become one of the leading treatments in the world for prostate and ovarian conditions, including benign prostate hypertrophy (BPH), prostatitis, adenoma, uterine fibroids, ovarian cysts and tumors polycystic ovarian syndrome. It was believed that Crinum may enhance the cellular communication so maintaining the balance between cellular proliferation and apoptosis. Lately, it was shown that Crinum encourages cells to produce a substance called neopterin, which is responsible for communicating with immune cells calling them into action against foreign invaders and unhealthy or proliferative cells.

This suggests that Crinum may not only be valuable for prostate and ovarian conditions, but may also be beneficial for other conditions in which unhealthy, proliferative cells are involved. Another potentially valuable benefit of Crinum is that it may be used as a preventative agent, offering protection against prostate and ovarian conditions before they start ⁹. These findings were in agreement with the study carried out by Jenny et al. which showed dose-dependent inhibitory effects of the aqueous C. latifolium extract on cell proliferation of highly metastatic human prostate carcinoma PC3 cells, androgen-sensitive prostate adenocarcinoma LNCaP cells and BPH-1 cells ²⁹.

Antioxidant, Hepatoprotective and Metabolic effects: The ethanolic extract of C. defixum Ker Gawl bulbs showed significant dose dependent free radical scavenging property in different in vitro models such as DPPH, nitric oxide, superoxide and hydroxyl radical models. The antioxidant activity was attributed to the presence of high phenolic content ²⁶. Similarly, the 80% methanolic extract of C. jagus bulbs possessed a significantly high antioxidant activity in DPPH test, the effect was more pronounced when compared with vitamin C at increased concentrations (50-400 μg/ml) ⁹⁰. In addition, good to moderate DPPH radical scavenging and ferric-reducing activities were observed in some extracts of C. moorei bulbs ²⁷.

In the same way, the ethanolic extract of C. ornatum bulbs as well as some isolated alkaloids e.g. lycorine, haemanthamine and crinamine showed significant DPPH scavenging effects ⁴⁹. Equally, the aqueous extract of C. latifolium leaves revealed potent in vitro antioxidant activity by an oxygen radical absorbance capacity ²⁹. The aqueous leaves' extract of C. bulbispermum also displayed a dose-dependent moderate antioxidant activity (EC₅₀= 203.76 μg/ml) ²⁰. In a study by Indradevi et al., the ethanolic extract of C. asiaticum leaves demonstrated hepatocyte protective nature by attenuating markers of hyperglycemia-mediated oxidative stress and antioxidant competence in hepatic tissues of diabetic rats ⁹¹. A significant decrease in blood sugar, cholesterol, triglycerides, LDL levels and an increase in HDL level were observed. Besides, the activities of AST, ALT, ALP, ACP and LDH enzymes were diminished, whereas the activity of superoxide dismutase, catalase and reduced glutathione was increased by the extract.

Increases in liver lipid peroxides also improved. Similarly, ⁹²,Ilavenil et al. showed that C. asiaticum L. ethanolic extract and lycorine possess human erythrocyte protective effects against oxidative damage induced by 2-amidinopropane. The effects were attributed to their antioxidant nature. In addition, the aqueous leaves extract of C. bulbispermum (3 g/Kg, orally) significantly increased SGOT, SGPT and creatinine levels but did not significantly alter serum urea and haematological parameters (haemoglobin, RBC and WBC counts) ²⁰.

Lately, lycorine exhibited significant hepatoprotective effects against CCl₄-induced oxidative stress in Swiss albino mice at 5 mg/Kg which were comparable to Silymarin. It effectively normalized the increased generation of lipid peroxidation products, high levels of malondialdehyde, glucose, urea, serum bilirubin, and hepatic marker enzymes.

It also normalized the reduction of glutathione, vitamin C content and activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase. The histological and ultrastructural observations evidenced that lycorine effectively rescued the hepatocyte from CCl₄-induced oxidative damage without disturbing its cellular metabolic function and structural integrity ^{93, 94}.

Anti-genotoxic activity: (E)-N'-[(E)-2-butenoyl]-2-butenoylhydrazide from C. defixum was found to have anti-genotoxic activity when assayed by onion root tip assay. It imparted a clear dose dependent protective effect against the genotoxic effect of H₂O₂. Furthermore, it was more effective against clastogenic aberrations than physiological aberration at the highest concentration tested (250 ppm) ⁹⁵.

Antimicrobial activities: The methanolic extract of C. jagus possesses antibacterial activity and the alkaloid crinamine isolated from the bulbs showed a strong activity against Bacillus subtilis and Staphylococcus aureus. On the other hand, lycorine, hamayne and 6-hydroxycrinamine were found to be inactive. Neither the extract nor the isolated alkaloids possessed any antifungal activity ⁹⁶. Crinamine and lycorine showed also activity against Bacillus cereus and Pseudomonas aeruginosa ⁶⁰. Extracts of C. macowanii demonstrated weak antifungal properties against Candida albicans in vitro ⁹⁷, whereas Chaumont et al., had reported the activity of C. moorei extracts against several fungi pathogenic to man ⁹⁸. In addition, the total ethanolic extracts of C. augustum bulbs and C. asiaticum leaves and their fractions (I-V) showed inhibitory effects on both Gram +ve (S. aureus) and Gram -ve (E.coli) bacteria at 50 mg/ml, while that on the former was markedly greater.

The total extracts of both plants and their fractions had no inhibitory effects at a concentration of 5 mg/ml, whereas fractions containing alkaloids (II, III and IV) of both plants were the most effective especially at 10 and 50 mg/ml ²⁴. Ghosal et al. also reported a marked antibacterial action of ungeremine against ten bacterial isolates ⁶². Moreover, three flavonol glycosides quercetine-3-O-glucoside, kaempferol-3-O-β-D-xylopyranosyl(1→3)β-D-gluco pyranoside and quercetin-3-O-β-D-(6-O-acetyl gluco pyranosyl)(1→3)β-D-glucopyranoside were found to be inactive against S. aureus, E.coli and P. aeroginosea, but showed moderate antifungal activity against Candida albicans in vitro  ⁹⁹.

In another study, the CH₂Cl₂/MeOH (1:1) leaf extract of C. purpurascens, hippadine as well as sitosterol-3-O-β-D-glucopyranoside demonstrated antibacterial activity against P. aeruginosa, E. coli, K. pneumoniae, S. aureus, S. typhi and S. paratyphi B, whereas pratorimine did not show any antimicrobial activity against these bacteria strains ¹⁰⁰. Likewise, the ethanolic extract of C. asiaticum has a broad spectrum activity against Gram +ve and Gram -ve bacteria. It showed greater activity than the aqueous extract against K. pneumoniae, S. aureus, B. subtilis, P. aeruginosa and E. coli ¹⁰¹.

Regarding the antiviral activities, it was reported that lycorine exerts powerful antiviral effects on several RNA and DNA viruses such as Coxsackie B₂, poliomyelitis and Herpes type I ⁵⁷. This is accomplished by delaying virus production and decreasing the virus amount by blocking viral protein synthesis ¹⁰².

Lycorine is also effective against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) ¹⁰³. On the other hand, narciclasine inhibits protein synthesis at the step of peptide bond formation. Pretazettine strongly inhibits the activity of RNA-dependent DNA polymerase from various oncogenic viruses by binding to the enzyme as well ³⁰. Moreover, Duri et al. reported that C. macowanii bulbs extract caused 100% reduction of viral cytopathic effects in VERO cells infected with yellow fever virus. The same extract also resulted in 70% inhibition of viral replication and cytopathic effects in cells infected with Japanese encephalitis virus ¹⁰⁴. In addition, the methanolic extract of C. asiaticum var. japonicum roots showed significant anti-HIV-1 activity (ED₅₀ = 12.5 μg/ml) ¹⁰⁵. Tris phaeridine, lycorine, homolycorine, and haemanthamine showed high antiretroviral activities (IC₅₀= 0.4-7.3 μg/mL) as well ¹⁰⁶.

Antiparasitic activity: Four groups of Amaryllidaceae alkaloids (lycorine, crinine, tazettine and galanthamine-types) exhibited antimalarial activity at different potencies when evaluated in vitro for their ability to inhibit Plasmodium falciparum growth. 6-hydroxy-haemanthamine, haemanthamine and lycorine were found to be the most potent, while galanthamine and tazettine had the least potent activity against P. falciparum ¹⁰⁷. Likewise, lycorine, augustine and crinamine from C. amabile bulbs showed moderate antimalarial activity against P. falciparum ⁷⁸. In addition, Nair et al. reported that lycorine has a mild in vitro activity (IC₅₀= 0.34 μg/ml) against P. falciparum (strain NF-54), whereas 4a-dehydroxycrinamabine, crinamidin, macowine and undulatine were inactive ¹⁰⁸.

On the other hand, 4a-dehydroxycrinamabine demonstrated a very mild activity (IC₅₀= 11.07 μg/ml) against Trypanosoma brucei rhodesiense (strain STIB-900, stage trypomastigotes), the parasite associated with sleeping sickness ¹⁰⁸, while 1,2-diacetyllycorine and 3-O-acetylsanguinine from C. kirkii bulbs showed activity against T. brucei rhodesiense. 3-O-acetylsanguinine also exhibited some activity against T. cruzi ¹⁰⁹. Furthermore, crinamine and lycorine were active against Entamoeba hystolitica with IC₅₀ of 0.53 and 0.23 μg/ml, respectively. Dihydrolycorine has been used as a substitute for emetine in amoebic dysentery due to its protozoacidal effects ¹¹⁰.

Insecticidal and molluscicidal activities: In their search for new bioactive alkaloids from C. powellii bulbs, Velten et al. isolated two novel insecticidal alkaloids, cripowelline A and B. The aqueous solution of the former (0.1% w/v) revealed a considerable activity against diamondback moth on cabbage plants which lasted for seven days ¹¹¹.

On the other hand, the aqueous and ethanolic extracts of C. zeylanicum were tested against molluscan intermediate hosts of schistosomiasis and fascioliasis. Both extracts showed remarkable mortality rates against Biomphalaria pfeifferii (IC₅₀=50 mg/ml) and Lymnaea natalensis (IC₅₀=10 mg/ml) ¹¹².

Anti-snake venom activity: The methanolic extract of C. jagus bulbs has a significant anti-snake venom activity against Echis ocellatus venom. It significantly protected mice from death, myonecrosis and haemorrhage induced by the lethal effects of venoms. The extract showed a very minimal effect against Naja nigricollis venom, which is neurotoxic. These results have confirmed the ethnomedical use of the extract for treating snakebite victims among the rural Nigerian population ^{90, 113}.

Antifouling activity: Biofouling or biological fouling is the undesirable accumulation of micro-organisms, plants, algae, and/or animals on wetted surfaces. Settlement of higher organisms such as macroalgae and invertebrates may threaten the survival of individuals of benthic invertebrates.Therefore, benthic invertebrates have developed various defense systems against biofouling, such as biofilm. Accordingly, biofouling on ships hulls, offshore structures or aquaculture equipment represent a major global economic and technical problem.

Antifouling is the process of preventing or removing the accumulation of such biofoulers. Many types of antifoulig agents including organotins (e.g. tributyltin, triphenyltin) as well as copper and its derivative compounds have been found to be toxic to marine organisms. Therefore, the development of environmentally safe antifouling substances is urgently needed¹¹⁴.

Recently, seven compounds isolated from C. augustum Rox. bulbs were evaluated for their antifouling effects using larvae of barnacle Balanus amphitrite.Only 6-methoxy-crinamine, crinamine and buphanisine showed significant activity with EC₅₀ of 1.8, 1.2 and 0.75 µg/ml, respectively, versus the positive control copper sulphate (EC₅₀ = 0.23 µg/ml). In contrast to copper sulphate, the three alkaloids were nearly safe at all the tested concentrations ¹¹⁵.

Other Biological activities: Crinums contain strong emetic and diaphoretic alkaloids. Ingestion of raw bulbs causes nausea, vomiting and diarrhea ⁵. Crinamine was found to inhibit hypoxia inducible factor-1 (HIF-1) activity but not activity of hypoxia inducible factor-2. It showed potent dose-dependent inhibition (IC₅₀= 2.7 muM) of HIF-1alpha in a cell-based reporter gene assay, whereas lycorine, norgalanthamine and epinorgalanthamine showed no significant inhibition of HIF-1alpha induced transcriptional activity ¹¹⁶.

Additionally, the 80% methanolic extract of C. yemense bulbs showed a potent inhibitory effect on nitric oxide (NO) production in lipopolysaccharide-activated macrophages. Yemenine A, bulbispermine, crinamine, 6-hydroxycrinamine and lycorine also inhibited both NO production and induction of inducible nitric oxide synthase (iNOS) ¹¹⁷. The pyran derivative, 6-hydroxy-2H-pyran-3-carbaldehyde isolated from C. yemense was found to be a more potent tyrosinase inhibitor in treatment of hyperpigmentation than kojic acid ¹¹⁸. Lately, the ethanolic extract of C. asiaticum and norgalanthamine showed important potential to promote hair growth via the proliferation of dermal papilla ¹¹⁹.

Bioassay-guided study of the methanol extract of C. latifolium yielded 4-senecioyloxymethyl-3,4-dimethoxycoumarin which was found to be a strong inhibitor to the in vitro tube-like formation of human umbilical venous endothelial cells (HUVECs), while manifesting no cytotoxicity in tumor cell lines (B16F10, HCT116). In contrast, 5, 6, 3'-trihydroxy-7, 8, 4'-trimethoxyflavone showed a modest inhibitory effect on tube-like formation of HUVECs ¹²⁰.

Galanthamine has been reported to have blockade effects on ion channels of excitable membranes such as K⁺ and Na⁺ channels ¹²¹. Unlike tacrine, galanthamine was found to have no affinity for muscarinic or nicotinic cholinoceptors in radioligand binding assays ¹²². However, galanthamine (l-10 μM) was found to activate nicotinic acetylcholine receptors in cultured hippocampal neurons. Galanthamine and physostigmine also activated fibroblast cells that had been transfected with the α₄ β₂ form of the neuronal nicotinic receptor ¹²³.

Furthermore, Galanthamine eye drops significantly reduced intraocular pressure in rabbits' eyes. The effect was slow in onset and long-lasting peaking at two hours ¹²⁴. On the other hand, lycorine is a powerful inhibitor of growth and cell division in higher plants, algae and yeasts ¹²⁵, while lycorine-1-O-glucoside and lycoriside are potent promoters of root growth and seed germination in higher plants e.g. Allium cepa ^{75, 126}.

Conversely, palmilycorine, lycorine and lycorine-1-O- glucoside were shown to inhibit the emergence of plants' leaves ⁷⁵. Furthermore, Niño et al. reported that lycorine displayed moderate topoisomerase I inhibitory activity when tested utilizing genetically engineered mutants of the yeast Saccharomyces cerevisiae strains RAD+, RAD52Y and RS321 ¹²⁷. Lycorine (1 mg/kg) also induced a marked choleretic effect in rats anaesthetized with urethane ¹²⁸.

Recently, crinumin, a glycosylated serine protease with chymotrypsin-like activity was purified from the latex of C. asiaticum. It showed activity over a wide range of pH (4.5-11.5 and optimum at 8.5), temperature (75 °C and optimum at 70 °C) and is also functional against organic solvents and detergents even after prolonged exposure ¹²⁹.

Toxicological studies: Due to their alkaloidal content, Crinum plants are known to be toxic. Raw bulbs ingestion results in nausea, vomiting and diarrhea ⁵. In addition, C. zeylanicum was used in the Moluccas as a violent poison ¹³⁰.  As a result, some Crinum species were subjected to toxicological studies in order to evaluate their safety. For example, the acute toxicity of the aqueous extract of C. glaucum bulbs was studied by Okpo et al.. The LD₅₀ were 119 mg/Kg, i.p. and 1420 mg/Kg, p.o. in mice ¹⁹. Likewise, the i.p. and oral LD₅₀ of C. giganteum aqueous extract in mice were found to be 627 and 1468 mg/kg, respectively ⁴⁷.

In an analogous study on the same extract, the i.p. and p.o. LD₅₀ were 627 ± 5.8 and 1486 ± 18.9 mg/Kg in mice, and 520 ± 10.2 and 1023 ± 4.3 mg/Kg in rats, respectively ⁸. Similarly, the p.o. LD₅₀ of the total ethanolic extract of C. augustum bulbs was observed to be 1600 mg/Kg in mice ²⁴.

On the other hand, Ratnasooriya et al. evaluated the subchronic toxicity of the aqueous leaves extract of C. bulbispermum. The extract induced mild to moderate toxicity in rats which developed diarrhea and postural abnormalities on the second day, and two rats died by the fourth day. Liver and renal toxicities (increase of serum SGOT, SGPT, creatinine and urea) were also reported. The authors attributed this toxicity to the lycorine- and crinine-types of alkaloids present in the extract ²⁰.

Different extracts of C. bulbispermum bulbs were tested in a brine shrimp bioassay. The most effective one were the butanol fraction of the acidic extract of the non-flowering bulbs (LD₅₀= 63.1 μg/ml) followed by the ether fraction of the alkaline extract of the flowering bulbs (LD₅₀= 73 μg/ml) ¹³¹. Likewise, the essential oil of C. ornatum bulbs was found to be toxic through the brine shrimp assay (LC₅₀ = 1.701 μg/ml) ⁴⁹.

Considering the toxicological studies on the individual compounds, the acute LD₅₀ values of galanthamine by various routes were reported to be 18.7 (oral), 8 (i.v.), 14.4 (i.p.) and 6.2 (s.c.) mg/kg in mice ¹³², while they were 2-4 times higher in rats ¹³³. Micov and Georgievreported that daily administration of 1 or 4 mg/kg (equivalent to 0.5 and 2 mg galanthamine HBr) did not cause significant changes in weight, blood count, or in the morphology and histology of brain, liver, kidneys, adrenal glands, heart, and skeletal muscles. In addition, there was no evidence for teratogenicity ¹³⁴. Doses from 0.5 μg/kg to one-tenth the LD₅₀ were administered orally and s.c. to rats and rabbits. The highest doses had embryotoxic activity on pregnant animals, although they did not produce fetal malformations ¹³³.

CONCLUSION: Since about 1950s, Crinums have been subjected to extensive chemical and biological investigations due to their richness in pharmacologically active principles. Phytochemical investigations have resulted in isolation of diverse classes of compounds and have been focused predominantly on alkaloids. About 180 alkaloids belonging to different types of Amaryllidaceae alkaloids have been isolated from Crinum species.

In the same way, the enormous cytological, pharmacological and toxicological studies of Crinums exhibited a wide range of interesting biological activities. Analgesic, anti-inflammatory, anticholinestrase, CNS, CVS, antiallergic, antiana-phylactic, immunological, anti-tumor, anti-microbial and anti-parasitic activities are the most important reported effects.

Among the various classes of phytocompounds isolated from Crinums, alkaloids seem to be the main bioactive constituents. As a result, the above reviewed findings doubtlessly present Crinum as an endless source of bioactive principles. This fact can also be substantiated by four evidences. Firstly, only about thirty-five of 130 species have been phytochemically studied, whereas the largest number of species remains out there waiting to be visited. Secondly, hybridization among different species, that is considered a common phenomenon among Crinums.

Both the unstudied species and new hybrids open the gate towards isolation of further new compounds. Furthermore, chemical investigation of these unstudied species will be of high chemotaxonomic value to resolve the complicated taxonomic situation within this genus.

The third evidence comes from the lesser attention paid to both the non-alkaloidal and polar constituents, which could open new chemical and biological horizons if studied in the future. Fourthly, from a biological point of view, a large number of different Crinums' extracts as well as the purified compounds are still calling for further pharmacological screening. Both positive and new activities are strongly expected. Thus, the aforementioned results undoubtedly recommend Crinum and its Amaryllidaceous relatives -especially for their exclusive alkaloidal types- to be considered as candidates for development of new potent drugs, which can provide beneficial solutions against the every day challenging diseases.

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     How to cite this article:

     Refaat J, Kamel MS, Ramadan MA and Ali AA: Crinum; an endless source of Bioactive principles: a review. Part V. Biological profile. Int J Pharm Sci Res 2013; 4(4); 1239-1252.

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