Mitragyna Speciosa Korth Alkaloid Extract And Cytotoxicity Biology Essay

Nonstandard abbreviations: MSE, Mitragyna speciosa alkaloid infusion, DMSO, dimethyl sulfoxide ; HEK293, human embryologic kidney cells ; HepG2, human hepatocellular carcinoma cells ; HCT116, colon carcinoma cells ; SH-SY5Y, neuroblastoma cells ; A498, nephritic cell carcinoma ; SK-MEL-28, melanoma cells ; MEM, Eagle ‘s Medium Essential Medium ; FBS, foetus bovine serum ; DMEM, dulbecco ‘s modified Eagle ‘s medium ; IC50, half maximum repressive concentration.

Mitragyna speciosa has great possible to be used as opium replacement and handling opioid abstention syndrom & gt ; Cytotoxicity of the methanolic infusion was examined utilizing XTT check against 6 human malignant neoplastic disease cell lines & gt ; Tested with 10 concentrations of works infusion from 0.05 to 300Aµg/ml & gt ; Acute and Chronic in vitro survey & gt ; Acute dermatoxicity, chronic and moderate hepatotoxicity, nephrotoxicity and neurotoxicity were reported.

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Ethnopharmacological relevancy: Kratom, or Mitragyna speciosa Korth, has been reported as a mu- and delta- opiate receptor agonist bring forthing similar effects as those produced by morphia. It has been traditionally utilised as an opium replacement or to relieve opiate backdown symptoms. In malice of its comparatively common use, toxicological information of this works is still missing.

Purpose: The purpose of the survey is to find the ague and chronic cytotoxicity of Mitragyna speciosa Korth alkaloid infusion in vitro in assorted human malignant neoplastic disease cell lines.

Methodology: Cells were treated with 0.05 to 300Aµg/ml of Mitragyna speciosa Korth alkaloid infusion. The consequence of the alkaloid infusion on proliferative responses of the malignant neoplastic disease cell lines were assessed by cell proliferation cytotoxicity assay ( XTT ) after 24, 48 and 72 hours. Untreated cells were used as control.

Consequences and decision: Significant growing repressive activity of the alkaloid infusion on malignant neoplastic disease cell lines were noted. The Mitragyna speciosa alkaloid infusion showed acute possible toxicity to the tegument melonoma cells ( SK-MEL-28 ) after 24 hours incubation with IC50 value of 16.15 A± 1.45Aµg/ml. Furthermore, moderate chronic hepatotoxicity, nephrotoxicity, neurotoxicity, embryotoxicity and colon toxicity was reported in this survey.

Cardinal words: Mitragyna Speciosa, cytotoxicity, XTT, malignant neoplastic disease cell lines, IC50.


Mitragyna speciosa Kroth ( from the Rubiaeceae household ) is an autochthonal works which is found chiefly in the northern Malaysia peninsula and in cardinal and southern parts of Thailand ( Suhanya et al. , 2009 ) . It is known as “ Biak-biak ” and “ Ketum ” in Malaysia, and as “ Kratom ” in Thailand ( Saidin et al. , 2008 ) . The major indole-alkaloid component found in the Mitragyna speciosa is mitragynine, a mu- and delta-opioid receptor agonist. ( Juzaili et al. , 2010 ) . Surveies have shown the psychotropic belongingss of mitragynine is linked with it ‘s high affinity for opiate receptors, therefore bespeaking it ‘s possible for handling opiate dependence as replacing therapy ( Babu et al. , 2008 ) .

Traditionally, leaves from this works have been consumed by the Thai and Malayan indigens chiefly to handle diarrhea, and to bring forth a stimulation and euphoric consequence to battle weariness and to increase tolerance to the hot Sun ( Kavita et al. , 2008 ) . Furthermore, the foliages have been reported to possess opium-like belongingss in which it ‘s exciting consequence was found in low dosage. High doses of the Mitragyna speciosa infusion can do analgesia and hallucination ( Suchitra et al. , 1998 ) . Due to its alone medicative belongingss, kratom has been widely used to handle hurting and opium backdown symptoms since the 19th century. However, dependence and several opioid abstention syndrome such as crossness, oscitance, rhinorrhoea, myodynia, diarrhea, shudder, sickness, nystagmus, and arthralgia were reported among the chronic users ( Kavita et al. , 2008 ) . Anorexia, weight loss, skin blackening and psychosis have besides been reported ( Suwanlert, 1975 ) .

Although, the pharmacolofical effects of Kratom in human and experimental animate beings are good established, the doses required to bring forth toxicity still remain ill defined. The lone toxicological survey by Harizal et Al. ( 2010 ) , standardized methyl alcohol infusion of Mitragyna speciosa Korth caused mild nephrotoxicity and terrible hepatotoxicity at doses higher than 1000 mg/kg ( Harizal et al. , 2010 ) . No acute neurotoxicity effects were found in the cerebral mantle and hippocampus of the rat ( Harizal et al. , 2010 ) .

The issue of abuse of the works by the drug addicts has caused major concerns in Malaysia and Thailand. Consequently, Kratom works has been listed as a controlled substance in Malaysia, Thailand and Australia. However, in other parts of the universe, kratom is presently non strongly regulated. The availbility of kratom over the cyberspace has caused important drug maltreatment issue, such as self-treatment in opioid backdown and in chronic hurting ( Kavita et al. , 2008 ; Somsmorn et al. , 2008 ) . The chief aim of this survey is to look into the possible ague and chronic in vitro cytotoxicity of Mitragyna Speciosa Korth alkaloid infusions against different human malignant neoplastic disease cell lines in order to gauge its possible toxicity to worlds.

2. Methodology

2.1 Preparation of Mitragyna speciosa Korth alkaloid infusion

Fresh foliages of Mitragyna Speciosa Korth were collected from the wood in Perlis, Malaysia. Methanol-chloroform extraction method was used to pull out the alkaloid compounds. The foliages ( 5kg ) were dried and soaked in methyl alcohol for 3 yearss. The methanol mixture was filtered and the filtrate evaporated utilizing a rotary evaporator. The extraction and vaporization process was repeated three times. Following that, the petroleum methyl alcohol infusion was re-dissolved in 10 % acetic acid and so washed with hexane. The acidic bed was basified to pH 9 utilizing ammonia hydrated oxide and extracted with trichloromethane. The gathered organic bed was filtered through Na sulphate anhydrous and the filtrate was extracted utilizing a rotary evaporator to obtain 5.86g of petroleum alkaloid infusion. The akaloid infusion was so identified utilizing Dragendorf trial ( Juzaili et al. , 2010 ) . Last, the infusion was redissolved in dimethylsulphoxide ( DMSO ) to organize the stock solution, following which, it was filtered utilizing 0.2I?m syringe filter before being tested on the different cell lines.

2.2 Cell Culture

The consequence of alkaloid infusion on cell viability was assessed in a cell civilization system utilizing cells from human embryologic kidney cells ( HEK293 ) , human hepatocellular carcinoma cells ( HepG2 ) , colon carcinoma cells ( HCT116 ) , neuroblastoma cells ( SH-SY5Y ) , nephritic cell carcinoma ( A498 ) and melanoma cells ( SK-MEL-28 ) which were obtained from American Type Culture Collection ( ACTT ) and Aurigene Accelerating Discovery Ltd, Bangalore, India. HepG2, A-498, and SK-MEL-28 cell lines were grown in Eagle ‘s Medium Essential Medium ( MEM ) ; with 10 % fetus bovine serum ( FBS ) and supplmented with 1 % of 100x penicillin-streptomycin antibiotic, 2mM glutamine, 1 % non-essential amino acid and 1 % Na pyruvate. Meanwhile, HEK293 cell line was grown in Dulbecco ‘s modified Eagle ‘s medium ( DMEM ) with high glucose content ( 4.5gL1 ) . SH-SY5Y cell line was cultured in 1:1 mixture of MEM and Ham ‘s F12 medium. HCT116 cell line was grown in McCoy ‘s 5a medium. The medium for cryopreservation contained 20 % FBS and 5-8 % DMSO in growing medium. All the cell lines were maintained at 370C in a 5 % CO2 atmosphere with 95 % humidness. At 60-70 % meeting, cells were trypsinized and seeded in 96-well home bases in several optimum cell denseness. Twenty-four hours after the cell seeding, cells were treated with assorted concentrations of works infusions.

2.3 cell proliferation ( XTT ) checks

The cell viability of the malignant neoplastic disease cell lines were determined by utilizing cell proliferation XTT checks in which the feasible cells were determined by the sum of mitochondrial dehydogenases released in the life cells. Mitochondrial dehydrogenese of the variable cells cleave the tetrazolium ring of XTT ( 2,3-bis [ 2-Methoxy-4-intro-5-sulfophenyl ] -2 H-tetrazolium-5-carboxyanilide interior salt ) giving orange formazan cystals which can be measured by a spectrophotometer ( Scudiero, 1988 ; Weislow, 1989 ) . An addition or lessening in life cell Numberss result in a attendant alteration in the sum of formazan formed, bespeaking the degree of cytotoxicity caused by the works infusion. The cells were seeded into 96 Wellss home bases in several optimum cell denseness and incubated for 24 hours to acquire 70 % feeder. Following that, cells were incubated with assorted concentrations of works infusion ( 0.05-300Aµg/ml ) for 24 hours, after which, works infusion was removed from each well and so washed with phosphate-buffered saline ( PBS ) . After incubation, for every 100Aµl of serum free medium, 50Aµl of XTT solution with phenazine methosulphate ( PMS ) was added, and incubated for a farther 4 hours at 37oC. The optical density of the samples were checked at 465nm utilizing fluorescence spectrophotometry. Untreated cells were used as control. Cell viability was determined after 24, 48 and 72 hours incubation clip and defined as a per centum of cell endurance ( ratio of optical density of treated cells to untreated cells ) . All informations were recorded as average A± SEM of triplicate measurings.

3. Consequences

3.1 Preparation of alkaloid infusion

One kg of dried powdery mitragyna speciosa leaves ( about 5kg of fresh foliages ) gave a rough infusions of 105g methanolic infusion. From the petroleum methanolic infusion, 5.86g of alkaloid infusion was obtained ( 0.12 % output of fresh leave weight ) . The sum of mitragynine exist in the alkaloid infusion was approxiamately 22-24 % ( Juzaili et al. , 2010 )

3.2 Cytotoxic activity of Mitragyna speciosa alkaloid extracts on malignant neoplastic disease cell lines

The consequences depicted in Table 1, Figure 1 and 2 summarize the cytotoxic effects of the alkaloid infusion on HepG2, A498, SK-MEL-28, HEK293 HCT116, and SH-SY5Y cell lines. Table 2 indicated the categorization of cytotoxicity for natural merchandises. Untreated cells were used as control. The IC50 consequences ( average A±SEM ) are listed as the per centum of cell endurance after exposure to different concentrations of MSE and determined at three incubation times. Based on the figure 1 consequences, XTT assay showed that alkaloid infusion caused a important cytotoxic consequence from 0.05 to 300 Aµg/ml.

3.2.1 HCT116 cells

Within 24hrs, there was a clear dose-dependent suppression of cell proliferation compared to the control ( Fig. 2a ) and the consequence become pronounced at the doses higher than 11Aµg/ml. By 48 hour, proliferation of cells treated with lower concentration of alkaloid infusion ( a‰¤ 33Aµg/ml ) has recovered. However, cell proliferation suppression remained observed at other extract concentration. Approximately 100 % of mortality rate was observed with highest concentration of alkaloid infusion, 200 Aµg/ml. The IC50 for the cell at 24 and 72 hrs period were 62 and 43Aµg/ml severally ( Table 1 ) .

3.2.2 HEK293 cells

With HEK293 cells, alkaloid infusion of Mitragyna speciosa caused a dose-dependent suppression of cell proliferation at all incubation times. As with other cells, cell proliferation recovering was observed at lowest concentration after 48 hour. At higher doses of infusion ( & gt ; 33Aµg/ml ) , cell proliferation was inhibited. After 72 hour, about 100 % cells were dead at highest dosage, & gt ; 100Aµg/ml ( Fig. 2b ) . The IC50 value in this cell was estimated as 46 Aµg/ml in 24 hour and 27 Aµg/ml in 72 hour ( Table 1 ) .

3.2.3 SH-SY5Y cells

With SH-SY5Y cells, low doses of alkaloid infusion ( 1.23 – 11.11 Aµg/ml ) somewhat increased the cell proliferation up to 48 hour ( Fig. 2c ) . However, higher doses ( & gt ; 33Aµg/ml ) inhibited the cell growing well within 24 hour. The IC50 of the cells after 24 and 72 hour intervention were 66 and 34Aµg/ml, severally ( Table 1 ) .

3.2.4 HepG2 cells

Alkaloid infusion treated HepG2 cells had shown the same form of cell proliferation suppression as other cell lines. At concentration higher than 33Aµg/ml, there was a outstanding cell decease throughout the experiment ( Fig. 2d ) . Low or no suppression was found at the two lowest dosage. The IC50 for HepG2 cells at 24 and 72 hour intervention were about 49 and 27Aµg/ml, severally ( Table 1 ) .

3.2.5 SK-MEL-28 cells

For SK-MEL-28 cells, acute cytotoxicity was observed within 24 hour intervention ( Fig. 2e ) . Almost 100 % of cell decease was reported at 2 highest doses ( 100 and 200Aµg/ml ) . Low to chair suppression was noted at concentration lower than 33Aµg/ml. Simultaneously, cells were recovered at low concentration of infusion. The IC50 for the cells was 16Aµg/ml about at all the clip points ( Table 1 ) .

3.2.6 A498 cells

With A498 cells, dose-dependent suppression of cell proliferation became outstanding after 100 Aµg/ml ( Fig 2f ) . Low or no suppression of cell proliferation was observed at concentration lower than 33Aµg/ml. However, there was a sudden decreasing in cell proliferation after 48 hour of high dose-treatment ( 100Aµg/ml ) . The IC50 reported in this cells were approximately 82 and 15Aµg/ml in 24 and 72 hour intervention ( Table 1 ) .


The usage of traditional medical specialties from natural merchandises have become popular all over the universe particularly in the development states. Herbs were ever presumed to be safe because of its “ naturality ” . However, as quoted famously by Paracelsus, “ All substances are toxicants ; there is none that is non a toxicant. The right dosage differentiates a toxicant and a redress ” . Self medicine among the user without cognizing its toxicity and non being cognizant of the dosage that is being consumed is highly unsafe. On the other manus, most of herbal medical specialties are given without a proper prescription, high doses or in combination with other medicines may do toxic effects.

Assorted types of pharmacological benefit from Mitragyna speciosa had been reported. However, due to it morphine-like belongingss, it has been sharply misused among the drug addicts as opium replacement or to relieve opiate backdown symptoms. Several states such as Malaysia, Thailand, Myammar and Australia have legislated this works due to its narcotism. Even throught, the possible toxicity of the mitragyna speciosa is still remain ill-defined. Therefore, toxicological appraisal of this alkaloid infusion was investigated in this survey. The alkaloid compounds were extracted utilizing the methanol-chloroform extraction method. Previous surveies have identified that mitragynine was the most abundant alkaloid found. It has been good established to possess morphine-like effects on in vitro and in vivo surveies ( Idid et al. , 1998 ; Matsumoto et al. , 1996 ) . Other indole alkaloids such as speciofoline, rhychophylline, stipulatine, isomitraphylline and ajmalicine have besides been isolated from the foliages every bit good ( Beckett et al. , 1965 ) . Besides, there was an interesting determination in which another alkaloid compound, 7-hydroxymitragynine, had been reported to hold a stronger analagesic consequence and a powerful GI theodolite suppression in the mice when compared to morphine ( Matsumoto et al. , 2004a, 2006b ) . Tolerance and morphine-like backdown symptoms by 7-hydroxymitragynine and mitragynine have besides been reported.

In the present survey, the cytotoxic effects of Mitragyna speciosa alkaloid infusion was evaluated utilizing several human malignant neoplastic disease cell lines in vitro. Due to its fast, easy growth and other particular mammlian feature, malignant neoplastic disease cell lines were widely used as primary showing for the in vitro cytotoxicity. For illustration, HepG2 cells are the transfected cells which demoing metabolic activities such as cytochrome P450s metamorphosis and hydroxylation ( Wu et al. , 2006 ) . Besides, Aµ- and I?- types opiate binding side are presented on SH-SY5Y cells. HEK293 cells are the kidney embryonic cells which consist of many cell features.

In add-on, the usage of the XTT assay greatly simplifies the process for mensurating proliferation as compared to the MTT check, whereby the solubilization measure that is present in the MTT check was avoided. There was a restriction to the high concentrations of Mitragyna speciosa alkaloid infusion used since highly high concentrations of Mitragyna speciosa interfered with assay fluoroscence measuring ( Saidin et al. , 2008 ) . However, there was a dose dependent toxicity tendency seen with the alkaloid infusion at the doses of concentration a‰¤ 300Aµg/ml.

Regardless of incubation times, In vitro showing of alkaloid infusion demonstrated that skin melanoma cell SK-MEL-28 was the most sensitive cell line examined. The IC50 after 24hrs intervention of SK-MEL-28 was 16.9Aµg/ml. Epidemiology survey showed that blackening of the tegument was found in chronic consumers the of Mitragyna speciosa infusions, but the mechanism remain ill-defined. However, most decently, it was caused by the dark pigment, melanin which produced by activation of melanocytes in response to the melanocyte-stimulating endocrine ( MSH ) stimulation. MSH was released by pituatray secretory organ as response to the mitragynine-estimulating corticotropin-releasing endocrine ( CRH ) release from hypothalamus.

Apart from the acute cytotoxicity effects ( 24 hour intervention ) , another major determination in this survey was the chronic cytotoxicity effects by Mitragyna speciosa infusion as determined by 72 hour intervention. Moderate cytotoxicity on kidney, liver, colon and nervus malignant neoplastic disease cell lines were observed. However, cell proliferation was wholly inhibited when treated with the dose a‰?200Aµg/ml. There were some differences in the IC50 values for the HepG2, HEK293 and SH-SY5Y in this survey from those of Saidin et al. , ( 2008 ) . The survey of Saidin et Al. ( 2008 ) showed that mitraynine was most toxic to SH-SY5Y and moderate cytotoxicity to HEK293 and HepG2. The IC50 consequences in Saidin ‘s survey was comparatively higher than the present survey. Variation such as mode of readying and extraction methods can besides contributed to the information variableness. Uncontrollable factors such as clime, growing and storage status might impact the quality of the substrates as good ( Akansha et al. , 2008 ; Hanapi et al. , 2010 ) . Besides, the usage of comparative IC50 and absolute IC50 remain controversial among the research workers. Relative IC50 showed the concentration required to convey the curve down to the point half manner of the upper limit and minimal tableland of the curve. Whilst, absolute IC50 is defined as the concentration required to give 50 % of suppression. However, comparative IC50 was more normally used comparison to absolute IC50 because the authority of the drug is ignored in the finding of absolute IC50.

Harizal ‘s survey showed that unwritten disposal of standardised methanolic infusion of Mitragyna speciosa resulted in terrible hepatotoxicity and mild nephrotoxicity in high doses. This had been proven by histological and biochemical scrutiny of Mitragyna speciosa infusions treated liver cells. Kupffer cells, enlargment of karyon ( karyomegaly ) , and important lift of ALT degree was found ( Harizal et al. , 2010 ) . Metabolic activation of xenobiotic was believed to be one of the factors that had caused the hepatotoxicity. Besides, ingestion of the the Mitragyna speciosa infusion by gnawers has been found to do an addition in blood force per unit area ( Harizal et al. , 2010 ) . There is a likeliness that the effects produced are due to I±2-adrenoceptor adversaries consequence, which is the instance with yohimbine ( Verwaerde et al. , 1997 ) . On the other manus, in vivo acute treamtment of Mitragyna speciosa did non convey any harm in axons and dendrities of the hippocampal nerve cells ( Harizal et al. , 2010 ) . High dosage of disposal among the chronic user are categorized as high hazard user because of the drug tolerance and dependence effects.

It has been noticed that mitragynine is structurally similar to yohimbine, an alkaloid with stimulation and aphrodisiac effects which is found of course in Pausinystalia yohimbe ( from Rubiaeceae household ) ( Fig.3 ) . Yohimbine is an I±2-adrenoceptor adversary which has been used to handle idiopathic and medication-induced erectile upset ( Benjamin et al. , 2007 ) . Several similarities in side effects mitragynine and yohimbine such as shudder, crossness, hallucination, giddiness, tegument flushing, ictus and nephritic failure have been reported in the literature ( Quinton, 1963 ; Eric et al. , 1989 ; Lydia et al. , 2001 ) . An interesting antinociceptive survey had demonstrated that pre-treatment of yohimbine wholly blocked the opiate receptor agonist such as morphia ( mu-opioid receptor agonist ) , U-50,488 ( kappa- opioid receptor agonist ) and SNC80 ( delta- opioid receptor agonist ) ( lydia et al. , 2001 ) . Based on the chemical construction of yohimbine, ester group and hydroxy group at the C-17 place have the stronger affinity to the I±2-adrenergic receptor, 5-hydroxytryptamine and Dopastat receptor. Therefore, this indicated that yohimbine have high potency to be competitory agonist to the opiate receptor. Minor differences in their chemical constructions such as the molecular weight, mutual opposition, indoloquinolizidine construction, sum of rings, might given some alterations in the pharmacological effects every bit good.

In decision, Mitragyna speciosa alkaloid infusion showed most sensitive cytotoxic consequence on tegument melanoma cells after 24hr of drug intervention. Chronic and moderate cytotoxicity was reported in the human liver, kidney, colon and nervus malignant neoplastic disease cells. This determination supports the consequence of the hepatotoxic and nephrotoxic effects from in vivo survey of Harizal et Al. ( 2010 ) . Futher surveies on other active compounds in the alkaloid infusion are neccessary in order to place the most toxic chemical constituents in the infusion. Besides, metabolic activation, particularly cytochrome P450 metamorphosis activation by the infusion must be carried out in order to acquire a better apprehension of the mechanism of toxicity.


This research was supported in portion by Aurigene Discovery Technologies ( M ) Sdn. Bhd and University of Malaya Grant PS460/2010A. The work of my co-workers who assisted me in the undertakings is appreciatively acknowledged.


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