Nonstandard abbreviations: MSE, Mitragyna speciosa alkaloid extract, DMSO, dimethyl sulfoxide; HEK293, human embryonic kidney cells; HepG2, human hepatocellular carcinoma cells; HCT116, colon carcinoma cells; SH-SY5Y, neuroblastoma cells; A498, renal cell carcinoma; SK-MEL-28, melanoma cells; MEM, Eagle’s Medium Important Medium; FBS, fetus bovine serum; DMEM, dulbecco’s modified Eagle’s medium; IC50, half maximal inhibitory focus .
Mitragyna speciosa has nice potential for use as opium substitute and treating opioid abstinence syndrom > Cytotoxicity of the methanolic extract was examined utilizing XTT assay in opposition to 6 human most cancers cell strains > Examined with 10 concentrations of plant extract from zero.05 to 300µg/ml > Acute and Power in vitro examine > Acute dermatoxicity, continual and reasonable hepatotoxicity, nephrotoxicity and neurotoxicity have been reported.
Ethnopharmacological relevance: Kratom, or Mitragyna speciosa Korth, has been reported as a mu- and delta- opiate receptor agonist producing related results as these produced by morphine. It has been historically utilized as an opium substitute or to alleviate opiate withdrawal signs. Regardless of its comparatively frequent utilization, toxicological information of this plant remains to be missing.
Intention: The goal of the examine is to find out the acute and continual cytotoxicity of Mitragyna speciosa Korth alkaloid extract in vitro in varied human most cancers cell strains.
Methodology: Cells have been handled with zero.05 to 300µg/ml of Mitragyna speciosa Korth alkaloid extract. The impact of the alkaloid extract on proliferative responses of the most cancers cell strains have been assessed by cell proliferation cytotoxicity assay (XTT) after 24, 48 and 72 hours. Untreated cells have been used as management.
Outcomes and conclusion: Vital progress inhibitory exercise of the alkaloid extract on most cancers cell strains have been famous. The Mitragyna speciosa alkaloid extract confirmed acute potential toxicity to the pores and skin melonoma cells (SK-MEL-28) after 24 hours incubation with IC50 worth of 16.15 ± 1.45µg/ml. Moreover, reasonable continual hepatotoxicity, nephrotoxicity, neurotoxicity, embryotoxicity and colon toxicity was reported on this examine.
Key phrases: Mitragyna Speciosa, cytotoxicity, XTT, most cancers cell strains, IC50.
Mitragyna speciosa Kroth (from the Rubiaeceae household) is an indigenous plant which is discovered primarily within the northern Malaysia peninsula and in central and southern components of Thailand (Suhanya et al., 2009). It is named “Biak-biak” and “Ketum” in Malaysia, and as “Kratom” in Thailand (Saidin et al., 2008). The most important indole-alkaloid constituent discovered within the Mitragyna speciosa is mitragynine, a mu- and delta-opioid receptor agonist. (Juzaili et al., 2010). Research have proven the psychoactive properties of mitragynine is linked with it’s excessive affinity for opiate receptors, therefore indicating it’s potential for treating opiate dependancy as alternative remedy (Babu et al., 2008).
Historically, leaves from this plant have been consumed by the Thai and Malaysian natives primarily to deal with diarrhoea, and to supply a stimulant and euphoric impact to fight fatigue and to extend tolerance to the new solar (Kavita et al., 2008). Moreover, the leaves have been reported to own opium-like properties by which it’s stimulating impact was present in low dose. Excessive doses of the Mitragyna speciosa extract could cause analgesia and hallucination (Suchitra et al., 1998). On account of its distinctive medicinal properties, kratom has been broadly used to deal with ache and opium withdrawal signs for the reason that nineteenth century. Nonetheless, dependancy and a number of other opioid abstinence syndrome comparable to irritability, yawning, rhinorrhoea, myalgias, diarrhoea, tremor, nausea, nystagmus, and arthralgia have been reported among the many continual customers (Kavita et al., 2008). Anorexia, weight reduction, pores and skin darkening and psychosis have additionally been reported (Suwanlert, 1975).
Though, the pharmacolofical results of Kratom in human and experimental animals are nicely established, the doses required to supply toxicity nonetheless stay poorly outlined. The one toxicological examine by Harizal et al. (2010), standardized methanol extract of Mitragyna speciosa Korth brought on delicate nephrotoxicity and extreme hepatotoxicity at doses increased than 1000 mg/kg (Harizal et al., 2010). No acute neurotoxicity results have been discovered within the cortex and hippocampus of the rat (Harizal et al., 2010).
The difficulty of misuse of the plant by the drug addicts has brought on main issues in Malaysia and Thailand. Consequently, Kratom plant has been listed as a managed substance in Malaysia, Thailand and Australia. Nonetheless, in different components of the world, kratom is at present not strongly regulated. The availbility of kratom over the web has brought on important drug abuse subject, comparable to self-treatment in opioid withdrawal and in continual ache (Kavita et al., 2008; Somsmorn et al., 2008). The principle goal of this examine is to analyze the potential acute and continual in vitro cytotoxicity of Mitragyna Speciosa Korth alkaloid extracts in opposition to completely different human most cancers cell strains with a view to estimate its potential toxicity to people.
2.1 Preparation of Mitragyna speciosa Korth alkaloid extract
Contemporary leaves of Mitragyna Speciosa Korth have been collected from the forest in Perlis, Malaysia. Methanol-chloroform extraction technique was used to extract the alkaloid compounds. The leaves (5kg) have been dried and soaked in methanol for Three days. The methanol combination was filtered and the filtrate evaporated utilizing a rotary evaporator. The extraction and evaporation process was repeated thrice. Following that, the crude methanol extract was re-dissolved in 10% acetic acid after which washed with hexane. The acidic layer was basified to pH 9 utilizing ammonia hydroxide and extracted with chloroform. The collected natural layer was filtered via sodium sulphate anhydrous and the filtrate was extracted utilizing a rotary evaporator to acquire 5.86g of crude alkaloid extract. The akaloid extract was then recognized utilizing Dragendorf check (Juzaili et al., 2010). Lastly, the extract was redissolved in dimethylsulphoxide (DMSO) to type the inventory resolution, following which, it was filtered utilizing zero.2Î¼m syringe filter earlier than being examined on the completely different cell strains.
2.2 Cell Tradition
The impact of alkaloid extract on cell viability was assessed in a cell tradition system utilizing cells from human embryonic kidney cells (HEK293), human hepatocellular carcinoma cells (HepG2), colon carcinoma cells (HCT116), neuroblastoma cells (SH-SY5Y), renal cell carcinoma (A498) and melanoma cells (SK-MEL-28) which have been obtained from American Sort Tradition Assortment (ACTT) and Aurigene Accelerating Discovery Ltd, Bangalore, India. HepG2, A-498, and SK-MEL-28 cell strains have been grown in Eagle’s Medium Important 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% sodium pyruvate. In the meantime, HEK293 cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) with excessive glucose content material (four.5gL1). SH-SY5Y cell line was cultured in 1:1 combination 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-Eight% DMSO in progress medium. All of the cell strains have been maintained at 370C in a 5% CO2 environment with 95% humidity. At 60-70% confluence, cells have been trypsinized and seeded in 96-well plates in respective optimum cell density. Twenty-four hours after the cell seeding, cells have been handled with varied concentrations of plant extracts.
2.Three cell proliferation (XTT) assays
The cell viability of the most cancers cell strains have been decided through the use of cell proliferation XTT assays by which the viable cells have been decided by the quantity of mitochondrial dehydogenases launched within the residing cells. Mitochondrial dehydrogenese of the variable cells cleave the tetrazolium ring of XTT (2,Three-bis[2-Methoxy-4-intro-5-sulfophenyl]-2 H-tetrazolium-5-carboxyanilide interior salt) yielding orange formazan cystals which will be measured by a spectrophotometer (Scudiero, 1988; Weislow, 1989). A rise or lower in residing cell numbers end in a concomitant change within the quantity of formazan fashioned, indicating the extent of cytotoxicity attributable to the plant extract. The cells have been seeded into 96 wells plates in respective optimum cell density and incubated for 24 hours to get 70% confluent. Following that, cells have been incubated with varied concentrations of plant extract (zero.05-300µg/ml) for 24 hours, after which, plant extract was faraway from every nicely after which washed with phosphate-buffered saline (PBS). After incubation, for each 100µl of serum free medium, 50µl of XTT resolution with phenazine methosulphate (PMS) was added, and incubated for an additional four hours at 37oC. The absorbance of the samples have been checked at 465nm utilizing fluorescence spectrophotometry. Untreated cells have been used as management. Cell viability was decided after 24, 48 and 72 hours incubation time and outlined as a share of cell survival (ratio of absorbance of handled cells to untreated cells). All information have been recorded as imply ± SEM of triplicate measurements.
Three.1 Preparation of alkaloid extract
One kilogram of dried powdered mitragyna speciosa leaves (roughly 5kg of contemporary leaves) gave a crude extracts of 105g methanolic extract. From the crude methanolic extract, 5.86g of alkaloid extract was obtained ( zero.12% yield of contemporary depart weight). The quantity of mitragynine exist within the alkaloid extract was approxiamately 22-24% (Juzaili et al., 2010)
Three.2 Cytotoxic exercise of Mitragyna speciosa alkaloid extracts on most cancers cell strains
The outcomes depicted in Desk 1, Determine 1 and a couple of summarize the cytotoxic results of the alkaloid extract on HepG2, A498, SK-MEL-28, HEK293 HCT116, and SH-SY5Y cell strains. Desk 2 indicated the classification of cytotoxicity for pure merchandise. Untreated cells have been used as management. The IC50 outcomes (imply ±SEM) are listed as the share of cell survival after publicity to completely different concentrations of MSE and decided at three incubation occasions. Based mostly on the determine 1 outcomes, XTT assay confirmed that alkaloid extract brought on a major cytotoxic impact from zero.05 to 300 µg/ml.
Three.2.1 HCT116 cells
Inside 24hrs, there was a transparent dose-dependent inhibition of cell proliferation in comparison with the management (Fig. 2a) and the impact turn out to be pronounced on the doses increased than 11µg/ml. By 48 hrs, proliferation of cells handled with decrease focus of alkaloid extract (â‰¤ 33µg/ml) has recovered. Nonetheless, cell proliferation inhibition remained noticed at different extract focus. Roughly 100% of mortality fee was noticed with highest focus of alkaloid extract, 200 µg/ml. The IC50 for the cell at 24 and 72 hrs interval have been 62 and 43µg/ml respectively (Desk 1).
Three.2.2 HEK293 cells
With HEK293 cells, alkaloid extract of Mitragyna speciosa brought on a dose-dependent inhibition of cell proliferation in any respect incubation occasions. As with different cells, cell proliferation recovering was noticed at lowest focus after 48 hrs. At increased doses of extract (>33µg/ml), cell proliferation was inhibited. After 72 hrs, roughly 100% cells have been useless at highest dose, >100µg/ml (Fig. 2b). The IC50 worth on this cell was estimated as 46 µg/ml in 24 hr and 27 µg/ml in 72 hrs (Desk 1).
Three.2.Three SH-SY5Y cells
With SH-SY5Y cells, low doses of alkaloid extract (1.23 – 11.11 µg/ml) barely elevated the cell proliferation as much as 48 hrs (Fig. 2c). Nonetheless, increased doses (>33µg/ml) inhibited the cell progress considerably inside 24 hrs. The IC50 of the cells after 24 and 72 hrs remedy have been 66 and 34µg/ml, respectively (Desk 1).
Three.2.four HepG2 cells
Alkaloid extract handled HepG2 cells had proven the identical sample of cell proliferation inhibition as different cell strains. At focus increased than 33µg/ml, there was a outstanding cell demise all through the experiment (Fig. second). Low or no inhibition was discovered on the two lowest dose. The IC50 for HepG2 cells at 24 and 72 hrs remedy have been roughly 49 and 27µg/ml, respectively (Desk 1).
Three.2.5 SK-MEL-28 cells
For SK-MEL-28 cells, acute cytotoxicity was noticed inside 24 hrs remedy (Fig. 2e). Nearly 100% of cell demise was reported at 2 highest doses (100 and 200µg/ml). Low to reasonable inhibition was famous at focus decrease than 33µg/ml. Concurrently, cells have been recovered at low focus of extract. The IC50 for the cells was 16µg/ml roughly at on a regular basis factors (Desk 1).
Three.2.6 A498 cells
With A498 cells, dose-dependent inhibition of cell proliferation grew to become outstanding after 100 µg/ml (Fig 2f). Low or no inhibition of cell proliferation was noticed at focus decrease than 33µg/ml. Nonetheless, there was a sudden reducing in cell proliferation after 48 hrs of excessive dose-treatment (100µg/ml). The IC50 reported on this cells have been about 82 and 15µg/ml in 24 and 72 hrs remedy (Desk 1).
Using conventional medicines from pure merchandise have turn out to be common everywhere in the world particularly within the growing international locations. Herbs have been at all times presumed to be secure due to its “naturality”. Nonetheless, as quoted famously by Paracelsus, “All substances are poisons; there may be none that isn’t a poison. The precise dose differentiates a poison and a treatment”. Self medicine among the many person with out figuring out its toxicity and never being conscious of the dose that’s being consumed is extraordinarily harmful. However, most of natural medicines are given with no correct prescription, excessive doses or together with different medicines could trigger poisonous results.
Varied kinds of pharmacological profit from Mitragyna speciosa had been reported. Nonetheless, on account of it morphine-like properties, it has been aggressively misused among the many drug addicts as opium substitute or to alleviate opiate withdrawal signs. A number of international locations comparable to Malaysia, Thailand, Myammar and Australia have legislated this plant on account of its narcotism. Even throught, the potential toxicity of the mitragyna speciosa remains to be stay unclear. Subsequently, toxicological evaluation of this alkaloid extract was investigated on this examine. The alkaloid compounds have been extracted utilizing the methanol-chloroform extraction technique. Earlier research have recognized that mitragynine was probably the most considerable alkaloid discovered. It has been nicely established to own morphine-like results on in vitro and in vivo research (Idid et al., 1998; Matsumoto et al., 1996). Different indole alkaloids comparable to speciofoline, rhychophylline, stipulatine, isomitraphylline and ajmalicine have additionally been remoted from the leaves as nicely (Beckett et al., 1965). In addition to, there was an fascinating discovering by which one other alkaloid compound, 7-hydroxymitragynine, had been reported to have a stronger analagesic impact and a potent gastrointestinal transit inhibition within the mice when in comparison with morphine (Matsumoto et al., 2004a, 2006b). Tolerance and morphine-like withdrawal signs by 7-hydroxymitragynine and mitragynine have additionally been reported.
Within the current examine, the cytotoxic results of Mitragyna speciosa alkaloid extract was evaluated utilizing a number of human most cancers cell strains in vitro. On account of its quick, simple rising and different particular mammlian attribute, most cancers cell strains have been broadly used as main screening for the in vitro cytotoxicity. For instance, HepG2 cells are the transfected cells which displaying metabolic actions comparable to cytochrome P450s metabolism and hydroxylation (Wu et al., 2006). In addition to, µ- and Î´- varieties opiate binding facet are introduced on SH-SY5Y cells. HEK293 cells are the kidney embryonic cells which include many cell traits.
As well as, using the XTT assay drastically simplifies the process for measuring proliferation as in comparison with the MTT assay, whereby the solubilization step that’s current within the MTT assay was prevented. There was a limitation to the excessive concentrations of Mitragyna speciosa alkaloid extract used since extraordinarily excessive concentrations of Mitragyna speciosa interfered with assay fluoroscence measurement (Saidin et al., 2008). Nonetheless, there was a dose dependant toxicity pattern seen with the alkaloid extract on the doses of focus â‰¤ 300µg/ml.
No matter incubation occasions, In vitro screening of alkaloid extract demonstrated that pores and skin melanoma cell SK-MEL-28 was probably the most delicate cell line examined. The IC50 after 24hrs remedy of SK-MEL-28 was 16.9µg/ml. Epidemiology examine confirmed that darkening of the pores and skin was present in continual shoppers the of Mitragyna speciosa extracts, however the mechanism stay unclear. Nonetheless, most correctly, it was attributable to the darkish pigment, melanin which produced by activation of melanocytes in response to the melanocyte-stimulating hormone (MSH) stimulation. MSH was launched by pituatray gland as response to the mitragynine-estimulating corticotropin-releasing hormone (CRH) launch from hypothalamus.
Aside from the acute cytotoxicity results (24 hr remedy), one other main discovering on this examine was the continual cytotoxicity results by Mitragyna speciosa extract as decided by 72 hrs remedy. Reasonable cytotoxicity on kidney, liver, colon and nerve most cancers cell strains have been noticed. Nonetheless, cell proliferation was utterly inhibited when handled with the dose â‰¥200µg/ml. There have been some variations within the IC50 values for the HepG2, HEK293 and SH-SY5Y on this examine from these of Saidin et al., (2008). The examine of Saidin et al. (2008) confirmed that mitraynine was most poisonous to SH-SY5Y and reasonable cytotoxicity to HEK293 and HepG2. The IC50 ends in Saidin’s examine was comparatively increased than the current examine. Variation comparable to method of preparation and extraction strategies may contributed to the information variability. Uncontrollable components comparable to local weather, progress and storage situation may have an effect on the standard of the substrates as nicely (Akansha et al., 2008; Hanapi et al., 2010). In addition to, using relative IC50 and absolute IC50 stay controversial among the many researchers. Relative IC50 confirmed the focus required to convey the curve all the way down to the purpose half approach of the utmost and minimal plateaus of the curve. While, absolute IC50 is outlined because the focus required to provide 50% of inhibition. Nonetheless, relative IC50 was extra generally used examine to absolute IC50 as a result of the efficiency of the drug is ignored within the willpower of absolute IC50.
Harizal’s examine confirmed that oral administration of standardized methanolic extract of Mitragyna speciosa resulted in extreme hepatotoxicity and delicate nephrotoxicity in excessive doses. This had been confirmed by histological and biochemical examination of Mitragyna speciosa extracts handled liver cells. Kupffer cells, enlargment of nucleus (karyomegaly), and important elevation of ALT degree was discovered (Harizal et al., 2010). Metabolic activation of xenobiotic was believed to be one of many components that had brought on the hepatotoxicity. In addition to, consumption of the the Mitragyna speciosa extract by rodents has been discovered to trigger a rise in blood strain (Harizal et al., 2010). There’s a probability that the results produced are on account of Î±2-adrenoceptor antagonists impact, which is the case with yohimbine (Verwaerde et al., 1997). However, in vivo acute treamtment of Mitragyna speciosa didn’t convey any harm in axons and dendrities of the hippocampal neurons (Harizal et al., 2010). Excessive dose of administration among the many continual person are categorized as excessive threat person due to the drug tolerance and dependancy results.
It has been seen that mitragynine is structurally just like yohimbine, an alkaloid with stimulant and aphrodisiac results which is discovered naturally in Pausinystalia yohimbe (from Rubiaeceae household) (Fig.Three). Yohimbine is an Î±2-adrenoceptor antagonist which has been used to deal with idiopathic and medication-induced erectile dysfunction (Benjamin et al., 2007). A number of similarities in unwanted effects mitragynine and yohimbine comparable to tremor, irritability, hallucination, dizziness, pores and skin flushing, seizure and renal failure have been reported within the literature (Quinton, 1963; Eric et al., 1989; Lydia et al., 2001). An fascinating antinociceptive examine had demonstrated that pre-treatment of yohimbine utterly blocked the opiate receptor agonist comparable to morphine (mu-opioid receptor agonist), U-50,488 (kappa- opioid receptor agonist) and SNC80 (delta- opioid receptor agonist) (lydia et al., 2001). Based mostly on the chemical construction of yohimbine, ester group and hydroxy group on the C-17 place have the stronger affinity to the Î±2-adrenergic receptor, serotonin and dopamine receptor. Thus, this indicated that yohimbine have excessive potential to be aggressive agonist to the opiate receptor. Minor variations of their chemical buildings such because the molecular weight, polarity, indoloquinolizidine construction, whole of rings, may given some adjustments within the pharmacological results as nicely.
In conclusion, Mitragyna speciosa alkaloid extract confirmed most delicate cytotoxic impact on pores and skin melanoma cells after 24hr of drug remedy. Power and reasonable cytotoxicity was reported within the human liver, kidney, colon and nerve most cancers cells. This discovering helps the results of the hepatotoxic and nephrotoxic results from in vivo examine of Harizal et al. (2010). Futher research on different energetic compounds within the alkaloid extract are neccessary with a view to determine probably the most poisonous chemical elements within the extract. In addition to, metabolic activation, particularly cytochrome P450 metabolism activation by the extract have to be carried out with a view to get a greater understanding of the mechanism of toxicity.
This analysis was supported partially by Aurigene Discovery Applied sciences (M) Sdn. Bhd and College of Malaya Grant PS460/2010A. The work of my colleagues who assisted me within the tasks is gratefully acknowledged.