Synthesis And Pharmacological Activites Of Mangiferin Esterified-derivatives

1 BackgroundMangiferin, a naturally occurring xanthone glucoside, is widely distributed in herbals such as Mangifera indica L., Anemarrhena asphodeloides Bge and Swertia mussotii Franch and has recently attracted much attention due to its anti-diabetic, anti-inflammatory, expectorant, antitussive and anti-asthma activities.Mangiferin now can be produced in large scale and with very high purity (more than 95%). Unfortunately, to date mangiferin has not yet been developed to clinical drug, just because it has too many activities to focus on a stronger one.According to scientists, mangiferin has certain bio-activities; its weak pharmacological activity is due to its low solubility, low permeability and low bio-availability. In other words, mangiferin is a BCS-4 drug.In order to improve the bio-availability of mangiferin so as to improve its pharmacological activity, some studies were involved in improvement of solubility, and had little effects. Improvement of permeability was another way, but only few studies were involved. These studies all focused on preparation of highly lipid-soluble derivatives, which were still not good enough to be put into application even though they showed certain pharmacological activities.In this paper we tried to prepare lipid-soluble derivatives too. But one thing was important:we prepared esterified-derivatives, which have not yet been mentioned in existing literatures. These novel esterified-derivatives may lead to some breakthroughs.2 Methods and resultsThis paper reported the chemical synthesis, separation and structure confirmation of 7 novel mangiferin esterified-derivatives. In addition, the physicochemical properties and pharmacological activities of 3 main target compounds (PAM, HPM and HBM) were studied.(1) Synthesis. Mangiferin as original compound, H+ as catalyst, anhydride as solvent, mangiferin respectively reacted with acetic anhydride, propionic anhydride and butyric anhydride to produce esterified-derivatives. The reaction selectivity was so low that the reaction yielded 3 to 5 multi-acylation compounds.(2) Separation, purification and structure confirmation. Silica gel chromatography was employed to separate target compounds from the reaction product.7 novel compounds were separated and confirmed by 1H NMR,13C NMR and HMBC spectrum, their chemical structure seen in the following figure. Out of these 7 novel compounds,3 main target compounds were as follow: (a)7,2',3',4',6'-penta-acetyl-mangiferin(PAM), formula:C29H28O16; (b)3,6,7,2',3',4',6'-hepta-propionyl-mangiferin(HPM), formula:C40H46O18; (c)3,6,7,2',3',4'-hexa-butyryl-mangiferin(HBM), formula:C43H54O17. These 7 compounds were reported for the first time.(3) Physicochemical properties. PAM, HPM and HBM were easily soluble in ethyl acetate, hardly soluble in water, stable in aq. solution at pH＜9; they partly decomposed in aq. solution at pH＞10, especially at high temperature. Their melting points decreased sharply with a rate of about 100℃. They had obvious features of UV absorption. (4) O/w distribution coefficient. The concentrations of mangiferin, PAM, HPM and HBM in water and n-octanol, by which to form a liquid-liquid extraction system, were determined respectively so as to calculate the o/w distribution coefficient. The o/w distribution coefficient of mangiferin was smaller than that of PAM, HPM and HBM, and it increased when pH decreased. The distribution coefficients of PAM, HPM and HBM were all bigger than 100, with a max at pH7 (＞270); and with the increase of acidity or alkalinity, they decreased.(5) Coughing mice induced by strong ammonia were employed to study the antitussive activity. Mangiferin's high- and mid-dose group (1.0,0.5 mmol·kg-1) showed certain antitussive effect because they could significantly decrease the cough frequency(P＜0.01) and prolong the cough incubation period(P＜0.05). The antitussive effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to decrease the cough frequency and to prolong the cough incubation period.(6) Phenol red secreting mice were used to study the expectorant activity. Mangiferin's high- and mid-dose group (1.0,0.5 mmol·kg-1) showed certain expectorant effect because they could significantly increase the secretory of phenol red (P＜0.05 or P＜0.01); the expectorant effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to increase the secretory of phenol red.(7) Asthma guinea pigs induced by histamine-Ach were used to study the anti-asthma activity. Mangiferin's high-and mid-dose group (1.0,0.5 mmol·kg-1) showed certain anti-asthma effect because they could significantly prolong the asthma incubation period (P＜0.05 or P＜0.01); the anti-asthma effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to prolong the asthma incubation period.(8) Bacteria culture in vitro was employed to study the bacteriostatic activity. Although PAM, HPM and HBM showed bacteriostatic effect for MRSA(sensitive) and Bacillus paratyphosus at the max concentration (75 mg·mL-1) they didn't show that for other bacteria, or at other concentration levels.(9) HSV-1 inhibition test was used to study the anti-virus activity. Cytotoxicity of mangiferin, PAM, HPM and HBM was all not so strong; they could not obviously inhibit HSV-1, and the CPE inhibition rate was less than 50%.(10) Swelling of mice ear and permeability of mice celiac capillary were used to evaluate the anti-inflammatory activity. Mangiferin's high-and mid-dose group (1.0,0.5 mmol·kg-1), PAM's high- and mid-dose group (0.25,0.125 mmol·kg-1), HPM's high-, mid- and low-dose group (0.25,0.125, 0.063mmol·kg-1) and HBM's high-, mid- and low-dose group (0.25,0.125, 0.063mmol·kg-1) could significantly inhibit the ear swelling, showed obvious anti-inflammatory effect; although mangiferin's low-dose group (0.25mmol·kg-1) and PAM's low-dose group (0.063mmol·kg-1) had the trend to inhibit the ear swelling, the inhibition effect was not significant (P＞0.05).In another test, mangifrin's high-, mid- and low-dose group(1.0,0.5, 0.25mmol·kg-1), PAM's high-and mid-dose group(0.25,0.125mmol·kg-1), HPM's high-, mid- and low-dose group(0.25,0.125,0.063mmol·kg-1) and HBM's high- and mid-dose group(0.25, 0.125mmol·kg-1) could significantly decrease the permeability of mice celiac capillary (P＜0.01 or P＜0.05), showed obvious anti-inflammatory effect; although PAM's low-dose group (0.063mmol·kg-1) and HBM's low-dose group (0.063mmol·kg-1) had the trend to decrease the permeability of mice celiac capillary their anti-inflammatory effect was not significant (P>0.05).(11) MTT and tumor bearing mice were used to evaluate the anti-tumor activity. In MTT test, PAM, HPM and HBM showed the effect to enhance the apoptosis of 7901, hella and 7404 tumor cell. In another test, HBM high-dose group (1.0mmol·kg-1) could significantly inhibit the S180 tumor cell in vivo (P＜0.05). Although HBM mid-dose group (0.5 mmol·kg-1) had the trend to inhibit tumor cell its inhibition effect was not significant (P＞0.05).(12) PTP1B inhibition test and hyperglycemia model mice were used to evaluate the hypoglycemic activity and to investigate the hypoglycemic mechanism.In hyperglycemia mice induced by STZ, the mangiferin's high- and mid-dose group (1.0,0.5 mmol·kg-1) and all dose levels group (0.25,0.125, 0.063 mmol·kg-1) of PAM, HPM and HBM significantly showed hypoglycemic effect(P＜0.01 or P＜0.05). Although mangiferin's low-dose group (0.25 mmol·kg-1) had the trend to decrease the mice glucose level its effect was not significant (P＞0.05).In hyperglycemia mice induced by adrenaline, high-dose group (1.0 mmol·kg-1) of PAM, HPM and HBM could not supress the sharp increase of mice glucose level (P＞0.05). In PTP1B inhibition test in vitro, at 5\ig-mL'1, PAM, HPM and HBM showed obvious PTP1B inhibition effect; precipitates appeared at 20μg·mL-1, this indicated that PAM, HPM and HBM had very high PTP1B inhibition activity.3 Conclusions(1) Esterified-derivatives not only can be easily prepared from mangiferin by direct acylation reaction, but also can be easily, efficiently separated and purified from the reaction product. Novel compound IⅠ～Ⅳ, PAM, HPM and HBM in this paper are reported for the first time.(2) The physicochemical properties and the o/w distribution coefficient of PAM, HPM and HBM are different from those of mangiferin.(3) PAM, HPM and HBM do not show any antitussive, expectorant, anti-asthma, bacteriostatic and antiviral effect even though mangiferin certainly showes these activities. This indicates that esterified-derivatives lose these activities because of being acylated. On the other hand, PAM, HPM and HBM can slightly enhance the apoptosis of 7901, hella and 7404 tumor cell in vitro, and have slight inhibition effect on S180 tumor cell in vivo. This indicates that esterified-derivatives inherite the slight tumor inhibition activity from mangiferin. Third, in the case of showing similar anti-inflammatory and hypoglycemic effect, mangiferin requires four times the molar dose that derivatives neede; in addition, PAM, HPM and HBM can significantly inhibit PTP1B. This indicates that esterified-derivatives have stronger hypoglycemic activity than mangiferin.(4) For the mangiferin esterified-derivatives, there exists a trend that the longer esterification moieties (non→acetyl→propionyl→butyryl), the higher lipid-solubility, and the stronger anti-inflammatory and hypoglycemic activity. In other words, there exists a certain structure-activity relationship.(5) Mangiferin, PAM, HPM and HBM neither have the insulin-like activity nor have the activity to stimulate the islet cell to secrete insulin heavily in short time. The hypoglycemic mechanism of PAM, HPM and HBM may lie in PTP1B inhibition and islet cell repair. First, PTP1B inhibition can increase the concentration of insulin receptor as to increase the availability of insulin; on the other way, high lipid-solubility means high transmembrane permeability, more drugs get into the islet cell and make the cell recover from the STZ damage as to increase the insulin secretion.(6) By analyzing the corresponding relation of the chemical structure changes and the pharmacological activity changes, it indicates that-OHs on the aglycon exist or not would directly make the compound to have antitussive, expectorant, anti-asthma, bacteriostatic and antiviral activity or not; in addition, PTP1B inhibition, anti-inflammatory and hypoglycemic activity only get related with the chemical structure and the structure features of the aglycon, or to say, these activities have nothing to do with other moieties on the aglycon.(7) The aglycon is more valuable than mangiferin itself. The pharmacological activity of mangiferin depends on its aglycon; and the aglycon reasonably has stronger activity than mangiferin, for example, the anti-inflammatory and hypoglycemic activity. It would be certain that acylated aglycon of mangiferin had very strong anti-inflammatory and hypoglycemic activity.4 Innovations(1)Took BCS principles as guidelines, the chemical structure of mangiferin was modified to increase the lipid-solubility of the target compounds. High lipid-solubility led to high transmembrane permeability, so that more drugs got into the cell to increase the bio-availability, and as a result, the pharmacological activity significantly strengthened. The study results in this parer indicated that the anti-inflammatory and hypoglycemic activity of mangiferin esterified-derivatives (PAM, HPM and HBM) achieved the expected purpose.(2)For the first time, esterified-derivatives of mangiferin were chemically prepared. The esterified-derivatives (compoundⅠ～Ⅳ, PAM, HPM and HBM) mentioned in this paper were novel compounds. The anti-inflammatory and hypoglycemic activities of PAM, HPM and HBM were far more stronger than these of mangiferin, their parent compound.