Comparative Analysis Of Saponins From Different Phytolaccaceae Species And Their Antiproliferative Activities

Herbal medicine contains a number of active compounds depending on the part of the plant used.Moreover,some of these compound’s activities have not been analysed hence their therapeutic significance may or may not be known.Traditional herbalist argues that the use of herbal medicine with the conjoint ingredients is more effective than when used as isolated active compounds.The quality and the efficacy of herbal medicine are of great concern especially in their nature as multi-ingredient medicines.For the efficacy testing to be effective,approaches,for example,ensuring high yields of the active principles and targeting the active components are of great significance.Although standardisation of the quality may be done,it is imperative to know the cause of their variations since they assist in the reproducibility of herbal medicines.A number of factors that have been investigated have shown to lead to the variation in chemical constitutes.Environmental changes being one of the major conditions for this variation have been studied in this study.Since plant have no control to any changes in the environmental conditions(biotic and abiotic factors),they have therefore to adapt to the different changes.Adaptation may include changes in the type and amount of the chemical constitutes.Medicinal plants of different species collected from different geographical regions have shown variations in both their chemical contents and pharmacological activities due to the differences in the environmental conditions of the collection sites.In this study,phytochemicals and antiproliferative activities of roots of Phytolacca acinosa found in different provinces of China(Sichuan,Henan and Shandong),a species of Phytolacca americana(P.americana)collected from Wuhan botanical garden and Phytolacca dodecandra(P.dodecandra)collected in Kenya-Aberdare’s ranges were investigated.The plant species were collected during the fruiting season depending on the seasons of the specific location site.Phytolacca species are known to contain toxic substances in their roots,seeds and leaves at maturity.Hence,the collection period is certainly essential to ensure the toxic components have not accumulated.The major components that are known to have potency in bioactivities in the roots of this family are the triterpenoid saponins.To ensure a maximum yield of the targeted compounds the extraction method and the specific conditions such as the solvent concentration,sample:solvent ratio,number of times of extraction and lastly the duration of the extraction were optimized.The preeminent method of extraction in this analysis was determined to be the ultrasound-assisted method with specific conditions as follows:ethanol-H2O(1:1,v/v),with a sample:solvent ratio of 1:8 and extraction was performed 3 times each for 30 min.The method was used for the extraction of the five samples after which quantitative analysis was carried out using the UV-Vis spectrometry.Among the samples investigated,P.dodecandra gave the highest percentage yields of the triterpenoids saponins.Comparing samples collected from the different location though from the same species(P.acinosa),Sichuan detected higher yields of the triterpenoids as compared to Shandong and Henan.P.americana samples collected in Wuhan botanical garden gave the least saponins content.Liquid Chromatography-Mass Spectrometry was applied to comprehensively study the quantitative and qualitative variations in the chemical constitutes in the five samples.The crude samples were first purified using solvents which resulted in three fractions(water,Petroleum ether and saturated 1-butanol).Separation using column chromatography loaded with AB-8 microporous resin was done on a concentrated fraction of saturated 1-butanol.Elution of the column was done using 70%ethanol.Optimisation of sample preparation to improve peak separation and fragmentation was done and two methods were used to prepare the samples for LC-MS studies.In one of the methods,samples were prepared using absolute methanol while in the other with a ratio of 3:2 of methanol:water.Peak separation of the samples prepared with solvents at a ratio gave better separation and fragmentation as compared to that which was prepared using methanol only.For the analysis of peak identification in the five samples,60 triterpenoid saponins were detected within the four samples from China,among which 22 were identified as common within at least three samples.As for the species collected in Kenya,Phytolacca dodecandra,a total of 35 peaks were identified and only nine peaks were similar to the ones detected from samples from China.Moreover,a number of isomers were detected and their structure elucidated using previous studies.An assessment of the common and different compounds was done in three samples(representing variation in samples collected in different geographical location and representing the variation in species)to expansively illustrate the variation in their quantity and qualityTo investigate the anti-cancer potent of these samples,two cancer cell lines were used namely,Hep G2;Hepatocellular carcinoma),(SGC-7901;Gastric carcinoma).The Sulforhodamine B(SRB)Growth Assay was used in this analysis.In all the samples,growth inhibition of the cell lines was detected.However,their growth inhibition varied across the five samples.P.dodecandra from Kenya showed the highest IC50 values in both cell lines(9.71±0.55 and 9.14±0.17 μg/mL)in Hep G2 and SGC-7901 respectively as compared to those of the other four samples.Comparing samples of same species(P.acinosa)but different collection sites in China,the sample from Sichuan gave IC50 values of 25.59± 1.63 and 27.20 ± 1.60μg/mL)in Hep G2 and SGC-7901 respectively as the highest values.Further,qualitative correlations were done to show compound-activity relationship using multivariate analysis of PLS regression.The percentage relative peak areas of the base peaks of most profuse peaks in the different samples were used in this analysis.The prediction analysis firstly proved the experimental cytotoxicity values from the different cell lines against the inhibition rate linearity was accurate.The correlation coefficient(R)was calculated.Since the optimal value is 1,the model gave R2 values of 0.999 for Hep G2 cell line and 0.997 for the SGC-7901 cell line.Other analysis on the standardised residue also further showed the accuracy of the model.Compounds that influenced most growth inhibition were also illustrated and the relationship between the samples was calculated using the PLS loading scores.The standardisation coefficient was used to identify further the specific different compounds in the samples that give more influence on the cytotoxicity of the different cell lines.Seven compounds were found to be more potent to cytotoxicity activity whereby those with a VIP value of>1.5 were factored out as m/z 809,973,1045,825,853,663 and 1329.Four of the compounds had been previously isolated and tentatively identified as 3-[Xyl-(1→4)-Glc]-phytolaccagenin(m/z 825),3-[Glc-(1→3)-Ara]-28-Glc-phytolaccagenic acid(m/z 973),3-[Xyl]-phytolaccagenin(m/z 663)and 3-[Xyl]-28-Glc-phytolaccagenic acid(809),and the other three were termed as novel compounds.Of the seven compounds,only four(m/z 825,m/z 663,m/z 853 and m/z 1329)were more potent in the SGC-7901,while Hep G2 growth rate inhibition was influenced more by six compounds.In summary,this study demonstrated that to achieve a maximum targeted secondary metabolite,it is essential to enhance the method by optimising the specific conditions.Further,it can be concluded that the optimised method may be used in the extraction of saponins in this family Phytolaccaceae and also in other plant families that contain saponins as the major chemical constitutes.Both UV-vis spectrometry and the LC-MS fingerprinting analysis may be used to investigate the quantitative and qualitative studies.There was major disparities in the secondary constitutes and their bioactivities in herbal plants due to the environmental changes where the herbal plants are collected.More so,the species that belong to the same family may have differences in their chemical constituents of which the more effective components could also be identified.