Research Article - Journal of Natural Product and Plant Resources ( 2017) Volume 7, Issue 2
Twenty-one species belonging to 13 genera of Acanthaceae obtained from Saudi Arabia were studied using electrophoretic analysis for protein patterns and two isoenzymes polymorphism in addition the phytochemical screening by TLC and HPLC. A total number of twenty-three protein bands observed between the species ranged 10 and 250 K Da, nine esterase and six peroxidase polymorphisms were detected. The biochemical data (protein profiles and isoenzymes polymorphisms) considered a significant tools for the relationships between the studied species. Furthermore, the phytochemical screening including the total contents of alkaloids, flavonoids, terpenoids and saponins were demonstrated the presence of alkaloids, flavonoids in all studied taxa with a variable contents and absence of total terpenoides and saponins in some species. As well, fourteen phenolic acids have been screened by high-performance liquid chromatography (HPLC) method. Gallic acid, phenol, protocatechuic acid, p-coumaric acid and o-coumaric acid in substantial amounts. Both the biochemical and the phytochemical data were conducted by means of the numerical analyses based on in total 38 electrophoretic characters and 18 phytochemical contents. On the basis of UPGMA clustering analysis, the two recognition distinct taxonomic groups and several clusters were distinguished. The existent results are useful for evaluating the relationships between the studied Acanthaceae species both at subfamilies and tribe levels.
Acanthaceae, Protein profiles isoenzymes polymorphism, Phytochemical assay, Numerical analysis
The Acanthaceae Juss. ex Bercht and J. Presl are a relatively large family comprising of 3900 species related to 200-205 genera . It is currently placed in order Lamiales close to the Bignoniaceae . Bentham and Hooker  recognized tribes Ruellieae, Justicieae and Acantheae in the family and Bremekamp  also recognized these lineages but united the first two as subfamily Ruellioideae. Acanthaceae represented in Saudi Arabia by 14 genera and 35 species, many of them are economically important for both traditional medicine and horticulture . Phylogenetic variations and taxonomic relationships among Acanthaceae were previously investigated using chloroplast DNA sequences by Lucinda et al.,  and using iridoids and quaternary amines by Henrik, 1988.
A conventional key and its tabular version to the 36 taxa from 21 genera of the Acanthaceae s.l. in Egypt are provided by Adel El-Gazzar et al., . Seed protein and isozyme electrophoresis have been the most widely employed molecular genetic markers during the last quarter century [8,9]. Some taxa of this family are studied phytochemically by several authors, Daniel and Sabnis 10, Lucinda and Michael , Wamtinga et al.,  and Vijayalakshmi and Kripa . The main objective of this study is to clarify the relationships among 21 taxa related to two Acanthaceae subfamilies through polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC) and to discuss whether these characters can provide an additional fundamental tool which helps in the future the explanation of the taxonomic trends at specific and infra-specific level within the family.
Twenty-one species that have been assigned to Table 1 were collected from the natural habitats of Jazan of Saudi Arabia and identified according to Chaudhary , Alfarhan et al.,  and Masrahi .The voucher specimens are deposited at the Jazan University Herbarium, KSA (JAZUH).
|1||Anisotes triculcus (Forssk.) Nees||Acanthoideae||Acantheae||Wadi razan|
|2||Blepharis maderaspatensis (L.) Hayne||Jabal Abadil|
|3||Blepharis ciliaris (L.) B.L. Burtt||Abu Arish|
|4||Crossandra wissmanii Schwartz||Jabal Fayfa|
|5||Lepidogathis scariosa Nees||Wadi Al Abadil|
|6||Justicia flava (Vahl) Vahl||Ruellioideae||Justicieae||Wadi Abadil,|
|7||Justicia heterocarpa T. Anders||Jabal Fayfa|
|8||Justicia caerulea Forssk||Wadi Abadil,|
|9||Monechma debile (Forssk.) Nees||Jabal Abadil|
|10||Ecbolium gymnostachyum (Nees) Milne-Redh||Jazan|
|11||Ecbolium viride (Forssk.) Alston||Al Ardha|
|12||Asystasia gangetica (L.) Anders||Ruellieae||Al Aridah|
|13||Barleria hochstetteri Nees in DC||Jazan|
|14||Barleria trispinosa (Forssk.) Vahl,||Jabal Abadil|
|15||Barleria bispinosa (Forssk.) Vahl,||Jabal Al|
|16||Hypoestes forskalei (Vahl) Soland.||Baysh|
|17||Peristrophe cernua Nees||Jabal Abadil|
|18||Peristrophe paniculata (Forssk.) Bru.||Ad Darb|
|19||Phaulopsis imbricata (Forssk.) Sweet||Bani Malik|
|20||Ruellia patula Jacq||Jabal Fayfa,|
|21||Ruellia grandiflora (Forssk.) Blatt||Jazan Herbarium|
Table 1: The distribution of the studied species of the Acanthaceae in jazan area and their distribution in the family according to (Hansen 1985).
PAGE (polyacrylamide gel electrophoresis) analysis Seed protein: 0.2 g of extracted seeds meal were homogenized with 0.2 ml of tris-HCl buffer containing 2% SDS and 2% β-mercaptoetahnole at pH 6.8, centrifuged at at 6000 rpm for 10 minutes. 100 μl were injected onto gel (12% thickness) with bromophenol dyes as tracking dye. Electrophoresis process carried out in separating buffer containing Tris-gylycin buffer pH 8.3. After electrophoresis process; the gel stained with commassie brilliant blue-R250. Characterizations of seed protein were carried out using one dimensional sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli . In isoenzymes analysis: an extracted seed, separated on PAGE was conducted as the method outlined Wendel and Weeden . The gels (8 % thickness) were stained after the electrophoresis process; the specific staining solution used according to Graham et al.,  and Jonathan and Wendel  as follows.
Esterase isoenzyme (EC.184.108.40.206): Gel staining, 1 ml of 1% α- naphthyl acetate dissolved in 60% acetone added to 25 ml 0.1 phosphate buffer (pH 6.5). 20 mg of fast blue RR were added to 25 ml of the same buffer. Both protein and esterase gels are scanned by using Hoefer Scanning densitometer GS 300.
Peroxidase isoenzyme (EC.220.127.116.11): Gel soaked in o-dianisidine dissolved in 150 ml of 95% ethanol, 20 ml of acetate buffer (0.88 M sodium acetate, 0.62 glacial acetic acid, pH 4.7), 20 ml of distilled water, and 5 ml of 3% hydrogen peroxide which added just before using. The gel was incubated at room temperature and washed and filtered .
Total alkaloids, flavonoids, saponins and terpenoids were estimated as follows: 20 g of extracted leaves are carried out according to method of Ben-Hammouda et al. . Alkaloids determined according to methods of Harborne . Flavonoid contents excluded according to methods of Boham and Kocipai . Saponin estimated according to methods of Obadoni and Ochuko . Total terpenoids contents estimated according to Olayiwola . Dried flavonoids extracts dissolved in chloroform: methanol (95:5) according to methods of Olayiwola and subjected to thin layer chromatography (TLC) which performed on silica gel plates (DC-Alufolien 60 F254). The chromatograms of the phenolic compounds were observed under UV before and after sprayed with aluminium chloride (AlCl3). Dried flavonoids dissolved in ethyl acetate: methanol: water (30: 5: 4). Developing solvent systems carried out according to Wagner and Bladt  and Nicola . Toluene: chloroform: acetone (40: 25: 35). Phenolic compounds of the plant samples was performed on a Hewlett-Packard HPLC (Model 1100), using a hypersil C18 reversed-phase column (250 x 4.5 mm) with 5 μm particle size. Injection carried out by Redone injection valve (Model - 7125) with 50 μl fixed loop was used.
Different protein, isoenzyme bands by PAGE technique and the phytochemical analysis were scored as either absent (0) or present (1) for all taxa (Table 5). Only reproducible and clear bands were scored for the construction of the data matrix. The data matrix thus prepared was the input file for using the NTsys-Pc program (version 2.02) (Rohlf, 2000). The similarity matrices were used for the construction of dendrograms with unweighted pair-group method on arithmetic averages (UPGMA).
In this study, all taxa were investigated under electrophoretic process by means of PAGE and the phytochemical screening carried out by TLC and HPLC .Total contents of alkaloids, flavonoids, terpenoids and saponins. The examined species are listed with the classification of the family by Bertel Hansen in Table 1.
Figure 1 illustrates a typical gel in which different patterns are shown for 21 taxa. The total number of 20 protein bands observed for each species between 10 and 250 KDa. The most similar band pattern was observed in the region of 20 and 37 kDa. Two bands around 37 kDa and 10 kDa observed among all studied species. The bands bigger at 37 kDa were variable among all those species. 13 protein bands found in A. triculcus. In two Blepharis species, 15 bands of protein profiles are detected both two species shared in 12 bands. In C. wissmanii, 13 protein profiles are observed. Also, 15 protein bands are present L. scariosa. Three species of Justicia have protein 17 bands, the lowest number of 13 found I in J. flava and J. heterocarpa, the highest one (15 bands) found in J. caerulea, three taxa shared in 9 protein. In M. debile, 13 bands of protein profiles are detected in this species. In two Ecbolium species, 15 bands of protein profiles are detected E. gymnostachyum has 12 bands while E. viride has 13 bands. In A. gangetica, 12 bands of protein profiles are detected in this species. In three species of Barleria species 14-15 bands of protein profiles are detected the highest number of protein (15 bands) in B. trispinosa while the lowest one of 14 bands found in B. hochstetteri and B. bispinosa. H. forskallei, 14 protein bands and 2 isoenzyme bands are found. In Peristrophe taxa, 13 protein bands found, two species shared in. In the same sense, in P. imbricate, 12 protein bands and 7 esterase group. 11-12 protein bands in Reullia species, 12 bands noticed in R. patula and 11 bands detected in R. grandiflora.
Five esterase and 4 peroxidase bands are found in A. triculcus, 6 esterase and three peroxidase isoenzymes found. C. wissmanii and L. scariosa have 6 esterase and 2-3 peroxidase bands. Three taxa of Justicia have five esterase and five peroxidase groups, the highest number of 5 bands found in J. flava and the lowest one of three groups estimated in J. hetercarpa; three species shared in four bands. M. debile has 6 peroxidase and two esterase profiles. In two species of Ecbolium in total eight esterase and peroxidase groups present in E. gymnostachyum and nine isoenzymes polymorphisms recorded in E. viride, both two species shared in total seven bands. In A. gangetica, five esterase and peroxidase bands are found. In three taxa of Barleria six Esterase groups and four peroxidase are found, shared in Est 5, Est 7, Est 9, Prx 3, Prx 4 and Prx 6. In H. forskalei two esterase and three peroxidase are detected. 6 esterase and three peroxidase found in two taxa of Peristrophe and shred in eight bands. 6 esterase and four peroxidase found in Phaulopsis. In two taxa of Reullia, five esterase and peroxidase found and shared in five bands, namely EST 7, EST 8, EST 9, Prx 2 and Prx 6.
(mg/g d. wt)
(mg/g d. wt)
(mg/g d. wt)
(mg/g d. wt)
|6||Justicia flava||3||0.464||1.047||0. 055||0.046|
|7||Justicia heterocarpa||3||0. 021||1. 0.32||0. 590||0. 0.32|
|8||Justicia caerulea||3||0. 055||1. 054||0.032||0. 080|
|9||Monechma debile||3||2.085||0.032||0. 008||0.821|
|10||Ecbolium gymnostachyum||2||0. 06||0. 018||0.022||0. 356|
|11||Ecbolium viride||2||0.203||0.045||0. 054||0.721|
|12||Asystasia gangetica||2||0. 654||0. 050||0.0091||0. 050|
|13||Barleria hochstetteri||3||0.301||0.054||0.058||0. 540|
|14||Barleria trispinosa||3||2.292||0.023||0. 036||0.023|
|15||Barleria bispinosa||3||0. 020||0. 03||0.531||0. 039|
|16||Hypoestes forskalei||3||0. 904||0.87||0.653||1.875|
|18||Peristrophe paniculata||2||0. 711||0.042||----||---|
|20||Ruellia patula||3||0.403||0.62||0.9 70||0.651|
Table 2: Flow rates (RF) and total contents of alkaloids, saponins and terpenoids in the family.
Current dendrogram of Figure 2 demonstrates two main groups. The upper (group A) contains A. gangetica, B. maderaspatensis, B. ciliaris, A. triculcus and C. wissmanii. Group B comprising of two subgrops, the first included the studied taxa of Justicia and Peristrophe. The second included cluster two taxa of Ecbolium, the studied taxa of genus Barleria and last level has two species of Reullia linked with L. scariosa. and P. imbricate.
From TLC analysis, the large number of flavonoids spots which appeared by spraying by aluminum chloride (ALCL3) found in L. scariosa followed by most taxa while the lowest one appeared in E. gymnostachyum, E. viride A. gangetica and P. paniculata (Figure 3, Table 3). TLC plate analysis gave a positive reaction for the flavonoid compounds and gave purple, yellow to brown colors because the aluminium chloride (AlCl3) served as a developer to make visible the secondary metabolite on the TLC plate. Alkaloids, flavonoids and terpenoids were detected in all the studied taxa with a variable contents while terpenoids and saponins are absent in some taxa, the highest value of alkaloids in three species of Justicia also, the highest contents of flavonoids has been estimated in three species of M. debile and B. trispinosa and P. cernua. From Table 4, no saponins are observed in C. wissmanii and two species of Peristrophe. Also, no terpenoids detected in A. triculcus and P. paniculata. Most the studied species contain a degree amount of flavonoids, The highest content of 2.085 mg/g was noticed in M. debile while the lowest one was estimated in B. bispinosa and J. heterocarpa. 14 phenolic contents are assayed by HPLC from them three unknown compounds (Table 3). Also high content alkaloids in three taxa of Justicia. Phenolic assayed revealed five phenolic compounds are found In Anisotes triculcus, synapic acid, phenol, p-coumaric, o-coumaric and unknown compounds. In Blepharis species, most studied compound recorded in this two species gallic acid, phenol, protocatechuic acid, o-coumaric acid and two unknown contents. C. wissmanii has ferulic acid, gallic, phenol and two unknown phenolic compounds. In L. scariosa most phenolic contents are present except synaptic acid, protocatechuic acid, p. hydroxy benzoic acid and unknown one. Justicia taxa have the most phenolic contents; all taxa are shared in presence of gallic acid, phenol, p-coumaric acid and o-coumaric acid. Also, five phenolic contents are determined M. debile; Phenol, protocatechuic acid, phloretic acid, o-coumaric acid and p-coumaric acid.
|S.No.||ferulic acid||sinapic acid||gallic acid||Phenol||vanillic||Protocatechuic||Phloretic||p- OH- benzoic||p-coumaric||o-coumaric||chlorogenic||Unknown||Unknown||Unknown|
(1) A. triculcus, (2) B. maderaspatensis, (3) B. ciliaris, (4) C. wissmanii. (5) L. scariosa. (6) J. flava, (7) J. heterocarpa, (8) J. caerulea, (9) M. debile, (10) E. gymnostachyum, (11) E. viride , (12) A. gangetica, (13) B. hochstetteri, (14) B. trispinosa, (15) B. bispinosa, (16) H. forskalei, (17) P. cernua , (18) P. paniculata, (19) P. imbricate, (20) R. patula and (21)R. grandiflora.
Table 3: Relative percent of phenolic contents (µg/mg d.wt extracts) of using HPLC
Figure 3: TLC of methanolic extract sprayed by aluminium chloride (AlCl3) detected by UV light; (1) A. triculcus, (2) B. maderaspatensis, (3) B. ciliaris, (4) C. wissmanii. (5) L. scariosa. (6) J. flava, (7) J. heterocarpa, (8) J. caerulea, (9) M. debile, (10) E. gymnostachyum, (11) E. viride , (12) A. gangetica, (13) B. hochstetteri, (14) B. trispinosa, (15) B. bispinosa, (16) H. forskalei, (17) P. cernua , (18) P. paniculata, (19) P. imbricate, (20) R. patula and (21) R. grandiflora.
In E. gymnostachyum has synaptic acid, phenol, p-hydroxy benzoic acid, chlorogenic acid and unknown compound in E. viride has synaptic acid, phenol, p-hydroxy benzoic acid and p-coumaric acid. Shared in four contents, synaptic acid, phenol, p-hydroxy benzoic acid and unknown one. A. gangetica has four phenolic compounds ferulic acid, gallic acid, phenol and chlorogenic acid. Three species of Barleria have a variable contents of phenolics and shared in two compounds, phenol and unknown one; B. hochstetteri and B. trispinosa shared in phenol, phloretic acidand unknowen compound while protocatechuic acid and unknown compound are estimated only in B. bispinosa. H. forskalei has most phenolic contents such as ferulic acid, phenol, protocatechuic acid, vanillic acid, P. hydroxy benzoic acid, p-coumaric acid, o-coumaric acid, chlorogenic acid and unknown compound. In the two species of Peristrophe, three phenolic contents. Phenol, phloretic and p-coumaric have been estimated. In phaulopsis imbricate, phenol, vanillic acid and p-coumaric acid were recorded. Ferulic acid, synaptic, phenol, protocatechuic acid, phloretic acid and p- coumaric acid, o-coumaric acid and unknown compound have been determined in R. patula whereas R. grandiflora has only p. hydroxy benzoic acid. In R. grandiflora ferulic acid, synaptic acid, phenol, protocatechuic acid and p-hydroxy benzoic acid have been detected. The two species shared in four compounds, ferulic acid, synaptic acid, phenol, protocatechuic acid. Phonogram of Figure 4 resulted in numerical analysis of 18 phenolic compounds showed there are two main groups have been recognized, the first (group A) included the studied taxa of family Acanthoideae linked with three species of Justicia, the second one (group B) included the most studied taxa of subfamily Ruellioideae.
Second levels divided into two sub levels, the first included two Ecbolium taxa grouped with H. forskalei the second level in which two Reullia species separated in alone cluster at 0.70, the remainders comprising of the two taxa of Peristrophe with three taxa of Barleria and the fourth cluster inclusive M. debile, L. scariosa, P. imbricata and A. gangetica.
|Seed Protein profiles|
|Total Pro. bands||13||14||13||13||15||13||13||14||13||12||13||12||14||15||14||14||12||12||12||12||11|
|Total Est bands||5||6||6||6||6||5||3||5||6||5||4||5||5||4||4||2||5||6||6||4||3|
|Total Prx bands||4||2||3||2||3||5||4||4||2||3||5||5||4||3||4||3||3||3||4||3||4|
Table 4: The presence and absence of the 56 electrophoretic characterizations and the phytochemical data for each species as numbered in Table 1 used in numerical analysis.
As storage proteins are not affected by environmental fluctuations, SDS-PAGE technology is particularly considered as a reliable tool for economic characterization of germplasm (Javid et al., 2004; Iqbal et al., 2005). Two bands around 37 kDa and 10 kDa observed among all studied species (monomorphic). May be considered positive markers, other bands consider polymorphic because they are present in some species and absent in another. 56 characters (Table 4) used in statiscal program (38 protein and two isoenzymes and 18 phytochemical contents). Our electrophoretic data of protein profiles and esterase polymorphism considers good support for inclusion, of modern classification of four major lineages within Acanthaceae s.s., as suggested previously by Hansen (1985) and Scotland et al. (1995). Figure 2. Reveales a dendrogram of phenolic compounds showed there are two main groups to be distinguished. The upper cluster contains A. triculcus, B. maderaspatensis, B. ciliaris, C. wissmanii, and three species of genus Justicia where the lower cluster comprising the remainders. The phenolic compound considers intermediate between two subfamily Acanthoideae and subfamily Rulleoideae which is agreement with Lucinda and Michael (1999). Dendrogram of Figure 2 obtained the numerical analysis of the electrophoretic protein and an isoenzymes pattern demonstrates two main clusters. the upper cluster (group A) contains A. gangetica, B. maderaspatensis, B. ciliaris, A. triculcus and C. wissmanii. The lower one (group B) contains most studied taxa of subfamily Reulloideae which in accordance with Lucinda and Michael (1999). Presence of flavonoids, saponins and phenolic contents in some taxa of the family agreed with studies of Vijayalakshmi and Kripa (2016). 14 phenolic compounds are assayed by HPLC, phenol found in all species, o-coumaric and p-coumaric were present in more than 70% of the taxa, p-OH benzoic and gallic acid was recorded in about 50%, such results are fairly agreed with Daniel and Sabnis (1987). Current phytochemical works revealed, gallic acid and p-hydroxy benzoic acid and two unknown compounds are more common in Acantheae and Justicieae of sub family Acanthoideae more than subfamily Relluoideae also, p-coumaric is common in most studied taxa of tribe Ruellieae of subfamily Relluoideae. It is clear that, three taxa of Justicia are linked with two species of Peristrophe shared in three flavonoids spots. Also, three Barleria species clustered in same group with Lepidagathis with the three Barleria species ranged between 28-36.3 %, also they have the highest number of protein profiles bands. These results in agreement with in agreement with Lucinda and Michael (1999).
The author would like to thank the Biology Department, Faculty of Science, Jazan University, KSA and Jazan University Herbarium (JAZUH) and Chemistry Department for all their efforts and facilities and thanks to staff members of Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jazan University, KSA.