Novel Cycloartan derivative with genetic and metabolic pro(cid:976)iling oftwo Crassulaceae species

seven steroidal components in both species. The investigation of fatty acid methyl ester (FAME) revealed the presence of 12 components in C. tetragona and 9 components in C. ovata, and a novel triterpene, namely, 28 Methyl-5 (cid:11) -cycloart12, 20, 24-trien-15 (cid:12) -Ol was isolated and identi(cid:976)ied from MeCl together with 5 known compounds.


INTRODUCTION
For many years, natural products had a signi icant role in the cure of widespread ailments and have improved the quality of human beings lives. (Akanda et al., 2014). Crassula is one of the largest genus of family Crassulaceae that comprises about 150 species (El-Hawary et al., 2016). Botanical description of closely allied plant species in these days is greatly supported via investigation of various powerful genetic principles such as DNA ingerprinting. It is reported as a favourable tool for the authentication of medicinal plant species and especially  useful in varieties or species that can't be distinguished morphologically and/or phytochemically (Kady et al., 2015). DNA ingerprinting following successful PCR ampli ication of tandemly repeated sequences is well-known as polymorphic and widespread in plant genomes (Mcgregor et al., 2000). Thus, PCR based on various techniques which include Random Ampli ied polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) are playing great rule in plant improvement (Manimekalai et al., 2006). The purpose of the present investigation was to evaluate the bene-it of the RAPD and the ISSR marker systems to distinguish the two Crassula species, and it is considered the irst assessment of genomic DNA of the two species as well as to igure out the dissimilarity in their chemical composition by determination of the lipid content with respect to their chemical composition, the mineral content and amino acid composition and identi ication of their terpenoidal compounds using GLC as well as quantitative determination of triterpenes, and isolation from the methylene chloride fraction. There was no much work concerning the genus Crassula or species tetragona  nor ovata, so we will shed light on both plants' constituents using different techniques.

Plant Material
C.tetragona and C.ovata collected from EL Orman botanical garden, Giza, Egypt and identi ied by Dr. Mohamed Gibali (senior botanist, National Research Center, Cairo, Egypt).

Genetic pro iling
Was accomplished at Biotechnology Research Lab, Horticulture Research Institute, Agriculture Research Center.

DNA isolation and sample preparation
The plant material (0.2 g) was kept in 1.5 ml microfuge tubes for DNA extraction utilizing the method illustrated by Williams et al. (Williams et al., 1990). Plant tissue was crushed with liquid nitrogen, 400 µl of AP1 (Activator protein-1) buffer and 4 µl stock solution of RNase (100 mg/ml) then vor-texed strongly, admixture was incubated and mixed during incubation. Then, 130 µl of AP2 (Activator protein-2) buffer was combined to the lysate, mixed and incubated, Lysate was administrated to the QIAshredder spin and was added in centrifugation for 2 min. Typically, Then, 0.5 volume of AP3 buffer and 1 volume of ethanol (96-100%) were combined to the cleared lysate and mixed. Then, the mix was applied over DNeasy Tiny spin column. Then, centrifuged. Then, 500 µl buffer AW (Column wash buffer) was combined to the column of DNeasy and centrifuged. Then, 500 µl AW buffer was combined to a column of DNeasy and centrifuged. A column of DNeasy was then moved to a 1.5 ml microfuge tube and 100 µl of preheated (65ºC) AE buffer was pipetted precisely onto the column of DNeasy sheath. Then, incubated and centrifuged to elute. A fresh microfuge can be applied for primary elute. Otherwise, the microfuge cylinder can be used again for the next elution stage to mix the elutes.

Oligonucleotide primers (RAPD and ISSR PCR analysis)
Two methods were accomplished for worthy statistical inquiry. The DNA magni ications were achieved in an automatic thermal round (model Techno 512) automated for one round at 94º C for 4 min displaced by 45 rotations of 1 min at 94º C, 1 min at 37º C, and 2 min at 72º C. the reaction was lastly kept at 72º C for 10 min. PCR ampli ication was done using six random deca-mer arbitrary primers and six ISSR primers produced by (Operon Biotechnologies, Inc. Germany).

Polymerase chain reaction (PCR)
The PCR ampli ication in both RAPD and ISSR anal

Total triterpenes
Spectrophotometric method using vanillin reagent for total saponin content (Hiai et al., 1976). Brie ly, one mg of the ethanol extract of both plants were separately dissolved in 10 ml 80% aqueous methanol to obtain a inal concentration of 100 µg/ml. 1 ml of the sample solution was moved, and 0.25 ml of vanillin reagent (8%, w/v in absolute alcohol) were placed in an ice bath, and then 2.5 ml of 72% v/v of sulphuric acid was slowly added to the wall of each test tube and left for 3 min. Finally, the test tubes were warmed in a water bath at 60 ºC for 10 min and cooled in an ice bath. The absorbance of the color produced was measured at λ max 544 nm against a blank prepared by the same procedure except ursolic acid. The total triterpene content of the analyzed sample was expressed as µg of ursolic acid /mg of sample.

Preparation of lipoidal matter
Round 50 g of the air-dried powder of each plant was extracted with n-hexane till exhaustion (no residue was left after evaporation of the last 5 ml of extract). Each extract was iltered, and the iltrate was evaporated under reduced pressure and temperature at about 60°C. The residue left was kept for an examination of total lipoidal matter (3 g and 2.89 g) for C. tetragona and C. ovata respectively.

Preparation of unsaponi iable Matter (USM)
One gram of each residue of n-hexane was saponiied separately by heating under re lux state for 24 hours with a combination of alcoholic potassium hydroxide 5% solution (200 ml) and benzene (20 ml). The heated mixtures were concentrated, diluted with 100 ml distilled water and extracted with consecutive portions of ether (each of 50 ml) till exhaustion, then washed with water several times till the washed extracts were neutral to litmus paper, dehydrated over anhydrous sodium sulphate and the solvent was distilled off. Each residue of unsaponi iable matter (USM) obtained was waxy, yellowish-white in color and weighed 0.83 g and 0.67 g for plants of C. tetragona and C. ovata respectively. (Vogel, 1957) Isolation of the fatty acids from the saponi iable The alkaline aqueous solution was left after extraction of USM, acidi ied with 10% hydrochloric acid and the liberated fatty acid were extracted with ether (5 x 50 ml) the mixed ethereal extract was washed with water till neutral to litmus, dried over anhydrous sodium sulphate, evaporated till dryness under reduced pressure. The fatty acids, isolated from the Hexane extracts of the plants of C. tetragona and C. ovata, amounted to 0.19 g and 0.3 g respectively.

GLC analysis of the USM and FAME
Detection of hydrocarbon and sterol content of USM was held out on GLC Hewlett Packard.
HP-6890, G.C network GC system equipped with an FID detector. The analysis was achieved on an HP-5 column; using N2 as a carrier gas, injection temperature 240 o C, detector temperature 280 o C. Aliquots, 2 µL each, of 10% chloroform solutions of the analyzed USM and reference samples were co-chromatographed and rough identi ication of the hydrocarbons and sterols of the USM was held out by a comparison of their retention time with the available reference compounds. Total fatty acids fractions for both species were imperiled to methylation. The fatty acids methyl esters were examined by GLC under the same conditions like USM except for detector temperature 300 o C. Identi ication of the fatty acids methyl esters was carried out by direct comparison of retention time of each of the separated components with those of available reference fatty acid methyl esters analyzed under the same conditions.

Sample Preparation
Fresh plants of C. tetragona and C. ovata were subjected to air drying, and the dried plants were percolated with absolute ethanol till exhaustion, the crude extracts in each case were suspended in distilled water, and the aqueous suspension of each plant was successively extracted by a partition with methylene chloride and n-butanol, the methylene chloride fraction was subjected for isolation.

DNA Pro ile
The data represented in this paper were important in that RAPD and ISSR ingerprinting has been ef icacious in distinguishing variation in a species thought being devoid of a molecular variant (Raina et al., 2001). The ef iciency of a molecular marker technique depends on the amount of polymorphism it can detect among the set of attainments under investigation. In the present study, ISSR and RAPD primers not only generated the highest number of polymorphic markers, but the polymorphism was suf iciently well distributed to enable discrimination between accessions by each primer pair. Table 1 The RAPD Technique In order to evaluate the ef iciency of RAPD-PCR ingerprinting for the two species, the DNA was used as a template for the 6 RAPD primers banding pattern. Total of 94 bands template for all primers, 41 of them were polymorphic (43.617%). The highest band number was produced with primer OP-A02 (22 bands), while the lowest was produced with primer OP-B02 (10 bands), Primer OP-D01 showed the highest polymorphic percent (68.421%) while primer OP-B07 showed the lowest polymorphic percent (7.143%). Figure 1, Tables 2, 3 and 4

The ISSR Technique
The results of ISSR ingerprinting by using 6 ISSR primers banding pattern showed 60 bands for all primers, 28 of them were polymorphic (46.666%) The highest band number was produced with primer HB-15 (15 bands) while the lowest was produced with primer 44B (7 bands). Primer HB-14 showed the highest polymorphic percent (81.818%) while the primer, while primer HB-15 showed the lowest polymorphic percent (13.333%). Figure 2,Tables 2, 3 and 4.

Total triterpenes
Total triterpene was extracted according to the spectrophotometric method using vanillin reagent for total saponin content found to be 63.18889 µg/100µg ± 0.01 in C. tetragona and 87.06667µg/100µg ± 0.01 in C. ovata. The triterpene content of the tested sample, expressed as urosolic acid equivalent, was deduced from the pre-established calibration curve in and calculated using the following equation: y=0.0046x+0.0027 R 2 = 0.9619 Where, y=absorbance, x= corresponding concentration (µg/ml) and R 2 =correlation coef icient.

Lipid content investigation
Investigation of the unsaponi iable matters of nhexane extract of C. tetragona revealed the presence of 13 identi ied hydrocarbons with the presence of n-tricontane (57.65%) as the major hydrocarbon. In addition to Four identi ied steroidal components; with the presence of campesterol (19.77%)  as the major steroidal compound. On the other hand, investigation of the unsaponi iable matters of n-hexane extract of C. ovata revealed the presence of 15 identi ied hydrocarbons with n-tricontane (57.44%) as the major hydrocarbon. In addition to Three identi ied steroidal components; with the presence of β-sitosterol (13.98%) as the major steroidal compound. Table 5. FA metabolism have signi icant physiological consequences for many medical disorders such as obesity, cardiovascular disease, and diabetes melli- Investigation of FAME revealed the presence of 9 identi ied components in C. tetragona and 5 identi ied components in C. ovata, respectively. Table 6 showed that C. ovata has a higher percent of total unsaturated fatty acid 73.42% than that in C. tetragona 32.56%.
C. tetragona contains the monounsaturated fatty acid Oleic acid (omega-9); which has great importance to protects cell membranes from free radicals (Haug et al., 2007). Moreover, both species showed the presence of different long-chain mono and polyunsaturated fatty acids. The very longchain polyunsaturated fatty acids (PUFAs) (C-18-C-22) have great importance in modern nutrition as they possess a protective effect on the cardiovascular system, in lammatory diseases and cancer (Gogus and Smith, 2010). The unsaturated   H NMR and 13 C spectral data of compounds 1, 2, 3, 4, 5and 6
(1) Continued on next page  (Lunn and Theobald, 2006). Table 6 showed that C. tetragona has a higher percent of total saturated fatty acid 59.46% than that in C. ovata. 23.49%; the major saturated fatty acid is palmitic acid (21.50%) in C. tetragona and lignoceric acid (10.66 %) in C. ovata. Saturated fatty acids have altered effects on the concentration of plasma lipoprotein cholesterol fractions. For example, myristic (C14:0) and palmitic (C16:0) acids increase LDL cholesterol (C et al., 2003) those fatty acids were found in both plants.
Previous studies stated that oleic acid has a role in preventing in lammation of skeletal muscle cells (Coll et al., 2008). puri ied palmitoleic acid has shown anti-in lammatory activity (Bernstein et al., 2014). Moreover both linoleic acid and γ-linolenic acid play an important role in in lammation suppression through two distinct pathways; the irst one is through competitive inhibition of the activity of cyclooxygenase and lipoxygenase enzyme, resulting in decrease the production of pro-in lammatory substances prostaglandins and leukotrienes; while the second mechanism aid in the biosynthesis of prostaglandin E1 that has an inhibitory effect on pro-in lammatory cells (C et al., 2003).

Isolation
A phytochemical investigation was performed on the MeCl extract of the aerial parts of C. tetragona and C. ovata. Six compounds were isolated by different isolation methods illustrated in (Figure 4). Structure elucidation was achieved by NMR and mass spectrometry (Table 7). Compound 1 was found to be a novel cycloartan derivative identi ied as 28 Methyl-5α-cycloart12, 20, 24-trien-15β-ol., namely melilof icinaside. In addition to 5 known compounds, namely Campesterol (2) (Figure 6 ). The cycloartan nucleus was con irmed from the 1 HNMR and 13 CNMR data (Table 7 , Figures 7  and 8 ). All carbons and protons positions were con irmed by HSQC shown in Figure 9 The longrange coupling was observed in HMBC between    Figure 10. This compound was a novel compound identi ied as 28 Methyl-5α-cycloart12, 20, 24-trien-15β-ol.

CONCLUSION
This is the comparative study that shed light on the C. tetragona and C. ovata in the comparative demonic DNA investigation using 6 Primers for RAPD, and ISSR techniques showed a similarity in both species by 56.4% and 53.4% respectively. And on making a quantitative estimation showed the highest triterpene content. The investigation of the unsaponi iable matters of n-hexane extract of the species revealed the presence of 22 hydrocarbons and n-tricontane was the major hydrocarbon. In addition to seven steroidal components with the presence of campesterol as the major steroidal compound in C. tetragona and the presence of βsitosterol as the major steroidal compound in C. ovata. The investigation of FAME revealed the presence of 21 components in C. tetragona and C. ovata, and the major unsaturated fatty acid is Oleic /Elaidic (30.90%) found in C. tetragona and Arachiodonic acid (73.42%) in C. ovata. And a novel triterpene, namely, 28 Methyl-5α-cycloart12, 20, 24-trien-15β-Ol was isolated and identi ied from MeCl together with 5 known compounds.