Isolation and Identification of Flavonoids in Iraqi Rheum ribes Root

The aim of the study was isolate and identify the flavonoids in Iraqi Rheum ribes root, the results obtained from the HPLC analysis indicate that the Iraqi Rheum ribes root contain different types and concentration of flavonoids which include 29.9056 μg/g aloe-emodin, 252.880 μg/g emodin, 76.8493 μg/g chrysophanol, 56.1317 μg/g physician and 49.2098 μg/g rutin, while the types of flavonoids in the isolated extract of flavonoids from the Iraqi Rheum ribes root were 146.096 μg/g aleo-emodin, 37.174 μg/g emodin, 11.525 μg/g chrysophanol, 20.010 μg/g physcion, 30.271 μg/g rutin. The identification the all types of flavonoids in isolated flavonoids from the Iraqi Rheum ribes roots as compared with the flavonoids in the crude roots refers to the efficiency of the method for isolation of flavonoids from the roots of plant.


INTRODUCTION
Thousands of newer compounds and organic molecules in phytochemistry has been identified every year, different isolation methods, Pharmacological testing, biochemical methods were used to discovering and developing new drugs from secondary metabolite in plants, which used as drugs or to prevent various diseases, this compounds includes flavonoids, alkaloids and terpenoids, etc. [1,2].
Flavonoids are low molecular weight [3], bioactive poly-phenols [4], widely distributed among the plant kingdom, which play a vital role in photosynthesising cells [5].Flavonoids have been reported to exert a wide range of biological activities.These include: anti-inflammatory, antibacterial, antiviral, anti-allergic and anti-oxidant [5,6].Many types of flavonoids are used to treat common diseases, such as quercetin which used to treat herpes virus, and also to reduce the plasma bad fat [Low density lipoprotein-cholesterol] [7], while Cyandidanol-3, meciadanol and catechins used to treat Ulcer by its action to Gastric H+/K+ ATPase [8], also Genistein and Quercetin have anticancer effect [9], Fisetin and Quercetinare used as anti-diabetes [10].
Rheum ribes Linn (Polygonaceae) is widely distributed in the north of Iraq, Turkey, Iran, Afghanistan, Russia and Pakistan.Young shoots of the plants and petals are eaten as a vegetable in Iraq and also used to improve appetite and treat diarrhea [11,12].Roots of the plants are widely used in traditional medicine to treat ulcers, diarrhea, hemorrhoids and reported to have an antidiabetic effect [13], antibacterial activity [14] and antioxidant properties [15] due to the present of phenolic compounds (good natural antioxidants) [16], and also flavonoids such as Aloe-emodin, Emodin, Rhein, Chrysophanol, Kaempferol, …., etc. [15].
So that, the present study aims to isolate flavonoids from the roots of the plants, and evaluate the isolation method by identify the types of flavonoids in the isolated part and compare with flavonoids in crude roots.

MATERIAL AND METHODS
The general laboratory chemicals with highest purity are used in this study, which obtained from Sigma-Aldrich, BDH and GCC.

Plant Collection:
The Rheum ribes Linn roots were obtained from the local market of Kirkuk in north of Iraq.Authentication was done at the herbarium of the Faculty of Science, Tikrit University.

Extraction of Flavonoids:
The extraction of flavonoids from Rheum ribes was done according to the method of (Chen et al, method) with some modification [17].Firstly must remove the fatty contents by using soxhlet apparatus, in which 50g of the dried roots powder were extracted with 150ml of diethyl ether for 3 hours.The defatted powder was dried at 40°C in an air oven, and then extracted twice with condenser by using an ethanol solution as solvent for extraction [200ml (70%)] at 90℃ for 2h, filtration and then centrifugation at 3000 rpm for 15 min, evaporated the solvent and the aqueous extract was condensed under reduced pressure and then the extract was stored at 4℃ until used.

Quantitative determination of total flavonoid:
The quantitative determination of flavonoids in dried roots of Rheum ribes was done according to the aluminum chloride method (colorimetric method) [18].

Sample Preparation
One gram of Rheum ribes Linn roots powder was dissolved in 5ml of 80% ethanol in a test tube, mixed and used ultrasonic for 25min at 25°C, and then the supernatant was obtained after centrifugation at 7500rpm for 15min, after that the charcoal was used to removed dye from the extract, The solvent was evaporated to dryness by rotary evaporator at 35°C, and then dissolved with 1ml of ethanol and mixed well by vortex, 20μl was subjected to analysis by HPLC.

Method of Analysis
The sample flavonoids for the roots and for the isolated flavonoids were done according to the method of [19,20] with some modification, by using C-18 column (250 × 4.6 mm).The mobile phase consisted of linear graduated of solvent A(0.1% formic acid), solvent B(1:3:6)v/v acetonitrile, methanol and 0.1%formic acid, with flow rate1.2ml/min and the column temperature was kept at 40°C.The UV detection wavelength was 264 nm.

RESULTS AND DISCUSSION
The results indicate that the total flavonoid content in Iraqi Rheum ribes Linn roots was 22.680mg/gm which assessed by aluminum chloride method.This result was more than the results of Ibrahim et al. [21], which found that the ethanolic extract of Rheum ribes roots contain 687mg/100g of flavonoids and 149.01mg/100g in water extract.
The analysis of flavonoids by HPLC in Iraqi Rheum ribes Linn roots and its isolated flavonoids was carried out, Figure 1 showed the peaks of the standard flavonoids, with different Retention times -Rt, Table 1.
While the chromatograms of isolated flavonoids from the roots of Rheum ribes was shown in Figure 3, in which ten peaks of flavonoids were obtained with different Rt(1.23, 1.888, 2.398, 3.078, 3.318, 4.003, 4.208, 4.877, 5.14 and 5.807) min and the area under curves were (62348, 15119, 117329, 14506, 138104, 17186, 110687, 15595, 105869 and 22566).The results obtained from chromatograms of the crude roots and for the isolated flavonoids as compared with chromatogram of five standard flavonoids, as shown in Figure 1 and its Rt value in Table 1, indicate that crude roots may contain 29.9056μg/g aloe-emodin, 252.880 μg/g emodin, 76.8493μg/g chrysophanol, 56.1317μg/g physician and 49.2098 μg/g rutin.The other peaks were unknown, which may indicate to other types of flavonoids, so that we need to use more modern techniques such as HPLC-MS, GC-MS, or NMR or another type of flavonoids for HPLC analysis to identify all types of flavonoids in the roots.
The all types of flavonoids in crude roots were identified in isolated flavonoids, which include 146.096 μg/g aleo-emodin, 37.174 μg/g emodin, 11.525 μg/g chrysophanol, 20.010 μg/g physcion, 30.271 μg/g rutin and four unknown peaks.No information were available in the literature about the isolation or extraction of flavonoids from the roots of Rheum ribes, but this method of extraction was widely used to isolate of flavonoids with high yield, which may use in experimental study to identify its effect as anticancer, antidiabetic, antioxidant, antihyperlipidemic, …, etc., Al-Salihi et al. [24], extracted and isolated flavonoids from Iraqi date palm pollen by the same method used in the present study, and then study the hypolipidemic effect of the isolated flavonoids.
The identification the all types of flavonoids in isolated flavonoids from the Iraqi Rheum ribes roots as compared with the flavonoids in the crude roots refers to the efficiency of the method for isolation of flavonoids from the roots of plant.

CONCLUSION
The anti-oxidant properties of Rheum ribes roots, may be due to the high contain of flavonoids, which identified by HPLC.These types of flavonoids include aloe-emodin, emodin, chrysophanol, physcion and rutin which identified in crude roots or in isolated flavonoids from the roots.So that the isolation method, consider an appropriateness method for extraction of flavonoids from Rheum ribes roots.
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Figure 3 .
Figure 3. HPLC Analysis of Isolated Flavonoids from Rheum ribes roots

Table 1 .
Retention Times and Area Under Curves for Standard Flavonoids

Figure 2 .
HPLC Analysis of flavonoids in crude Iraqi Rheum ribes roots

Table 2 .
Retention Times and Area Under Curves for Iraqi Rheum ribes roots flavonoids Conc

Table 3 .
Retention Times and Area Under Curves for isolated flavonoids from Iraqi Rheum ribes Linn roots Conc.