|Ahmad Wasim1, Mujeeb Mohd1, Ahmad Sayeed1*, Zaidi S.M.A2*
|Corresponding Author: Dr. S. M. Arif Zaidi Department of Surgery, Faculty of Medicine, Jamia Hamdard, New Delhi-110062, India Email: [email protected]|
|Received: 22 January2013 Accepted: 20 February 2013|
|Citation: Ahmad Wasim, Mujeeb Mohd., Ahmad Sayeed1*, Zaidi S.M.A.* “Current strategy for research on quality identification of Rheum emodi Wall. rhizome” Int. J. Drug Dev. & Res., January- March 2013, 5(1): 297-304.|
|Copyright: © 2013 IJDDR, Ahmad Sayeed et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.|
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Pharmacognostical standardization of dried rhizome of Rheum emodi Wall. (Polygonaceae) was carried out as per the WHO, and Pharmacopeia guidelines for morphological, physicochemical (ash and extractive values) parameters, preliminary phytochemical screening, assay of total phenolics and flavonoids, determination of contaminants as well as fingerprint profiling using HPTLC and HPLC. The present study reveals standardization profile for Rheum emodi Wall., matching with the limits and standards of Pharmacopiea. The new parameters added by this communication for quality control of plant drugs are assay of phenolic and flavonoids, HPTLC and HPLC fingerprinting as well as cochromatography of R. emodi extracts with standards emodin and chrysophanic acid. The present communication may help in accurate identification of plant material and also to check adulteration, which will provide safer and better bio-efficacy of plant material.
Rheum emodi, Emodin, Chrysophanic acid, Standardization, Pharmacognosy, Physicochemical standards.
During the past decade, the therapeutic use of herbal medicine is gaining considerable momentum in the world. The use of herbal medicine, due to toxicity and side effects of allopathic medicines, has led to sudden increase in the number of herbal drug manufactures. Herbal medicines, as the major remedy in traditional system of medicine, have been used in medical practices since antiquity. The practices continue today because of its biomedical benefits as well as place in cultural beliefs in many parts of the world and have made a great contribution towards maintaining human health. Therefore, reproducible standards of each plant are necessary for effective quality control to prevent adulteration (1). Standardization is a system that ensures a predefined amount of quantity, quality and therapeutic effect of ingredients in each dose (2). Herbal products cannot be considered scientifically valid if the drug tested has not been authenticated and characterized in order to ensure reproducibility in the manufacturing of the product. Moreover, many dangerous and lethal side effects have recently been reported, including direct toxic effects, allergic reactions, effects from contaminants, and interactions with herbal drugs (3). Therapeutic activity of an herbal formulation depends on its phytochemical constituents. Thus, the present study deals with standardization of medicinal plant i.e. Rheum emodi Wall. Ex Meissn. (Polygonaceae), which is a perennial herb, commonly known as revand chini. It is the Himalayan species of Indian rhubarb found wild at an altitude of 4000-12000 feet in Kashmir, Nepal, Sikkim and Bhutan (4). The drug has been recommended as a purgative, stomachic, astringent, and as general tonic, also used in chronic bronchitis, asthma and in certain skin diseases (5,6). It contains a large number of anthraquinone derivatives such as physcion, chrysophanol, emodin, aloe emodin, emodin glycoside, rhein, etc (6-9), which are reportedly known for various biological activities (9- 10) such as anti-oxidant (11), anti-microbial (12), anti-fungal (13), cytotoxic (14), nephroprotective (15) and anti-viral activities (16). This plant has been included in Indian Pharmacopeia (18), since it is an important medicinal plant and explored traditionally in many system of medicine like Ayurveda and Unani etc. Keeping in mind, the importance of Rheum emodi rhizome and quality control parameters discussed till date, it was thought worthwhile to propose a new protocol by using conventional as well as modern analytical techniques, which will be useful in future and might become the part of Pharmacopeias/formulary for quality checking of R. emodi or the formulations containing it as an ingredient.
Folin Ciocalteu reagent was purchased from Sisco Research Laboratories Pvt. Ltd., India. Catechin and rutin were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and solvents used were of analytical grade available commercially.
The rhizomes of R. emodi were collected from Khari Baoli, local drug market, New Delhi, and were authenticated by Dr. H. B. Singh, Ref. NISCAIR/RHMD/1327/129, New Delhi. The rhizomes were dried and powdered in an electric grinder. The R. emodi powder (1.0 g) was extracted separately with 15 mL of methanol by sonication for 30 min at 45ºC. The process was repeated twice to ensure complete extraction. The extract obtained were pooled and dried under reduced pressure. The dried extract, was dissolved in HPLC grade methanol to get the concentration of 1.0 mg/mL and subjected to total phenolic and flavonoid content analysis, and for the fingerprint profile by HPTLC and HPLC. For HPTLC finger printing profile of chloroform and petroleum ether extract R. emodi powder (1.0 g) was extracted separately with 15 mL of chloroform and petroleum ether, respectively by sonication for 30 min at 45ºC temperature. Prior to use, all samples were filtered through a 0.45 μm nylon membrane filter.
Morphological observation of rhizomes of R. emodi was done for shape, size, surface characteristics, texture, color, consistency, odour, taste, etc
Physicochemical parameters were determined as per the WHO guidelines. Total ash value, loss on drying, water soluble ash, acid insoluble ash, alcohol soluble extractive and water soluble extractive values were determined (17-19).
The term “foreign matter” is used to designate any matter, which does not form a part of the drug as defined in the monograph. Hundred g of the powdered drug was taken and spread out as a thin layer. Plant material collected should be free from foreign matters like soil, insect parts or animal excreta. They were separated and weighed and the percentage was calculated.
The freshly prepared extract of R. emodi was qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using standard procedures (20-21).
The method of AOAC official method of analysis was followed for the determination of aflatoxins (21). The methanolic acidic extract was taken and partitioned with NaCl and hexane. In aqueous layer, dichloromethane was added. Process was repeated for 2-3 times to collect dichloromethane extract and then evaporated up to 2.0-3.0 mL. This extract was passed through silica gel column followed by washing the column with a mixture of benzene: acetic acid (9:1, v/v) and ether: hexane (3:1, v/v). Finally aflatoxin was eluted with 100 mL of dichloromethane: acetone (90:10, v/v). The eluted aflatoxins were concentrated upto 5.0 mL and were dried by passing nitrogen gas. Dried extract was taken and 200 μL of hexane followed by 50 μL of trifluoroacetic acid was added. This solution was then vortexed in a vortex mixture exactly for 30 seconds and allowed to stand for five minutes (exact). Finally, 1.95 mL mixture of water and acetonitrile (9:1, v/v) was added to this solution. Known concentration (20 ppb, 40 ppb, and 80 ppb) of standard aflatoxin B1, G1, B2 and G2 were taken and derivatized in the same manner as for sample. The analysis was carried out on a Waters Alliance e2695 separating module (Waters, USA). The derivatized samples (Both extract and standards) were injected into HPLC column (C18; 15 cm x 4.6 mm) and analysed using fluorescent detector. The peaks of aflatoxin in drug samples were compared with peak of standards (B1, G1, B2 and G2).
The 50 mg of sample was dissolved into methanol, 1.0 gm of sodium oxalate was added followed by addition of diethyl ether and petroleum ether 50 mL each. It was shaken for 1.0 min. Organic layer was transferred into separating funnel and 600 ml of distilled water was added with saturated solution of sodium chloride solution. Aqueous layer was discarded and the process was repeated thrice. Organic layer obtained was then passed over sodium sulphate and evaporated upto 2-5 mL. This concentrated solution was again mixed with acetonitrile (30 mL) and petroleum ether (30 mL), which was eluted with diethyl ether by passing over the column. The elute obtained was concentrated up to 5.0 mL using rotavapor (Buchi, R-215, Switzerland) and analysed in GC-MS (Agilent 7890A GC system, USA) by AOAC method (22).
Aluminum chloride colorimetric method was used for flavonoids determination (23). Plant extract (0.5 mL of 10 mg/mL) in methanol was separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1.0 M potassium acetate and 2.8 mL of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm (Shimadzu UV-Vis 1601). The calibration curve of rutin was prepared by preparing different dilutions in the concentrations range of 10-100 μg/mL in methanol.
Total phenols were determined by Folin Ciocalteu method (23). A dilute extract of plant material (0.5 ml of 10 mg/mL) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5.0 mL, 1:10 diluted with distilled water) and 1.0M aqueous sodium carbonate 4.0 mL. The mixtures were allowed to stand for 15 min and the total phenols were determined by colorimetry at 765 nm (Shimadzu UV-Vis 1601). The standard curve was prepared using 25, 50, 100, 150, 200, 250 and 300 μg/mL solutions of gallic acid in methanol.
Chromatography was performed on 5 × 10 cm aluminum HPTLC plates coated with 0.2 μm layers of silica gel 60F-254. Samples were applied as bands of 4.0 mm wide, 8.3 mm apart by the use of a CAMAG (Switzerland) Linomat V sample applicator fitted with a microlitre syringe. A constant application rate of 120 nL/s was used. Linear ascending development, to a distance of 80 mm, with toluene: ethyl acetate (9:1, v/v) for petroleum ether extract, toluene: ethyl acetate (8:1, v/v) for chloroform extract and for methanol extract using toluene: ethyl acetate: formic acid (7:3:1, v/v/v) used as mobile phases, was performed in a 10 × 10 cm twin-trough glass chamber. Before chromatography, the chamber was saturated with mobile phase vapors for 15 min; 10 mL mobile phase was used for each development. After the development, plates were dried in a current of air by means of an air dryer. Densitometric scanning at 440, 254 and 254 nm petroleum ether, chloroform and methanol extract, respectively was performed with a CAMAG TLC scanner III operated by winCats software. The source of radiation was a tungsten lamp, the slit dimensions was kept at 4.0 × 0.30 mm, and the scanning speed was 10 mm/s.
The HPLC-PDA method for the fingerprint profile was carried out on a Waters Alliance e2695 separating module (Waters Co., MA, USA) using photo diode array detector (Waters 2998) with autosampler and column oven. The instrument was controlled by use of Empower software installed with equipment for data collection and acquisition. Compounds were separated on a C18 reverse phase column (25 x 4.6 mm, particle size 5.0 μm, Merck, Germany) maintained at room temperature. The mobile phase consisted of acetonitrile and water in the ratio of 50:50 (v/v). The flow rate was 1.0 mL/min; and column was maintained at room temperature. Analysis was performed at a wavelength of 296 nm using 10 μL of injection volume.
Quantitative estimation of emodin and chrysophanic acid in Rheum emodi was carried out in-house developed simultaneous method using HPTLC and HPLC as described above.
The pharmacognostic study is the major and reliable criteria for identification of plant drugs. The pharmacognostic parameters are necessary for confirmation of the identity and determination of quality and purity of the crude drug. The detailed and systematic pharmacognostic evaluation would give valuable information for future studies.
R. emodi rhizome was solid, compact and cylindrical shaped, 7.5-10.0 cm in length and 2.5-3.5 cm in diameter. Outer surface was mostly irregular longitudinally wrinkled, furrowed or ridged but few of them showed transverse wrinkles and usually covered with brownish or yellowish brown or dark brown cortex. It has bitter taste and aromatic odor.
The quantitative determination of some pharmacognostic parameters is useful for setting standards for crude drugs. The physical constant evaluation of the drugs is an important parameter in detecting adulteration or improper handling of drugs. The moisture content of the drug was not too high, thus it could discourage bacterial, fungi or yeast growth. Equally important or the evaluation of crude drugs, is the ash value and the values for total, water soluble and acid?insoluble ash are given in Table 1. The total ash is particularly important in the evaluation of purity of drugs, i.e. the presence or absence of foreign inorganic matter such as metallic salts and/or silica.
Preliminary phytochemical screening of the extract of R. emodi revealed the presence of various bioactive components like flavonoids, phenolics, tannins, saponins, glycosides, resins, proteins and amino acids.
After the comparison of GC-MS chromatogram of samples with retention time of 31 standard pesticides, this was observed that R. emodi was not containing any pesticide. The aflatoxins (B1, B2, G1 and G2) analysis of R. emodi was also carried out by HPLC method and found freed from any type of aflatoxins (Limit NMT 4ppb for all B1, B2, G1 and G2).
Phenolics are the most wide spread secondary metabolite in plant kingdom. These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelator. It has been reported that the antioxidant activity of phenol is mainly due to their redox properties, hydrogen donors and singlet oxygen quenchers (24). Therefore, in the present study, total phenolic content present in extract was estimated using modified Folin- ciocalteau method. In R. emodi extract, the total phenolic content was found 0.945 ± 0.18% w/w.
It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable. The mechanisms of action of flavonoids are through scavenging or chelating process (25-26). Therefore, in the present study, total flavonoid content present in extract was estimated using Aluminum chloride colorimetric method. In R. emodi extract, the flavonoid content was found 2.79±0.43% w/w.
Well resolved HPTLC fingerprint profiles were recorded for the future reference and identity of the plant material. Fingerprint profile of petroleum ether extract showed seven spots after derivatization using anisaldehyde sulphuric acid at 440 nm, the chloroform extract showed eleven spots and seven spots in methanol extract under UV at 254 nm (Fig 1). Whereas HPLC chromatogram showed presence of 14 peaks in the methanolic extract of R. emodi rhizome at 296 nm (Fig 2).
Emodin and chrysophanic acid are hydroxy anthraquinone derivatives with potential biological significance, could be considered as a chemical marker for the standardization of R. emodi rhizomes. Methanolic extract of Rheum emodi rhizome contained 0.33%, 0.70% w/w of emodin and chrysophanic acid, respectively using simultaneous in-house developed HPTLC method, and 0.33%, 0.78% w/w by HPLC method.
The present study may be useful to supplement the information with regards to standardization and identification of R. emodi and in carrying out further research and its use in Unani/Ayurvedic system of medicine.
Authors are thankful to the CCRUM, Department of AYUSH, Ministry of Health and Family Welfare, Government of India, New Delhi India, for the financial support.
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