Phamacognostical Evaluation and Determination of Total Phenols of Paeonia sinjiangensis K . Y .

Paeonia sinjiangensis K. Y. Pan is a perennial herb belonging to the family Ranunculaceae which is one of the most important crude drugs in traditional Chinese medicine, used as an anti-inflammatory, analgesic and sedative agent. This paper deals with the detailed pharmacognostical evaluation of the crude drug P. sinjiangensis K. Y. Pan. The microscopic, physico-chemical, preliminary physicochemical parameters, total phenols contents presented in this paper may be proposed as parameters to establish the authenticity of P. sinjiangensis K. Y. Pan and can possibly help to differentiate the drug from its other species.


Introduction
Paeonia sinjiangensis K. Y. Pan is belong to family Ranunculaceae which is a perennial herb.The plant is native of Xinjiang in China and naturalized in the Altai mountain area, mostly in the western Xinjiang region.As one of the most important crude drugs in traditional Chinese medicine, it has functions of inhibiting aggregation of platelet 1 and stimulating hepatic cell regeneration 2,3 , stabilizing erythrocyte membrane structure 4 , removing thrombus, preventing coagulation 5 , avoiding hepatic fibrosis 6 , stopping atherosclerosis, protecting heart and liver and antitumor 7 , etc.It is also frequently used as a remedy for diseases of women 8 .
In recent years, a large number of articles have been published on the study of the contents of paeoniflorin from Radix paeoniae rubra [9][10][11][12] .Our research group has studied the contents of paeoniflorin by rapid resolution liquid chromatography and polysaccharide with orthogonal test design from P. sinjiangensis K. Y. Pan 13,14 .However, from the large amount of concerned literature, it was found that few reports have been published regarding the phamacognostical evaluation and total phenols content of P. sinjiangensis K. Y. Pan from Xinjiang of China.So the aim of this study was to carry out a more exhaustive analysis of P. sinjiangensis K. Y. Pan.Thus, phamacognostical evaluation and determination of total phenols of P. sinjiangensis K. Y. Pan as the research object.In the present study, we initiated this study to phytochemical characterize and total phenols content of P. sinjiangensis K. Y. Pan from Xinjiang and reported the results in this paper.

Experimental
The plants were collected in October 2010, locally from the Altai mountain area of Xinjiang Province, China.The voucher specimen was authenticated as P. sinjiangensis K. Y. Pan by Yonghe Li, a chief apothecary of the Traditional Chinese Medicine Hospital of Xinjiang and accessioned into the herbarium of Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University for future reference (the voucher specimen number: 2010-356.) Solvents, namely, Folin-Ciocalteu phenol reagent, petroleum ether, chloroform, ethanol (95%), methanol, and reagents, namely, ammonia, iodine, ferric chloride, acetic acid, nitric acid, sulfuric acid, silicowolframic acid, hydrochloric acid, bromocresol green, α-naphthol, ninhydrin, gelatin, and so on, were purchased from Tianjin Fu-Yu Meticulous Chemical Reagent Company, China.

Microscopic studies
Microscopic studies were done by transferring the plants to powder (# 60).Observe powder features of hand sample slides 15 .

Ash values
Physicochemical Analysis was performed using standard procedures which are helpful in determining the quality and purity of crude drugs, especially in powder form 16 .
Total ash About 3 g powder was accurately weighed and taken in a crucible, which was previously ignited and weighed.The powder was spread as a fine, even layer on the bottom of the crucible.The crucible was incinerated gradually by increasing temperature to make it dull red hot until free from carbon.The crucible was cooled and weighed.The procedure was repeated to get constant weight 16 .

Acid insoluble ash
The ash obtained as described above was boiled with 10 ml of 2N HCl for ten minutes.The insoluble ash was collected on an ash less filter paper and washed with hot water.The insoluble ash was transferred into a crucible, ignited and weighed.The procedure was repeated to get a constant weight 16 .

Water soluble ash
The ash obtained as described in the determination of total ash was boiled for 5 minutes with 25 mL of water.The insoluble matter was collected on ash less filter paper and washed with hot water.The insoluble ash was transferred into crucible, ignited for 15 minutes and weighed.The procedure was repeated to get a constant weight.The weight of insoluble matter was subtracted from the weight of the total ash.The difference of weight was considered as water-soluble ash 16 .Fluorescence analysis was carried out according to the method of 16 and Chase and Pratt 17 .

Preliminary physicochemical screening
The powder of dried plants was subjected to continuous soxhlet extraction with various organic solvent such as petroleum ether, benzene, chloroform, methanol and ethanol respectively.Extractive values of crude drugs are useful for their evaluation, especially when the constituents of a drug can not be readily estimated by any other means.Further, there values indicate the nature of the constituents present in a crude drug 18 .After concentration and drying of each extract in vacuum desiccator, identification of phytoconstituents was carried out using chemical test.

Determination of polyphenols
Total phenols content in the ethanol extract was determined by the modified Folin-Ciocalteu method 19 .An aliquot of extract was mixed with 0.5 ml of Folin-Ciocalteu reagent and 1.5 mL of sodium carbonate (20 %).The tubes were vortexed for 20 sec and allowed to stand for 10 min at 75℃ for color development.Absorbance was then measured at 760 nm using UV-VIS spectrophotometer.The amount of total polyphenols in the extract was calculated from the calibration curve in terms of gallic acid equivalents (y=0.09221+137.25x,R=0.999).

Results and Discussion
The powder microscopy of the plant revealed the presence of fiber, non-glandular hairs, pollen grain, catheter, stomata, glandular scales and hairs, palisade cells (Figure 1).The proximate analysis result shown that the total ash value, acid insoluble ash value, water soluble ash value were 9.54±0.03%,0.83±0.14%and 5.59±0.03%,respectively (Table 1).
*Average of three determinations ±SEM.
*Average of three determinations.